Our previous biochemical data showed that the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP but inhibited by high concentrations of ATP; however, the molecular mechanism was unknown. Here, we report the crystal structures of the αγ heterodimer with ATP binding only to the allosteric site (αMgγMg+CIT+ATP) and to both the allosteric site and the active site (αMg+ATPγMg+CIT+ATP). Structural data show that ATP at low concentrations can mimic ADP to bind to the allosteric site, which stabilizes CIT binding and leads the enzyme to adopt an active conformation, revealing why the enzyme can be activated by low concentrations of ATP. On the other hand, at high concentrations ATP is competitive with NAD for binding to the catalytic site. In addition, our biochemical data show that high concentrations of ATP promote the formation of metal ion-ATP chelates. This reduces the concentration of free metal ion available for the catalytic reaction, and thus further inhibits the enzymatic activity. The combination of these two effects accounts for the inhibition of the enzyme at high concentrations of ATP. Taken together, our structural and biochemical data reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) is responsible for the catalytic conversion of isocitrate into α-ketoglutarate in the Krebs cycle. This enzyme exists as the α The crystal structures of human cytosolic NADP-IDH  and porcine mitochondrial NADP-IDH  have been determined, and together with biochemical data, these enzymes have been shown to exist and function as homodimers in which both subunits are catalytic6. The activity of human IDH1 is regulated through conformational changes of the active site upon substrate binding, and the other mammalian NADP-IDHs appear to share a similar regulatory mechanism7.The Krebs cycle is a central part of the metabolic pathway to generate ATP through the oxidation of acetyl-CoA derived from the chemical breakdown of carbohydrates, fats and proteins. Separately, the Krebs cycle also provides metabolic intermediates for biosynthetic reactions leading to the de novo synthesis of proteins, lipids and nucleic acids. Isocitrate dehydrogenases (IDHs) are a family of enzymes responsible for the oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (α-KG) using NAD or NADP as coenzyme with the concomitant production of NADH or NADPH. In mammals, NAD-dependent IDHs  are localized to the mitochondria and participate in the Krebs cycle. In addition, mammalian cells contain two NADP-dependent IDHs (NADP-IDHs) located to both the cytosol and the mitochondria, which have been demonstrated to play important roles in cellular defense against oxidative damage, detoxification of reactive oxygen species, and synthesis of fat and cholesterol9. The α and β subunits form a heterodimer (αβ) and the α and γ subunits form another heterodimer (αγ), which are assembled into a heterotetramer (α2βγ) and further into a heterooctamer 11. In addition, the activities of mammalian NAD-IDHs can be regulated by many metabolic intermediates and products of the Krebs cycle via feedback activation and inhibition mechanisms. Early biochemical studies showed that the α subunit is the catalytic subunit in the holoenzyme, whereas the β and γ subunits are the regulatory subunits, and the activity of the holoenzyme is positively regulated by citrate (CIT) and ADP and negatively regulated by ATP and NADH15. Recently, we carried out a comprehensive biochemical study on the enzymatic properties of the α2βγ holoenzyme and the αβ and αγ heterodimers of human NAD-IDH16. We found that the αγ heterodimer exhibits similar enzymatic properties as the α2βγ holoenzyme and can be allosterically activated by CIT and ADP, whereas the αβ heterodimer cannot; moreover, both heterodimers can be inhibited by NADH16. In addition, our biochemical data showed that in the holoenzyme, both α subunits have catalytic activity; in contrast, the γ subunit plays a regulatory role, whereas the β subunit plays a structural role to facilitate the assembly of the holoenzyme16. Our detailed structural and biochemical studies of the αγ and αβ heterodimers further revealed the molecular mechanisms by which the αγ heterodimer can be allosterically activated by CIT and ADP and inhibited by NADH, and the αβ heterodimer cannot be regulated by CIT and ADP but can be inhibited by NADH20. Intriguingly, our biochemical data also showed that the activities of the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP, but inhibited by high concentrations of ATP16. However, the molecular mechanism underlying the dual regulatory roles of ATP on the αγ heterodimer and the holoenzyme remained elusive.Compared to the NADP-IDHs, the composition and regulation of NAD-IDHs are much more complex. Mammalian NAD-IDHs are comprised of three types of subunits in the ratio of 2α:1β:1γ with similar molecular masses of 37 kDa, 39 kDa and 39 kDa, respectively2+ alone or Mg2+ and ATP, and the allosteric site bound with Mg2+, CIT and ATP (αMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP). In the αMgγMg+CIT+ATP structure, there is ATP bound to the ADP binding site of the allosteric site, which stabilizes CIT binding through Mg2+-mediated interactions and leads the enzyme to adopt an active conformation. This provides the structural basis for why the enzyme can be activated by low concentrations of ATP. In the αMg+ATPγMg+CIT+ATP structure, there is ATP bound to the NAD binding site of the active site, which hinders NAD binding and thus inhibits the enzymatic activity. In addition, our biochemical data show that high concentrations of ATP facilitate the formation of metal ion-ATP chelates and decrease the concentration of free metal ion available for the catalytic reaction, thus inhibiting the enzymatic activity. The combination of these two effects explains why the enzyme can be inhibited by high concentrations of ATP. Our structural and biochemical data together reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.In this work, we determined two crystal structures of the αγ heterodimer of human NAD-IDH with the active site bound with either Mg2+ and the allosteric site bound with Mg2+, CIT and ATP from a crystallization solution containing CIT and a relatively low concentration of ATP (2 mM). This led to the determination of the structure of the αMgγMg+CIT+ATP heterodimer. Crystallization of the αγ heterodimer in solutions containing CIT and relatively high concentrations of ATP (10–20 mM) yielded crystals of the αγ heterodimer in which there is residual electron density at the NAD binding site of the active site, indicating that the NAD binding site is partially occupied by a ligand . Soaking the crystals of the αMgγMg+CIT heterodimer in a solution containing a very high concentration of ATP (200 mM) yielded crystals of the αγ heterodimer with the active site bound with Mg2+ and ATP and the allosteric site bound with Mg2+, CIT and ATP, which led to the determination of the structure of the αMg+ATPγMg+CIT+ATP heterodimer. Of note, the very high concentration of ATP (200 mM) in the soaking solution is unlikely to be physiologically relevant. Nevertheless, the structure of the αMg+ATPγMg+CIT+ATP heterodimer can provide structural insights into the mechanism by which high concentrations of ATP might compete with NAD to bind at the NAD binding site and thus inhibit the enzymatic activity of the αγ heterodimer. The αMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures were determined at 2.30 Å and 2.26 Å resolution, respectively  than the inactive αMgγ structure . In particular, the key residues and structure elements at the allosteric site, the active site, and the heterodimer interface all exhibit very similar conformations to those seen in the αMgγMg+CIT+ADP structure, rather than to those seen in the αMgγ structure  at the allosteric site is oriented towards the CIT-binding subsite and forms a hydrogen bond with CIT, and the side chain of Tyr126A at the active site is oriented towards the ICT-binding subsite and in a proper position to interact with ICT. In both the α and γ subunits, the N-terminal region of the α7 helix at the heterodimer interface assumes an α-helical conformation to form a long α7 helix, and the β7 strand bends towards the β5-β6 loop and the α7 helix to form a network of hydrogen bonds. These results indicate that the two structures adopt an active conformation.Our previous structural and biochemical studies demonstrated that the binding of CIT at the allosteric site induces conformational changes at the allosteric site, which are then transmitted to the active site via the heterodimer interface. This leads the active site to assume an active conformation favorable for substrate (ICT) binding, and thus decreases ure Fig. . For exaMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures, there are Mg2+, CIT and ATP bound at the allosteric site, and the residues involved in the binding of the metal ion and ligands assume almost identical conformations as those seen in the αMgγMg+CIT+ADP structure . Structural analysis shows that ATP occupies the ADP binding site, and particularly the adenine and α-phosphate of ATP make very similar interactions with the surrounding residues as those of ADP. In detail, the adenine of ATP forms two hydrogen bonds with the main-chain amine and carbonyl of Asn285G, and the α-phosphate forms two hydrogen bonds with the main-chain amines of Thr274G and Gly275G. However, unlike the β-phosphate of ADP, which is oriented towards the Mg2+-binding site and forms two hydrogen bonds with the side chains of Asn273G and Thr274G and a coordination bond with the Mg2+, the β- and γ-phosphates of ATP are oriented away from the Mg2+-binding site and make different interactions with the enzyme due to potential steric conflicts with the