Escherichia coli has four SPOR proteins that bind peptidoglycan, of which FtsN is essential for cell division. DamX and DedD are suggested to have semiredundant functions in cell division based on genetic evidence. Here, we solved the structure of the SPOR domain of DedD, and we show that both DamX and DedD interact with and stimulate the synthetic activity of the peptidoglycan synthases PBP1A and PBP1B, suggesting that these class A PBP enzymes act in concert with peptidoglycan-binding proteins during cell division. Escherichia coli contains four SPOR proteins, DamX, DedD, FtsN, and RlpA, of which FtsN is essential for septal peptidoglycan synthesis. DamX and DedD may also play a role in cell division, based on mild cell division defects observed in strains lacking these SPOR domain proteins. Here, we show by nuclear magnetic resonance (NMR) spectroscopy that the periplasmic part of DedD consists of a disordered region followed by a canonical SPOR domain with a structure similar to that of the SPOR domains of FtsN, DamX, and RlpA. The absence of DamX or DedD decreases the functionality of the bifunctional transglycosylase-transpeptidase penicillin-binding protein 1B (PBP1B). DamX and DedD interact with PBP1B and stimulate its glycosyltransferase activity, and DamX also stimulates the transpeptidase activity. DedD also binds to PBP1A and stimulates its glycosyltransferase activity. Our data support a direct role of DamX and DedD in enhancing the activity of PBP1B and PBP1A, presumably during the synthesis of the cell division septum.Sporulation-related repeat (SPOR) domains are present in many bacterial cell envelope proteins and are known to bind peptidoglycan.  Escherichia coli, the PG sacculus forms a thin, mostly single layer in the periplasm. PG is composed of linear glycan strands made of alternating N-acetylglucosamine and N-acetylmuramic acid residues which are connected by short stem peptides containing l- and d-amino acids (d-Ala at position 4 of one peptide with meso-diaminopimelic acid (mDAP) at position 3 of another. These cross-links are synthesized by dd-transpeptidases called penicillin-binding proteins (PBPs), which are the primary target of β-lactam antibiotics. Most β-lactams target several PBPs, but some are more specific; for example, aztreonam selectively inhibits PBP3 (The peptidoglycan (PG) sacculus is an essential net-like polymer that surrounds the cytoplasmic membrane in most bacteria , 2. Althno acids . The glyits PBP3 , albeit During the cell cycle, the PG sacculus is first enlarged during cell elongation and then split into two during cell division. Sacculus growth and division require the coordinated actions of synthetic and hydrolytic PG enzymes, while the structural integrity of the sacculus and the whole cell envelope has to be preserved at all times , 7. The dd-endopeptidases also contribute to septal PG hydrolysis; these cleave within the glycan strands and hydrolyze dd-cross-links, respectively , a partially functional allele that supports cell viability , 31E. co strands . The bes strands , the cel strands , 35, and strands . Geneticentation . DedD beiability .How DamX and DedD affect cell division and what their function is during septation are currently unknown. In this work, we provide the first genetic and biochemical evidence supporting a direct role of DamX and DedD in enhancing the activity of PBP1B and, in the case of DedD, the activity of PBP1A.E. coli: FtsN (PDB ID 1UTA) (2LFV)  (Bacillus subtilis (PDB ID 1X60)  spectroscopy to provide structural context for our functional study of the role of SPOR proteins in E. coli.SPOR domain protein structures have been determined for two of the four proteins in ID 1UTA)  and DamX) (2LFV) . Additiond 6I0A) , and theID 1X60)  have bee1H-15N correlation spectrum of 15N-labeled DedD (residues 28 to 220 with a single transmembrane region removed) displayed both disperse peaks and intense narrow peaks with a very low 1H chemical shift dispersion, confirming the presence of structured and unstructured regions (1H-15N nuclear Overhauser effect (NOE) relaxation measurements displayed positive NOE values (>0.6) for residues 144 to 220, in agreement with the presence of a globular and stable SPOR domain  to the C terminus (red). Download Copyright © 2020 Pazos et al.2020Pazos et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02796-20.5FIG S21H-15N heteronuclear NOE recorded with and without initial proton saturation. The saturation experiment is light purple (positive) and dark purple (negative). The interleaved reference experiment, recorded without 1H saturation, is in black. Download FIG S2, PDF file, 1.1 MB.Overlay of Copyright © 2020 Pazos et al.2020Pazos et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02796-20.6FIG S31H-15N BEST-TROSY spectrum of DedD 28-220 is shown, with the peak assignments indicated. Download FIG S3, TIF file, 2.7 MB.The Copyright © 2020 Pazos et al.2020Pazos et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa RlpA (6I0A)  . We see A (6I0A) . This biA (6I0A) , such asA (6I0A) . Overall10.1128/mBio.02796-20.7FIG S46I0A)  binding by DedD was generated via superposition of the NMR structure of DedD (this work) on the liganded crystal structure of RlpA (PDB ID 6I0A) . Sequenc6I0A) , are map6I0A) , are mapCopyright © 2020 Pazos et al.2020Pazos et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli, based on the decrease in fitness of the single-knockout mutants when grown in the presence of cefsulodin, as revealed by a chemical genomics screening . Inducer, 10 μg ml−1 sodium salicylate. Download FIG S5, TIF file, 2.8 MB.DamX structural domains and constructs tested in the present work. Tenfold serial dilutions of wild type and ΔCopyright © 2020 Pazos et al.2020Pazos et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02796-20.9FIG S6FIG S6, TIF file, 2.4 MB.Overproduction of the different DamX constructs. Coomassie-stained SDS-PAGE gel (upper gel) and Western blot using anti-His tag antibody to detect the overproduction of the indicated DamX variants. Whereas the SDS-PAGE gel contained both noninduced and induced samples of each strain, the Western blot contained only the induced ones, with an empty lane between samples. The detected proteins are marked with asterisks. Download Copyright © 2020 Pazos et al.2020Pazos et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the −1 for cells lacking PBP1B (mrcB) and 30 μg ml−1 for cells lacking PBP1A (mrcA). The overproduction of DamX or DedD increased the resistance to cefsulodin of cells lacking PBP1A (mrcA) but not of cells lacking PBP1B (mrcB)  , supportdamX mutant manifests specifically in the presence of cefsulodin and is independent of PBP3 activity.PBP1A and/or PBP1B are required to incorporate new peptidoglycan at the lateral cell wall of the future cell division site during the preseptal cell elongation phase . The SPO10.1128/mBio.02796-20.10FIG S7mrcA or ΔmrcB strains. Growth inhibition of the indicated strains by concentration gradient strips of aztreonam. Download FIG S7, TIF file, 2.1 MB.The absence of DamX does not alter the MIC of aztreonam for ΔCopyright © 2020 Pazos et al.2020Pazos et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli host, solubilized with DDM  detergent and copurified by affinity chromatography on a nickel-nitrilotriacetic acid (NTA) column, making use of an N-terminal His tag on the SPOR protein. The purified fractions were labeled with Bocillin FL and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorescence imaging and Coomassie blue staining. The results show that PBP1B coeluted with His-DamX, while PBP1A was found only in the wash fraction . We observed no significant changes in the percentage of cross-links produced by PBP1A or PBP1B  cells under nonpermissive conditions, suggesting that DamX antagonizes FtsQ function  medium  for protein production. When appropriate, antibiotics were added to the medium .Unless stated otherwise, The following proteins were purified following published protocols: PBP1A , PBP1B , and Fts(i) RlpA. LOBSTR (low-background strain) E. coli cells containing plasmid pPZW25 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an optical density at 578 nm (OD578) of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG  to the cell culture, which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation , and the pellet was resuspended in buffer I . After addition of 200 μM phenylmethylsulfonylfluoride (PMSF), a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich) and DNase, the cells were disrupted by sonication . The cell lysate was centrifuged , and the supernatant was incubated overnight with 4 ml of nickel-nitrilotriacetic acid (Ni-NTA) Superflow (Qiagen), which had been pre-equilibrated in buffer I containing 20 mM imidazole, at 4°C with gentle stirring. The resin was poured into a gravity column and washed with 25 volumes of wash buffer . Bound protein was eluted with elution buffer . The Ni-NTA-eluted protein was dialyzed against 1 liter of dialysis buffer I  for 30 min; 500 ml of dialysis buffer I was replaced with 500 ml dialysis buffer II  and further dialyzed for 30 min. Restriction-grade thrombin  was added to remove the oligohistidine tag during overnight dialysis against 1 liter of dialysis buffer II at 4°C. The sample was diluted 1:1 with buffer no salt  and applied in AKTA A buffer  to a 5-ml HiTrap Q HP column using an ÄKTA Prime system  for anion-exchange chromatography . Although a gradient from 150 mM to 1 M NaCl was applied, the protein was present in the flowthrough and wash. The protein was dialyzed against 3 liters of storage buffer  and stored at −80°C.(ii) His-DamX. LOBSTR cells containing plasmid pPZW23 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an OD578 of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG to the cell culture, which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation , and the pellet was resuspended in buffer I . After addition of 200 μM PMSF, a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich) and DNase, the cells were disrupted by sonication , and the cell lysate was centrifuged . The supernatant was discarded, and the membrane pellet was resuspended in extraction buffer  and incubated overnight with mixing at 4°C. Resuspended sample was centrifuged , and the supernatant was incubated with Ni-NTA Superflow (Qiagen) as described for RlpA. The protein did not bind to the resin and was collected from the flowthrough. The flowthrough containing the protein was dialyzed against 3 liters of dialysis buffer I  overnight. The sample was applied in AKTA A buffer  to a 5-ml HiTrap Q HP column using an ÄKTA Prime system , and the protein was collected in the flowthrough. The protein was dialyzed against 3 liters of dialysis buffer II  overnight. The sample was applied in dialysis buffer II containing 0.2% Triton X-100 reduced to a 5-ml HiTrap SP HP column using an ÄKTA Prime system  for cation-exchange chromatography . The protein was eluted using a gradient from 150 mM to 2 M NaCl. Protein-containing fractions were dialyzed against 3 liters of storage buffer  and stored at −80°C.(iii) DamX-His and sDamX-His. LOBSTR cells containing plasmid pPZW26 and pPZW27 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an OD578 of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG to the cell culture which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation , and the pellet was resuspended in buffer I . After addition of 200 μM PMSF, a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich), and DNase, the cells were disrupted by sonication , and the cell lysate was centrifuged . For DamX-His, the supernatant was discarded, and the membrane pellet was resuspended in extraction buffer  and incubated overnight with mixing at 4°C. Resuspended sample was centrifuged , and the supernatant was incubated with Ni-NTA Superflow (Qiagen), washed, and eluted as described for RlpA using buffers containing 0.2% Triton X-100 reduced. The eluted protein was dialyzed against 3 liters of storage buffer  and stored at −80°C. sDamX-His was purified from the supernatant of the cell lysate centrifugation using the protocol for DamX-His but omitting detergent in buffers. The eluted protein was dialyzed against 3 liters of dialysis buffer I  for 1.5 h and against 3 liters of dialysis buffer II  overnight. The sample was diluted 1:1 with buffer no salt  and applied in AKTA A buffer  to a 5-ml HiTrap SP HP column using an ÄKTA Prime system  for cation-exchange chromatography . The protein was eluted in a gradient from 100 mM to 2 M NaCl. Protein-containing fractions were dialyzed against storage buffer  and stored at −80°C.