Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3′ end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3′ end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits. Synthesis of eukaryotic ribosomes relies on a large array of ribosome biogenesis factors (RBFs) that coordinate the multiple steps of pre-ribosomal RNA (pre-rRNA) modification, cleavage, and folding, together with ribosomal protein (RP) assembly . ProgresAfter initial assembly in the nucleolus, pre-40S particles are rapidly transported to the cytoplasm , where tUp to this final stage, the 3′ end of the 18S rRNA is extended with remnants of the internal transcribed spacer 1 (ITS1). In human cells, this last precursor to the 18S rRNA  is generated in the nucleolus by endonucleolytic cleavage of earlier pre-rRNAs 78 or 81 nucleotides downstream of the mature 3′ end . This 3′The RIO1 ATPase was shown to play a critical function in the last maturation step of the 18S rRNA , but theRibosomal protein RPS26/eS26, which is in close contact with the 18S rRNA 3′ end on the mature small ribosomal subunit, was also shown to be necessary for efficient cleavage by NOB1 in yeast and in human cells , but itsHere, we have used cryo-EM to solve the structures of human pre-40S particles trapped with a catalytically inactive form of RIO1 in this final maturation stage. Image analysis revealed two distinct structural states, prior and after 18S rRNA cleavage. In the pre-cleavage state, DIM2 and NOB1 are still in place, while the postion of RIO1 is not clearly defined and RPS26 is absent. In contrast, the post-cleavage state displays RIO1 and RPS26 stably associated with 40S particles, which contain mature 18S rRNA. In vivo assays confirmed the central role of RPS26 for triggering both processing of the 18S-E pre-rRNA and the release of DIM2, NOB1, and RIO1. In vitro, cleavage of the 18S-E pre-rRNA was partially stimulated by ATP binding to RIO1 in RPS26-depleted pre-40S particles and was enhanced by adding back purified RPS26. These data suggest a model in which ATP-bound RIO1 and RPS26 cooperatively displace DIM2 to activate the final cleavage of the 18S rRNA 3′ end by NOB1.Chaetomium thermophilum D281A ortholog  . Autophoortholog . Proteinortholog . SDS–PAGortholog . Bottom-ortholog , in addiortholog . Of noteortholog . Other pWe then performed cryo-EM and single-particle analysis on the particles purified using RIO1(kd)-StHA as bait. 2D classification assays performed with RELION  yielded State A harbored an rRNA scaffold closely resembling that of the mature 18S rRNA. On the head region, the three-way junction formed by rRNA helices h34, h35, and h38 is fully formed , while ROn the contrary, the cryo-EM map corresponding to state B harbored a density located on the intersubunit side, at the back of the head region, into which the X-ray structure of RIO1 could unambiguously be fitted . Of noteAs previously observed in the structure of wild-type RIO1 solved by X-ray crystallography , our cryIn state B, the rRNA scaffold harbors a fully mature conformation. Contrary to what was observed on the platform in state A, no density corresponding to nucleotides belonging to the ITS1 could be detected, suggesting that the 18S rRNA 3′ end is mature. Furthermore, neither NOB1 nor DIM2 could be found in this area; instead, a cryo-EM density sheathing the 18S rRNA 3′ end revealed the presence of RPS26 . These oWe conclude that states A and B of the pre-40S particles here isolated with RIO1(kd)-StHA correspond to two very late maturation stages, just before and right after cleavage of the 18S rRNA 3′ end. These data indicate that rRNA cleavage and release of NOB1 and DIM2 coincide with the association of RIO1 in a stable conformation within the pre-40S particle and the incorporation of RPS26 in its final location. RIO1 enzymatic activity, and more specifically the release of the ATP hydrolysis products, seems required for its own dissociation from the particle rather than for the 18S-E pre-rRNA processing by NOB1.States A and B of the RIO1(kd)-StHA pre-40S particles harbor two distinct conformations of the platform domain: in state A, the 18S-E pre-rRNA 3'-end is protected by DIM2, which prevents endonucleolytic cleavage by NOB1; in state B, the platform displays a mature conformation, with RPS26 replacing DIM2. Both RPS26 and RIO1 were shown to be necessary for processing of the 18S-E pre-rRNA to occur in human cells , which aIn order to assess the role of RPS26 binding to pre-40S particles in 18S rRNA maturation as well as DIM2 and NOB1 release, we analyzed the RNA and protein composition of intermediate and late pre-40S particles purified from cells depleted of RPS26 using a specific siRNA (siRPS26). The level of 18S-E pre-rRNA cleavage in pre-40S particles was measured by RNase H analysis in pre-40S particles isolated using different baits (RIO1(kd)-StHA or RIO1(wt)-StHA, HASt-LTV1) . The amoWe then used western blot analyses to monitor how RPS26 depletion influences the release of NOB1 and DIM2 . As expeComparative proteomics analyses of RIO1(kd)-StHA-associated pre-40S particles in the absence or presence of RPS26 confirmed that no other RPS was lost upon RPS26 depletion . This exSaccharomyces cerevisiae have shown that rRNA cleavage by Nob1 in cytoplasmic pre-40S particles purified with tagged Rio1 was stimulated in vitro by ATP binding to Rio1  triggers the recruitment of RPS26 to the pre-40S particle, which in turn further displaces DIM2 from cleavage site 3. This cooperative action of RIO1 and RPS26 would then lead to a putative, probably short-lived cleavage-competent state , in whicIn yeast, expression of the Rio1-D244A mutant  was shown to promote the accumulation of 80S-like particles, with which it co-purifies. In these 80S-like particles, pre-40S particles containing unprocessed 20S pre-rRNA, the last precursor to the 18S rRNA in yeast, are associated with large ribosomal subunits . Such paThe function of RIO1 in the release of DIM2 and NOB1 echoes the roles that other NTPases play in pre-ribosomal particle remodeling along the maturation pathway. Their nucleotide-hydrolyzing activity was shown to power the release of other RBFs, as well as their own release, contributing to the unidirectionality of the maturation . NTPasesCVCL_U427) and expressing HASt-DIM2, HASt-LTV1, RIO1(wt)-StHA, and RIO1(D324A)-StHA have been described previously  or EIF1AD (5′-ACCGCAGACAGUAUCAUGAGA-3′) (Eurogentec) was added at a final concentration of 500 nM to 100 µL of cell suspension . After electrotransformation at 240 V, cells were plated and collected 48 hr later. Control cells were electro-transformed with a scramble siRNA. Knockdown efficiency of siRNAs was assessed by quantitative PCR, using HPRT1 as an internal control.To knockdown expression of the corresponding human protein genes, siRNA duplex of RPS26  using a dounce homogenizer. Lysed cells were centrifuged , and the lysate was incubated with EZview Red Anti-HA Affinity Gel (Sigma-Aldrich) for 2 hr in an overhead shaker. For electron microscopy studies, beads were washed six times with TAP buffer  and eluted by incubation in TAP buffer supplemented with 0.2 mg/mL HA peptide (Sigma-Aldrich). Eluates were washed and concentrated by Vivacon 2, 100 kDa MWCO centrifugal devices (Sartorius). For subsequent analyses by silver staining, western blotting, and mass spectrometry, beads were washed three times with TAP buffer and bound material was eluted with SDS sample buffer.To purify pre-ribosomal particles, the protocol described in Following TAP purification, 100 μL of concentrated RIO1 pre-40S particles, corresponding to 3.5 µg of protein, was reduced by incubation for 5 min at 95°C with 5 µL of Laemmli buffer containing 30 mM DTT and then alkylated with 90 mM iodoacetamide for 30 min at room temperature in the dark. The reduced/alkylated sample was then loaded onto SDS–PAGE gel (stacking 4% and separating 12% acrylamide). For one-shot analysis of the entire mixture, no fractionation was performed, and the electrophoretic migration was stopped as soon as the protein sample entered the separating gel as one single band. The proteins, revealed with Instant Blue (Expedeon) for 20 min, were found in one blue band of around 5 mm width. Single slice containing the whole sample was cut and washed before the in-gel digestion of the proteins overnight at 37°C with a solution of modified trypsin . The resulting peptides were extracted from the gel by one round of incubation  in 1% formic acid–acetonitrile (40%) and two rounds of incubation  in 1% formic acid–acetonitrile (1:1). The extracted fractions were air-dried. Tryptic peptides were resuspended in 14 µL of 2% acetonitrile and 0.05% trifluoroacetic acid (TFA). Nano-LC-MS/MS analysis was performed in duplicate injections using an Ultimate 3000 nanoRS system  coupled with an LTQ-Orbitrap Velos mass spectrometer  operating in positive mode. Five microliters of each sample was loaded onto a C18-pre-column (300 μm inner diameter × 5 mm) at 20 μL/min in 2% ACN, 0.05% TFA. After 5 min of desalting, the pre-column was switched online with the analytical C18 nanocolumn  equilibrated in 95% solvent A  and 5% solvent B . Peptides were eluted by using a 5–50% gradient of solvent B for 105 min, at a flow rate of 300 nL/min. The LTQ-Orbitrap Velos was operated in data-dependent acquisition mode with the XCalibur software. Survey scans MS were acquired in the Orbitrap, on the 300–2000 m/z (mass to charge ratio) range, with the resolution set to a value of 60,000 at m/z 400. Up to 20 of the most intense multiply charged ions (>2+) per survey scan were selected for CID (collision-induced dissociation) fragmentation, and the resulting fragments were analyzed in the linear ion trap (LTQ). Dynamic exclusion was used within 60 s to prevent repetitive selection of the same peptide.Each protein sample corresponding to RIO1(kd)-StHA pre-40S particles purified from RPS26 depleted cells and control cells  was reduced with 6 µL of 1 M DTT  during 10 min at 95°C under agitation, cooled to room temperature, and alkylated with 19 µL of 1 M iodoacetamide  during 30 min in the dark. Then, each sample was digested with trypsin using the S-Trap Micro spin column protocol  according to the manufacturer’s instructions . The poohttp://www.matrixscience.com) database search engine was used for peptide and protein identification using automatic decoy database search to calculate a false discovery rate (FDR). MS/MS spectra were compared to the SwissProt H. sapiens database (supplemented with the sequence of the D324A RIO1 mutant). MS mass tolerance was set at 5 ppm and MS/MS mass tolerance was set at 0.8 Da and 0.02 Da for data acquired on LTQ-Orbitrap Velos and Orbitrap Fusion Tribrid mass spectrometers, respectively. The enzyme selectivity was set to full trypsin with two missed cleavages allowed. Protein modifications were fixed carbamidomethylation of cysteines, variable phosphorylation of serine, threonine and tyrosine, variable oxidation of methionine, variable acetylation of protein N-terminus. Proline software  according to the log2 ratio -StHA particles (with RNA concentrations of ~60 ng/µL as estimated by Nanodrop measurement) was deposited onto QUANTIFOIL holey carbon grids . Grids were plunge-frozen using a Leica EM-GP automat; temperature and humidity level of the loading chamber were maintained at 20°C and 95%, respectively. Excess solution was blotted with a Whatman filter paper no. 1 for 1.7–1.9 s, and grids were immediately plunged into liquid ethane (−183°C).Images were recorded on a Titan Krios electron microscope  located at EMBL, Heidelberg, Germany. The cryo-electron microscope was operating at 300 kV and was equipped with a