surrounding residues. Specifically, the β-phosphate of ATP forms two hydrogen bonds with the main-chain amines of Lys276G and Ser277G, and the γ-phosphate forms a hydrogen bond with the side chain of Ser277G. Meanwhile, a water molecule occupies the position of the β-phosphate of ADP and mediates the interaction between the Mg2+ and the α-phosphate of ATP. These results indicate that ATP can mimic ADP to bind to the allosteric site, and this ATP binding stabilizes the CIT and Mg2+ binding and thus the related conformational changes, leading the enzyme to adopt an active conformation. This provides the structural basis for why the αγ heterodimer can be activated by low concentrations of ATP. Since ATP has a larger volume than ADP, the ADP binding site cannot accommodate the three phosphates of ATP, and hence the β- and γ-phosphates of ATP are positioned away from the Mg2+-binding site. As a result, the interaction between ATP and Mg2+ is mediated via a water molecule instead of the direct interaction between ADP and Mg2+, suggesting that the stabilization of the CIT and Mg2+ binding by ATP is weaker than the stabilization by ADP. This explains why ATP has a weaker activatory effect than ADP17.In both the αure Fig. . Structu75GFig. . Howeverg2+Fig. , the β- 77GFig. . MeanwhiMgγMg+CIT+ATP structure, there is Mg2+ bound at the active site, and in the αMg+ATPγMg+CIT+ATP structure, there are both Mg2+ and ATP bound at the active site. Structural comparison shows that in both structures, the key residues at the active site assume very similar conformations, and the Mg2+ maintains identical interactions with the surrounding residues as those seen in the αMgγMg+CIT+ADP structure, rather than those seen in the αMgγ structure . As there is no structure of the αγ heterodimer bound with an NAD at the active site thus far, we compared the αMg+ATPγMg+CIT+ATP structure with the αMg+NADHγNADH structure to dissect the effects of the ATP binding at the active site. The structural comparison shows that ATP binds to the NAD binding site, and the adenine, ribose, and α-phosphate of ATP occupy the positions of the corresponding moieties of NADH in the αMg+NADHγNADH structure and maintain similar interactions with the surrounding residues19. In detail, the adenine of ATP forms two hydrogen bonds with the main-chain amine and carbonyl of Asn276A, the ribose 2′-OH forms a hydrogen bond with the side chain of Asp268A, and the α-phosphate forms two hydrogen bonds with the main-chain amines of Gly264A and Thr265A. On the other hand, the β- and γ-phosphates of ATP occupy a slightly different position from the nicotinamide moiety of NADH and make no interactions with any residues of the enzyme; however, there is a water molecule mediating the interaction between the α- and β-phosphates of ATP. The observation that ATP makes fewer interactions with the enzyme compared to NADH (and NAD) suggests that ATP has a weaker binding with the enzyme than NADH (and NAD). These results indicate that ATP at high concentrations can mimic NADH (and NAD) to bind to the NAD binding site and prevents NAD binding which accounts for in part why high concentrations of ATP can inhibit the enzymatic activity of the αγ heterodimer.In the αure Fig. . As thers19Fig. . In deta2+)23. In cells, about 90% of total intracellular ATP (in the range of 1–10 mM depending on the cell types and determination methods) exists in the form of MgATP, and about 50% of total intracellular ADP (in the range of 0.1–1 mM) exists in the form of MgADP27. Thus, the concentrations of ATP and ADP can affect the proportion of the metal ion that exists in chelate form versus free ion form, thereby modulating the enzymatic activity in many metal dependent catalytic reactions. We speculated that high concentrations of ATP may promote the formation of MnATP or MgATP and decrease the concentration of free Mn2+ or Mg2+ available for the catalytic reaction, leading to the inhibition of the enzyme. To investigate this possibility, we analyzed the dual regulatory roles of ATP on the activity of the αγ heterodimer at different concentrations of Mn2+ or Mg2+  and in the absence of the activators CIT and ADP , ATP has activatory effect at low concentrations but inhibitory effect at high concentrations. Moreover, as the metal ion concentration increases, the ATP concentrations required to reach the optimal activation and the complete inhibition of the enzyme are elevated; additionally, the ATP concentration range for activation is also extended  with other ligand(s), we also analyzed the regulatory effect of ATP on the αγ heterodimer at different concentrations of the substrate ICT, the cofactor NAD, the inhibitor NADH, and the activators CIT and ADP Fig. . As expe19. This might be explained as follows: when the CIT concentration reaches the optimal activation level (2 mM), more CIT would compete with ICT to bind to the active site, and thus inhibit the enzymatic activity. In the presence of lower concentrations of ADP , ATP plays dual regulatory roles, and the ATP concentration required to reach the optimal activation is gradually decreased from 1.5 mM ATP (at 0.5 mM ADP) to 0.5 mM (at 2 mM ADP); meanwhile, the enzymatic activity is also increased accordingly , a further increase in ADP and ATP would compete with NAD to bind to the active site or reduce the concentration of free metal ion, thus inhibiting the enzymatic activity. As expected, in the presence of both CIT and ADP, ATP exhibits a regulatory pattern similar to a combination of the patterns seen in the presence of CIT and ADP alone  to 1 mM ATP in the presence of higher concentrations of CIT (2 mM and 4 mM) Fig. . Intrigugly Fig. . In the one Fig. . In the 17. Intriguingly, while the early biochemical studies showed that ATP has inhibitory effect on the activity of mammalian NAD-IDHs20, our biochemical studies showed that ATP can activate the activities of the αγ heterodimer and the holoenzyme of human NAD-IDH at low concentrations but inhibit the enzymatic activity at high concentrations. To investigate the molecular basis for the dual regulatory roles of ATP on the αγ heterodimer, we determined the crystal structure of αMgγMg+CIT+ATP, which was obtained from crystals grown from a crystallization solution containing a low concentration of ATP (2 mM), and we also determined the crystal structure of αMg+ATPγMg+CIT+ATP, which was obtained from soaking the crystals of αMgγMg+CIT in a crystallization solution containing a high concentration of ATP (200 mM). In addition, we performed biochemical studies to analyze the effects of the concentrations of metal ion and ligands on the dual regulatory roles of ATP on the αγ heterodimer. The structural and biochemical data together reveal the molecular mechanisms of the activation and inhibition of the αγ heterodimer by ATP.In cells, the ATP/ADP ratio is considered to be a key factor for controlling respiration and metabolism in response to energy demand. A low ATP/ADP ratio corresponds to a low energy level of the cell and thus attenuates cellular respiration, whereas a high ATP/ADP ratio corresponds to a high energy level of the cell and thus ramps down cellular respiration. As a rate-limiting step of the Krebs cycle, the activity of NAD-IDH is also regulated by ADP and ATP. Our previous biochemical and structural studies and the early biochemical studies of other groups showed that ADP can positively regulate the activity of mammalian NAD-IDHs via an allosteric mechanismMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures, there are Mg2+, CIT and ATP bound to the allosteric site. The ATP mimics ADP in binding to the ADP binding site; this ATP binding stabilizes the CIT and Mg2+ binding and the associated conformational changes at the allosteric site, the active site, and the heterodimer interface, leading the enzyme to adopt an active conformation similar to that of αMgγMg+CIT+ADP. As the ADP binding site cannot accommodate the three phosphates of ATP, the β- and γ-phosphates of ATP are oriented away from the Mg2+-binding site, and the stabilization of the CIT and Mg2+ binding by ATP is mediated via a water molecule, which is apparently weaker than the stabilization by ADP. The structural results are consistent with our previous and current biochemical data showing that both ADP and ATP have a synergistic activatory effect with CIT on the αγ heterodimer; however, the activatory effect of ATP is weaker than that of ADP, and thus can be compromised by ADP. The structural and biochemical data together provide the molecular basis to explain why the αγ heterodimer can be activated by low concentrations of ATP.In both the αMg+ATPγMg+CIT+ATP structure, there are Mg2+ and ATP bound to the active site. The ATP mimics NADH in binding to the NAD binding site; this binding prevents the binding of NAD and leads to the inhibition of the enzyme. Nevertheless, although the adenine, the ribose, and the α-phosphate of ATP occupy the same positions as the corresponding moieties of NADH in the αMg+NADHγNADH structure and maintain similar interactions with the surrounding residues19, the β- and γ-phosphates of ATP occupy a slightly different position from the nicotinamide moiety of NADH and make no interactions with the enzyme. This suggests that ATP has a weaker binding with the enzyme compared to NADH (and NAD). These results are also consistent with the structural data demonstrating that ATP can only bind to the NAD binding site at a very high concentration. Moreover, our biochemical data show that increases in the metal ion concentration can increase the ATP concentration required to reach the optimal activation and the complete inhibition of the enzyme, as well as broaden the ATP concentration range for activation. This indicates that increasing the metal ion concentration