(iv) DedD. LOBSTR cells containing plasmid pPZW24 were grown in 2 liters of LB medium supplemented with kanamycin at 37°C to an OD578 of 0.4 to 0.5. Protein overproduction was induced by addition of 0.5 mM IPTG to the cell culture, which was further incubated for 4 h at 37°C. Cells were harvested by centrifugation  and the pellet was resuspended in buffer I . After addition of 200 μM PMSF, a 1-in-1,000 dilution of protease inhibitor cocktail (Sigma-Aldrich) and DNase, the cells were disrupted by sonication , and the cell lysate was centrifuged . The supernatant was discarded, and the membrane pellet was resuspended in extraction buffer  and incubated overnight with mixing at 4°C. Resuspended sample was centrifuged , and the supernatant was incubated with Ni-NTA Superflow (Qiagen), washed, and eluted as described for RlpA using buffers containing 0.2% Triton X-100 reduced. Restriction-grade thrombin  was added to the Ni-NTA-eluted protein to remove the oligohistidine tag during dialysis against 3 liters of dialysis buffer I  for 20 h at 4°C. Sample was dialyzed against 3 liters of dialysis buffer II  for 4 h at 4°C and 3 liters of dialysis buffer III  for 18 h at 4°C. The sample was diluted 1:1 with no-salt buffer  and applied in AKTA A buffer  to a 5-ml HiTrap SP HP column using an ÄKTA Prime system  for cation-exchange chromatography . The protein eluted in a gradient from 150 mM to 2 M NaCl. Protein-containing fractions were dialyzed against storage buffer  and stored at −80°C.−1) at 37°C to an A600 of 0.8. Protein expression was induced for 3.5 h by addition of 0.5 mM IPTG. Cells were collected by centrifugation at 4,000 × g for 20 min at 15°C and resuspended in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, and EDTA-free protease inhibitor cocktail (Roche). The cells were lysed by three passages through a cell homogenizer . After centrifugation at 4,000 × g for 20 min at 4°C, the supernatant was recovered and spun down at 150,000 × g for 1 h at 4°C. The pelleted membranes were solubilized in 25 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10% (vol/vol) glycerol, 40 mM DDM  and 1 tablet of complete EDTA-free protease inhibitors (Sigma) per 50 ml of buffer. The mixture was incubated for 1 h at room temperature under gentle agitation followed by centrifugation at 150,000 × g for 1 h at 4°C. The supernatant containing the solubilized membrane proteins was loaded onto a HisTrap column  conditioned in buffer B . After a wash with buffer B supplemented with 80 mM imidazole, the proteins were eluted in 0.5- to 1-ml fractions using a linear gradient of imidazole from 80 to 500 mM. A 15-μl portion of each fraction was incubated with 2 μM Bocillin FL for 30 min at 37°C. The fractions were analyzed by SDS-PAGE, followed by fluorescence imaging and Coomassie blue staining.C43(DE3) cells transformed with pETDuet plasmids  were gro600 of 0.6, at which point 1 mM IPTG was added to induce protein overproduction overnight at 25°C. Harvested cell pellets were resuspended in lysis buffer  and lysed by processing twice with a homogenizer . Cellular debris was pelleted by centrifugation at 125,000 × g for 1 h. The resultant supernatant was loaded onto 10 ml Ni2+-saturated Ni-NTA Superflow beads (Qiagen) and washed with 65 mM imidazole in 20 mM HEPES (pH 8.0)–300 mM NaCl, and the protein was eluted with 300 mM imidazole in the previous buffer. Fractions containing pure DedD were pooled and desalted into a buffer of 20 mM HEPES (pH 8.0) and 300 mM NaCl. Protein was frozen rapidly in liquid nitrogen and stored at −80°C until required.For labeled DedD (residues 28 to 220) purification, pYS001 was transformed into BL21(DE3). This strain was cultured in 1 liter of M9 containing 1 g/liter of ammonium chloride and 2 g/liter of glucose at 37°C to an OD−1 chloramphenicol when strains carrying pKG110-derived plasmids were assayed. When appropriate, 10 μM sodium salicylate (inducer) was added to the plates. Unless stated otherwise, 30 μg ml−1 cefsulodin was used.Cells were grown overnight at 30°C, the optical density was normalized for each strain assayed in the plate, and the cells were spotted in a 10-fold dilution series on Lennox LB plates , which were incubated overnight at 30°C. Plates were supplemented with 20 μg ml578 of 0.3 to 0.4. A 1-ml portion of each strain was centrifuged for 1 min at 13,000 × g and resuspended in 1 ml of 0.9% NaCl. Resuspended cells were diluted to an OD578 of 0.125 using 0.9% NaCl. Cells were distributed onto LB Lennox plates using a cotton swab soaked with the cell suspension. Once the plates were dried, an aztreonam MIC test strip (Liofilchem) was applied to each plate, and all the plates were incubated overnight at 30°C.Overnight cultures of the test strains were grown at 30°C in LB Lennox, reinoculated 1:100, and grown to an OD14C]GlcNAc-labeled lipid II . An endpoint GTase-TPase activity assay was performed as described elsewhere  of [14C]GlcNAc-labeled lipid II was dried in a glass vial using a vacuum concentrator and resuspended in 5 μl of 0.2% Triton X-100. To start the reactions, the assayed proteins were added to the resuspended lipid II and further incubated for 60 min at 37°C with shaking (800 rpm). Reactions were stopped by boiling for 5 min, and further cellosyl digestion, reduction, and analysis by HPLC were performed as described in reference 18) differs between samples due to differences in peak 1, the phosphorylated disaccharide pentapeptide. Peak 1 is generated by acid hydrolysis of unused lipid II (or glycan strands ends carrying the C55-PP moiety) after the GTase-TPase reaction, because lipid II (without hydrolysis) does not elute from the C18 HPLC column used to separate the muropeptides. In samples with abundant unused lipid II, peak 1 varies due to differences in the efficiency of the acid hydrolysis of lipid II between samples. This effect does not impair the quantification of other peaks (PG products). Tris-Tricine SDS-PAGE was used to separate glycan strands , at 298 K on 1.08 mM 13C,15N-labeled DedD sample prepared in a 3-mm-diameter NMR tube. All NMR spectra for backbone, side chains, and NOE assignments were recorded on Bruker spectrometers operating at 700, 850, and 950 MHz 1H NMR frequencies and equipped with 1H,13C,15N-labeled cryoprobes.NMR data were collected in 20 mM HEPES, 300 mM NaCl, 10% D1H,15N-BEST-TROSY (BT), 3D BT-HNCANH, 3D BT-HNCO, 3D BT-HNCACO, 3D BT-HNCACB and 3D BT-HN(CO)CACB spectra. Manual side chain assignment was then achieved with conventional 2D 1H,13C-HSQC (gradient heteronuclear single quantum coherence), 3D (H)C(CCO)NH, 3D H(CCCO)NH, and 3D 15N-NOESY-HSQC, as well as 3D aliphatic and aromatic 13C-NOESY-HSQC experiments. Spectra were analyzed with CcpNmr Analysis 2.4.1.Resonance assignments of the backbone was performed using two-dimensional (2D) For structural restraints, dihedral angles (phi and psi) were predicted from backbone chemical shift with the neural network program TALOS+, and distance constraints were determined after manual peak-picking and automatic assignment of the 3D NOESY-HSQC experiments reported above by Unio′10 version 2.0.2. Structures were subsequently calculated from these restraints by Aria 2.3.1, with 80 structures from runs 0 to 5, 200 for runs 6 and 7, and 600 for the last run. The 20 lowest-energy structures were further refined in water. Ramachandran analysis showed 86.1%, 13.9%, 0.0%, and 0.0% of the residues of DedD in most favored, additional allowed, generously allowed, and disallowed regions, respectively.1H-15N NOE relaxation data were collected at 25°C on Bruker spectrometers operating at 700 MHz and equipped with 1H,13C,15N-labeled cryoprobes. 1H-15N NOE values were determined by the comparison of the intensities of each amide resonance with and without a 3-s saturation period and using the BEST-HETNOE sequence (sequence . Standar6ZTG. All other data supporting the findings of this study are included in the main text and supplemental material.The 20 lowest-energy structures were deposited in the PDB with accession number


Eucommiaulmoides in terms of usefulness as the aligner-type orthodontic device. Erkodur, Essix C+®, Eucommia elastomer, and edgewise brackets were used . The orthodontic force on the upper right incisor was measured every 24 h for two weeks using a custom-made measuring device. The force of the Eucommia elastomer (4.25 ± 0.274 N) and multi bracket system (5.32 ± 0.338 N) did not change from the beginning to the end (p > 0.01). The orthodontic force exerted by the Eucommia elastomer was lower than that of the multi-bracket orthodontic appliance from the beginning to the end. The force of Erkodur significantly decreased from the beginning to 24 h (6.47 ± 1.40 N) and 48 h (3.30 ± 0.536 N) (p < 0.01). The force of Essix C+® significantly decreased from the beginning (13.2 ± 0.845 N) to 24 h (8.77 ± 0.231 N) (p < 0.01). The thermoplastic orthodontic appliance made of Eucommia elastomer continuously exerted a constant orthodontic force for two weeks under water immersion conditions. The orthodontic force of Eucommia elastomer was found to be similar to the orthodontic force exerted by the multi-bracket orthodontic appliance with 0.019 × 0.025 in nickel–titanium wire. These results suggest that the Eucommia elastomer has possibly become one of the more useful materials to form thermoplastic orthodontic appliance exerting low continues orthodontic force.The aim of this study was to investigate the orthodontic force exerted by thermoplastic orthodontic appliances incorporating  Orthodontic treatment using a transparent thermoplastic orthodontic appliance, called aligner-type orthodontic devices, has recently increased with the aesthetic demands of patients. Aligner-type orthodontic devices appeared in the early 1970s as a retainer for preventing relapse of the teeth after orthodontic treatment . To dateHowever, the amount of tooth movement using an aligner is limited to a range of 0.25 mm to 1.0 mm ,12, so aTherefore, to make orthodontic treatment with an aligner more efficient, the development of a new thermoplastic materials that can continuously exert suitable orthodontic force for tooth movement are required.Eucommia elastomer (EE), a trans-1,4-polyisoprene extracted and purified from Eucommia ulmoides, is a highly biosafety polymer exhibiting high tensile properties and large breaking strain [g strain . As a teg strain ,16,17,18g strain ,21,22,23Eucommia ulmoides in terms of usefulness as the aligner-type orthodontic device.The aim of this study was to investigate the orthodontic force exerted by thermoplastic orthodontic appliances incorporating ® Plastic (EC) , and (EE)  were used. Trans-1,4-polyisoprene powder extracted and purified from Eucommia ulmoides was fused and was formed as EE by mold injection. All of these materials were 1.0 mm thick single-layer materials with 120.0 mm in diameter (Erkodur (ER) , Essix C+diameter .n = 3, each). The ER was heated to 160 °C, pressed to the model, and cooled for 45 s using a vacuum-forming machine   was duplicated using a 3D scanner  and 3D printer  as the normal dentition. The aligners were formed using ER and EC according to the instructions of the manufacturer (Germany) . The EC Germany) . The EE Germany) .n = 5). Test specimens were prepared similar to the water absorption test and subjected to X-ray diffraction (XRD) analysis  in dry condition The XRD analysis was conducted at 2θ range between 2° and 60° with a step size of 0.02° in a continuous mode of 4.0°/min. Fourier transformation infrared spectroscopy (FTIR) was conducted. Part of each material (6.0 × 6.0 × 1.0 mm) was prepared and the spectra were recorded with an FTIR spectrometer  and averaged over 20 scans between 700–4000 cm−1. Ten specimens of 1.0 mm thickness were prepared according to ISO 527-2 type 5B [Five round specimens (ϕ15 × 1.0 mm) of EE was prepared and water absorption test was conducted according to ISO 4049:2019  (n = 5). type 5B . Five sp type 