Gatan K2 summit direct electron detector using counting mode. Automatic image acquisition was performed with SerialEM, at a magnification corresponding to a calibrated pixel size of 1.04 Å and a total electron dose of 29.88 e-/Å2 over 28 frames. Nominal defocus values ranged from −0.8 µm to −2.8 µm.Nine thousand four hundred and ninety-four stacks of frames were collected at EMBL. Frame stacks were aligned to correct for beam-induced motion using MotionCor2 . ContrasAtomic models of pre-40S particle (PDB-ID 6G51)  or the mFinal atomic models of states A (pre-cleavage) and B (post-cleavage) pre-40S particles were refined using REFMAC5  and PhenMaps and models visualization was done with Coot and UCSF Chimera; figures were created using UCSF Chimera and ChimeraX .DNA/RNA reverse probe hybridizing in the 3′-end of 18S rRNA . After annealing by cooling down to room temperature for 20 min, the reaction mixture was diluted to 30 µL with a reaction mix containing 1X RNase H reaction buffer, 25 µM DTT, 0.5 U/l RNasin (Promega), and 50 U RNase H (New England Biolabs), and incubated at 37°C for 30 min. The reaction was then blocked by addition of 0.3 M sodium acetate, pH 5.2 and 0.2 mM EDTA, and the RNAs were recovered by ethanol precipitation after phenol–chloroform–isoamylalcohol (25:24:1) extraction.For RNase H digestion assays, 250 ng of pre-40S purified RNAs were denatured at 95°C for 6 min with a RNA/GATCCTTCCGCAGGTTCACCTACG-3′) was added after 1 hr and incubated overnight at 45°C. Membranes were washed twice for 10 min in 2X SSC and 0.1% SDS and once in 1X SSC and 0.1% SDS, and then exposed. Signals were acquired with a Typhoon Trio PhosphorImager and quantified using the ImageLab software.RNAs were then separated on a 12% polyacrylamide gel (19:1) in 1X TBE buffer containing 7 M urea. RNAs were transferred to Hybond N + nylon membrane  and crosslinked under UV light. Membrane pre-hybridization was performed at 45°C in 6X SSC , 5X Denhardt’s solution, 0.5% SDS, and 0.9 mg/mL tRNA. The 5′-radiolabeled oligonucleotide probe 3′18S (5′-The RNA ladder corresponds to a 131 nucleotides sequence comprising the 3’18S probe and the T7 promoter sequence . Using tEscherichia coli BL21-CodonPlus (DE3) strain carrying pET29b-hRPS26-HisTag was induced by addition of 0.5 mM IPTG and incubated O.N at 20°C. Cells were pelleted and lysed by sonication in 20 mM HEPES, 50 mM NaCl, 2 mM EDTA, 10% glycerol, 20 mM Imidazole, and (EDTA)-free protease inhibitor . The extract was treated with 750 U of Benzonase 30 min at room temperature under agitation and then clarified by centrifugation and filtration (0.22 µm). The recombinant protein was purified from the soluble fractions using FPLC , by a nickel-attached HiTrap chelating column  with a linear gradient of 20–500 mM imidazole. The eluted fractions containing His-tagged proteins were pooled, dialyzed, and concentrated on Amicon 10 kDa MWCO centrifugal devices (Sartorius) with dialysis buffer . The purity of the protein was finally checked by SDS–PAGE 12% followed either by Coomassie blue staining or by western blot (hRPS26-HisTag construct was cloned into pET29b vector using NdeI/KpnI (GenScript). ern blot .The method of pre-40S particle purification described above was followed, and instead of elution, beads were then washed three times with 1 mL of TAP buffer. Most of the supernatant was discarded, and 50 μL of buffer X  added to the beads (suspended in approx. 20 μL of remaining TAP buffer) to reach a final volume of 70 μL. Nucleotides at a final concentration of 1 mM and/or 2 µg of hRPS26 were added when required. Reactions were incubated at 37°C for 1 hr, and RNAs were then immediately extracted with Tri Reagent. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study is significant in providing high-resolution cryo-EM images of the final two steps of 40S biogenesis in human cells, which provide structural insights into the roles of ribosome biogenesis factor Dim2 in blocking cleavage of the pre-rRNA by the endonuclease Nob1, and of the kinase Rio1 and 40S subunit protein Rps26 in displacing Dim2 to allow 18S rRNA cleavage by Nob1. Rps26 knockdown experiments add important biochemical evidence that Rps26 is critical for 18S rRNA cleavage and that it also enhances dissociation of Dim2 and Nob1; and in vitro cleavage experiments reconstitute the stimulatory function of Rp26 in 18S rRNA processing and confirm that ATP binding to Rio1 is sufficient to stimulate 18S rRNA cleavage by Nob1. The work also highlights a difference between human and yeast, as Nob1 is shown here to function on 40S subunits rather than on 80S ribosomes.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Cynthia Wolberger as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Arlen W Johnson (Reviewer #2); John Woolford (Reviewer #3).Thank you for submitting your article "The final step of 40S ribosomal subunit maturation is controlled by a dual key lock" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:This paper presents cryo-EM structures of immature 40S subunits affinity-purified with a catalytically defective form of Rio1, in two major states. State A contains Nob1 and Dim2 but lacks Rps26 as well as density for the mutant Rio1-kd protein itself; and it has unprocessed 18S rRNA. Dim2 is seen to interact with the site of Nob1 cleavage in this state. State B lacks Nob1 and Dim2 but contains both Rps26 and Rio1-kd, and the 18S rRNA is processed. These findings suggest that these structures represent the final two steps in 18S rRNA processing, which appear to occur on 40S pre-ribosomes rather than in 80S complexes as seen in yeast. Comparing the two structures predicts a steric clash between Rio1 and Dim2, which might initiate Dim2 dissociation and attendant Rps26 binding to replace Dim2, as Rps26 occupies the same position on the 40S as Dim2. A role for Rps26 in stimulating 18S rRNA cleavage, presumably by preventing Dim2 association and steric hindrance of Nob1 access to the cleavage site, was provided by si-RNA knockdown of Rps26, which decreases the cleavage of 18S rRNA, while increasing occupancy of Nob1 and Dim2 in the pre-40S particles. In state B, Rio1-kd is bound to ADP and its Asp-D341 carries a phosphate group, suggesting that the kd mutation does not prevent ATP binding and hydrolysis, but rather release of the products of ATP hydrolysis. Supporting this inference, they show that adding ATP or non-hydolyzable AMP-PNP confers similar amounts of 18S rRNA cleavage in the pre-ribosomes affinity purified in association with Rio1-kd. Together, these results suggest the model that Rio1 and Rps26 function in a coordinated manner to displace Dim2 and enable access of Nob1 to the cleavage site, and that completion of ATP hydrolysis is necessary for release of Rio1 from the final, mature 40S subunit.Essential revisions:The reviewers had some concerns about the novelty of the main conclusions and the final model in Figure 7, which seemed to be fairly well supported by previous results already obtained in yeast; however, they agreed that it was important to address the mechanism in humans, and noted that the finding that cleavage apparently occurs on 40S rather than 80S pre-ribosomes represents an important difference with yeast. Obviously, providing data from high-resolution structures in support of the model is also quite valuable. At the same time, the reviewers felt that you should attempt to add more scientific value to the paper by performing a few additional experiments to support a direct, critical role for Rps26 in the proposed mechanism. This would entail (i) demonstrating that 40S particles affinity-purified with Rio1 from Rps26-repressed cells are defective for Nob1 cleavage compared to those purified from Rps26-replete cells, and (ii) attempt to restore Nob1 cleavage and evoke release of Dim2 and Nob1 to the Rps26-depleted particles by adding back purified Rps26.Second, it is important to perform mass-spectrometry on the Rio1-associated 40S subunits following repression of Rps26 to insure that no other Rps proteins have been lost, in an effort to support the conclusion that Rps26 alone is required in conjunction with Rio1 for stimulating rRNA cleavage and dissociation of Dim2 and Nob1.A third major point is to explain how you can differentiate between ADP/D341-P versus ATP in the EM density map.Fourth, it is requested that you plot the cleavage data in a more straightforward and less misleading way, by providing the fraction cleaved (18S/(18S+18SE)).Fifth, you should address whether the data in Figure 6A-B indicate significantly greater 18S rRNA cleavage in the 40S ribosomes containing WT Rio1 vs. the Rio1-kd mutant, which, if so, would seem to be at odds with your conclusion that ATP binding and hydrolysis, but not release of the hydrolysis products by Rio1 is sufficient for 18 rRNA cleavage.Finally, you should attempt to revise the text to address all four major comments of Referee # 3; and also comment on whether your results can be reconciled with previous findings indicatingTsr2 loading of Rps26 in the nucleus-eg. might Rps26 associate with nuclear pre-40S particles and then undergo a rearrangement in the cytoplasm to help displace Dim2?Reviewer #1:Previous work has implicated NOB1 in the final processing of the 18S rRNA precursor in cytoplasmic 40S pre-ribosomes, and showed that DIM2 restricts this endonucleolytic cleavage activity. It was also shown previously that Rio1 and Rps26 are necessary for cleavage by Nob1, and for release of Dim2 and Nob1 from the mature 40S particle. This paper presents cryo-EM structures of 40S subunits affinity-purified with a catalytically defective form of Rio1, in two major states. State A contains Nob1 and Dim2 but lacks Rps26 as well as density for the mutant Rio1-kd protein itself; and it has unprocessed 18S rRNA. Dim2 is seen to interact with the site of Nob1 cleavage in this state. State B lacks Nob1 and Dim2 but contains both Rps26 and Rio1-kd, and the 18S rRNA is processed. These findings suggest that these structures represent the final two steps in 18S rRNA processing, which appear to occur on 40S pre-ribosomes rather than in 80S complexes as seen in yeast. Comparing the two structures predicts a steric clash between Rio1 and Dim2, which might initiate Dim2 dissociation and attendant Rps26 binding to replace Dim2, as Rps26 occupies the same position on the 40S as Dim2. A role for Rps26 in stimulating 18S rRNA cleavage, presumably by preventing Dim2 association and steric hindrance of Nob1 access to the cleavage site, was provided by si-RNA knockdown of Rps26, which decreases the cleavage of 18S rRNA, while increasing occupancy of Nob1 and Dim2 in the pre-40S particles. In state B, Rio1-kd is bound to ADP and its Asp-D341 carries a phosphate group, suggesting that the kd mutation does not prevent ATP binding and hydrolysis, but rather release of the products of ATP hydrolysis. Supporting this inference, they show that adding ATP or non-hydolyzable AMP-PNP confers similar amounts of 18S rRNA cleavage in the pre-ribosomes affinity purified in association with Rio1-kd. Together, these results suggest the model that Rio1 and Rps26 function in a coordinated manner to displace Dim2 and enable access of Nob1 to the cleavage site, and that completion of ATP hydrolysis is necessary for release of Rio1 from the final, mature 40S subunit.The results appear to be significant in providing high resolution cryo-EM images of the likely final two steps of 40S biogenesis, and provide structural insights into the roles of Dim2 in blocking cleavage of the pre-rRNA by Nob1, and of Rio1 and Rps26 in displacing Dim2 to allow 18S rRNA cleavage by Nob1. The Rps26 knockdown experiments add further evidence