can compensate for the inhibitory effect of ATP. These results suggest that high concentrations of ATP facilitate the formation of MgATP or MnATP and reduce the concentration of free metal ion available for the catalytic reaction, leading to the inhibition of the enzyme. The combined effects of the competitive binding of ATP to the NAD binding site and the formation of ATP-metal chelate account for the inhibition of the αγ heterodimer by high concentrations of ATP.On the other hand, the inhibition of the αγ heterodimer by high concentrations of ATP occurs through two different mechanisms. In the α16. Briefly, the DNA fragments encoding the α and γ subunits of human NAD-IDH were cloned into co-expression vector pQlinkN with the C-terminal of the γ subunit attached with a TEV protease cleavage site and a His6 tag following the pQlinkN cloning procedure28. The pQlinkN-α-γ-tev-His6 plasmid was transformed into E. coli BL21(DE3) CodonPlus strain (Novagen), and protein expression was induced by 0.4 mM IPTG for 20 hours at 23 °C. The target protein was first purified by affinity chromatography using an Ni-NTA column (Qiagen). The His6-tag of the target protein was cleaved by TEV protease, and the protein mixture was reloaded on an Ni-NTA column and the flow-through fraction containing the target protein was further purified by gel filtration using a Superdex 200 10/300 GL column . The purified protein has high purity (>95%) as analyzed by 12% SDS-PAGE, which was concentrated to about 10 mg/ml and stored in the storage buffer  for the structural and biochemical studies.The αγ heterodimer of human NAD-IDH was prepared as described previouslyMgγMg+CIT+ATP heterodimer were grown from drops consisting of equal volumes (1 μl) of the protein solution supplemented with 0.2 mM Mg2+ and 2 mM ATP, and the reservoir solution containing 0.2 M sodium citrate, pH 8.0, and 20% (w/v) PEG3350. Crystals of the αMgγMg+CIT heterodimer were grown from drops consisting of the protein solution supplemented with 0.2 mM Mg2+, and the reservoir solution containing 0.2 M sodium citrate, pH 5.5, and 24% (v/v) PEG400. Crystals of the αMg+ATPγMg+CIT+ATP heterodimer were obtained by soaking the crystals of αMgγMg+CIT in the crystallization solution supplemented with 200 mM ATP for 10 mins. The pH values of the stock solutions of the metal ion and ATP were adjusted to 7.4, which is the same as that of the protein stock solution, before addition into the crystallization or soaking solutions. Prior to diffraction data collection, the crystals were cryoprotected using the reservoir solution supplemented with 25% ethylene glycol and then flash-cooled into liquid N2. Diffraction data were collected at 100 K at BL17U1 of Shanghai Synchrotron Radiation Facility (SSRF) and BL19U1 of National Facility for Protein Science in Shanghai (NFPSS) and processed with HKL200029. Statistics of the diffraction data are summarized in Table Crystallization was performed using the hanging drop vapor diffusion method at 20 °C. Crystals of the α volumes  μl of thMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures were solved with the molecular replacement method as implemented in program Phaser30 using the αMgγ structure (PDB code 5GRH) as the search model. Structure refinement was carried out initially with program Phenix31 and finally with program REFMAC532. Manual model building was carried out with program Coot33. Stereochemistry and quality of the structure models were analyzed using programs in the CCP4 suite34. Structure figures were prepared using PyMol35. Statistics of the structure refinement and final structure models are summarized in Table The α16. The standard reaction solution (1 ml) consisted of 33 mM Tris-acetate, pH 7.4, 2 μg enzyme, 0.6 mM ICT, 2 mM Mn2+, and 3.2 mM NAD. The activity is defined as the velocity (V) at the specific conditions, which is expressed as μmol of NADH produced per min per mg of enzyme (μmol/min/mg). To analyze the dual regulatory roles of ATP on the activity, the activity was measured at the standard conditions with varied concentrations of ATP (0–10 mM). The pH value of the ATP stock solution was adjusted to 7.4, which is the same as that of the protein stock solution, before addition into the reaction mixture. The effect of ATP is presented as the ratio (V/Vab) of the activity of the enzyme in the presence of ATP (and regulators) (V) verse that in the absence of ATP and any regulators at the standard conditions (Vab which is defined as 1).Enzymatic activity of the αγ heterodimer was assayed using a method as described previously2+ and Mg2+ on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of Mn2+ or Mg2+ . To analyze the effects of the substrate ICT and the cofactor NAD on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of ICT  or NAD . To analyze the effect of the inhibitor NADH on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of NADH . To analyze the effects of the activators CIT or/and ADP on the dual regulatory roles of ATP, the activity was measured at the standard conditions with varied concentrations of CIT , or ADP , or both CIT and ADP. The pH values of the stock solutions of the metal ion and ligands were adjusted to 7.4, which is the same as that of the protein stock solution, before addition into the reaction mixture. All experiments were carried out at 25 °C and repeated at least twice under the same conditions.To analyze the effects of MnMgγMg+CIT+ATP and αMg+ATPγMg+CIT+ATP structures of human NAD-IDH have been deposited in the Protein Data Bank with accession codes 6L57 and 6L59, respectively.The α


The FT-IR data show that the –CH3 peak intensity decreases while the –CH2– and C=N peak intensities increase as the temperature is increased from 100 to 250 °C. This result is attributed to decomposition of the dimethylamido ligands. Based on the FT-IR data, it can also be assumed that a new decomposition product is formation at 250 °C. While in situ QMS analysis demonstrates that vaporized CpZr(NMe2)3 decomposes to N-ethylmethanimine rather than methylmethyleneimine. The in situ monitoring with FT-IR spectroscopy and QMS provides useful information for understanding the behavior and decomposes of CpZr(NMe2)3 in the gas phase, which was not proven before. The study to understand the decomposition of vaporized precursor is the first attempt and can be provided as useful information for improving the reliability of a high- advanced ultra-thin film deposition process using atomic layer deposition in the future.We developed a newly designed system based on in situ monitoring with Fourier transform infrared (FT-IR) spectroscopy and quadrupole mass spectrometry (QMS) for understanding decomposition mechanism and by-products of vaporized Cyclopentadienyl Tris(dimethylamino) Zirconium (CpZr(NMe To solvtability .2 films are typically deposited by chemical vapor deposition (CVD) [Ultra-thin ZrOon (CVD) , 6 or aton (CVD) , 8. Of ton (CVD) . As suchBecause thin film growth based on ALD proceeds via the chemical reactions of vaporized precursors and co-reactants, it is important to select the appropriate precursor for a successful process , 11. In Thermal stability of the precursor for ALD process has to be considered in two points. Precursors are exposed to consistently thermal stress because precursors are heated to be vaporized during ALD process. Also, gas line for feeding vaporized precursors into the chamber is heated above the vaporizer temperature to prevent condensation or coagulation of the vaporized precursors and facilitate diffusion. At this time, the gas line is heated to a higher temperature as it is closer to the chamber from the evaporator. It is generally heated to a temperature between 100 and 200 °C. Therefore, high thermal stability should be ensured so that thermal decomposition does not occur in this temperature range. But, the precursor that has too high thermal stability does not decompose ligand at process temperature and it leads to degradation of film reliability . So, theThe thermal stability and the decomposition mechanism of precursor have been studied by thermogravimetric analyzer (TGA), differential scanning calorimeter (DSC), Fourier transform infrared (FT-IR) and spectroscopy, and quadrupole mass spectroscopy (QMS) –22. TGA Thermal decomposition in moving vaporized precursors due to thermal stress exposure has not received attention. But, understanding this mechanism can provide very useful information for process optimization and new precursor design because thermal decomposition of unwanted precursors can affect process reliability.2)3; Cp: cyclopentadienyl (C5H5), Me: methyl (-CH3) in gas phase by using newly designed system based on in situ monitoring using Fourier transform infrared (FT-IR) spectroscopy and quadrupole mass spectrometry (QMS).In this study, we try to understand about the thermal decomposition mechanism of the typical precursor Cyclopentadienyl Tris(dimethylamino) Zirconium  having a 10-Torr range. In each trial, the precursor was placed inside the bubbler (which was held at 80 °C), after which the vaporized material was supplied to the feed line, which was constructed of stainless steel (SUS) and had stainless steel gaskets at each connection in addition to manual valves that allowed for the application of vacuum. Both the feed line and FT-IR gas cell were held at 100 °C to prevent the precursor from condensing. The gas cell was constructed of KBr windows, had a volume of 45.5 mL, and was also held at a vacuum of 10−3 Torr. The FT-IR employed a Hg-Cd-Te detector and cooled with liquid nitrogen. The spacing between the IR source and KBr window is 5 cm. A specially designed heating element allowed the temperature of the vaporized precursor to be controlled up to 500 °C.This apparatus comprises a vacuum system