5B  and were type 5B  and compUsing 3D data of the standard model , the stainless-steel-made dentition model was prepared where the upper right central incisor was tipped 3° labially  as the crowded dentition. A force sensor  was attached to at the middle of root of the upper right central incisor a. AligneTo compare the force, the orthodontic force exerted by multi-bracket orthodontic appliances using nickel titanium wire and metal brackets was measured using the same dentition model. The 0.022 × 0.028 inch slot brackets  were attached to the dentition model using light-cure orthodontic adhesive system  and metal primer . The 0.019 × 0.025 in nickel titanium wire  were prepared and inserted to the dentition model . They wet-test. The mean elastic modulus of EE after 24 h without strain and two weeks with 1% constant strain were analyzed by the Student’s t-test. The p-values of less than 0.05 were considered as statistically significant. The mean force in each group were statistically analyzed by one-way analysis of variance (ANOVA) and Dunnett’s test . The p-values of less than 0.01 were considered as statistically significant.The mean amount of water absorption of EE after 24 h and two weeks were analyzed by the Student’s 3 and 0.8761 ± 0.3730 μg/mm3, respectively, and a significant difference was found (p < 0.05). The XRD patterns of EE are shown in p > 0.05).The amount of water absorption of EE after 24 h and two weeks were 0.0344 ± 0.4291 μg/mmp > 0.01). The orthodontic force exerted by EE was lower than that of multi-bracket orthodontic appliance from the beginning to the end. The force of ER was significantly decreased from the beginning to 24 h (6.47 ± 1.40 N) and 48 h (3.30 ± 0.536 N) (p < 0.01). The force of EC was significantly decreased from the beginning (13.2 ± 0.845 N) to 24 h (8.77 ± 0.231 N) (p < 0.01).Various measuring devices have been reported for orthodontic force ,21,22,23The EE demonstrated a lower amount of water absorption than both ER and EC . Also, tIn ER, it has been reported that the force that exerts as tooth displacement increases also increases ,21,22. OThe optimal correction force for multi-bracket devices is said to be in the range of 0.343 to 0.980 N  or not e2 [Schwarz  has repo2 , it can 2  exceeded2  and Igle2  reported2 , dynamic2 , tooth m2 ,36. For In addition, the thermoplastic materials’ wear, perforation, and cracking of the aligner has been reported as an issue ,38, and Furthermore, the EE is made from a natural plant and biocompatibility was confirmed according to ISO 10993 ,41; it iEucommia ulmoides continuously exerted a constant orthodontic force for two weeks under water immersion conditions. The orthodontic force of Eucommia elastomer was found to be similar to the orthodontic force exerted by the multi-bracket orthodontic appliance with 0.019 × 0.025 in nickel–titanium wire. Those results suggest that the Eucommia elastomer is possibly become one of useful materials to form thermoplastic orthodontic appliance exerting low continues orthodontic force.The thermoplastic orthodontic appliances incorporating


Vitamin D deficiency has recently become a global public health problem. However, it is still unclear if gene polymorphisms in the vitamin D pathway influence vitamin D levels among pregnant women in Eastern and Central China. The objective of this study was to assess factors influencing vitamin D levels in pregnant women. A total of 326 participants in Shandong and Henan provinces in China were enrolled from August 2017 to April 2019. Serum 25(OH)D levels and single nucleotide polymorphisms (SNPs) in the vitamin D pathway were measured using the blood samples collected in the first trimester, second trimester, and third trimester. Data on demographics, lifestyle, and health behavior were collected using a questionnaire. Statistical analyses were performed using the R software. The prevalence of 25(OH)D deficiency was significantly more severe in pregnant women. The average 25(OH)D value of all enrolled pregnant women was 14.57 ± 7.21 ng/ml (deficiency). Only 15 (4.60%) participants had a 25(OH)D concentration ≥30 ng/ml (sufficient). The prevalence of four ranks of vitamin D levels from severe 25(OH)D deficiency to 25(OH)D sufficiency  was 29.14%, 52.45%, 13.80%, and 4.60%, respectively. Variants of GC (rs1155563) and CYP24A1 (rs6013897) were significantly associated with both 25(OH)D concentrations and vitamin D deficiency among pregnant women, respectively. Our findings suggest that pregnant women in Eastern and Central China are at high risk of vitamin D deficiency. Genetic mutants in the vitamin D pathway (GC and CYP24A1) were significantly associated with 25(OH)D levels in pregnant women in Eastern and Central China. Results of this study showed that pregnant women were at high risk of vitamin D deficiency in Eastern and Central China. Serum 25(OH)D level was influenced by BMI, physical activity time, vitamin D intake, and gene polymorphisms in vitamin D pathway (rs1155563 in GC and rs6013897 in CYP24A1) among pregnant women. Vitamin D supplementation could modify this effect of gene polymorphisms. The ris3 to 1,25(OH)2D3 is present in the kidney. Calcitriol conversed in the kidney binds to the vitamin D binding protein (DBP), which is encoded by the GC vitamin D binding protein (GC) gene. 24‐hydroxylase (CYP24A1) is capable of transforming 25(OH)D and 1,25(OH)2D to their inactive forms to protect target organs or tissues from excessive vitamin D signaling , 24‐hydroxylase (CYP24A1), and the vitamin D receptor (VDR) genes. Other previous studies have also reported significant associations of variants in VDR, CYP27B1, CYP24A1, DHCR7/NADSYN1, CYP27A1, and CYP3A4 . The CYP27B1 (cytochrome P450 monooxygenase 25(OH)D 1αhydroxylase) that metabolizes 25(OH)D Carter, . Thus, t Carter, . Variant Carter, . Barry e Carter,  revealed2 and 25(OH)D3, antibody cross‐reactivity, and high inter‐laboratory variation (up to 38%) D3 separately D levels and SNPs during pregnancy to guide pregnant women in order to elevate their 25(OH)D levels.22.1A total of 326 pregnant women who resided in Tengzhou and Dengzhou cities for at least 1 year were enrolled from several community centers between 2017 and 2019. Clinical information including age, weeks of gestation, body mass index (BMI), sun exposure, dietary and drinking habits, physical activities, and medical records during pregnancy was completed by all subjects.2.2g, 5 min, 4°C) to separate serum (used for measuring 25(OH)D levels) and EDTA anticoagulant tubes used for DNA extraction. The participants' age, sex, height, and body weight were recorded. Other baseline characteristics such as the use of medication, smoking habits, level of physical activity, drinking habits, sleeping time, and lifestyle were obtained via a standardized questionnaire.The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Medical Ethics Committee of Central People's Hospital of Tengzhou, Jining Medical University, and the Research Ethics Committee of the Central Hospital of Dengzhou. Informed consent was obtained from all participants. With the participants' consent, a fasting blood sample (8 hr) was drawn into a serum separator tube and an EDTA anticoagulant tube. Serum separator tubes were centrifuged ; GC ; CYP2R1 ; CYP24A1 ; CYP27B1 ; CYP3A43 ; NADSYN1/DHCR7 ; RXRA . A Rapid DNA Extraction and Detection Kit (Tiangen) was used to extract DNA from peripheral blood leukocytes according to the manufacturer's protocol, and SNP genotyping was conducted and analyzed using the MassARRAY iPLEX Gold platform (Sequenom).2.42 and 25(OH)D3 levels in the serum were measured using a modified LC‐MS/MS method. Briefly, 100 μl aliquots of calibrators or serum samples were spiked with 500 μl isotope‐labeled internal standard solution. Samples were vortexed for 1 min and centrifuged for 5 min at 9,250 g and 4°C. After centrifugation, 400 μl of sample supernatant was transferred into a 96‐deep well plate and then dried under nitrogen at 60°C. Using 100 μl acetonitrile solution of PTAD, we performed derivatization for 60 min and then added 50 μl methanol for reconstitution. Subsequently, the residuals were injected into the UPLC‐MS/MS system (Waterscorporation). Chromatographic separation on the Waters ACQUITY UPLC I‐class was performed under binary gradient conditions with a mobile phase A of water with 0.1% formic acid (volume fraction) and 5 mM ammonium formate and mobile phase B of methanol with 0.1% formic acid. The column temperature was maintained at 40°C. Xevo TQD was used for MS/MS detection in positive electrospray ionization mode and multiple reaction monitor (MRM) mode. The accuracy of this method was validated by analyzing the National Institute of Standards and Technology SRM 972a. Comparing the measurement results with the reference values of SRM 972a, the accuracy of this method was 90%–115% and 95%–110% for 25(OH)D2 and 25(OH)D3, respectively.The 25(OH)D2.5We used the commonly accepted cutoffs for severe 25(OH)D deficiency (<10 ng/ml), deficiency (10–20 ng/ml), and insufficiency (20–30 ng/ml). Levels ≥30 ng/ml were considered sufficient or optimum, while levels >150 ng/ml were considered vitamin D intoxication Holick, .2.6t test or analysis of variance (ANOVA) was used to compare the mean values of continuous variables. Continuous variables were expressed as mean ± SD, while categorical variables were expressed as frequency and percentage. Body mass index was divided into four subgroups  according to the standard in China D deficiency were re‐analyzed using the Kruskal–Wallis H test modified by vitamin D supplementation and sunshine exposure. Differences were considered statistically significant at a p value <.05.Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) 17.0 (SPSS Inc.) and R software. Student's 33.1SD, 23.81 ± 3.85). Almost half of them experienced fewer physical activities . Most of them had less sunshine exposure  during pregnancy. None of the participants had drinking or smoking behavior during pregnancy.The study included 326 pregnant women based on biochemical tests, physical examinations, and detailed questionnaires. The levels of biochemical indicators and clinical characteristics are shown in Table 3.2Although nearly half of the participants had a habit of vitamin D intake Table , we foun3.3To further determine the correlation between 25(OH)D levels and other related clinical characteristics, the influence factors including daily vitamin intake, age, body mass index (BMI), and physical activity were analyzed. As shown in Table 3.4To evaluate the possible contribution of variations in 25(OH)D status among pregnant women, 40 SNPs in the genes encoding the vitamin D pathway enzymes  were analyzed. In the variance analysis, rs1155563 in GC and rs6013897 in CYP24A1 were associated with 25(OH)D levels among the pregnant women. Both SNPs with their minor alleles had lower 25(OH)D concentrations than those with major alleles D. We completed all test procedures in one single‐center analysis by a single experienced technician to eliminate the probability of inter‐laboratory variation. Based on this method, we identified that severe 25(OH)D deficiency or insufficiency was prevalent among pregnant women in Shandong and Henan Provinces, China. This finding is in line with previous studies, indicating that low 25(OH)D levels are a global problem  could not separately test all of the epimers of 25(OH)D, such as 3‐epi 25(OH)DIn summary, the 25(OH)D status was tested using a reliable LC‐MS/MS method. The results indicate that pregnant women in Eastern and Central China are at high risk of vitamin D deficiency, suggesting the need for advanced training to improve food safety knowledge, attitudes, and practices. rs1155563 in GC and rs6013897 in CYP24A1 influenced 25(OH)D levels in pregnant women. This study also revealed that 25(OH)D levels were influenced by BMI, physical activity time, and vitamin D intake among pregnant women. Repletion in this population may require vitamin D dietary supplementation, increasing physical activity, and appropriate BMI. To improve the vitamin D nutritional status of pregnant women, the dosage and dietary structure of pregnant women's vitamin D should be appropriately determined.The authors declare that they do not have any conflict of interests.