supporting previous findings that Rps26 is important for 18S rRNA cleavage and provide evidence that it also enhances dissociation of Dim2 and Nob1; and the in vitro cleavage experiments support previous findings from yeast that ATP binding to Rio1 is sufficient for 18S rRNA cleavage by Nob1.1. While the cryo-EM structures are certainly valuable, there is a concern that the major conclusions of the paper, embodied in the model in Figure 7, were already very likely to be true based on previous findings from yeast, cited in the Introduction, that Dim2 restricts endonucleolytic cleavage by Nob1 by masking the cleavage site, that Nob1 and Dim2 dissociate only after 18S rRNA cleavage, that Rio1 and Rps26 are both necessary for cleavage by Nob1, and that Rio1 is also necessary for the release of Dim2 and Nob1 from the mature 40S subunit.2. It might be necessary to perform MS analysis on the Rio1-associated 40S subunits following knock-down of Rps26 to insure that no other Rps proteins are lost in order to bolster the conclusion that Rps26 alone is required in conjunction with Rio1 for stimulating rRNA cleavage and dissociation of Dim2 and Nob1.3. Results in Figure 6A-B seem to indicate significantly greater 18S rRNA cleavage in the 40S ribosomes containing WT Rio1 vs. the Rio1-kd mutant, which seems at odds with the conclusion that ATP binding and hydrolysis, but not release of the hydrolysis products by Rio1 is sufficient for 18 rRNA cleavage. This needs to be addressed.4. It may be important to show directly that Rps26 is required for 18S rRNA cleavage by carrying out in vitro cleavage assays like those shown in Figure 6 following knock-down of Rps26.Reviewer #2:Ribosome assembly is a complex pathway that is thought to involve quality control checks to ensure the correct assembly of ribosomes. In yeast, a major event in assessing the assembly of small subunits is the formation of an 80S-like complex to promote final cleavage of 18S rRNA in the cytoplasm. However, less is known about the pathway of ribosome assembly in humans. In this manuscript, the authors use cryo-EM to analyze particles affinity purified from human cells with a catalytic mutant of RIO1 kinase. The comparable mutant in yeast associates with 80S-like particles. The authors resolve two apparently sequential states of 40S maturation that represent intermediates before and after cleavage at site 3 to generate the mature 3'-end of 18S. Surprisingly, the authors find no evidence for an 80S-like particle. Consistent with earlier work from yeast, the authors show that cleavage at site 3 depends on RIO1 and is stimulated by ATP. Their data also strongly implicate the loading of RPS26 in displacement of DIM2 to allow cleavage by NOB1. A role for Rps26 in cleavage of pre-18S rRNA has also previously been reported in yeast. In general the data are clearly presented and the manuscript is well-written.1. The authors present the model that RIO1, together with RPS26, displace DIM1 to promote cleavage at site 3 by NOB1. The authors have nearly all the tools to test this in vitro. Does the addition of RPS26 to particles affinity purified with RIO1 from RPS26-repressed cells promote NOB1 cleavage and release of DIM2?2. In Figure 3C the authors suggest that RIO1 contains ADP and phospho D341. It is not clear from the figure that this is the case and not ATP. A supplemental figure showing the fit of ADP and P-D341 vs ATP in the density map would help make this point.3. In Figures4C, 5B and 6B the data are not displayed appropriately. The authors plot the ratio of 18S/18SE. However, this misleadingly exaggerates the results: as the efficiency of cleavage approaches 100%, this ratio goes to infinity. The authors should plot this as fraction cleaved (18S/(18S+18SE)).Reviewer #3:During late stages of eukaryotic ribosome assembly the final step of pre-rRNA processing occurs and the last four ribosome biogenesis factors are released. Work described here provides a more detailed understanding of this process for human pre-40S particles: assembly factor DIM2 prevents access of the NOB1 endonuclease to its cleavage site. DIM2 is displaced by entry of RIO1 and RPS26, enabling cleavage of the 20S pre-rRNA by NOB1. A conformational change in RIO1 triggers its dissociation. Experiments in this well written manuscript provide strong support for this model, which extends our understanding of these steps from what had previously been found in yeast.The manuscript could be improved by addressing the following:1. In the Introduction, or at the beginning of Results, the authors might provide a little more information about RIO1 autocatalytic activity and its putative role, before describing its use as an affinity purification bait. Can they estimate how much activity remains in the D324A mutant that they use?2. That the bait protein Rio1 cannot be visualized in State A particles might be confusing to some readers. The authors suggest that it is loosely bound and falls off during purification. They should provide a slightly more detailed explanation of when during purification this might occur…certainly after attachment to the affinity gel. It also seems reasonable that RIO1 is present but particularly flexible in state A particles.3. Page 11: Is there any evidence for how RPS26 initially contacts binds to preribosomes? Might it bind near DIM2, then undergo a series of conformational shifts to help displace DIM2?4. In my opinion, an interesting question in ribosome assembly is how is each assembly factors is released from preribosomes. There are more than 200 assembly factors , but many many fewer NTPases. Does each NTPase help many AFS depart or are there additional release mechanisms, or both? The authors might highlight this issue a little more, e.g., by comparing what they found with a few other examples from the literature, especially if there are any cases where NTPases are not yet known to be involved in an AF release. How does binding of one protein displace another? Essential revisions:The reviewers had some concerns about the novelty of the main conclusions and the final model in Figure 7, which seemed to be fairly well supported by previous results already obtained in yeast; however, they agreed that it was important to address the mechanism in humans, and noted that the finding that cleavage apparently occurs on 40S rather than 80S pre-ribosomes represents an important difference with yeast. Obviously, providing data from high-resolution structures in support of the model is also quite valuable. At the same time, the reviewers felt that you should attempt to add more scientific value to the paper by performing a few additional experiments to support a direct, critical role for Rps26 in the proposed mechanism. This would entail:(i) Demonstrating that 40S particles affinity-purified with Rio1 from Rps26-repressed cells are defective for Nob1 cleavage compared to those purified from Rps26-replete cells.As shown in Figure 5c, particles purified with tagged wild-type RIO1 are devoid of RPS26 even in cells expressing RPS26, and contain DIM2 and NOB1 like the particles purified from RPS26-depleted cells. The new mass spectrometry data provided here further show that the composition of pre-40S particles isolated from RPS26-depleted cells is not different from that purified in control cells, beyond the absence of RPS26. RPS26 appears to be the trigger of cleavage, and we could not isolate uncleaved particles containing RPS26. Similarly, Ameismeier et al. (2020) did not identify particles containing DIM2 and NOB1 together with RPS26. Therefore, we felt that the only way to directly show the function of RPS26 was to use the in vitro approach.(ii) Attempt to restore Nob1 cleavage and evoke release of Dim2 and Nob1 to the Rps26-depleted particles by adding back purified Rps26.As requested, we have performed in vitro cleavage experiments which show that adding back purified S26 to RIO1-containing/S26-depleted particles stimulates 18S-E cleavage. This effect is potentialized by the combined addition of ATP. We have modified the manuscript text accordingly: in the introduction section (last two sentences):“in vitro, cleavage of the 18S-E pre-rRNA was partially stimulated by ATP binding to RIO1 in RPS26-depleted pre-40S particles, and was enhanced by adding back purified RPS26. These data suggest a model, in which ATP-bound RIO1 and RPS26 cooperatively displace DIM2 to activate the final cleavage of the 18S rRNA 3’ end by NOB1.”In the Results section, p. 10, the Results section title was modified as follows:“ATP binding by RIO1 and addition of RPS26 stimulate in vitro rRNA cleavage by NOB1 in RPS26-depleted pre-40S particles”, and within the section (p.10):“We next sought to assess the role of RPS26 and produced recombinant human RPS26 .[…] These data show that in vitro cleavage by NOB1 in pre-40S-particle is stimulated both by RIO1 and RPS26 and support the hypothesis of a cooperative action of RPS26 and RIO1 in triggering the final step of 18S rRNA maturation..”Second, it is important to perform mass-spectrometry on the Rio1-associated 40S subunits following repression of Rps26 to insure that no other Rps proteins have been lost, in an effort to support the conclusion that Rps26 alone is required in conjunction with Rio1 for stimulating rRNA cleavage and dissociation of Dim2 and Nob1.We have performed the required proteomics experiments, and compared the proteic composition of RIO1(kd)-StHA purified particles in presence or in absence of RPS26. The corresponding figure is presented as Figure 5—figure supplement 1. Overall, this confirmed that RPS26 depletion does not affect the abundance levels of other RPS within RIO1(kd)-associated particles. However, we noticed that together with RPS26, EIF1AD was also significantly less purified upon RPS26 depletion, suggesting a putative regulation link between these two proteins. Since the purification bait, RIO1, is known to be associated with both methylosome and pre-40S particles, we have also monitored the level of EIF1AD within pre-40S particles by measuring the EIF1AD/RPS19 ratio on western blot experiments. This shows only a mild reduction (~25%) of EIF1AD within pre-40S particles upon RPS26 depletion.To summarize these observations, we have added the following sentence to the Results section, p.9 : “However, upon RPS26 depletion, RIO1(wt)-StHA co-purified with late cytoplasmic pre-40S particles, as attested by the presence of NOB1 and DIM2 , as well as that of 18S-E pre-rRNA . These data indicate that RIO1 association to late cytoplasmic pre-40S particles is stabilized in the absence of RPS26, while release of NOB1 and DIM2 is inhibited.