connected to a bubbler, a feed line with a valve, a heating element, and a gas cell attached to an FT-IR spectrometer . The lab-made vacuum system was held at a pressure of 10−8 Torr using a combination of a turbomolecular pump and a rotary pump, and this pressure was monitored with a CDG.A QMS instrument  was also connected to the vacuum system to allow for additional analysis of the vaporized precursor and was maintained at 150 °C with a heating jacket. The internal pressure in the QMS instrument was kept at 102)3  was vaporized by heating the stainless steel bubbler. During the initial 10 min, all valves of in situ monitoring systems were opened and the vaporized CpZr(NMe2)3 was passed to get identical vaporized precursor. Valves 2 and 3 were closed and the resulting vapors were transferred to the high-temperature zone. Valves 1, 2, and 3 were closed to isolate the vaporized CpZr(NMe2)3 for 5 min in heating element to give enough thermal stress. At that time, the heating element was heated to temperatures from 100 to 250 °C in increments of 25 °C. Valves 2 and 3 were opened, and 4, 5, and 6 were closed to move decomposition products to FT-IR and QMS. Valves 2 and 3 were closed and the resulting decomposition products were assessed using both the FT-IR and QMS instrumentation. The FT-IR acquired spectra over the range from 500 to 3500 cm−1 with a spectral resolution of 8 cm−1, summing 32 scans to obtain each spectrum. The QMS analyzed ions over the range from 1 to 300 atomic mass units (amu), operating in the electron impact ionization mode with an ionization energy of 30 eV. After measured, Valves 4, 5, and 6 were opened to remove the heated CpZr(NMe2)3 for 10 min.The CpZr(NMe2)3 is typically processed at temperatures from 300 to 380 °C [The precursor including cyclopentadienyl moieties tends to exhibit high thermal stability owing to the strong bonding between these groups and metal atoms, and CpZr(NMeo 380 °C –31.2)3 was expected to proceed primarily at the trimethylamine ligands rather than at the cyclopentadienyl-Zr bonds. Based on this assumption, the expected main decomposition products are summarized in Table Therefore, the thermal decomposition of vaporized CpZr2N-N(CH3)2•NMe1,2-elimination reaction of Tetramethylhydrazine3)2N-M(NCH2)3 → N(CH3)3 + H3C-N = M(NMe2)2(CH2)3, while Fig. Figure 2)3. The occurred temperatures in which the mass loss are0.5% and 5% are 87.6 °C and 132.6 °C, respectively. We also confirm that the weight became not zero at high temperature. It means that the Cp Zr(NMe2)3 is not completely decomposed and non-volatile materials are generated and remain.The TGA analysis result shows the approximate thermal decomposition temperature of CpZr(NMe2)3 were associated with alkane C–H stretching (around 3000–2840 cm−1), N–CH3 symmetric stretching (2776 cm−1), C=N stretching (around 1500–1400 cm−1), –CH3 deformation (1241 cm−1), –CH3 rocking (1142 cm−1), NC2 symmetric stretching (939 cm−1), and –CH2– stretching (794 cm−1). As the temperature of the vapor was increased from 100 to 250 °C, the intensities of these main absorption peaks all decreased, except for the peak related to –CH2– stretching 3, the trimethylamine ligand is more readily thermally decomposed than the cyclopentadienyl ligand, due to the difference in the dissociation energies of the Zr–N and C–N bonds. Therefore, it was expected that dimethylamine would be produced by the hydrogenation reaction of the dimethylamido ligand as a result of cleavage of the Zr–N bonds in the compound. It is also likely that C=N bonds were formed by the hydrogen elimination reaction of the dimethylamido ligands, because the intensity of the FT-IR peak associated with C=N bond stretching at 1450 cm−1 was increased with increasing temperature 3 at 250 °C differ from those in the previously proposed mechanism, in which the decomposition reactions of the dimethylamido ligands, such as hydrogenation and hydrogen elimination, predominate 3 and its fragments have masses of 228 and 144 amu, respectively. However, the CpZr(NMe2)3 parent ion at 144 amu was only detected at a heating temperature of 100 °C. Above this temperature, fragments with masses less than 80 amu were observed as a result of the rapid decomposition of the vaporized CpZr(NMe2)3 3. The ions appearing at 5 amu and below are attributed to hydrogen and hydrogen ionization, from 10–20 amu to methane and methane ionization, from 25–32 amu to N-ethylmethanimine, trimethylamine fragments and ionization, from 40–50 amu to dimethylamine and N-ethylmethanimine ionization, and from 60–75 amu to cyclopentadienyl and cyclopentadienyl ionization  increase with increasing heating temperature, while the masses associated with methane and methyl ionization  decrease with increasing heating temperature. Therefore, these results confirm that both the FT-IR and QMS analyses provide similar results regarding the decomposition reactions of vaporized CpZr(NMe2)3.The FT-IR spectra suggest that –CH2)3 on gas phase 3 is exposed to heating temperatures under 250 °C, it has different two decomposition mechanisms. Methylmethyleneimine that may fail to react further due to insufficient thermal energy and N-ethylmethanimine are mixed at temperatures 100 °C and below, and N-ethylmethanimine is present only at temperatures above 100 °C. When CpZr(NMe2)3 is exposed to heating temperatures 250 °C and above, N-ethylmethanimine and unknown decomposition products containing CH2 bonding are produced as decomposition products.When CpZr(NMe2)3 is exposed to thermal stress, methylmethyleneimine is preferentially generated, N-ethylmethanimine is generated through additional reactions, and unknown decomposition products containing CH2 bonding are generated when exposed to higher thermal energy above 250 °C. Therefore, we can easily predict methylmethyleneimine at temperatures lower than 100 °C and unknown decomposition products containing CH2 bonding at temperatures above 250 °C exist as a major decomposition.It can confirm from the analysis that when CpZr(NMe2)3, which has been confirmed by using our newly developed in situ monitoring systems, is expected to provide very useful information for ALD process optimization and new precursor design. Also, we try to suggest that expected mechanisms, and it showed that the in situ monitoring systems are useful to understand the reaction mechanism including thermal decomposition in the gas phase of various materials.The thermal decomposition mechanism of vaporized CpZr(NMe2)3. FT-IR analysis determined that the trimethylamine ligands were decomposed and that dimethylamine was formed via a hydrogenation reaction. The data also demonstrate that –CH2– and C=N groups were generated by elimination reactions as the temperature was increased. However, the in situ FT-IR analysis was unable to confirm which product having C=N bonds were obtained from the decomposition of dimethylamido ligands. QMS data demonstrated that N-ethylmethanimine was produced to a much greater extent than methylmethyleneimine via decomposition of the dimethylamido ligands as the temperature was increased.This work developed an in situ monitoring system using FT-IR and QMS and applied this new technique to assess the decomposition of vaporized CpZr(NMe2)3 by thermal stress using FT-IR and confirmed that the decomposition product with C=N groups is N-ethylmethanimine by using QMS analysis. The thermal decomposition mechanism of vaporized CpZr(NMe2)3 provides very useful information for optimization of ALD process and new precursor design.As the result, we estimated the reaction mechanism by analyzing the decomposition products of vaporized CpZr(NMe


However, the mechanism by which phonons in cuprates acquire chirality in a magnetic field is still unknown. Here, we report a similar thermal Hall conductivity in two cuprate Mott insulators with significantly different crystal structures and magnetic orders – Nd2CuO4 and Sr2CuO2Cl2 – and show that two potential mechanisms can be excluded – the scattering of phonons by rare-earth impurities and by structural domains. Our comparative study further reveals that orthorhombicity, apical oxygens, the tilting of oxygen octahedra and the canting of spins out of the CuO2 planes are not essential to the mechanism of chirality. Our findings point to a chiral mechanism coming from a coupling of acoustic phonons to the intrinsic excitations of the CuO2 planes.The heat carriers responsible for the unexpectedly large thermal Hall conductivity of the cuprate Mott insulator La 2 planes.What makes the phonons in cuprates become chiral, as measured by their thermal Hall effect, is an unresolved question. Here, the authors rule out two extrinsic mechanisms and argue that chirality comes from a coupling of acoustic phonons to the intrinsic excitations of the CuO In insulators, the carriers of heat are not charged, but neutral, and so the electrical Hall effect is zero. The thermal Hall conductivity κxy is measured by sending a heat current along the x-axis and detecting a transverse temperature gradient along the y-axis, in the presence of a perpendicular magnetic field . It has been shown that in certain conditions, spins can produce such chirality2. For example, magnons give rise to a thermal Hall signal in the antiferromagnet Lu2V2O7 (ref. 3). As a result, a measurement of the thermal Hall effect can in principle provide access to various topological excitations in insulating quantum materials, such as Majorana edge modes in chiral spin liquids4. Recently, the thermal Hall conductivity κxy seen in α-RuCl3 below T ≃ 80 K (refs. 6) has been attributed to the excitations of a Kitaev spin liquid7. Similarly, the κxy signal observed in some frustrated magnets—in which there is no magnetic order down to the lowest temperatures—has been attributed to spin-related