CYP) 2C9 is known to cause significant inter-individual differences in drug response and adverse effects. The frequencies of CYP2C9*2 and CYP2C9*3, both of which are responsible for the low activity of the enzyme, are not known in the Pakistani population. Therefore, we screened various ethnic groups residing in Pakistan for these polymorphisms. A total of 467 healthy human volunteers were recruited from six major ethnicities of Pakistan after written informed consent. Our results indicate that about 20% of the Pakistani population has a genotype containing at least one low activity allele. Ethnic Punjabi and Pathan populations had the highest frequencies of wild type genotypes while Urdu, Seraiki, and Sindhi populations showed higher rates of both low activity genotypes. The Baloch population showed the highest rates of low activity genotypes with less than 50% of the samples showing wild type genotypes, suggesting that more than half of the Baloch population possesses low activity genotypes. The frequencies found in various ethnic groups in Pakistan were comparable with ethnicities in the South Asian region except for the Baloch population. These results suggest that pharmacogenetics screening for low activity genotypes may be a helpful tool for clinicians while prescribing medications metabolized by CYP2C9.Genetic variation in cytochrome P450 ( In recent years, these CYP polymorphisms have become important pharmacogenetic biomarkers for drug dosing, efficacy, and safety. However, there is considerable variation in the distribution of CYP alleles in different populations around the world, which may result in variation in drug response and has important implications for personalized treatment.Genetic polymorphisms in cytochrome P450(CYP) genes can lead to altered enzymatic activity with consequent variation in the pharmacokinetics of a metabolized drug. This variation in the pharmacokinetics of the drug may lead to a change in the response of the drug. It is usually the result of single nucleotide polymorphisms (SNPs) or copy number variations in CYP genes. Therefore, these genetic variants play a crucial role in determining drug pharmacokinetics, non-responder phenomenon, and toxicity2. Another important site of expression is the gastrointestinal tract3. CYP2C9 is responsible for the metabolism of approximately 13% of the drugs metabolized by the cytochrome-catalyzed reactions4. Many of these drugs are clinically very important but have a narrow therapeutic index . Therefore, inter-individual variation in CYP2C9 expression and activity may result in inter-individual variation in drug response and safety. Genetic polymorphisms in CYP2C9 and drug-drug interactions further increase the variation in drug response6.One of the most important subfamilies of CYP is CYP2C, which consists of four enzymes: CYP2C8, CYP2C9, CYP2C18, and CYP2C19. Of these, the enzyme expressed most abundantly is CYP2C9, which is significantly involved in the metabolism of many important clinical drugs. Based on quantification by mass spectrometry, CYP2C9 is about 20% of the total hepatic P450 protein contentCYP2C9 gene contains several variants, but two of the most prevalent, investigated, and influential of them are rs1799853 and rs1057910, also known as CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. CYP2C9*2 results in an amino acid substitution from Arg to Cys as a consequence of C > T transition in the CYP2C9 gene at position 430 . CYP2C9*3 causes an amino acid substitution from Ilu to Leu as a result of a transversion, A > T in the CYP2C9 gene 7. The activity of CYP2C9 is significantly reduced as a result of these polymorphisms, and in the Caucasian population, they are responsible for a majority of decreased CYP2C9 activity phenotypes7. Both of these variations decrease the rate of phenytoin hydroxylation8. Hydroxylation of S-warfarin is impaired by CYP2C9*26, while the metabolism of tolbutamide is decreased by CYP2C9*39. Benzo[a]pyrene, an important lung carcinogen, is also metabolized by CYP2C9, and therefore, SNPs in this gene also carry the risk of lung cancer10. Therefore, these SNPs not only affect drug response and adverse effects, but they are also associated with certain disease phenotypes.CYP2C9 allele distribution have been described for various populations. Pakistan is a culturally diverse country, but little is known about the distribution of CYP2C9 genetic polymorphism in this country of over 200 million people. Therefore, we intended to determine the frequencies of these polymorphisms in the Pakistani population, with samples drawn from six of its most populous ethnic groups. We specifically investigated the samples of various ethnic populations from Pakistan to examine the frequencies of CYP2C9*1, CYP2C9*2, and CYP2C9*3 and then compared them with previous findings in other populations.Interpopulation differences in drug responses are well known, and in some cases, they correspond to differences in the frequency of associated genetic markers, especially CYP genes. That is why differences in CYP2C9*1, CYP2C9*2, and CYP2C9*3 in the Pakistani population were found to be 0.876, 0.059, and 0.064, respectively . In Urdu speaking population, the rate of CYP2C9*1, CYP2C9*2, and CYP2C9*3 were slightly different at 0.820, 0.078, and 0.023, respectively. The Seraiki population was found to have CYP2C9*1, CYP2C9*2, and CYP2C9*3 at frequencies of 0.882, 0.46, and 0.73, respectively. The results obtained from the Baloch population diverged significantly. In the Baloch population, the frequencies of CYP2C9*1, CYP2C9*2, and CYP2C9*3 were found to be 0.620, 0.160, and 0.220, respectively, whereas, in the Sindhi population, the frequencies were 0.830, 0.090, and 0.080 respectively  were homozygous for CYP2C9*2*2. This suggests that the homozygous polymorphism is rare in the Pakistani population. The frequency of these polymorphisms was found to be slightly different in different ethnic populations in Pakistan except for Baloch population samples, which showed an unusually high frequency of these polymorphisms. We recommend that genotyping of the CYP2C9 gene in patients on drugs such as warfarin, phenytoin, etc., may help to overcome the drug toxicity, chose the right alternative, and guide in therapeutic drug monitoring.In conclusion, both the οC. Genomic DNA was isolated using Gene Jet Genomic DNA extraction Kit (ThermoScientific) and was quantified using 1% agarose gel electrophoresis. Isolated genomic DNA was stored at – 20 °C until further processing33.The study was approved by the Institutional Review Board and Ethics Committee of Shifa Tameer-e-Millat University, Islamabad, Pakistan, through approval number IRB#990–265-2018. Informed written consent was obtained from all participating individuals. All experiments were performed in accordance with relevant guidelines and regulations. A total of 467 unrelated individuals from a healthy population were recruited for the present study. The study cohort consists of six major ethnicities of Pakistan, including Punjabis, Pathan, Sindhi, Balochi, Seraiki, and Urdu speaking. Ethnicity was self-reported. Five milliliters of venous blood was drawn into a sterile tube containing EDTA as an anti-coagulant and were stored at 4CYP2C9*2 and *3 were genotyped using ARMS-PCR (Allele Refractory Mutation System- Polymerase Chain Reaction) using a pair of outer primers and a pair of inner primers as described previously 34. PCR for both the SNPs was performed in a single tube with a total reaction volume of 25 µl containing 12.5 µl of 2X Dream Taq Master mix (ThermoScientific), 0.5 pM of 2C9*2 wild type reverse primer, 1.5 pM of 2C9*2 mutant reverse primer, 3.0 pM of common forward primer, 1.0 pM of 2C9*3 wild type forward primer, 2.0 pM of 2C9*3 mutant forward primer, 3.0 pM of common reverse primer and 3 µl of template DNA (20–50 ng/μl). Thermal profile was as follows: initial denaturation at 95οC for 10 min followed by 37 cycles with denaturation at 95οC for 45 s, 45 s of primer annealing at 58οC, initial extension at 72 οC for 45 s, and a final extension at 72 οC for 7 min. For visualization, 12 µl of PCR product was directly loaded onto 4% agarose gel. The PCR products for 2C9*2 had 105 bp fragment for the wild type allele and 114 bp fragment for the mutant allele, whereas 2C9*3 had 159 bp fragment for the wild type allele and 168 bp fragment for the mutant allele. Individuals were genotypes 2C9*1 when neither 2C9*2 nor 2C9*3 was detected. More than one-quarter of the total samples were sent for sequencing to validate further the results obtained through ARMS-PCR.Allelic Data were compiled according to the genotype and allele frequencies estimated from the observed numbers of each specific allele. The frequency of each allele and genotype in our samples is given together with the 95% confidence interval. The confidence interval for proportions was calculated using the formula (CI = p ± (1.96 × SE), SE = qrt [ p(1—p) / n ], p = proportion, n = sample size). Chi-squared test and p values were calculated using observed and expected frequencies as per the Hardy–Weinberg equation.The study was approved by the Institutional Review Board and Ethics Committee of the Shifa International Hospital and Shifa Tameer-e-Millat University, Islamabad, Pakistan.