“Comparative proteomics analyses of RIO1(kd)-StHA associated pre-40S particles in the absence or presence of RPS26 confirmed that no other RPS was lost upon RPS26 depletion . […] Altogether, these data lead us to conclude that RPS26 intervenes directly in the mechanism triggering rRNA cleavage by NOB1 at site 3 and dissociation of NOB1, DIM2 and RIO1 from pre-40S particles.”A third major point is to explain how you can differentiate between ADP/D341-P versus ATP in the EM density map.We have added a supplementary figure  showing the cryoEM map fitted with either ADP and P-D341 or ATP, which better illustrates this point.Fourth, it is requested that you plot the cleavage data in a more straightforward and less misleading way, by providing the fraction cleaved (18S/(18S+18SE)).All figures showing cleavage analysis have been modified according to this request.Fifth, you should address whether the data in Figure 6A-B indicate significantly greater 18S rRNA cleavage in the 40S ribosomes containing WT Rio1 vs. the Rio1-kd mutant, which, if so, would seem to be at odds with your conclusion that ATP binding and hydrolysis, but not release of the hydrolysis products by Rio1 is sufficient for 18 rRNA cleavage.2+ instead of Mn2+ as a divalent cation. The new plots do not indicate greater cleavage in pre-40S containing RIO1(wt) vs. the RIO1(kd) mutant in the presence of ATP . We do observe however that cleavage is somewhat more efficient with ATP than with AMP-PNP in pre-40S particles containing wild-type RIO1 . Such a difference is also visible for RIO1(kd) containing pre-40S particles, even though it fails to be statistically significant in these experiments (p=0.063 with a stringent paired non-parametric test). This result fits with our cryo-EM observation that the catalytic site of RIO1 contains ADP+Pi rather than ATP in the post-cleavage state, which strongly suggests that cleavage is favored by ATP hydrolysis. Overall, the results of these in vitro experiments do support our conclusion that ATP binding and hydrolysis is sufficient for 18S rRNA cleavage, while release of hydrolysis products appears to be dispensable (or at least not strictly required), there again consistent with the presence of ADP-Pi in RIO1 catalytic site in post-cleavage state B.We added a new series of cleavage experiments with more points to gain statistical power (figure 6c), and modified figure 6 to represent the fraction of cleaved rRNA as suggested above (18S/(18S+18SE)). Furthermore, we improved cleavage efficiency by using MgFinally, you should attempt to revise the text to address all four major comments of Referee # 3; and also comment on whether your results can be reconciled with previous findings indicatingTsr2 loading of Rps26 in the nucleus-eg. might Rps26 associate with nuclear pre-40S particles and then undergo a rearrangement in the cytoplasm to help displace Dim2?In our opinion, the timing of association of RPS26 to pre-ribosomal particles is still a matter of debate in yeast, and to our knowledge, has never been studied in human cells. This chronology of association might differ between human and yeast. However we acknowledge that the work of Vikram Panse’s lab on the interplay between Rps26 and Tsr2 should clearly be discussed in this manuscript. Thus, in the introduction section (p.4) , we have added the following paragraph:“Like RIO2, another ATPase of the same family that intervenes earlier in pre-40S particle maturation, RIO1 adopts different conformations depending on its nucleotide binding state . […] One cannot exclude that association of RPS26 to nuclear precursors might be highly labile and becomes more stable only during later maturation steps, but evidence is still lacking to date for the presence of human RPS26 in early precursors.”Reviewer #1:Previous work has implicated NOB1 in the final processing of the 18S rRNA precursor in cytoplasmic 40S pre-ribosomes, and showed that DIM2 restricts this endonucleolytic cleavage activity. It was also shown previously that Rio1 and Rps26 are necessary for cleavage by Nob1, and for release of Dim2 and Nob1 from the mature 40S particle. This paper presents cryo-EM structures of 40S subunits affinity-purified with a catalytically defective form of Rio1, in two major states. State A contains Nob1 and Dim2 but lacks Rps26 as well as density for the mutant Rio1-kd protein itself; and it has unprocessed 18S rRNA. Dim2 is seen to interact with the site of Nob1 cleavage in this state. State B lacks Nob1 and Dim2 but contains both Rps26 and Rio1-kd, and the 18S rRNA is processed. These findings suggest that these structures represent the final two steps in 18S rRNA processing, which appear to occur on 40S pre-ribosomes rather than in 80S complexes as seen in yeast. Comparing the two structures predicts a steric clash between Rio1 and Dim2, which might initiate Dim2 dissociation and attendant Rps26 binding to replace Dim2, as Rps26 occupies the same position on the 40S as Dim2. A role for Rps26 in stimulating 18S rRNA cleavage, presumably by preventing Dim2 association and steric hindrance of Nob1 access to the cleavage site, was provided by si-RNA knockdown of Rps26, which decreases the cleavage of 18S rRNA, while increasing occupancy of Nob1 and Dim2 in the pre-40S particles. In state B, Rio1-kd is bound to ADP and its Asp-D341 carries a phosphate group, suggesting that the kd mutation does not prevent ATP binding and hydrolysis, but rather release of the products of ATP hydrolysis. Supporting this inference, they show that adding ATP or non-hydolyzable AMP-PNP confers similar amounts of 18S rRNA cleavage in the pre-ribosomes affinity purified in association with Rio1-kd. Together, these results suggest the model that Rio1 and Rps26 function in a coordinated manner to displace Dim2 and enable access of Nob1 to the cleavage site, and that completion of ATP hydrolysis is necessary for release of Rio1 from the final, mature 40S subunit.The results appear to be significant in providing high resolution cryo-EM images of the likely final two steps of 40S biogenesis, and provide structural insights into the roles of Dim2 in blocking cleavage of the pre-rRNA by Nob1, and of Rio1 and Rps26 in displacing Dim2 to allow 18S rRNA cleavage by Nob1. The Rps26 knockdown experiments add further evidence supporting previous findings that Rps26 is important for 18S rRNA cleavage and provide evidence that it also enhances dissociation of Dim2 and Nob1; and the in vitro cleavage experiments support previous findings from yeast that ATP binding to Rio1 is sufficient for 18S rRNA cleavage by Nob1.1. While the cryo-EM structures are certainly valuable, there is a concern that the major conclusions of the paper, embodied in the model in Figure 7, were already very likely to be true based on previous findings from yeast, cited in the Introduction, that Dim2 restricts endonucleolytic cleavage by Nob1 by masking the cleavage site, that Nob1 and Dim2 dissociate only after 18S rRNA cleavage, that Rio1 and Rps26 are both necessary for cleavage by Nob1, and that Rio1 is also necessary for the release of Dim2 and Nob1 from the mature 40S subunit.We and others have already shown significant differences between ribosome maturation processes in yeast and humans. The data reported here underline yet another divergence: as stated in the discussion, unlike in yeast we did not find any 80S-like particles despite purifying particles using the same bait. This suggests that the last maturation steps of the small ribosomal subunit differ between these two species, which calls for a specific characterization in human cells. Although RIO1 and RPS26 were both shown to be required in the processing of 18S-E pre-rRNA, a precise description of the underlying mechanism is still missing. To our knowledge this study is the first to show a direct and combined action of these two proteins in the cleavage of the 18S-E pre-rRNA by NOB1.2. It might be necessary to perform MS analysis on the Rio1-associated 40S subunits following knock-down of Rps26 to insure that no other Rps proteins are lost in order to bolster the conclusion that Rps26 alone is required in conjunction with Rio1 for stimulating rRNA cleavage and dissociation of Dim2 and Nob1.This analysis has been performed; please see above.3. Results in Figure 6A-B seem to indicate significantly greater 18S rRNA cleavage in the 40S ribosomes containing WT Rio1 vs. the Rio1-kd mutant, which seems at odds with the conclusion that ATP binding and hydrolysis, but not release of the hydrolysis products by Rio1 is sufficient for 18 rRNA cleavage. This needs to be addressed.We agree that our previous representation of in vitro cleavage analysis might have been misleading; we have now represented the fraction of cleaved rRNA as requested (18S/(18S+18SE)), and believe this new plotting lifts the ambiguity (see above).4. It may be important to show directly that Rps26 is required for 18S rRNA cleavage by carrying out in vitro cleavage assays like those shown in Figure 6 following knock-down of Rps26.We also performed the suggested experiments; see above.Reviewer #2:Ribosome assembly is a complex pathway that is thought to involve quality control checks to ensure the correct assembly of ribosomes. In yeast, a major event in assessing the assembly of small subunits is the formation of an 80S-like complex to promote final cleavage of 18S rRNA in the cytoplasm. However, less is known about the pathway of ribosome assembly in humans. In this manuscript, the authors use cryo-EM to analyze particles affinity purified from human cells with a catalytic mutant of RIO1 kinase. The comparable mutant in yeast associates with 80S-like particles. The authors resolve two apparently sequential states of 40S maturation that represent intermediates before and after cleavage at site 3 to generate the mature 3'-end of 18S. Surprisingly, the authors find no evidence for an 80S-like particle. Consistent with earlier work from yeast, the authors show that cleavage at site 3 depends on RIO1 and is stimulated by ATP. Their data also strongly implicate the loading of RPS26 in displacement of DIM2 to allow cleavage by NOB1. A role for Rps26 in cleavage of pre-18S rRNA has also previously been reported in yeast. In general the data are clearly presented and the manuscript is well-written.1. The authors present the model that RIO1, together with RPS26, displace DIM1 to promote cleavage at site 3 by NOB1. The authors have nearly all the tools to test this in vitro. Does the addition of RPS26 to particles affinity purified with RIO1 from RPS26-repressed cells promote NOB1 cleavage and release of DIM2?We have added purified S26 to late pre-40S particles and showed that indeed this potentialized rRNA cleavage, please see above.2. In Figure 3C the authors suggest that RIO1 contains ADP and phospho D341. It is not clear from the figure that this is the case and not ATP. A supplemental figure showing the fit of ADP and P-D341 vs ATP in the density map would help make this point.We have added a supplementary figure  showing the cryoEM map fitted with either ADP and P-D341 or ATP to prove this point.3. In Figures 4C, 5B and 6B the data are not displayed appropriately. The authors plot the ratio of 18S/18SE. However, this misleadingly exaggerates the results: as the efficiency of cleavage approaches 100%, this ratio goes to infinity. The authors should plot this as fraction cleaved (18S/(18S+18SE)).We have addressed this issue, please see above.Reviewer #3:During late stages of eukaryotic ribosome assembly the final step of pre-rRNA processing occurs and the last four ribosome biogenesis factors are released. Work described here provides a more detailed understanding of this process for human pre-40S particles: assembly factor DIM2 prevents access of the NOB1 endonuclease to its cleavage site. DIM2 is displaced by entry of RIO1 and RPS26, enabling cleavage of the 20S pre-rRNA by NOB1. A conformational change in RIO1 triggers its dissociation. Experiments in this well written manuscript provide strong support for this model, which extends our understanding of these steps from what had previously been found in yeast.The manuscript could be improved by addressing the following:1. In the Introduction, or at the beginning of Results, the authors might provide a little more information about RIO1 autocatalytic activity and its putative role, before describing its use as an affinity purification bait. Can they estimate how much activity remains in the D324A mutant that they use?As suggested, we have added several sentences to try to emphasize on the ATPase role of RIO1 in small ribosomal subunits maturation. In the introduction section, we have modified the following sentence (p.4) as follows:“Absence of RIO1 or suppression of its catalytic activity impairs both rRNA cleavage and release of NOB1 and DIM2 in yeast and human cells , suggesting that RIO1 association to pre-40S particles as well as its ATPase activity are both required to yield fully functional small ribosomal subunits.”Chaetomium thermophilum ATPase activity results presented in Ferreira-Cerca et al. (2014). Thus, in the first section of the results part (p.5), we have modified the first paragraph as follows:Because we could not retrieve the original data, we could not precisely quantify RIO1 D324A autophosphorylation activity from our previous results , so we based our estimation on Chaetomium thermophilum D281A ortholog ”.