heat carriers8.In the last decade, the thermal Hall effect has become a useful probe of insulators9. In the ferrimagnetic insulator Fe2Mo3O8, the large κxy signal is attributed to the strong spin–lattice coupling characteristic of multiferroic materials10. An extrinsic mechanism is the skew scattering of phonons by rare-earth impurities11, as in the rare-earth garnet Tb3Ga5O12 (refs. 13). Recently, a large phononic κxy has been observed in the nonmagnetic insulator SrTiO3 (ref. 14). A proposed explanation involves the large ferroelectric susceptibility of this oxide insulator, together with an extrinsic mechanism whereby phonons are scattered by the polar boundaries from the antiferrodistortive structural transition at 105 K (ref. 15). This interpretation is supported by the fact that κxy is negligible in the closely related material KTaO3 (ref. 14), which remains cubic and free of structural domains.However, phonons can also generate a nonzero thermal Hall conductivity if some mechanism confers chirality to them. For instance, an intrinsic mechanism is the Berry curvature of phonon bands acquired from a magnetic environmentκxy signal was observed at low temperature inside the pseudogap phase16, i.e., for dopings p < p*, where p* is the pseudogap critical doping17. Because it persists down to p = 0, in the Mott insulator state, this negative κxy cannot come from charge carriers, which are not mobile at p = 0. Therefore, it must come either from spin-related excitations  or from phonons (as in ref. 15). To distinguish between these two types of heat carriers, a simple approach was recently adopted: the thermal Hall conductivity was measured for a heat current along the c-axis, normal to the CuO2 planes, a direction in which only phonons move easily20. In La2CuO4, at p = 0, the thermal Hall signal was found to be just as large as for an in-plane heat current, i.e., κzy(T) ≈ κxy(T) (ref. 21). This is compelling evidence that phonons are the heat carriers involved in the thermal Hall signal of this insulator. Moreover, it was found that this phonon Hall effect vanishes entirely immediately outside the pseudogap phase, i.e., κzy(T) = 0 at p > p*, revealing that phonons only become chiral upon entering the pseudogap phase21.In cuprates, a large negative 2CuO4 and Sr2CuO2Cl2, and find in both a large negative thermal Hall conductivity similar to that of La2CuO4. While the three materials share the same fundamental characteristic of cuprates, namely they are a stack of single CuO2 planes, there are significant differences between them  and Nd2CuO4. We see that as in La2CuO4, both materials show a large negative thermal Hall signal. We also observe a certain field dependence of κxx, larger than the small one observed in La2CuO4 (ref. 16). In Sr2CuO2Cl2, the field increases κxx slightly, below T ≈ 20 K. In Nd2CuO4, the field decreases κxx, below T ≈ 40 K.In Fig. κxy vs. T (right panels) are similar in shape, peaking at T ≈ 25 K, a temperature close to that where κxx vs. T peaks (left panels). At low temperature, κxx is dominated by phonons. Indeed, because there is a gap in the magnon spectrum of these antiferromagnets22, their contribution to κxx becomes negligible at low T compared to the phonon contribution. At T = 35 K, the magnon conductivity is only 2% of the measured κxx (ref. 20), and it rapidly becomes vanishingly small below that temperature. At T = 20 K, the magnitude of κxx is 8 times larger in Nd2CuO4 compared to Sr2CuO2Cl2  larger in Nd2CuO4. This is strong evidence that phonons are the heat carriers responsible for the Hall response.In Fig. κxy/κxx vs T for the three materials. We see that not only is this ratio of similar magnitude in the three cuprates, but its temperature dependence is also very similar, growing with decreasing T to reach a maximal (negative) value at T ≈ 10–15 K, where |κxy/κxx| ≈ 0.3–0.4% (at H = 15 T).In Fig. κxy in both Nd2CuO4 and Sr2CuO2Cl2 that is very similar to that previously reported for La2CuO4  allows us to draw several conclusions about the underlying mechanism for chirality in the cuprate Mott insulators.Having observed a large negative thermal Hall conductivity 1. In the collinear Néel antiferromagnetic order of La2CuO4, no thermal Hall effect is expected theoretically, because of the so-called “no-go” theorem, which states that Néel order on a square lattice has zero chirality1. However, if the spins of the Néel order cant out of the plane, as they do in La2CuO4, due to some Dzyaloshinskii–Moriya (DM) interaction, then some chirality becomes possible. In this case, one could get a nonzero κxy signal, but it is expected to be much smaller than the measured κxy signal in La2CuO4 (ref. 23). Our data on Nd2CuO4 and Sr2CuO2Cl2 completely eliminate this possibility, because a κxy signal of similar or larger magnitude is found in these materials for which there is no canting of spins out of the plane (see “Methods”), and so no DM interaction. We conclude that magnons are not responsible for the thermal Hall effect in cuprate Mott insulators. This conclusion is consistent with the fact that in La2CuO4 a large κxy signal persists down to temperatures well below the smallest magnon gap, of magnitude 26 K (ref. 22), and up in doping well above the critical doping for the suppression of Néel order, i.e., p ≈ 0.02 in Lax2−SrxCuO4 (ref. 16).It has been shown theoretically that in ferromagnetic or antiferromagnetic insulators, under certain conditions, magnons can have chirality and should give rise to a thermal Hall effectAs for a phonon scenario whereby phonons would acquire chirality through their coupling to spins, spin canting also appears to be unimportant.Let us now consider two mechanisms known to confer chirality to phonons in other materials—skew scattering off rare-earth impurities and scattering off structural domain boundaries—and show that neither is relevant to cuprates.3Ga5O12 (refs. 13), has been attributed to the skew scattering of phonons by superstoichiometric Tb3+ ions11. This extrinsic mechanism depends crucially on the details of the crystal-field levels of the rare-earth ion. A different rare-earth ion will in general produce skew scattering of a very different strength. The fact that the ratio κxy/κxx is the same in all three cuprates considered here is compelling evidence that the underlying mechanism does not depend on the nature of the particular cation, whether La, Sr, or Nd.The initial observation of a phonon thermal Hall effect, in the garnet Tbκxx (ref. 24). In Tb3Ga5O12, 2% of Tb3+ impurities gives rise to both a finite κxy signal from phonons (whose magnitude is given in Table κxx (ref. 13), whose value at T = 15 K is then only κxx = 1.2 W/Km (ref. 11). In the pyrochlore oxide Tb2Ti2O7, a frustrated magnet with a sizable thermal Hall effect8  (ref. 25), pointing again to strong scattering of phonons by Tb3+ ions.  By comparison, the thermal conductivity in the cuprate Mott insulators is an order of magnitude larger 14 , although the precise mechanism whereby these confer chirality to phonons is still unclear. Our comparative study of the three cuprates allows us to rule out a similar role for structural domains. Indeed, whereas La2CuO4 undergoes a structural transition to an orthorhombic phase below 530 K, both Nd2CuO4 and Sr2CuO2Cl2 remain tetragonal down to T ≈ 0 K, and yet all three have a similar thermal Hall effect, in both T dependence  under three different conditions: (1) for a field H = 10.6 T applied along the c-axis, (2) for a field H = 15 T applied at an angle of 45° from the c-axis , applied at T = 2 K (zero-field cooling), and (3) same as for (2), but applied at T = 300 K > TN (in-field cooling). In the latter in-field cooling condition, the in-plane component of the field (of magnitude 10.6 T) applied at T > TN will ensure that a single antiferromagnetic domain is present below TN. .) Comparing conditions (2) and (3) amounts to comparing a multidomain sample vs a monodomain sample.Because the collinear spin order in Srκxy is identical in the three situations, within error bars. So, magnetic domains in Sr2CuO2Cl2, and any associated structural distortions, do not influence the thermal Hall response. Note that the noncollinear order in Nd2CuO4 does not break the fourfold symmetry of the lattice, so here no magnetic domains are expected.The results of this comparative study are displayed in Fig. We conclude that structural  domains are not the mechanism that confers chirality to phonons in cuprates. Moreover, the thermal Hall conductivity of cuprates is independent of whether the system has orthorhombic or tetragonal symmetry, or whether there are apical oxygens in the structure or not.2CuO4 and Sr2CuO2Cl2 exhibit a large negative thermal Hall conductivity κxy very similar to that found in La2CuO4. The fact that the magnitude of κxy scales with the magnitude of the phonon-dominated κxx as the latter varies by a factor 10 between Sr2CuO2Cl2 and Nd2CuO4 is further evidence in favor of phonons as the carriers of heat responsible for the thermal Hall effect in these materials. Given the different crystal structures and cations involved in those three materials, the similarity in κxy/κxx allows us to rule out two extrinsic mechanisms of phonon chirality proposed for other oxides, namely the scattering off rare-earth impurities—invoked for Tb3Gd5O12—and the scattering off structural domain boundaries—invoked for SrTiO3. This suggests that phonon chirality in the cuprates comes from an intrinsic coupling of phonons to their environment.In summary, our results show that the cuprate Mott insulators Nd9). This could involve a coupling to charge or a coupling to spin, for example. In ref. 15, a flexoelectric coupling of phonons to their charge environment was shown to generate a Hall response. However, even in the nearly ferroelectric insulator SrTiO3, where the electric polarizability is exceptionally large, this intrinsic mechanism is estimated to be much too small. The inclusion of some additional, extrinsic, scattering mechanism—possibly structural domain boundaries—is deemed necessary. Applied to cuprates, the intrinsic flexoelectric coupling is certainly much too small. It is not clear what extrinsic mechanism could be added to make this mechanism strong enough to account for the observed data in the cuprate Mott insulators.Phonons can acquire chirality through a coupling to their intrinsic environment   (ref. 29). In Fig. 2CuO2Cl2 and Nd2CuO4 to that form and find a good fit over the intermediate temperature range from 30 to 100 K. An equally good fit is found for La2CuO4 (ref. 21). Whether this implies that phonons acquire a Berry curvature through their coupling to the environment remains to be determined. At low temperature, we would expect phonons to eventually decouple from their environment, whether that be spins or other excitations of electronic origin. The temperature below which they do so would shed light on the nature of that coupling. In Fig. κxy| in Sr2CuO2Cl2 falls more rapidly to zero than κxx does.  