First-principles calculations verified its biaxial strain effect. Like oxide heterostructures, the oxynitride has a superlattice of insulating and metallic blocks. Given the abundance of perovskite families, this study provides new opportunities to design superlattices by chemically modifying simple perovskite oxides with tunable anion-vacancy patterns through epitaxial lattice strain.Perovskite oxides can host various anion-vacancy orders, which greatly change their properties, but the order pattern is still difficult to manipulate. Separately, lattice strain between thin film oxides and a substrate induces improved functions and novel states of matter, while little attention has been paid to changes in chemical composition. Here we combine these two aspects to achieve strain-induced creation and switching of anion-vacancy patterns in perovskite films. Epitaxial SrVO Properties of perovskite oxides can be changed by manipulating anion-vacancy order patterns, but they are difficult to control. Here the authors show strain-induced creation and switching of anion vacancies in perovskite films in which the direction or periodicity of anion-vacancy planes is altered depending on the substrate employed. If the anion-vacancy concentration (δ in ABOδ3–) is a rational fraction of the oxygen stoichiometry in the unit cell, the vacancies tend to aggregate to form linear or planar defects. For example, fast oxygen diffusion in BaInO2.5 (δ = 1/2) results from oxygen-vacancy chains4. In SrFeO2 (δ = 1), (001)p defect planes of the original perovskite cell allow metal-metal bonding between square-planar Fe(II) centers, leading to a half-metallic state under pressure5. The superconducting Tc in cuprates strongly depends on the number of CuO2 sheets made by introducing (001)p defect planes6. Compounds having such anion-defect chains or planes can be synthesized through a variety of approaches such as cationic substitution7, topochemical and electrochemical reactions9, and under appropriate conditions .In oxides, the introduction of anion vacancies brings about a diversity of chemical and physical properties; the most widely being studied in perovskite oxidesx1−SrxMnO313, improved ferroelectricity in BaTiO314, multiferroicity in EuTiO315, and superconductivity in La1.9Sr0.1CuO4 (ref. 16). Furthermore, a ferroelectric response in tensile-strained SrTaO2N films is ascribed to a change in local coordination geometry17. The lattice mismatch also allows for the introduction of random oxygen vacancies21. Controlling vacancy ordering of perovskite oxides have also been reported27, but these efforts are limited to controlling crystallographic orientation of the deposited films such as Ca2Fe2O5 brownmillerite.Concurrently, advances in the materials science of perovskite-based systems has been amplified with the development of single-crystal  thin films. In particular, lattice strain through a mismatch between the underlying substrate and the deposited film is a key parameter that has been extensively studied. Strain-driven phenomena has led to charge/orbital order in La3 (600 °C in NH3 gas) topochemically transforming to SrVO2.2N0.6 (δ = 0.2) with regular (111)p anion-vacancy planes. This is already a surprising observation as such anion-vacancy order has never been seen in oxynitrides. The crystal and electronic structure of SrVO2.2N0.6 is mainly two-dimensional, with conducting octahedral layers separated by insulating tetrahedral layers. Most surprisingly, the same ammonia treatment of an epitaxial SrVO3 film on different substrates can change the periodicity of the (111)p plane, or can even alter the direction of anion-vacancy plane to (112)p, which is distinct from the previous efforts27 where the crystallographic orientation of the film is altered depending on the substrates. This observation suggests that lattice strain can be used to induce and manipulate the anion-vacancy planes and provide a controllable parameter for the development of exotic structural and electronic states in perovskite films.In this study, we show a low-temperature reaction of SrVO28, but the highly reducing atmosphere of high-temperature reaction with ammonia (ammonolysis) often makes it difficult to obtain the desired structures29. Anion-vacancy order, which is common in oxides, has not been reported in oxynitrides. Recently, low-temperature ammonolysis (≤500 °C) using oxyhydrides has been proven to be a useful approach to access highly nitridized BaTiO3 through topochemical H/N exchange 30. Subsequently, ammonolysis at moderate temperatures (~800 °C) has been shown to promote topochemical O/N exchange (EuTiO2N from EuTiO2.8H0.2)31. The present study extends these approaches to a vanadium oxyhydride32. When reacted at low temperature, 300 °C, in NH3, SrVO2H is topochemically converted into a cubic perovskite SrVO2.7N0.2  exhibit attractive properties including visible-light responsive photocatalysis3 and SrVO2H become drastically different , similar to that of 15R-type perovskite SrCrO2.8 (Sr5Cr5O14) with oxygen-vacancy order along (111)p33. The topochemical nature of the reaction is verified by ammonolysis reactions of Sr2V2O7 above 600 °C, which yielded Sr3V2O8 as the main product  patterns of SrVOent Fig. , althoug33 for SrV2.8 within the R−3m space group (Supplementary Note g) was constrained to gO + gN = 1. This analysis concluded that the isolated tetrahedral (6c) site is fully occupied by oxygen (gO1 = 1), while nitrogen atoms are partially populated at the 18h sites (gN2 = 0.325(8) ≈ 1/3 and gN3 = 0.173(5) ≈ 1/6), resulting in the average chemical formula SrVO2.203(8)N0.597(8). X-ray and neutron refinement for the SrVO2H-derived sample gave similar results, with a composition of SrVO2.22(5)N0.58(5) (Supplementary Note 2.2N0.6 (Sr5V5O11N3).Rietveld refinement to the synchrotron XRD data was carried out assuming the 15R-structure2.2N0.6 contains one-third of the anion vacancies in every fifth SrO3 (111)p planes and the residual oxide anions in the SrO2 plane are re-organized to form a double-tetrahedral layer  in tetrahedral coordination33. Since the tetrahedral vanadium in our case is exclusively pentavalent36, the tetrahedral coordination preference should also be the origin of this phase stabilization where SrV4+O3 (or SrV4+Ox/33–2Nx) is oxidized by aliovalent N3–/O2– exchange. Although diffusion of nitride anions requires larger activation energy relative to oxide anion37, perovskite-based compounds with AO2 layers have shown rapid oxide ion conduction40. In this regard, the diffusion of the nitride ions might be promoted by “in situ” formation of SrO2 layers during the ammonolysis reaction at 600 °C.15R-SrVOyer Fig. . Bond vayer Fig. . The ave3 has a single V4+ site and is characterized as a Pauli paramagnetic metal with a 3D Fermi surface41. However, the insertion of periodic anion-vacancy layers in SrVO3 results in a dramatic change of its physical properties. The 51V-NMR spectrum of 15R-SrVO2.2N0.6 powder can be fitted with a sharp and a broad component  = 1.4270 T, which is identical to Sr3V5+2O8 and hence assigned to the tetrahedral V1. This site has an extremely slow relaxation time of 1/T1 = 0.06 s–1  has a much faster relaxation rate (1/T1 = 10.55 s–1), and is assigned to the octahedral  sites.The cubic perovskite SrVOent Fig. . The fors–1 Fig. , indicat3 with its 3D Fermi surface41. Closer inspection shows that there are three pairs of flat Fermi surfaces, implying weak overlap between different t2g orbitals. A similar feature can be seen in the (111) SrTiO3 surface 2D electron gas (2DEG)42. NMR spectra of the octahedral site exhibit splitting at 5 K, accompanied by a divergence in 1/T1T , indicating itinerant antiferromagnetism. The magnetic origin of the transition is also observed in field-dependent electrical resistance of the nitridized SrVO3 film on SrTiO3 (111)  calculations that show a 2D Fermi surface with two cylinder-like sheets  Fig. . These e2.2N0.6 possesses (111)p defect planes with the fivefold periodicity . In order to investigate the epitaxial strain effects on oxynitrides, we fabricated an epitaxial SrVO3 thin film using a (111)-oriented substrate for subsequent ammonolysis. Surprisingly, thermal treatment  of the SrVO3 film on an LSAT (111) substrate resulted in the creation of new anion-defect layers. Scanning transmission electron microscopy (STEM) clearly indicated a (112)p planar defect, as shown in Fig. p. Based on STEM and XRD along with simulation, we constructed a monoclinic structure 7M-SrV2.71 38) with tetrahedra and pyramids around anion defects (Supplementary Note 2.2N0.6. A similar (112)p planar defect has recently been reported in BaFeO2.33F0.33 (Ba3Fe3O7F) powder, but with a threefold periodicity44. Nuclear reaction analysis (NRA) and elastic recoil detection analysis (ERDA), respectively, gave the nitrogen content of x = 0.8(2) and 0.54(3) in SrVOx2.71–Nx. Note that the oxynitride films have threefold domain structures related by 120° rotation , it is naturally expected that the observed (111)p to (112)p switching arises as a result of the compressive biaxial strain from the substrate. For a proof-of-concept, we grew epitaxial SrVO3 films on LaAlO3 (111) (LAO) and SrTiO3 (111) (STO) substrates and nitridized under the same condition  versus 15R-SrVO2.2N0.6 in the bulk (SVON-111), we used DFT to calculate the relative energies of the two SVON orientations at various biaxial lattice strains. For these calculations, we used the SCAN metaGGA functional45, which is more accurate in computing thermochemical properties than the Perdew–Burke–Ernzerhof (PBE) functional47. For SVON-112, we assumed a plausible distribution of N and O (to give Sr7V7O14N5) with reference to SVON-111  flow rates at 200 mL min−1 at 600 °C, as benchmarked by Katsura et al. , consistent with the experiment. On the other hand, at the Sr–Sr distance of SrTiO3 (dSr–Sr = 5.579 Å), the minimum free energy SVON compound is SVON-111. Further details of the calculation can be found in Supplementary Note To rationalize the formation of 7M-SrV3 (111) exhibits sixfold superlattice reflections in TEM and XRD 3 sequence in the bulk . Recent first-principles study on LaAlO3 showed epitaxial strain in the (001) and (111) planes results in distinct phases with different octahedral rotation patterns47.Further insights are garnered from observation that the oxynitride film grown on SrTiOXRD Fig. , implyinnce Fig. , in contulk Fig. . This obulk Fig. , which wulk Fig.  compared2.5 films with tetrahedral/octahedral layers stacked parallel and perpendicular to a substrate display distinct reactivities with anisotropic oxygen diffusions25. Upon the reduction of CaFeO2.5 films to CaFeO2, oxide anions are prone to migrate perpendicular to the substrate. Our oxynitride films on STO (111) are overall epitaxial but the superlattice is slightly corrugated , leading to the discovery of novel phenomena such as superconductivity and quantum Hall effects in 2DEG51. Given the abundance of perovskite oxides forming linear and planar anion defects, it is clear that the present study offers exciting new opportunities to design perovskite superlattices by chemically modifying simple 3D perovskite oxides with controllable anion-vacancy planes through the lattice strain in order to obtain unique properties and phases such as 2DEG. Furthermore, in an even broader perspective not just limited to oxides, the vacancy engineering we demonstrated here