“Autophosphorylation of this mutant in the presence of ATP was previously shown to be strongly impaired , and free π release activity reduced by at least 50% in its We hope these few sentences will be useful to the reader.2. That the bait protein Rio1 cannot be visualized in State A particles might be confusing to some readers. The authors suggest that it is loosely bound and falls off during purification. They should provide a slightly more detailed explanation of when during purification this might occur…certainly after attachment to the affinity gel. It also seems reasonable that RIO1 is present but particularly flexible in state A particles.We believe that the latter hypothesis is true; thus, in the Results sections describing the state A cryo-EM map (p. 6), we have added the following sentences:“Furthermore, NOB1 and DIM2 are the only two RBFs that can clearly be distinguished, while RIO1(kd)-StHA, the protein used as purification bait, cannot be clearly positioned on this cryo-EM map. […] Indeed, while this manuscript was in revision, a cryo-EM study focusing on similarly late human pre-40S particles revealed significant changes in RIO1 position on maturing pre-40S particles .”3. Page 11: Is there any evidence for how RPS26 initially contacts binds to preribosomes? Might it bind near DIM2, then undergo a series of conformational shifts to help displace DIM2?Beyond V. Panse’s lab work on early association of RPS26 to nucle(ol)ar yeast pre-40S particles, which has now been briefly described in the introduction section of our manuscript, there is to our knowledge no evidence regarding how and where RPS26 initially binds to pre-ribosomes. In their recent cryo-EM study, Ameismeier et al. only detected RPS26 in pre-40S particles in its final position, despite the depth of their analysis. This does not necessarily plead for a stepwise assembly; what reviewer 3 proposes might be true, but we have no structural or functional data to sustain this hypothesis in human cells.4. In my opinion, an interesting question in ribosome assembly is how is each assembly factors is released from preribosomes. There are more than 200 assembly factors , but many many fewer NTPases. Does each NTPase help many AFS depart or are there additional release mechanisms, or both? The authors might highlight this issue a little more, e.g., by comparing what they found with a few other examples from the literature, especially if there are any cases where NTPases are not yet known to be involved in an AF release. How does binding of one protein displace another?Thank you for this very interesting observation, which could be the object of a review article. Indeed, many NTPases are currently seen as “power switches'', that help release other assembly factors from maturing pre-ribosomes, and allow progress to later assembly steps of these ribosomal particles. We believe this is indeed the case, but also that NTPases might have other functions. For instance, our data show a very close interaction between RIO1 and a functional site of the small ribosomal subunit (the N7-methylated Guanosine 1639), which suggests that RIO1 might also directly probe the functionality of this nucleotide). In order to support this idea, we have added the following sentences to the final part of the discussion:RIO1 probes tRNA translocation capacities of pre-40S particles'' (p.13) as follows:““The function of RIO1 in the release of DIM2 and NOB1 echoes the roles that other NTPases play in pre-ribosomal particle remodeling along the maturation pathway. […] Furthermore, in the post-cleavage state B herein described […]…”


These diseases involving fibrosis may share common mechanisms and therapeutic targets, and therefore common intervention strategies and medicines may be applicable for these diseases. For this reason, deliberately introducing anti-fibrosis characteristics into predictive modeling may lead to more success in drug repositioning. In this study, anti-fibrosis knowledge base was first built by collecting data from multiple resources. Both structural and biological profiles were then derived from the knowledge base and used for constructing machine learning models including Structural Profile Prediction Model (SPPM) and Biological Profile Prediction Model (BPPM). Three external public data sets were employed for validation purpose and further exploration of potential repositioning drugs in wider chemical space. The resulting SPPM and BPPM models achieve area under the receiver operating characteristic curve (area under the curve) of 0.879 and 0.972 in the training set, and 0.814 and 0.874 in the testing set. Additionally, our results also demonstrate that substantial amount of multi-targeting natural products possess notable anti-fibrosis characteristics and might serve as encouraging candidates in fibrosis treatment and drug repositioning. To leverage our methodology and findings, we developed repositioning prediction platform, drug repositioning based on anti-fibrosis characteristic that is freely accessible via  Fibrosis is defined as the process of excessive accumulation of fibrous connective tissue in most tissues or organs, where normal cell injury, transdifferentiation and abnormal proliferation, and deposition of extracellular matrix can disrupt tissue function. In the new era of 21st century, the morbidity and mortality rates of various fibrotic diseases have increased progressively, bringing an enormous global health burden. In developed countries, fibroproliferative diseases are responsible for nearly 45% of deaths . As dataTHBS2, TIMP1, COL1A2, COL3A1, HYOU1, MMP2 and MMP7 [The polypharmacology of most anti-fibrosis drugs could improve therapeutic efficacy. Recent studies have found that, firstly, fibrosis is the common pathogenic process in most diseases. For example, there are multiple common cellular processes between lung cancer and IPF, including inflammation, cell apoptosis and tissue infiltration . Secondland MMP7 . Thirdlyand MMP7 . Differeand MMP7 , 13. In de novo drug discovery, drug repositioning has obvious advantages such that it could significantly shorten drug development periods, reduce laboratory cost and minimize potential safety risk. Nowadays, drug repositioning is one of the most productive strategies in drug development [Drug repositioning or repurposing refers to the ‘reuse of old drugs’, recycling existing drugs for new medical indications. Compared with elopment . With thelopment , 16, incelopment  and connelopment . SEA cluelopment . CMap coelopment . Howeverelopment , there aAFC), to accelerate the exploratory process of repositioning drugs and contribute to the cutting-edge study of its underlying mechanisms.For the first time, we built the anti-fibrosis knowledge base upon anti-fibrosis targeted research. Based on the knowledge base, two repositioning models, Structural Profile Prediction Model (SPPM) and Biological Profile Prediction Model (BPPM) were constructed to achieve high prediction accuracy. Centered on these two models, we then developed a repositioning computing platform, drug repositioning based on anti-fibrosis characteristic   from Janials.gov  from JanStructural and biological profiles of compounds were collected from DrugBank  and CMapA total of 20 263 natural products from Traditional Chinese Medicine Integrated Database (TCMID) , 5968 DrIn raw chemical structures (from DrugBank approved drugs and the anti-fibrosis knowledge base) and biological profiles (from CMap) data, compounds that exist in the anti-fibrosis knowledge base were labeled as positive candidates, whereas the rest were labeled as negative candidates. Then, chemical structures were converted into chemical fingerprints (166-bits MACCS keys) for processing chemical information in a fast and convenient way using RDKit . As to bOne-class support vector machine (nu = 0.3) was performed to estimate sample quality, remove outliers and confirm final positive and negative samples. In total, 70% of final samples were used as training set for model selection and super-parameter determination, whereas the remainder as testing set for model validation.Four different machine learning algorithms were selected for modeling on training set, including logistic regression with l1 and l2 penalties, decision tree, random forest and gradient boosting. Among them, method with highest precision and area under the curve (AUC) calculated by 5-fold cross-validation was selected for subsequent analysis. Iterative feature elimination (IFE) algorithm was performed to select optimal feature set through one-by-one feature deletion. Finally, SPPM and BPPM were constructed based on optimal modeling algorithm and feature set, and further validated by testing set. The details of model construction are shown in Network-based inference approaches are wildly applied in the realm of drug repositioning , 21. HerAFC was constructed through PostgreSQL database and Django framework. This platform serves as a practical tool for prediction of drug repositioning potential based on compound structures (via SPPM) and biological profiles (via BPPM) as well as displaying compound-target-disease network of drug repositioning mechanisms. Meanwhile, Dr AFC also integrated toolkits such as quantitative estimate of drug-likeness from Silicos-it [Dr licos-it , and simThe overall process is shown in ClinicalTrials.gov [To construct the anti-fibrosis knowledge base, 7058 fibrosis-related references from PubMed, 302 from CTD  and 2664ials.gov  were colIn modeling session, 2885 compound structures (from DrugBank approved drugs and anti-fibrosis knowledge base compounds)  and 6100Four different machine learning classifiers were evaluated and compared to choose the most optimal modeling method . GradienIn the process of building SPPM and BPPM, we found that even a small number of features could reach certain stability and reasonably good performance . Models Finally, SPPM and BPPM were built based on the most optimal modeling method and the selected small feature subset (top 38 features in SPPM and top 47 features in BPPM). In testing set, the average AUC for SPMM reaches 0.814  recall .We used SPPM to predict anti-fibrosis drugs from DrugBank experimental drugs. The comparative analysis was performed between the CTD compound–gene interactions of the predicted anti-fibrosis and non-anti-fibrosis drugs. The results show that the anti-fibrosis group accommodates stronger interactions, presumably more genetic effects thus greater repositioning potential .In Drugbank experimental drugs, multiple drugs with great repositioning potential  using their gene profiles. The results show that a total of 66 natural products have anti-fibrosis characteristic and repositioning potential, including ginsenoside Re (repositioning score = 0.979), muscone (repositioning score = 0.974) and cinnamic acid (repositioning score = 0.948) . Among tAFC . Ginsenorophages . It was rophages . Apart frophages  and decrrophages . BesidesAFC, the main function and workflow of which