In Fig. T regime in Sr2CuO2Cl2. We see that whereas κxx/T3 rises monotonically as T → 0, κxy/T3 drops rapidly toward zero, starting roughly at 5 K. We identify 5 K as the approximate decoupling temperature between acoustic phonons and their chiral environment.In a scenario of phonons coupled to their environment, there could be two relevant regimes of temperature, namely above and below the peak in ely Fig. . At tempp = 0.24, in both Nd-LSCO and Eu-LSCO, the thermal Hall signal coming from phonons—as opposed to charged carriers—is zero21 (Table p < p* (p* = 0.23). The magnitude of κxy is relatively constant from p* down to p = 0 when measured relative to κxx Moreover, because the phononic 31.One intrinsic mechanism has recently been proposed whereby phonons couple to an electronic state that breaks time-reversal and inversion symmetries, which would be realized in the pseudogap phase of cupratesp* observed by angle-dependent magnetoresistance32 and the drop in carrier density across p* observed in the electrical Hall effect35, both consistent with spin modulations with a wavevector Q = . Solutions of the Hubbard model in the paramagnetic state find that, in doped Mott insulators such as the cuprates, local moments37 persist all the way from half-filling up to a critical doping where the pseudogap disappears40. In calculations within the pseudogap phase, superexchange between local moments naturally favors short-range antiferromagnetic43 or singlet correlations37. In such a scenario, the question becomes: how can the coupling of phonons to spins make these phonons chiral (in the presence of a magnetic field)?Another possible mechanism is the coupling of phonons to short-range antiferromagnetic correlations. Experimental evidence for such correlations includes the Fermi-surface transformation across La2CuO4: La2CuO4 is the parent compound of the most widely studied family of single-layer cuprates, Lax2–SrxCuO4. In La2CuO4, there is an  oxygen atom above the Cu atom, thereby forming an octahedron of O atoms around Cu  structure to an orthorhombic (Cmca) structure at 530 K (ref. 44), wherein the octahedra are tilted  whose boundaries can in principle scatter phonons. (The samples of La2CuO4 studied in refs. 21 were twinned.) Below TN = 270 K, the Cu spins order into a collinear antiferromagnetic arrangement, whereby all alternating moments point along the same direction ([110]) within every CuO2 plane inside a given orthorhombic domain. The tilting of the oxygen octahedra causes a slight canting of the spins out of the CuO2 plane (by 0.17°) (ref. 45), thereby producing a Dzyaloshinskii–Moriya (DM) interaction that could, in principle, be a source of chirality. Cu Fig. . Upon coNd2CuO4: Nd2CuO4 is the parent compound of the electron-doped family of cuprates Ndx2−CexCuO4. Unlike La2CuO4, it does not undergo any structural transition and remains tetragonal down to T = 0. A significant difference from La2CuO4 is the absence of apical oxygens, so that Cu atoms in Nd2CuO4 are not surrounded by oxygen octahedra . This breaks the fourfold symmetry within a single CuO2 plane. However, in the next CuO2 plane along the c-axis, the same spin configuration is rotated by 90°, thereby restoring the fourfold symmetry of the entire system. This noncollinear magnetic structure therefore preserves the tetragonal symmetry of the crystal.Magnetically, NdSr2CuO2Cl2: Sr2CuO2Cl2 has the same crystal structure as tetragonal La2CuO4 (T > 530 K), with La replaced by Sr and the apical O replaced by Cl , except with no spin canting out of the plane48. It remains in the same magnetic phase down to at least T = 10 K (ref. 48). Cl Fig. . Unlike 2CuO4 was grown at the University of Science and Technology of China by a standard flux method, annealed in helium for 10 h at 900 °C, and cut in the shape of rectangular platelets with dimensions 0.50 × 0.69 × 0.066 mm3 (length between contacts × width × thickness in the c direction). Contacts were made with silver epoxy, diffused at 500 °C for 1 h. The thermal conductivity κxx of similar samples was studied in detail at low temperature (T < 20 K) (refs. 51). Single crystals of Sr2CuO2Cl2 were grown at the University of British Columbia using a flux-growth method. Here, we report data on two samples (labeled A and B), cut in the shape of rectangular platelets with dimensions 0.6 × 0.11 × 0.03 mm3. Contacts were made using silver paint. In all cases, the heat current was made to flow along the a-axis of the tetragonal structure.Our single crystal of Ndκxx was measured applying a heat current Jx within the CuO2 plane, generating a longitudinal temperature difference ΔTx = T+ − T−. The thermal conductivity along the x-axis is given by κxx = (Jx/ΔTx) (L/wt), where L is the distance between T+ and T−, w is the width of the sample, and t its thickness. By applying a magnetic field along the c-axis of the crystal, normal to the CuO2 planes, a transverse temperature gradient, ΔTy, was generated . Here, the voltage (heat-off) background in the thermocouple is carefully subtracted from the heat-on signal of the thermocouple to give the correct ΔTy(H). Once the entire temperature range is covered, say from 10 to 100 K, the field direction is reversed to –H. The same procedure is now applied for these negative values of the field. We then define ΔTy(H) = [ΔTy − ΔTy]/2, thereby removing any symmetric contamination of the signal coming from the longitudinal thermal gradient and the possible misalignment of transverse contacts.The measurements were made with a steady-state method as a function of temperature, using differential type-E thermocouples for Δx-axis is generated by a heater stuck at one end of the sample. The other end is glued to a block that serves as a heat sink , this block was made of copper. To confirm that the Hall response of copper in a field does not contaminate the Hall response coming from the sample, we performed the same measurement twice, once with the copper block  and then with a block made of the insulator LiF (using insulating contacts made with GE varnish), using the same sample of Nd2CuO4 in both cases. The results are shown in Supplementary Fig. κxy curve is obtained with the two setups. We conclude that using copper for the heat sink does not lead to any detectable contamination of the thermal Hall signal.The heat current along the Supplementary InformationPeer Review File


A new concept of fluctuating storage and release of an active phase in heterogeneous catalysis is proposed that involves the transient generation of active sodium oxide species via a reversible reaction of Na2WO4 with Mn7SiO12. The process is enabled by phase transitions and melting at the high reaction temperatures that are typically applied.Structural dynamics of a Mn‐Na Give and take: Operando Raman spectroscopy in combination with in situ XRD and thermal analysis was applied to study a Mn‐Na2WO4/SiO2 catalyst at work during the oxidative coupling of methane. Melting of Na2WO4 and Mn7SiO12‐MnWO4 redox chemistry occur simultaneously, thereby enabling the transient release and storage of active sodium oxide species. Coupling of these processes is essential to avoid catalyst deactivation. Its crystalline fraction is constituted by α‐cristobalite (92 wt %), quartz (0.5 wt %), Na2WO4 (4.1 wt %), and mixed‐valent Mn7SiO12 (3.4 wt %) . The manganese silicate mostly contains the transition metal in the oxidation state +3 (Mn2+Mn3+6SiO12). Another phase, MnWO4, which only features Mn in the oxidation state +2, was sporadically detected at certain spots by Raman spectroscopy . An endothermic event, indicated by a DTA signal at 689 °C in the thermal analysis of the catalyst in synthetic air , further confirms the melting of Na2WO4 on the surface of the catalyst support. For the catalyst, the relatively small endothermic signal arises from the low concentration of Na2WO4 but is, nevertheless, clearly verified by its reversibility during cooling. Solidification of supported, molten Na2WO4 evidently results in the formation of an amorphous phase . Furthermore, the crystallization of two unidentified phases is observed in the temperature range of 660–600 °C while cubic Na2WO4 only reappeared at 450 °C. Similar results were reported by Hou et al.,2WO4 in air from 500 °C onwards and a complete disappearance at 700 °C. In addition to this, a significant broadening of the symν (W−O) stretching mode of Na2WO4 at 923 cm−1 was observed in the Raman spectrum of the catalyst above the melting point of Na2WO4 , a slightly increased concentration of quartz was observed, yet the