might also be utilized for phonon control and to strongly modify thermal transport properties given less controlled approaches to yield record thermoelectric performance in commercial materials such as B0.5Sb1.5Te3 and PbSe53.In the last two decades, there has been tremendous progress in the study of artificial perovskite superlattice of at least two sets of perovskite compositions  and V2O5  were ground, pelletized, and preheated at 900 °C in air for 48 h with intermediate grindings. SrVO3 was obtained by annealing the Sr2V2O7 pellet at 900 °C in flowing H2−Ar (10:90 vol%) gas at a rate of 100 mL min−1 for 24 h. SrVO2H was prepared by a topochemical reaction of SrVO3 with CaH2  as described elswhere32. Ammonolysis reactions were performed for SrVO2H, SrVO3, and Sr2V2O7; the powder samples on a platinum boat were placed in a tubular furnace (inside diameter of 26 mm) and were reacted under NH3 flow (200 mL min−1) at various temperatures (300–800 °C) for 12 h, at a heating and cooling rate of 100 °C h−1. Before reaction, the tube was purged with dry N2 gas at ambient temperature to expel oxygen and moisture.SrKα radiation. The nitrogen contents of the samples after different NH3 treatments were determined by the combustion method  at Analytical Services, School of Pharmacy, Kyoto University. Approximately 2 mg was used for each experiment, and three data sets were averaged. A high-resolution synchrotron XRD experiment for the nitridized samples was performed at room temperature using the large Debye–Scherrer cameras with a semiconductor detector installed at the beamline BL02B2 . Incident beams from a bending magnet were monochromatized to λ = 0.42073(1) and 0.78086(2) Å for the SrVO3 samples after NH3 treatment at 300 and 600 °C, respectively. Finely ground powder samples were sieved through a 32-μm mesh sieve and were packed into Pyrex capillaries with an inner diameter of 0.2 mm, which were then sealed. The sealed capillary was rotated during measurements to improve randomization of the individual crystallite orientations. Powder neutron diffraction data were collected at room temperature using the high-resolution powder diffractometer BT-1 (λ = 1.54060 Å) at the NIST Center for Neutron Research (NCNR), and time-of-flight (TOF) diffractometers, iMATERIA54, and NOVA at the Japan Proton Accelerator Research Complex (J-PARC). The collected synchrotron and neutron profiles were analyzed by the Rietveld method using RIETAN-FP program55 (SPring-8 and NIST) and Fullprof program56 (J-PARC).We conducted XRD measurements using a D8 ADVANCE diffractometer (Burker AXS) with a Cu 3 thin films with a thickness of 100 nm were deposited on single crystalline substrates of SrTiO3 (111), (LaAlO3)0.3(SrAl0.5Ta0.5O3)0.7 (LSAT) (111), LaAlO3 (111), (LSAT) (100), and LaAlO3 (100), using the pulsed laser deposition technique. We used a KrF excimer laser at λ = 248 nm, with a deposition rate of 2 Hz and an energy of ~2 J cm−2. The substrate temperature was kept at 950 °C during the deposition, with an oxygen partial pressure of 5 × 10–7 Pa. Subsequently, the oxide films were placed in a platinum boat in a tubular furnace and heated under an NH3 flow at 600 °C (LSAT (111)) and 620 °C (SrTiO3 (111), LaAlO3 (111), LSAT (100), and LaAlO3 (100)) for 12 h, in the same manner as in the case of the powder samples.Epitaxial SrVOKα radiation (Bruker AXS D8 DISCOVER), where the diffractometer in parallel beam geometry was equipped with one- and two-dimensional detectors. STEM images were acquired using an aberration-corrected microscope  operating at an acceleration voltage of 300 kV. The convergence semiangle of the incident probe was 18 mrad, while the detector collection semiangles ranged from 77 to 200 mrad for HAADF and 10–19 mrad for ABF imaging. The high-resolution STEM images in Fig. Structures of the films were examined by XRD with Cu 15N12C resonant nuclear reaction at 899.98 keV. NRA measurements were carried out with a 1-MV tandetron accelerator at Tandem Accelerator Complex, University of Tsukuba. The experimental error of around ±20% originates from instability of accelerator and small thickness of the film. Details of the measurement and calibration are given in ref. 17.The amount of nitrogen in the oxynitride film was evaluated by NRA method using the 57. The experimental errors were around 5% under a typical condition.The anion compositions of the films were also determined by ERDA with a 38.4 MeV Cl beam generated by a 5-MV tandem accelerator 51V-NMR for the powder sample of 15R-SrVO2.2N0.6. 51V-NMR spectra (the nuclear spin of I = 7/2 and the nuclear gyromagnetic ratio of 51γ/2π = 11.193 MHz T−1) were obtained by sweeping magnetic field in a fixed frequency of 15.9 MHz. Nuclear spin–lattice relaxation rate 1/T1 was determined by fitting the time variation of the nuclear magnetization m(t) after a saturation pulse to a theoretical function for a nuclear spin I = 7/2 relaxation at the central transition. The electrical resistance of the nitridized SrVO3 film on SrTiO3 (111) was measured down to 2 K by means of a standard four-probe method using a physical property measuring system  at 0 and 7 T.A conventional spin-echo technique was used to measure 2.2N0.6 was performed by using the PBE parametrization of the generalized gradient approximation58 and the projector augmented wave method (PAW)59 as implemented in the Vienna ab initio simulation package (VASP)63. In the PAW potentials used in this study, the following states are treated as core for each element: [Ar] 3d10 for Sr, [Ne] for V, and [He] for O and N, which is in common with the strain calculation described in the next paragraph and Supplementary Note 64 and then extracted the vanadium-d Wannier functions from the calculated band structures using the Wien2Wannier and Wannier90 codes68. For this purpose, we also used the PBE functional. Further details of these calculations are discussed in Supplementary Note Crystal structure optimization for 15R-SrVO2.71 and 15R-SrVO2.2N0.6 were generated in pymatgen69. Stability calculations were performed using VASP, with the SCAN metaGGA functional70. The relative free energies of the 7M and 15R phase for a given temperature, ammonia flow rate, and biaxial strain, are calculated using a thermodynamic grand potential, Φ, asμ, of oxygen and nitrogen are determined from benchmarked nitrogen activities by Katsura46; further details of this referencing is discussed in Supplementary Note –8 eV Å−1, and then we perform the slab transformation into (111) or (112) Miller indices and apply isotropic biaxial strain.For the biaxial strain calculations, ordered approximants for 7M-SrVSupprementary InformationPeer Review File


Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core. However, some PLC subfamilies, such as PLCε, also hydrolyze other phosphatidylinositol phosphate species at internal membranes (Phospholipase C (PLC) enzymes hydrolyze phosphatidylinositol lipids from cellular membranes in response to diverse cellular signals , 2. Thessphate IP and diacembranes . Thus, P2+-induced Ca2+ release in the cardiovascular system (A). Recent functional analysis of a catalytically active PLCε fragment containing the EF3-RA1 domains confirmed that the CDC25, PH, and RA2 domains, as well as EF1/2, are dispensable for expression and activity. The structure of this fragment revealed that the RA1 domain and the linker connecting the C2 and RA1 domains (C2-RA1) form extensive interactions with EF3/4, the TIM barrel, and the C2 domain. Thus, in PLCε, the core is expanded to encompass the PH-RA1 region.PLCε is required for maximum Car system , 8. In pr system , 9, 10. r system , 12, 13,r system A. RecentThe PLCε RA1 and RA2 domains also contribute to subcellular localization and activation via direct interactions with the scaffolding protein mAKAP (muscle-specific protein kinase A–anchoring protein) and the Rap1A and Ras GTPases, respectively , 17, 18.q-dependent activation of the related PLCβ enzyme   , which rFig. 1B, , which iFig. 1B, , 23, 25.G12V, but other regions of PLCε may also be required for activation. Given that PLCε domains EF3-C2 are essential for basal activity  and andG12V,G12V-dependent activation, we hypothesized that Rap1A allosterically activates PLCε by promoting or stabilizing potentially long range intra- and interdomain interactions within the lipase. Because both Ras and Rap1A bind to and activate the enzyme via the RA2 domain, the surface of the RA2 domain is the most likely candidate to mediate these interactions. To date, only two residues on the RA2 domain have been characterized with respect to activation by GTPases. Mutation of Lys2150 and/or Lys2152 (Rattus norvegicus numbering) decreases basal activity and eliminates G protein–dependent activation in cell-based studies  were individually mutated to alanine in the background of the PLCε PH-COOH variant, and their melting temperatures (Tm) and basal activities were determined. As controls, we also expressed, purified, and characterized the PH-COOH Lys2150 and Lys2152 point mutants, because they should be insensitive to Rap1A-dependent activation , we idetivation , 23.FiguTm values comparable with that of PH-COOH , 23Tm vaFig. 2C, . Thus, tG12V-dependent activation. To gain structural insight into how these elements that are distant in primary structure contribute to activation by Rap1AG12V, we used SAXS to compare the solution structures of the PLCε PH-COOH and EF3-COOH variants alone and in complex with Rap1AG12V. These variants were chosen because they formed stable complexes with Rap1AG12V that could be isolated by size-exclusion chromatography (SEC), but only PH-COOH has increased lipase activity upon binding of the GTPase  and a Dmax of ∼165 Å, with a largely globular structure with some extended features (G12V binding (B). In this plot, compact, globular proteins have bell-shaped curves that converge to the qRg axis at low values, whereas elongated and more rigid structures exhibit curves that extend out to higher qRg, and highly flexible structures do not converge at all .We first compared the SAXS solution structure of the Rap1A profile . Evolvin–PH-COOH  32, 33), 33G12V–± 0.12 Å , D and Efeatures . However binding B. In thie at all . The dats of qRg B.Figure G12V. EF3-COOH was also monomeric and monodisperse in solution, with an Rg of 41.9 ± 0.43 Å and a Dmax of ∼175 Å  function is also consistent with the protein having a mostly globular structure with a modest degree of extendedness and/or flexibility, consistent with the flexibly connected RA2 domain (I and 4C). These results also confirm that deletion of the PH domain and EF1/2 in this variant does not perturb the EF3-RA1 structure in solution  function revealed a much more compact and globular structure, as shown by the more bell-shaped curve and most clearly in the ∼30 Å decrease in Dmax to ∼145 Å . Because EF3-COOH is not activated by Rap1AG12V  eliminates G protein–stimulated activation in cells , we gentry 2C5L tivation . One expA) but may be in relatively close spatial proximity, based on the observed locations of the N and C termini in the recent structure of the PLCε EF3-RA1 fragment , and differences in flexibility  activation of PLCε and reveal that activation involves substantial conformational changes within the lipase. Whereas the PLCε RA2 domain is required for activation, this process also appears to be dependent on the PH domain, EF1/2, and hydrophobic surface residues