is shown in AFC platform, anti-fibrosis and potential repositioning could be predicted from compound structures and/or biological profiles. The supported ‘drug repositioning mechanism analysis’ could infer the relationships among compounds, fibrosis-related targets and diseases, which help researcher understand pathology. Furthermore, drug-likeness estimation, chemical similarity calculation and structure matching were integrated into Dr AFC to provide useful information for drug development.Based on SPPM and BPPM, we constructed a computing platform for repositioning research purpose, named Dr AFC allows users to upload compound structures or compound-induced biological profiles for repositioning potential prediction. As shown in AFC accepts simplified molecular-input line-entry system (SMILES) strings of compound structures for SPPM prediction and accepts gene profiles with row names in the format of Affymetrix U133A probe ID, Entrez ID or gene symbol for BPPM prediction. Both methods support .txt, .csv and .xlsx files  basAFC also contains functionalities including drug-likeness estimation, chemical similarity calculation and structure matching tools. Users could upload their compounds in SMILES and perform these ligand-based calculations. Drug-likeness estimation could evaluate and score the compound drug-likeness, ranging from 0 to 1 with higher score indicating higher potential for lead compound. Chemical similarity calculation and structure matching provide convenient ways for users to search compound with similar structures, same structures or substructures, supporting single compound calculation and simultaneous calculation for multiple compounds.Dr AFC data (https://www.biosino.org/drafc). This in silico platform also provides a highly accurate way to generate data for rational drug design via combining the advanced machine-learning algorithm.Fibrosis is the common mechanism underlying diseases that has attracted global attention. Anti-fibrosis characteristic of a compound could essentially infer its great repositioning potential. However, the anti-fibrosis characteristic has not been thoroughly considered and extensively applied into the realm of drug discovery till now. In this study, we first bridge the gap by developing a platform that can provide intensive information conveniently on Dr AFC was built upon the anti-fibrosis knowledge base, which pioneered the curation and deployment of fibrosis-related studies over the past decade. Structural profile (SPPM) and biological profile (BPPM) that show extraordinary capabilities in drug repositioning prediction  were integrated into Dr AFC. BPPM shows slightly higher performance than SPPM based on AUC metric. The possible reason could be that biological profile is more tolerant and could contain information reflecting an overall effect of compound in the body. Biological profile has shown its advantage in multiple repositioning algorithms previously, such as CMap [2 [Dr  as CMap , L1000CD CMap [2  and MANT CMap [2 . BesidesIn BPPM, 47 biological markers exhibited strong prediction capabilities. These genes are directly or indirectly linked to various fibrotic diseases. Notably, ribosomal proteins including RPL30, MRPL15, RPL32, RPS3A, RPLP0, RPL7, RPL23A and RPL13A are the main part of these biological markers. Ribosomes serve as significant regulators in immune signaling pathways, tumorigenesis pathways, and cardiovascular and metabolic diseases , 42. ForBased on external molecule sets, natural products are validated to have the most appealing anti-fibrosis characteristics and repositioning potential among chemicals from different sources. Natural products provide a wealth of valuable natural resources for modern medicine and are seen as promising and popular candidates for drug repositioning studies . Their nAFC (https://www.biosino.org/drafc), for studying fibrosis mechanisms and drug repositioning.In summary, based on anti-fibrosis characteristics, we constructed two predictive repositioning models, SPPM and BPPM, which predict the anti-fibrosis characteristics and repositioning potential from compound structures and/or compound-induced biological profiles. SPPM and BPPM take the advantage of therapeutic commonality and universality of fibrotic diseases, and dramatically increase the success rate of drug repositioning predications. This study not only established a highly efficient strategy of predicting repositioning, but also developed a convenient and user-friendly computing platform, Dr Fibrosis is the common mechanism of diseases, which could be applied in drug repositioning.AFC, for studying fibrosis mechanisms and drug repositioning.We developed a convenient and user-friendly computing platform, Dr AFC shows high performance on both cross-validation and external validation, which demonstrates its potential applications in drug discovery.Dr Natural compounds proved to be the better repositories for drug repositioning.Figure_S1_bbaa115Click here for additional data file.Figure_S2_bbaa115Click here for additional data file.Figure_S3_bbaa115Click here for additional data file.Table_S1_bbaa115Click here for additional data file.Table_S2_bbaa115Click here for additional data file.Table_S3_bbaa115Click here for additional data file.Table_S4_bbaa115Click here for additional data file.


Optoelectronic devices in the UV range have many applications including deep-UV communications, UV photodetectors, UV spectroscopy, etc. Graphene has unique exciton resonances, that have demonstrated large photosensitivity across the UV spectrum. Enhancing UV absorption in graphene has the potential to boost the performance of the various opto-electronic devices. Here we report numerical study of UV absorption in graphene on aluminum and magnesium hole-arrays. The absorption in a single-layer graphene on aluminum and magnesium hole-arrays reached a maximum value of 28% and 30% respectively, and the absorption peak is tunable from the UV to the visible range. The proposed graphene hybrid structure does not require graphene to be sandwiched between different material layers and thus is easy to fabricate and allows graphene to interact with its surroundings. Examples of graphene-based devices include high speed transistors, flexible electronics, high-speed electro-absorption modulators, transparent electrodes, photodetectors, molecule sensors, etc7. Enhancement of the light-matter interaction is crucial in boosting the performance metrics of the various optical devices based on graphene. Surface plasmon resonance (SPR) dramatically enhances the local electric fields and has been used to enhance the performance of graphene coupled devices in the terahertz (THz) and infra-red range (IR)9. Other examples of SPR enhanced graphene devices include photon detectors, gas sensors, and DNA sensors11.The remarkable optical, electronic and mechanical properties of graphene have drawn much interest in the research community since its discovery in 200014. In terms of biosensing, absorption and native fluorescence of biomolecules resides in the UV range. Therefore, enhancing UV absorption in graphene has the potential to boost the performance of these optoelectronic and biosensing devices. In the UV range, the exciton resonances at the saddle point in the band structure of graphene produce an abnormal absorption peak near 4.5 eV. The position and the shape of the exciton peak is a strong function of the free charge carrier density in graphene15, due to the screening effect of the many-body interactions at the saddle point. These unique exciton resonances have demonstrated large photosensitivity across the UV spectrum16. Graphene has been shown to boost the performance of photosensitivity of UV detectors, and enhance UV photoluminescence18. However, in-depth analysis of UV absorption in graphene is still at an early stage.Comparatively, when considering graphene devices, less attention has been focused in the ultraviolet (UV) region than in other regions of the electromagnetic spectrum. Opto-electronic devices in the UV range can be used for deep-UV communications, UV photodetectors, UV spectroscopy, etc24. All-dielectric absorbers have been proposed for perfect UV absorption in a single-layer graphene19. Metal-dielectric-metal plasmonic structures achieved numerically enhanced absorption of UV light in a single-layer graphene up to 50%23. Almost all investigated structures consist of a graphene layer sandwiched between a top dielectric or metal layer and a substrate that acts like a mirror. In practice, fabricating such sandwiched structures can be challenging since the fabrication steps associated with the top layers can alter the physical properties of graphene. Besides, having graphene embedded in between materials layers will limit its interactions with the environment, which is fundamental for the purpose e.g. of sensing applications. Here through FDTD simulations, we propose to use an aluminum and magnesium hole-array to enhance UV absorption in a single-layer graphene. These structures are easy to fabricate in practice; graphene can be transferred to the hole-arrays without additional deposition/lithography steps. The metals of the hole-arrays are aluminum or magnesium. These metals are chosen due to their excellent plasmonic properties in the UV range of the spectrum26. The graphene on aluminum and magnesium hole-arrays achieved 28% and 30% absorption at 300 nm, respectively, and the absorption peak is tunable from the UV to the visible range.Several research articles have studied enhancing UV absorption in graphene27, but large enough to exhibit large transmittance. Since no propagating modes are supported, the enhanced transmittance observed through the hole-array is associated with an excitation of plasmonic resonances on the front and back of the metallic film. One single-layer graphene covered the aluminum or magnesium hole-array and in the hole regions was immersed/suspended in air. A single unit cell of the array with graphene is modeled under an incident plane wave with transverse magnetic (TM) polarization towards the -z direction, and periodic boundary conditions used along x and y directions was taken from the literature30. Since aluminum form an oxide layer at the interface between aluminum and air, an Al2O3 layer with 5 nm thickness was added at the interface between the aluminum film and air. In addition, an Al2O3 layer with 4 nm thickness was inserted at the interface between the aluminum film and the quartz substrate. As for magnesium, a MgO layer with 5 nm thickness was added at the interface between the magnesium film and air. In addition, a MgO layer with 4 nm thickness was inserted at the interface between the aluminum film and the quartz substrate. The thicknesses of oxide layers are taken from our previous studies25.Numerical simulations of the transmission spectrum of an aluminum or magnesium hole-array with graphene were carried out using finite-difference time-domain method . As shown in Fig. mentclass2pt{minim31. Figure 32 and imported into Lumerical as the 2-dimensional material model for a single-layer graphene. In order to reduce the computation volume to a manageable size, the thickness of graphene layer in the 2-dimensional model was chosen to be 2 nm instead of 0.34 nm (the thickness of an atomic layer). The complex conductivity employed to model the 2 nm graphene is calculated by multiplying 34. The excellent agreement between the simulated transmission spectrum and the theoretical one validates the accuracy of the graphene model that was used in the simulations.The optical constants of an intrinsic single-layer graphene were taken from the literatureConvergence tests on the