general phase composition of the catalyst remained unaltered . Both the support  as well as the Mn2WO4 disappears at 600 °C in favor of an unknown transient phase  only revealed a significant oxygen release (m/z=32) at elevated temperatures for the Mn7SiO12 phase, with an onset at 807 °C . This could be associated with the phase transition of cubic Na2WO4 into unknown transient phases at 600 °C, the commencing formation of MnWO4 at 650 °C as well as the disappearance of Mn7SiO12, also observed from 650 °C onwards  to Mn2+ (in MnWO4), the release of molecular oxygen with its maximum at 927 °C ((3) and (4) in Figure Progressive surface mobility, initiated by restructuring of supported Na2‐TPD experiments performed by Gordienko et al.,The broad temperature range for the oxygen release covers the onset temperature of oxidative coupling of methane (650 °C) as well as the reaction temperatures that are typically applied 700–800 °C). A weight loss of 0.2 % (0.22 mg) was caused by the event, with the DTA curve indicating endothermicity (4). It is postulated that the process described in Equation (1) provides mobile lattice oxygen under steady‐state operation of the catalyst. This is in agreement with O00–800 °C−1 is dominated by a broad band of the β‐cristobalite support, which is formed via phase transition from α‐cristobalite at 225 °C.4/O2/N2 (4:1:4) feed at various temperatures  and Na2WO4 (920–930 cm−1). At the highest reaction temperature, the formation of MnWO4 becomes apparent, as indicated by a very weak band at 874 cm−1. Carbon oxides, ethane, and water were identified as the main reaction products via online mass spectrometry and gas chromatography in the effluent gas of the Raman cell . Significant formation of coke was not observed , which is attributed to consecutive reactions of the desired C2+ products on the hot stainless‐steel walls of the operando cell and the inadequate reactor geometry. However, comparison of the catalytic tests conducted with the empty operando cell and the same cell filled with the catalyst under identical reaction conditions clearly showed a lower activity and selectivity to C2+ products for the empty reactor  occurs under reaction conditions and is indeed reversible, was provided by operando Raman spectroscopy at different reaction temperatures and feed compositions Figure  A and B,4/N2 (4:5) feed at 700 °C , braunite is suddenly reformed at the expense of MnWO4 after approximately 8 h , thereby highlighting the significance of the Mn7SiO12 phase in maintaining catalytic activity. The presence of Mn7SiO12 is also clearly responsible for the formation of CO2 in place of CO . As can be seen in Figure 4/O2/N2 feed , active sodium oxide species, which are responsible for high activity and selectivity in the oxidative coupling of methane,2WO4 phase acts as storage phase responsible for transient generation of active sodium oxide species that would, in absence of the stabilizing Mn7SiO12–MnWO4 redox couple, suffer from steady sublimation,In conclusion, the present in situ and operando experiments disclosed reversible redox activity of the Mn2WO4 enable the generation of the active phase by providing mobile sodium species. Furthermore, the supported Mn7SiO12 phase was observed to function as oxygen‐donor at working temperatures, which further enhances sodium oxide formation and, thus, has implications for the reactivity. However, the availability of adsorbed or lattice oxygen due to the presence of redox‐active elements alone2WO4/SiO2 catalyst and does not explain the mechanistic role and importance of Na in this system. On the other hand, a pure silica‐supported sodium oxide would rapidly deactivate under the severe reaction conditions applied in the oxidative coupling of methane.2WO4/SiO2 catalyst guarantees long‐term stability. The synergistic element combination discovered by chance is so successful because melting and redox reactions occur in the same temperature window ] is largely impeded and generates only transient amounts of activet>8 h). Such a low concentration of oxygen in the gas phase is beneficial in terms of the selectivity. Only in total absence of oxygen is the system disturbed and MnWO4 formed in noticeable amounts. The oxygen donor Mn7SiO12  should be addressed to the authors.SupplementaryClick here for additional data file.


We calculated ECCO2R rate requirements to maintain arterial partial pressure of CO2 (PaCO2) at clinically desirable levels in mechanically ventilated ARDS patients using a six‐compartment mathematical model of CO2 and oxygen (O2) biochemistry and whole‐body transport with the inclusion of an ECCO2R device for extracorporeal veno‐venous removal of CO2. The model assumes steady state conditions. Model compartments were lung capillary blood, arterial blood, venous blood, post‐ECCO2R venous blood, interstitial fluid and tissue cells, with CO2 and O2 distribution within each compartment; biochemistry included equilibrium among bicarbonate and non‐bicarbonate buffers and CO2 and O2 binding to hemoglobin to elucidate Bohr and Haldane effects. O2 consumption and CO2 production rates were assumed proportional to predicted body weight (PBW) and adjusted to achieve reported arterial partial pressure of O2 and a PaCO2 level of 46 mmHg at a tidal volume of 7.6 mL/kg PBW in the absence of an ECCO2R device based on average data from LUNG SAFE. Model calculations showed that ECCO2R rates required to achieve mild permissive hypercapnia (PaCO2 of 46 mmHg) at a ventilation frequency or respiratory rate of 20.8/min during mechanical ventilation increased when tidal volumes decreased from 7.6 to 3 mL/kg PBW. Higher ECCO2R rates were required to achieve normocapnia (PaCO2 of 40 mmHg). Model calculations also showed that required ECCO2R rates were lower when ventilation frequencies were increased from 20.8/min to 26/min. The current mathematical model predicts that ECCO2R rates resulting in clinically desirable PaCO2 levels at tidal volumes of 5‐6 mL/kg PBW can likely be achieved in mechanically ventilated ARDS patients with current technologies; use of ultraprotective tidal volumes (3‐4 mL/kg PBW) may be challenging unless high mechanical ventilation frequencies are used.Extracorporeal carbon dioxide (CO These devices have generally been categorized as lower or higher CO2 extraction devices2R technologies has great potential as such devices can be integrated into a continuous renal replacement extracorporeal circuit2 removal may be increased by using physical principles other than diffusion such as electrodialysis2R requirements for achieving adequate blood gas chemistry or normocapnia in lung‐protective strategies have not been quantitatively evaluated except in preclinical studies.There are several ECCO2R on total body CO2 content and blood gas chemistry using a mathematical model of CO2 biochemistry and transport. In so doing, this study defines the ECCO2R requirements for extracorporeal devices to achieve adequate blood gas chemistry when using protective and ultraprotective strategies during mechanical ventilation in ARDS patients.In this report, we explore the effects of low tidal volumes and ECCO22 and oxygen (O2) whole‐body storage and transport employed in this study was that developed by others,2R device for extracorporeal veno‐venous removal of CO2. Only steady state conditions were considered. The compartments included were: lung capillary blood, arterial blood, venous blood, post‐ECCO2R venous blood, interstitial fluid, and tissue cells; the model separately calculated the acid‐base and O2 contents of each compartment. As proposed previously,2 contents could be calculated from those in venous blood only; thus, those compartments were not directly involved in formulating mass transport relationships. The volumes of each fluid compartment were assumed as 0.75% , 6.75% (venous blood), 0% (lung capillary blood and post‐ECCO2R venous blood), 12.6% , and 20% (tissue cells) of the predicted body weight. The latter volume was based on assuming muscle is the only store of CO2 from a well‐perfused tissue. Muscle tissue volume is an underestimate of total tissue volume containing CO2 and neglects the largest store in bone; it has been previously proposed to be an approximation for estimating changes in CO2 tissue content that occur over short periods of time.The six‐compartment model of CO2 whole‐body transport. The model for O2 whole‐body transport is identical to that proposed previously2 transport across the ECCO2R device was neglected. The lung was simply characterized by three separate elements: alveoli that are involved in gas exchange describing ventilation and perfusion of the lung, alveoli dead space (ventilation but no perfusion), and a pulmonary shunt (perfusion but no ventilation). The lung was considered to be mechanically ventilated with a given frequency or respiratory rate, a tidal volume per PBW and a ratio of dead space to tidal volume fixed at 0.60; the latter is the average value reported for patients with ARDS.2 and O2 in the alveoli and lung capillary blood were assumed in equilibrium, that is, no resistance to gas exchange across the alveoli/lung capillary membrane. Total concentrations of CO2 and O2 in arterial blood were the weighted average of their concentrations in lung capillary and pulmonary shunt blood flows. The ECCO2R device was characterized by a CO2 removal rate, expressed as mL of CO2 removed per min. The equations describing CO2 whole‐body transport are outlined in the Appendix; the complete model equations describing O2 whole‐body transport and other elements of the model were previously described.Figure 2 chemistry of blood, including separate chemical reactions in plasma and erythrocytes, were formulated as described previously.2 to hemoglobin in the erythrocytes. The model