on the RA2 domain. These observations are consistent with the following model . BecauseR. norvegicus PLCε variants were subcloned into pFastBac HTA . Site-directed mutagenesis in the PH-COOH background was performed using the QuikChange site-directed mutagenesis kit (Stratagene) or the Q5 site-directed mutagenesis kit (NEB). All subcloned PLCε variants contained an N-terminal His-tag and TEV cleavage site and were sequenced over the entire coding region. The proteins were expressed and purified as previously described  was subcloned into pFastBac HTA. The protein were expressed in baculovirus-infected High5 cells. Cell pellets were resuspended in lysis buffer containing 20 mm HEPES, pH 8.0, 100 mm NaCl, 10 mm β-mercaptoethanol, 0.1 mm EDTA, 10 mm NaF, 20 mm AlCl3, 0.1 mm leupeptin and Lima Bean trypsin inhibitor, 0.1 mm phenylmethylsulfonyl fluoride, and 20 μm GTP and lysed via a dounce homogenizer on ice. The lysate was centrifuged for 1 h at 100,000 × g, and the pellet was resuspended in lysis buffer supplemented with 1% sodium cholate, resuspended via dounce homogenization on ice, and solubilized at 4 °C for 1 h. The sample was then centrifuged for 1 h at 100,000 × g, and the supernatant was diluted 2-fold with lysis buffer.cDNA encoding N-terminally His-tagged constitutively active G12V was loaded on a Ni-NTA column pre-equilibrated with lysis buffer and first washed with lysis buffer containing 10 mm imidazole and 0.2% cholate, followed by a second wash with lysis buffer supplemented with 10 mm imidazole and 10 mm CHAPS. The protein was eluted with lysis buffer containing 250 mm imidazole, pH 8.0, and 10 mm CHAPS. His-tagged Rap1AG12V was then concentrated and applied to tandem Superdex S200 columns pre-equilibrated with G protein S200 buffer . Fractions containing purified protein were identified by SDS-PAGE, pooled, concentrated, and flash-frozen in liquid nitrogen. Rap1AG12V used for activation assays was purified in modified G protein S200 buffer containing 1 mm CHAPS.His-tagged Rap1AG12V was incubated with 5% (w/w) TEV protease and dialyzed overnight in 1.5 liters of dialysis buffer containing 20 mm HEPES, pH 8.0, 50 mm NaCl, 1 mm MgCl2, 10 mm β-mercaptoethanol, 1 mm CHAPS, and 20 μm GTP at 4 °C. The dialysate was applied to a Ni-NTA column, and the flow-through containing cleaved Rap1AG12V was collected and passed over the column two more times. The flow-through was collected, concentrated to 1 ml, applied to tandem Superdex S200 columns, and purified as described above.For SAXS experiments, His-tagged Rap1ATm) of PLCε variants were determined as previously described  ice-cold TCA and centrifuged. Free [3H]IP3 in the supernatant was quantified by scintillation counting. All assays were performed at least three times in duplicate.All activity assays were carried out using [escribed . Briefly–1 ng/μl . The PH-G12V-dependent increases in PLCε lipase activity were measured using the same approach with some modifications. The liposomes were first incubated with increasing concentrations of Rap1AG12V·GTP in 50 mm HEPES, pH 7.0, 3 mm EGTA, 1 mm EDTA, 100 mm NaCl, 5 mm MgCl2, 3 mm DTT, and 390 μm CHAPS at 30 °C for 30 min. The reaction was initiated by addition of the PLCε variant, incubated at 30 °C for 8 min, and processed as described above. All activation assays were performed in duplicate with protein from at least two independent purifications.Rap1AG12V, supplemented with 0.5 mm CaCl2, was incubated on ice for 30 min before being applied to a Superdex S200 column pre-equilibrated with complex S200 buffer . Fractions containing the purified complex were identified by SDS-PAGE, pooled, and concentrated for use in SAXS experiments.A 1:3 or 1:5 molar ratio of PLCε PH-COOH or EF3-COOH to Rap1AG12V–PLCε PH-COOH complex, and the Rap1AG12V–PLCε EF3-COOH complex were diluted to final concentrations of 2–3 mg/ml in S200 buffer (EF3-COOH) or complex S200 buffer and centrifuged at 16,000 × g for 5 min at 4 °C prior to data collection. SEC-SAXS was performed at the BioCAT Beamline at Sector 18 of the Advanced Photon Source  of individual frames were plotted with the scattering chromatograms, which show the integrated intensity of individual exposures as a function of frame number, and used to help determine appropriate sample ranges for subtraction. PRIMUS , and Dmax. GNOM (P(r)) functions via an indirect Fourier transform method.Protein samples were eluted from a Superdex 200 Increase 10/300 GL column using an ÄKTA Pure FPLC  at a flow rate of 0.7 ml/min. The eluate passed through a UV monitor followed by a SAXS flow cell consisting of a quartz capillary. The data were collected in two different setups at the beamline. The PH-COOH, EF3-COOH, and EF3-COOH complex data were collected in a 1.5-mm–inner diameter quartz capillary with 10 μgeometry  to preveprofiles . BioXTASanalysis  32) as  as m walanalysis . The radax. GNOM  was usedGraphical plots were generated from buffer-subtracted averaged data (scattering profile and Guinier plots)  or indirversus PLCε PH-COOH, as noted in the figure captions. All error bars represent standard deviation.GraphPad Prism v.8.0.1 was used to generate all plots. One-way analysis of variance was performed with Prism v.8.0.1 followed by Dunnett's post hoc multiple comparisons lyonam@purdue.eduData not provided within the article is available upon reasonable request to A. M. Lyon at


RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity. RecQ helicases constitute a unique subgroup of the SF2 (super-family 2) helicases and they play essential roles in the maintenance of genome integrity . Humans RECQ5-associated syndrome  and protection of stalled replication forks by HR is essential to maintain genome integrity. The RAD51 protein bound to ssDNA performs key functions in these processes, including the homology search, DNA strand exchange and ssDNA protection . Howeversyndrome . Howeverethality . In humaethality , and RECposition . Elevateposition , moreovebination . RECQ5 ibination ,21,24,25bination ,26–28, pbination , and actbination . In addibination . These fE. coli RecQ and the yeast BLM homolog Sgs1 suggest that the HRDC domain is not required for ATP hydrolysis or helicase activity, but it promotes stable DNA binding  domain ,4,5,31.  binding ,33. Cons binding . Interes binding ,34,35. T binding .Despite its clear links to genome integrity, the precise functions of RECQ5 have been difficult to fully define because it has been implicated in numerous processes, has numerous interacting partners, and may have partial functional redundancy with the other RECQ helicases . To helpThe GFP–RECQ5 expression plasmid was constructed by inserting the GFP sequence into the plasmid pTXB1–RECQ5 . The seqE. coli ARCTIC RIL strain (Agilent). The culture was grown to an OD 0.7 at 37°C in 2 × TY media supplemented with ampicillin (100 mg/l), shifted to 11°C and then induced over night with 1 mM IPTG. The bacterial pellet was resuspended in cell breakage buffer (CBB)  and 1 mM PMSF, sonicated and centrifuged at 100 000 × g for 1 h. The clarified extract was mixed with chitin beads (New England BioLabs) preequilibrated in T buffer  supplemented with 500 mM KCl. To release the RECQ5 protein from the intein tag, T buffer with 500 mM KCl and 50 mM DTT was added to the beads, and incubated O/N on a rotary shaker at 4°C. Cleaved RECQ5 protein was eluted with buffer T containing 150 mM KCl and 1 mM DTT. RECQ5 peak protein fractions were pooled and loaded onto a hydroxyapatite column (Sigma-Aldrich) equilibrated with T buffer containing 150 mM KCl and 1 mM DTT. RECQ5 protein was eluted by linear gradient of 0–1000 mM KH2PO4 gradient in T buffer. Pooled peak fractions were loaded on Mono S column  equilibrated with T buffer with 150 mM KCl and 1 mM DTT. RECQ5 protein was eluted with 0–800 mM KCl gradient in T buffer. Peak RECQ5 fractions were pooled, concentrated using Vivaspin Centrifugal Concentrator  and stored in small aliquots at −80°C.Human RECQ5, RECQ5–F666A (FA), GFP–RECQ5 were purified similarly to the previously described protocol, with few modifications . ExpressE. coli and purified as previously described (E. coli strain. The culture was grown to an OD 0.7 at 37°C in 2 × TY media supplemented with ampicillin (100 mg/L), and protein expression was induced by adding 1 mM IPTG. Cells were harvested 3 h after IPTG induction by centrifugation and lysed by sonication in buffer containing 50 mM Tris–HCl [pH 7.5], 10% sucrose, 0.5 mM EDTA, 600 mM KCl, 0.01% NP-40, 10 mM 2-mercaptoethanol, cocktail of protease inhibitors , and 1 mM PMSF. The cell lysate was centrifuged at 100 000 × g for 1 h and the supernatant was mixed with Ni-NTA agarose beads (Sigma) pre-equilibrated in T buffer supplemented with 200 mM KCl and 10 mM 2-mercaptoethanol for 1 h. DMC1 protein was eluted by step gradient of imidazole  in buffer T with 50 mM KCl. The fractions containing DMC1 protein were pooled and applied onto hydroxyapatite (Sigma-Aldrich) column equilibrated with T buffer containing 100 mM KCl and 10 mM 2-mercaptoethanol. The DMC1 protein was eluted by linear gradient of 100–1000 mM KH2PO4 gradient in T buffer. The DMC1 peak fractions were then loaded on Mono Q column  pre-equilibrated with T buffer containing 100 mM KCl and 10 mM 2-mercaptoethanol and eluted by 170–850 mM KCl gradient in T buffer. The peak DMC1 fractions were applied onto a Superdex 200  gel filtration column equilibrated with T supplemented with 150 mM KCl and 10 mM 2-mercaptoethanol. The protein was eluted using the same buffer, and fractions containing DMC1 were polled, concentrated using Vivaspin Centrifugal Concentrator (30 000 MWCO PES) and stored at −80°C.For, BLI measurements, Human wild-type (wt) RAD51, RAD51–K133R (KR), RAD51–K133A (KA) and RAD51–I287T (IT) were expressed in escribed . Brieflyescribed . The expE. coli cells in LB were grown at 37°C, induced 4 h at 37°C with 0.2 mM IPTG when OD600 reached 0.5, and then harvested by centrifugation. The cells were resuspended in 50 ml cell lysis buffer (50 mM Tris (7.5), 10% sucrose, 1 M NaCl, 10 mM EDTA, 1 mM tris(2-carboxyethyl)phosphine hydrochloride [TCEP]), 0.1% Triton, protease inhibitor cocktail ) and lysed by sonication. The extract was centrifuged for 1 h at 40 000 rpm at 4°C using a Ti-45 rotor. The supernatant was precipitated with 0.24 mg/ml ammonium sulfate for 1 h. The pellet was recovered by centrifugation at 10 000 rpm at 4°C for 10 min. Then, the pellet was dissolved in His loading buffer  for 1 h. The resuspension was cleared by centrifugation at 15 000 rpm at 4°C for 10 min. The protein solution was purified through a Ni-NTA column (Roche) and further treated with SUMO protease to cleave off the His6-SUMO tag while dialyzing into dialysis buffer , and the following protease inhibitors: aprotinin, chymostatin, leupeptin and pepstatin A at 3 mg/ml each, and 1 mM PMSF) for sonication. After centrifugation (100 000 × g for 90 min), the clarified lysate was loaded on 20 ml Q fast flow sepharose (Amersham Pharmacia Biotech) column and then the column was developed with 100–600 mM KCl gradient (200 ml) in buffer B buffer A . The fractions containing DMC1 were pooled together for incubation with 3 ml Ni2+ NTA-agarose (Qiagen) for 1 h. The matrix was poured into a column, washed with 50 ml buffer B with 1000 mM KCl and then with 10 ml each of 30 and 50 mM imidazole in buffer A before being eluted with 15 ml of 200 mM imidazole in buffer A. The 200 mM imidazole