thicknesses of the graphene model and mesh sizes were carried out. The differences of the transmission intensity through a 2 nm and 1 nm thickness graphene model was found to be less than 0.1%. Thus, the thickness of the graphene layer was set to 2 nm in the simulation to achieve an optimal balance between the simulation accuracy and the simulation size. An overall mesh of 2 nm was used to cover the hole-array and graphene layer and an additional mesh of 1 nm was used to cover just the graphene region. Convergence test on different mesh sizes on the graphene layer was also conducted by varying mesh size from 4 to 0.5 nm and less than 0.25% deviation was found below mesh size 1 nm , the first order air-metal SPR mode red-shifts to longer wavelengths, while multiple absorption peaks emerge at shorter wavelengths. For example, for aluminum hole-arrays with periodicity 360 nm, there are three absorption peaks at around 390 nm, 300 nm, 260 nm. The first order air-metal SPR mode corresponds to the absorption peak around 390 nm, while higher order SPR modes correspond to absorption peaks at shorter wavelengths. Figure Enhancing graphene absorption in the ultraviolet range can boost the performance of optoelectronic and biosensing devices. Here we demonstrated numerically that by coupling graphene with aluminum and magnesium hole-arrays, up to 30% absorption in graphene at a wavelength of 300 nm can be achieved. By tuning the periodicities of the hole-arrays, the graphene absorption peaks can be shifted towards the visible range. The positions of the graphene absorption peaks correspond to the UV SPR modes on the hole-arrays. Multiple graphene absorption peaks are found for hole-arrays with large periodicities. The proposed structures are easy to fabricate without additional lithography steps on top of graphene.Supplementary Information.


Yoga is an ancient Indian philosophy and way of life that is being used as a method of improving health and wellbeing. Evidence shows that yoga has several health benefits, such as managing many noncommunicable diseases, such as hypertension, and improving mental health. The popularity of yoga is growing in the UK, but it is mostly unregulated with little information available about yoga providers and their sessions and attendees. This study aimed to explore who is providing yoga; what sessions are available, where, and at what cost; and who attends these sessions in the UK and whether yoga providers were aware of health conditions in their sessions. A cross-sectional survey was undertaken among yoga providers in the UK. They were approached through four major UK yoga associations. In total, 407 yoga providers participated. Most providers were aged 45–64 years (69%), female (93%), and white (93%). The median number of group sessions and one-to-one sessions delivered per week was four and two, respectively. The most common styles were Hatha (28%), Iyengar (26%), and Vinyasa (15%). Sessions had a varying emphasis on different yogic practices, but 59% of providers allocated most time to yogic poses (asana), 18% to breathing practices (pranayama), and 12% to meditation (dhyana) and relaxation practices. Most (73%) reported that their attendees disclosed their health conditions to them, most commonly mental health issues (41%), hypertension (25%), and heart diseases (9%). This study showed that yoga sessions are widely available in the UK, often provided and practiced by women, and concentrate on yogic poses. Sessions concentrate on the asana and tend not to include many of the more holistic aspects of yoga that are practiced in South Asian countries. Yoga providers are often aware of health conditions but may benefit from training to deliver sessions suitable for specific health conditions. Yoga, an ancient system of health and way of life, originated in the Indian subcontinent over 5000 years ago . There aYoga helps discipline the body and mind through low- to moderate-intensity physical activities, breathing practices, meditation and relaxation practices, and a healthy lifestyle . It requYoga has a high acceptance rate in South Asian countries, such as India and Nepal, and it is widely practiced in these countries ,15. The Yoga is also growing in popularity in the UK ,29. A reWe conducted a cross-sectional study of yoga providers using an online survey and a descriptive approach.Yoga providers  were eligible if they were at least 18 years old and delivered a yoga session at least once in a fortnight during the last six months in the UK prior to the survey date.A web-based survey questionnaire was developed and piloted among yoga providers (three yoga teachers and one yoga therapist). The questionnaire was administered through Jisc Online Surveys, formerly known as Bristol Online Surveys software . The queYoga providers were approached through professional yoga associations in the UK. Five yoga associations were approached to circulate the survey among their members, and four agreed, namely Association for Iyengar Yoga in the UK and Republic of Ireland, British Wheel of Yoga (BWY), Complementary and Natural Health Council (CNHC), and Independent Yoga Network. They either emailed their members an invitation letter with the survey link or posted the study details on their website and Facebook page. Data collection took place from 3 March to 3 September 2020. The participant information sheet was provided at the beginning of the study, and those interested gave written informed consent. Participation in this study was entirely voluntary. Participants were entered into a prize draw of GBP 100 if they provided their email addresses. Participation in the prize draw was entirely voluntary, and confidentiality was protected in accordance with regulatory requirements. Email data were not downloaded, and participants were not identifiable in any process.The required sample size was estimated on the basis of the accuracy of estimates of proportions, for example, of the proportion delivering a certain style of yoga. A sample size of 384 was sufficient to estimate proportions to within 5% error using a 95% confidence interval. A larger sample was approached presuming that not all would respond.p ≤ 0.05. Mean and standard deviation (SD) were calculated for normally distributed continuous variables, median and interquartile range (IQR) for skewed continuous variables, and number and percentage for categorical variables. Responses of yoga teachers and therapists were compared using the Wilcoxon Rank Sum test for continuous variables and the chi-square test for categorical variables. If more than 20% of cells had a value <5, Fisher’s exact test was preferred over the chi-square test. Data were analyzed using Stata16  . RegionsThe socio-demographic characteristics of participants are presented in Yoga sessions were available as group and one-to-one sessions though the majority of participants (95%) delivered group sessions . The delSessions were available in all the ONS regions of the UK although South West (14%), South East (13%), and London (13%) were the most common regions among respondents . SessionParticipants reported using different styles of yoga, but the most common styles reported were Hatha (28%), Iyengar (26%), and Vinyasa (15%) yoga . Hatha yThe majority of participants (73%) reported that attendees disclosed their health conditions . They reWe have provided a contemporary description of yoga providers and their practice across the country; we found that yoga providers were mostly aged 45–64 years, female and white, and yoga was mostly practiced by women. The predominance of women in both delivery and practice in the UK is similar to that seen in previous surveys here ,38 and oYoga sessions were available in the evenings and on the weekends, which makes them accessible for those who are not able to attend sessions during day times on weekdays. Because of the wide availability of group yoga sessions and the significant difference between the cost of group and one-to-one sessions, group sessions appear to be more affordable and accessible for a wider range of people than one-to-one sessions.Different styles of yoga were reported in this study, with Hatha and Iyengar being the most commonly delivered styles. Hatha yoga is an overarching term for physically based yoga practices, and the high rate of the report of Hatha yoga possibly suggests that Hatha yoga appears to be a common UK parlance used for any type of yoga. Our study found that yoga, as practiced in the UK, concentrates on asana and to a lesser extent pranayama and dhyana and relaxation practices, and this is similar to that seen in other high-income countries, such as Australia  and GermThis study showed that attendees commonly disclosed their health conditions, and the high rate of disclosure suggests that yoga providers were generally aware of the health conditions of their attendees. The most commonly disclosed health conditions in yoga sessions were mental health issues and hypertension, both in our study and in studies from the UK and other countries ,38,39. TYoga is unregulated in the UK, and this might mean that yoga teachers with no proper training can deliver yoga sessions in the UK. People with health conditions attend these sessions; therefore, there is a need for an initiative to better regulate yoga practice in the UK. In addition, there is clearly a need for better training for yoga teachers in supporting specific health conditions, especially for mental health issues, hypertension, and heart disease, which were found to be the most commonly disclosed health conditions in this study. It is also important for yoga providers to be aware of their attendees’ health conditions to provide safer practice.Yoga is understudied in the UK, and our findings showed that further studies are warranted. One important question to be further investigated is the knowledge, experiences, and attitudes of yoga providers in relation to managing health conditions in their sessions and that of health professionals in recommending yoga for people with these conditions. In addition, future studies should aim to explore the motivations of the attendees and reasons for the popularity of yoga among women in the UK.To the best of our knowledge, this was the first study describing the characteristics of yoga providers and their sessions and attendees in the UK. The standard steps in questionnaire development (design and pretesting) were followed to ensure the validity and reliability of the questionnaire. Missing data were low in this study. An online survey was conducted, so we may have missed those who have accessibility issues to online platforms. It was not possible to know the exact number of yoga providers in the UK because registration with a yoga association is not mandatory in the UK, and yoga providers can register with more than one association, so we cannot estimate accurately our response rate. However, as far as we are aware, there are around 150 yoga therapists in the UK, and we were, therefore, able to access most yoga therapists delivering sessions in the UK. The survey was conducted during the COVID-19 pandemic, an unprecedented time for everyone, which is likely to affect the delivery of yoga sessions; however, we requested participants to report their usual yoga sessions. This study shows that yoga sessions are widely available in the UK and often provided and practiced by women. Sessions concentrate on the asana and tend not to include many of the more holistic aspects of yoga that are practiced in South Asian countries. Yoga teachers are often aware of health conditions but may benefit from training to deliver sessions suitable for specific health conditions.


Rxx) was affected by the scattering of the GB only in the low carrier concentration region, and the standard QHE of a monolayer graphene was observed regardless of the stitching angle of the GB. In addition, a controlled device with an added metal bar placed in the middle of the Hall bar configuration was introduced. Despite the fact that the equipotential lines in the controlled device were broken by the additional metal bar, only the Rxx was affected by nonzero resistance, whereas the Hall resistance (Rxy) revealed the well-quantized plateaus in the QH state. Thus, our study clarifies the effect of individual GBs on the QH states of graphenes.Charge carrier scattering at grain boundaries (GBs) in a chemical vapor deposition (CVD) graphene reduces the carrier mobility and degrades the performance of the graphene device, which is expected to affect the quantum Hall effect (QHE). This study investigated the influence of individual GBs on the QH state at different stitching angles of the GB in a monolayer CVD graphene. The measured voltage probes of the equipotential line in the QH state showed that the longitudinal resistance ( Rxx) accompanied by the quantized plateau of Hall resistance (Rxy), can be observed at up to room temperature [RK/ν (RK is the von Klitzing constant and ν is the filling factor with integer number), is utilized as the resistance standard in metrology [h-BN) substrate [Graphene is an atomically thin monolayer that has attracted considerable attention in many fields due to its excellent mechanical ,2 and elperature . Thus, tetrology ,13,14. Tetrology  and deteubstrate .Chemical vapor deposition (CVD) graphene often forms grain boundaries (GBs) during CVD growth ,18, consRxx becomes nonzero because the equipotential line condition along the edges in the QH state is broken. However, each region separated by the metal bar maintained the typical quantized Rxy. Our study provides an exemplary controlled experimental result for the effect of the GB on the QH state in a CVD graphene.In this study, we experimentally investigated the effect of the stitching angle of the GB on the QH state of graphene. It is remarkable that all the devices with a GB showed the standard QH states, which had the same sequence of filling factors as those of a single-crystal monolayer CVD graphene. This supports the fact that QH states are robust because of topological protection from scattering by the GB. In contrast, a controlled device with an additional electrode placed at the middle of the Hall bar structure was examined to show that the initial QH state is split into two QH states by the effect of the metal bar with different topologies. As a result, 2) at 1070 °C for 3 h and was polished using the chemical mechanical polishing method. Second, the polished copper foil was then heated up to 1070 °C with 200 sccm Ar gas and 100 sccm H2 gas in CVD chamber for annealing for 2 h to increase the crystallinity and remove residuals on the copper surface. The H2 was reduced to 40 sccm, and 15 sccm CH4 gas (0.1% diluted in Ar) was injected during graphene growth. Finally, CH4 gas was turned off after growth, and the chamber was cooled to room temperature.First, a 100 μm thick copper foil  was annealed in forming gases  PMMA-C4 was spin-coated on the graphene/Cu substrate (3000 rpm/m). The PMMA-supported graphene film was then detached from the copper foil using the electrochemical bubbling transfer method and was rinsed in distilled water a few times to clean the residuals. Second, the PMMA/graphene was picked up by SiO2/Si substrate and dried on hot plate at 100 °C for 10 min. The PMMA was then removed by acetone and isopropyl alcohol. Finally, the graphene was subsequently etched by oxygen plasma (3 mTorr and 30 W) for D1, D2, D3, and D4 to fabricate the Hall bar structure and the graphene electrode. Metal electrode patterning was performed using electron-beam lithography, followed by Ti/Au (5/50 nm) evaporation under high-vacuum conditions (approximately 10−6 Torr). The graphene device was annealed in forming gas  at 300 °C for 3 h to remove contamination on the graphene surface before performing electrical transport measurement.The graphene device was fabricated on a heavily −6 Torr) in a cryostat , Quantum Design Inc., San Diego, CA, USA) with a semiconductor parameter analyzer .The Raman spectra were recorded with a 532 nm laser under ambient conditions . The DC electrical characteristics of the graphene were analyzed under high vacuum (approximately 102) substrate using the electrochemical bubbling transfer method [FEμ = 12,000 cm−2 V−1 s−1. Raman spectroscopy was used to characterize the crystallinity of the graphene, as shown in All graphene flakes presented in this study were synthesized on a copper (Cu) foil using chemical vapor deposition (CVD) at 1070 °C with a mixture of hydrogen and methane gases. First, the graphene was transferred onto a silica  dependence of the longitudinal resistance (Rxx) and the Hall resistance (Rxy) at a magnetic field of 14 T and a temperature of 2 K. The Rxy in a monolayer graphene under a magnetic field is well known to be quantized according to e is the electron charge, h is Planck’s constant, n is a non-negative integer, and ± represents electrons and holes. This quantization can be translated into the quantized filling factor: Rxx  moving toward zero and well-quantized plateaus in Rxy  with filling factors of ν = ±2, ±6, and ±10. We also measured the magnetic field dependence of Rxx and Rxy at a fixed gate voltage: Vg = −30 V, corresponding to the region of the hole-type carrier, as shown in the inset of Rxx was constant, and Rxy showed a linear dependence. However, as the magnetic field increased, Rxx showed an apparent oscillation, and Rxy showed quantized plateaus, reflecting the Landau levels (LLs) and QHE. It is convenient to visualize the QHE using a Landau fan diagram, where Rxx is plotted as a function of the magnetic field B and Vg (+1/2e2/h  where gsB and Vg d. As a rVi) at each electrode probe measured when the current (Ids) was injected through electrodes 1–8 (schematic in Rxx = (Vi − Vj)/Ids, where i and j are the probes at the same edge, such as R23, and the Hall resistance: Rxy = (Vi − Vk)/Ids, where i and k are the probes facing each other, such as R25, as a function of the gate voltage sweep, as shown in Rxx and Rxy are very similar to those shown in Rxx, which occurred whenever the filling factor changed, as depicted in the inset of Rxx pairs had the same channel length and width. At the zero Landau level, the resistance peaks of the electrode pairs across the GB (R23 and R56) were higher than those that did not contain the GB (R34 and R67) because of scattering at the GB, which is consistent with previous reports [Rxy show well-quantized Hall plateaus accompanied by Rxx = 0 Ω at filling factors ν = ±2, ±6, and ±10, indicating the standard QHE of a monolayer graphene.The GB was obtained at the interface when two hexagonal graphene domains were merged without any additional treatment or fabrication process. In this situation, the location of the GB can be easily identified by simple optical microscopy , as indi reports ,37. Howe reports . HoweverV2= V3 = V4 when Vg > VDirac and V5 = V6 = V7 when Vg > VDirac, even though the GB existed between electrodes 2(5) and 3(6). The voltage probe showed that the potential line in the device was similar to the standard QH state, just as we drew the equipotential lines in blue and red, as shown in the schematic in To clarify the influence of the GB on the QHE, we focused on the potential profiles of the sample. In general, because backscattering is suppressed in the QH state, the equipotential along the edge is generated, which is distinguished from the metallic state. When the direction of the applied current and magnetic field is fixed, both holes and electrons accumulate at the same edge of the device. Therefore, the sign of the potential changes depends on the carrier type. Interestingly, in Rxy measured with two electrodes passing through the GB, such as R24 in Rxy results, which indicate the equipotential along the edge, regardless of the stitching angle. Therefore, this confirms that the topologically protected QH state is robust and rarely affected by GB scattering.As mentioned previously, the details of defect lines in the GB can be different from the stitching angle between two graphene domains. Therefore, it can be another parameter to consider in the scattering mechanism and its effect on the QH state. We fabricated three more devices with different stitching angles, as shown in h-BN) substrate. The optical microscopy image of the device is shown in Vi) measured at each electrode probe as a function of the gate sweep at 2 K and 14 T. Unlike the equipotential line of a typical QH state in the graphene device, as shown in V5 was not equal to V4 (Vg > VDirac) but equal to V2 at the first LL (ν = ±2), which made R45 nonzero in the QH state, as shown in V5 was not equal to V2 at higher LLs, indicating that the equipotential lines were broken at high carrier concentrations. The Rxy shows the standard sequence of the filling factors ν = ±2, ±6, and ±10, as shown in To contrast the effect of the edge states on the QHE, we broke the equipotential line along the edges by placing an additional metal electrode at the center of the Hall bar structure of a single-crystal monolayer CVD graphene device on a hexagonal boron nitride (Rxx) appears to be dominant only in the low carrier concentration region as we compared the peaks resistance at the Landau levels. Furthermore, it does not disrupt the dissipationless edge states, showing the standard QHE in the monolayer graphene, as evidenced by the ideal equipotential lines detected by the voltage measurement of each probe and the well-quantized Hall plateaus at filling factor ν = ±2, ±6, ±10, regardless of the stitching angles. We also compared the results of the device that artificially broke the QH states. As a result, the distortion of equipotential lines caused by the broken QH states only affects Rxx with nonzero resistance, while Rxy showed the well-quantized Hall plateaus. Therefore, we postulate that topologically protected QH states are robust, even when an individual GB is present.We studied the effect of a single GB in four different devices  at various stitching angles  on the electrical transport in a CVD graphene, especially in the QH regime. In the device D2 with the stitching angle of 7°, the scattering at the GB in the longitudinal resistance (