eluate were further fractionated in a 1 ml Mono Q column (Amersham Pharmacia Biotech), using a 15 ml gradient of 200–600 mM KCl in buffer B. The fractions containing DMC1 were diluted to 50 mM KCl and loaded on a 1 ml Mono S column for being devloped with 10 ml gradient of 100–500 mM KCl in buffer B. DMC1 containing fractions were pooled and concentrated in a Centricon-30 concentrator (Amicon) before small aliquots and storage at −80°C. Note that the His6 tag was not removed from the N-terminus of DMC1 prior to use.For DNA curtain measurements, human RAD51, RAD51–K133R, RAD51–I287T were purified as previously described  with min3.Samples were deposited on freshly cleaved mica  mounted on microscope slides (Marienfeld). After 2 min, the sample drop was rinsed with MilliQ water and dried with filtered air. Atomic force microscopy was done with NanoWizard 3 (JPK Instruments) mounted on the fluorescence microscope (Leica). Air-dried samples were scanned in intermittent contact mode (air). Super Sharp Silicon (SSS-MCHR-50) scanning probes by Nanosensors were used. Volume analysis was acquired with SFMetricsV4c1 software. Calibration of protein size was done using RPA (116 kDa) which had a mean volume of 130 nm2, 5 mM CaCl2, 50 mM KCl, 2 mM ATP, 1 mM DTT, 0.2 mg/ml BSA) at 37°C. All reactions contained either M13 ssDNA  or pUC19 dsDNA . Reactions were initiated by the addition of 10 nM RECQ5 or GFP–RECQ5, as indicated. Aliquots were removed at the indicated time points and quenched by addition of 50 mM EDTA. The quenched reactions were quantified by the ATPase/GTPase Activity Assay Kit as per the manufacturer's instructions .Comparison of unlabeled RECQ5 and GFP–RECQ5 ATP hydrolysis activities was performed in RECQ5 buffer  for 10 min at 37°C before the addition of 10 nM RECQ5, RECQ5–F666A, or GFP–RECQ5. Aliquots were removed at the indicated time points and quenched by addition of 50 mM EDTA and were quantified as described above.2, 2 mM CaCl2,100 μg/ml BSA, 20 mM creatine phosphate and 20 μg/ml creatine kinase) for 10 min at 37°C. RECQ5 (in indicated concentrations) and RPA (300 nM) were added to the nucleoprotein filament and incubated for 5 min, followed by the addition of HOP2-MND1 (300 nM) for 2 min. D-loop formation was initiated by addition of pBluescript SK(–) (500 ng) to bring the final reaction volume to 10 μl and incubated for another 10 min at 37°C. The reactions were deproteinized by addition of 10 μg proteinase K and 0.1% SDS and incubated for 10 min at 37°C and analyzed by electrophoresis in a 0.9% agarose gel in 1× TAE . Gels were imaged on a FLA-9000 scanner (Fujifilm) and quantified with Multi Gauge V3.2 (Fujifilm).To form the nucleoprotein filament, RAD51 (2 μM) or DMC1 (2 μM) was incubated with 40 nM 5′-fluorescein-labeled 90-mer oligonucleotide (5′-AAA TCA ATC TAA AGT ATA TAT GAG TAA ACT TGG TCT GAC AGT TAC CAA TGC TTA ATC AGT GAG GCA CCT ATC TCA GCG ATC TGT CTA TTT-3′) in D-loop buffer  for 10 min. BLI experiments consisted of 5 steps performed in BLI buffer supplemented with 2 mM ATP: (i) recording initial baseline with BLI buffer for 30 s; (ii) loading 5′ biotinylated dT43-mer (40 nM) for 120 s; (iii) washing with BLI buffer for 30 s; (iv) association step with 2 μM RAD51 protein for 240 s to form the presynaptic filament, (v) dissociation step with 40 nM RECQ5 and 2 μM BRC3 peptide for another 240 s. Please note that all BLI experiments looking at RECQ5-mediated removal of RAD51 from ssDNA include the BRC3 peptide to prevent RAD51 from rebinding to the ssDNA once it dissociates. For all experiments monitoring RECQ5 translocase activity, controls were performed with BRC3 peptide only (minus RECQ5). The data from the time period where BRC3 without RECQ5 was added to the RAD51 filament, were normalized to the starting point. The change in signal was then plotted as a function of time for each nucleoprotein filament.BLI experiments were performed using a single-channel BLItz instrument (ForteBio) in Advanced Kinetics mode at room temperature and with shaking at 2,200 rpm. For RAD51 filament disruption experiments, prior to the measurements the streptavidin biosensors  were pre-hydrated by incubation with BLI buffer  were incubated with BLI buffer for 10 min prior to use. These protein–protein interaction BLI experiments consisted of three steps: (i) recording initial baseline with BLI buffer; (ii) loading of 0.5 μg/ml rabbit polyclonal RECQ5 antibody pre-incubated with 1.5 μM RECQ5 for 10 min; (iii) association step with 2.5 μM RAD51, DMC1 or RPA protein. The binding affinity of the proteins was measured as an increase in the thickness of the biomolecule layer in nanometers (nm). All BLI measurements were performed in triplicate.As a control to test for RAD51 interactions with BRC3, BLI experiments using anti-GST biosensors  and immobilized GST–BRC3. Biosensors were coated by 1 μM GST–BRC3 for 120 s, washed and subsequently mixed with 1 μM Rad51 variants for 120 s (association phase). The dissociation phase was initiated by flushing the biosensor to BLI buffer for 120 s. Shaker speed was set to 1800 rpm.N-(biotinyl) ), and 8% mPEG 2000-DOPE -2000] ). All lipids were purchased from Avanti Polar Lipids. The ssDNA substrate was generated using rolling circle replication with a biotinylated primer, a circular M13 ssDNA template, and phi29 DNA polymerase, as described  microscope (Nikon) equipped with a 488-nm laser , a 561-nm laser , and two Andor iXon EMCCD cameras ,40. The escribed ,40. The escribed ,40. For escribed ,40.2, 1 mM DTT, 0.2 mg/ml BSA) at 37°C with a flow rate of 1 ml/min. Secondary structure was reduced with a 500 μl 7 M urea, immediately followed by 5–10 ml of BSA buffer containing 100 pM unlabeled RPA, RPA–GFP or mCherry–RPA, as indicated. RPA was flushed out with either RAD51 HR buffer  or DMC1 HR buffer  at 1 ml/min for 2 min. Then, either RAD51 (2 μM), RAD51–K133R (2 μM), RAD51–I287T (2 μM) or DMC1 (2 μM) in a 50 μl loop was injected into the flow cell, buffer flow was terminated when protein reached the flow cell, and the reactions were incubated at 37°C for 20 min. The loss of RPA fluorescence signal was monitored to verify recombinase filament assembly. The time-increase decrease in the normalized RPA–GFP fluorescence intensity (integrated over entire ssDNA molecules) reflects the disassembly of RPA–GFP, which is used for the calculation of RAD51 assembly , where k is the observed RPA association rate which is equal to RAD51 dissociation rate.For disassembly reactions, presynaptic complexes were formed as described above. Disassembly was then initiated by resuming buffer flow with HR buffer lacking both CaCl2, 5 mM CaCl2, 50 mM KCl, 2 mM ATP, 1 mM DTT, 0.2 mg/ml BSA) supplemented with RPA . 50 μl samples containing the indicated GFP–RECQ5 concentrations plus 0.5 nM RPA–mCherry, RPA–GFP or unlabeled RPA (as indicated) were injected into the flow cell at a rate of 0.2 ml/min, and RECQ5 activity was observed under constant buffer flow in reaction buffer  for a total period of 20–25 minutes. All data were collected as previously described for Srs2  is an intermediate in many aspects of DNA replication and repair. However, naked ssDNA is unlikely to exist in physiological settings, instead it quickly becomes sequestered by the abundant heterotrimeric ssDNA-binding protein complex RPA . For insin vivo . This fusion protein retains function in vivo ,30 and bin vivo ; note thin vivo . We concnditions  and in tom ssDNA . AlthougN = 54; Figure To visualize unlabeled RECQ5 interactions with RPA-bound ssDNA, we prepared ssDNA curtains bound by RPA–GFP, unbound RPA–GFP was then flushed out of the sample chambers followed by the addition of unlabeled 25 nM RECQ5 Figure ; note, tin vitro , however, most of the complexes exhibited two or more photobleaching steps , consistent with the conclusion that RECQ5 can form higher order multimers while translocating on ssDNA  and dissociates ∼76% more slowly  compared to RAD51  and 6300 ± 390 nt (N = 69) for RECQ5 and GFP–RECQ5, respectively  due to signal background  that were tethered by both ends to the flow cell surface ,61. In td Figure . Remarkad Figure . Interesd Figure  63). Th. The.g. N = 227 of 241) showed evidence of dim fluorescent RPA–mCherry foci by the end of the 20-minute duration measurements with ∼50% of the RPA–mCherry foci appearing at approximately the 6-min time point but the mCherry signal did not increase substantially in intensity over time , as described above, however, only one end of the DNA was anchored to the surface. The other end of the DNA was free in solution and was modified to harbor a 30-nt 3′ ssDNA overhang, as previously described . We rease Figure . These fN = 95/152; Figure N = 57/152; Figure N = 77/95) involving direct recruitment of RECQ5 to the heteroduplex joints, RECQ5 bound directly to the heteroduplex joint, but then failed to undergo any observable translocation  RECQ5 translocated along the RAD51–ssDNA, resulting in disruption of the heteroduplex DNA joint, and the ATTO565-labeled dsDNA fragments appeared to remain bound to RECQ5 as it translocated , the collision with the heteroduplex dsDNA caused RECQ5 to stall and stop translocating , RECQ5 bypassed the heteroduplex DNA joint  UvrD-like helicase that is considered a prototypical antirecombinase that acts by dismantling Rad51–ssDNA filaments ,66,67. SThe yeast mutant Rad51–I345T bypasses the need for the Rad55–Rad57 paralog complex to counteract the antirecombinase activity of Srs2 . SimilarSgs1 is one of the two RecQ helicases found in yeast and participates in DNA end resection and Holliday junction dissolution . Sgs1 caSrs2 and Sgs1 can efficiently strip the Rad51 from ssDNA but are unable to dismantle Dmc1 filaments ,62. HereInterestingly, although human BLM is considered an antirecombinase ,34,73, win vivo functional characteristics may be defined at the level of substrate specificity. For instance, we can readily detect human RECQ5 disruption of RAD51 and DMC1 filaments and we can also see extensive translocation of RECQ5 on RPA–ssDNA. However, we can find no evidence for RECQ5 translocation or helicases activity on dsDNA molecules, and only limited evidence for unwinding when RECQ5 is specifically targeted to an DNA end, even though RECQ5 can bind to dsDNA and dsDNA supports RECQ5 DNA-dependent ATP hydrolysis activity. These observations suggest the possibility that RECQ5 activities may be restricted to ssDNA–containing reaction intermediates. As indicated above, these findings for RECQ5 are in striking contrast to our results with BLM helicase, which we find readily unwinds naked dsDNA but exhibits no binding or translocation activity on either RPA–ssDNA, RAD51–ssDNA or even RAD51-bound dsDNA substrates , linking them to DNA repair, replication, recombination and transcription. In moving forward, it will be important to examine and compare the mechanistic attributes of the other human RecQ helicase family members and it will also be important to fully define how the various protein domains and interaction factors contribute to the properties of these enzymes.The differences in substrate specificity for RECQ5 and BLM may arise from the differences in protein domain structure for these two RecQ helicases. For instance, the RecQ C-terminal (RCQ) domain is present in most RecQ helicases, including BLM and Sgs1, but this domain is truncated in RECQ5 ,5,31. Thgkaa1184_Supplemental_FileClick here for additional data file.