included competitive binding of O2, CO2, and hydrogen ions to hemoglobin to elucidate Bohr and Haldane effects as described by others.2 chemistry in blood is comprehensive; it requires solving, in general, 28 equations and 12 parameters for each compartment containing blood . This model has been previously shown to accurately describe changes in acid‐base chemistry after addition to or removal of CO2 and strong acid from blood,2 and O2,Acid‐base and O2 concentrations of arterial blood, venous blood, and post‐ECCO2R venous blood were calculated as the volume‐weighted average of the CO2 contents in plasma and erythrocytes based on the calculated partial pressure of CO2 (PaCO2), bicarbonate concentration, and carbaminohemoglobin concentrations, the latter in erythrocytes only. The total amount of CO2 in each compartment was calculated by multiplying the total concentration by their volume.The total CO0.67.2 of 0.6. Using the reported average partial pressure of O2 to fraction of inspired O2 ratio of 161 and average PaCO2 in arterial blood of 46.0 mm Hg,2 tissue uptake rate as 4.0 mL of O2 per kg PBW and a respiratory quotient as 0.966. Therefore, for patients with an actual body weight of 78 kg, as in LUNG SAFE,2 tissue uptake rate was 265 mL of O2/min and the CO2 production rate was 256 mL of CO2/min. The pulmonary shunt fraction, O2 tissue uptake rate and respiratory quotient as reported above were fixed for all simulations in this study.In the current study, several parameters were assumed for average patients with ARDS. PBW was assumed as 85% of actual body weight, as approximated previously for critically ill patients.3All calculated results assumed an actual patient body weight of 78 kg.2R device; Table 2) at a tidal volume of 7.6 mL/kg PBW was identical to that reported in LUNG SAFE2 concentrations varied in the model compartments containing blood—the simulated total CO2 concentrations were 21.8 mM in arterial blood, 24.5 mM in venous blood, 27.3 mM in interstitial fluids, and 10.5 mM in tissue cells. When tidal volume decreased from 7.6 to 3 mL/kg PBW, PaCO2 increased and pHa decreased progressively as expected, and there was a maximal increase in total body mass of CO2 of approximately 50% when decreasing tidal volume from 7.6 to 3 mL/kg PBW. Note that the PaCO2 in venous blood (PvCO2) was calculated to be approximately 10 mm Hg higher than in arterial blood when the tidal volume was 7.6 mL/kg PBW, and this difference between PvCO2 and PaCO2 increased at lower tidal volumes. Figure 2 in the various model compartments in the absence of the ECCO2R device. As expected, the total amount of CO2 in arterial blood was negligibly small and that in interstitial fluids was approximately one‐half of the total body CO2. There was a progressive increase in total mass of CO2 in each compartment as tidal volume was reduced.The effect of reductions in tidal volume during mechanical ventilation in ARDS patients on acid‐base chemistry was first evaluated in the absence of the ECCO2 when altering tidal volume and respiratory rate during mechanical ventilation in the absence of the ECCO2R device. Higher ventilation frequencies significantly reduced PaCO2 when tidal volume was reduced. The increase in respiratory rate from 20.8/min to 26/min also resulted in higher arterial pH by 0.07 units at each tidal volume (results not shown).Figure 2R rate from the extracorporeal device to achieve a PaCO2 of 46 mm Hg (mild permissive hypercapnia), considered as standard of care based on LUNG SAFE,2R rate increased. Increasing ventilation frequency from 20.8 to 26/min substantially decreased the required ECCO2R rate. ECCO2R rates required to achieve normocapnia were correspondingly higher and were achieved at a PvCO2 of 48.7 mm Hg.The ECCO42 of 46 mm Hg, pHa of 7.33, and moderately low tidal volumes. Further reductions in tidal volume would be accompanied by elevated levels of PaCO2 and reductions in arterial pH, but permissive hypercapnia with PaCO2 ≥50 mm Hg during the first 48 hours after initiating mechanical ventilation is associated with an increased risk of intensive care unit mortality in ARDS patients.2R results in variable reductions in PaCO2 within a few hours2R device used, as well as other clinical variables. As noted however by others,2R device to the reduction in PaCO2. The approach taken in the current study was to quantify the relationship between the ECCO2R rate and changes in PaCO2 and other components of acid‐base chemistry in ARDS patients undergoing mechanical ventilation using a comprehensive biochemical and physiological simulation model.The current standard of care for mechanical ventilation in ARDS patients was identified in LUNG SAFE as mild permissive hypercapnia, a PaCO2 and O2 chemistry and whole‐body storage and transport2R rate requirements for mechanically ventilated ARDS patients treated with low tidal volumes. Others2 as a function of treatment time and the blood flow rate for one specific ECCO2R device and compared their model predictions with empirical data from preclinical studies in a porcine model. The current mathematical model is comparable to this latter model in overall design but differs in three major ways. First, the current model only predicts the relationship between acid‐base chemistry and the ECCO2R rate; thus, the current approach is general and not dependent on the performance characteristics of a specific device. In practical clinical applications, the predictions from the current model may require supplementary data relating the ECCO2R rate to the blood flow rate for a specific device.2 after abrupt, transient changes in the ECCO2R rate as well as time‐dependent changes in temperature and metabolic rate during the preclinical experiments. Those time‐dependent model elements were necessary to explain the data from their porcine experiments2, O2, and acid‐base chemistry; thus, it is more general and applicable to a greater variety of clinical conditions. We believe our model and the previous modelThis theoretical effort extends a previously published mathematical model of CO2 and O2 exchange in the lung that is unlikely to accurately represent the pathophysiological conditions in ARDS patients such as ventilation/perfusion inequalities.2 in muscle cells; thus, the estimate of tissue cell volume and mass of CO2 stored in tissues is likely an underestimate . ECCO2R rates were not however reported from that study. In addition, Schmidt et al.2 of 43 mm Hg and a pH of 7.39; again, the former level in arterial blood is similar to that in Figure 2R at a rate of 51 mL of CO2/min achieved a PaCO2 of 53 mm Hg. The calculated predictions in Figure 2 of 46 mm Hg if the ECCO2R was increased to 84 mL of CO2/min.A major limitation of this work is the lack of empirical confirmation of the accuracy of the model predictions, partially because there are few clinical studies to date that have measured both ECCO2R to allow reductions in tidal volume from 6 to 4 mL/kg PBW without a 20% increase in PaCO2 in approximately 80% of patients. Overall, reductions in tidal volume to 4 mL/kg PBW with simultaneous use of ECCO2R maintained PaCO2 at 46.7‐48.0 mm Hg with ventilation frequencies of 23.5‐27.4/min during the first 24 hours of treatment. A shortcoming of this trial was the inability to measure clearance and total amount of CO2 removal by ECCO2R as originally planned as a secondary endpoint of that trial.2R rate required to achieve approximately these same conditions (4 mL/kg PBW and ventilation frequency of 26/min) as 84 mL of CO2/min at a PvCO2 of 55.6 mm Hg . Obviously, higher ECCO2R rates would be required to achieve reductions in tidal volumes to 4 mL/kg PBW in closer to 100% of patients.As this study was being finalized to first submit for publication, the results from the SUPERNOVA trial were published.2 production, higher ECCO2R rates will likely be required. Most relevant parameters in the current model have been scaled to patient body weight; thus, a simple approach to extrapolate the reported required ECCO2R rates to other patients would be by body weight. Preliminary calculations from the current model have theoretically confirmed this hypothesis. Third, several other biochemical data were not reported in the LUNG SAFE publication, such as arterial bicarbonate concentration or base excess; those values could only be estimated based on values previously reported in the literature.Additional limitations to this work include the following. First, the PBW could only be estimated from the actual body weight because the data necessary for calculating PBW in the LUNG SAFE publication were not reported. Second, model predictions were only reported for patients with an actual body weight of 78 kg. Because larger patients have higher rates of CO52R rates resulting in clinically desirable PaCO2 levels at tidal volumes of 5‐6 mL/kg PBW can likely be achieved in mechanically ventilated ARDS patients with current technologies; use of ultraprotective tidal volumes (3‐4 mL/kg PBW) may be challenging unless higher mechanical ventilation frequencies are used. The recently reported secondary analysis from the SUPERNOVA trial that lower CO2 extraction devices required high mechanical ventilation frequencies to achieve clinically acceptable PaCO2 levels at ultraprotective tidal volumesThe current mathematical model predicts that ECCOJKL is a consultant to Baxter International and NxStage Medical Inc. . JG, DP, and KH are full‐time employees of Baxter International with ownership interests.JKL designed the study, developed the mathematical model, wrote the computer program, and wrote the first draft of the manuscript. DP provide advice on the development of the mathematical model. JG and KH provided clinical input into the study design. DP, JG, and KH also reviewed the manuscript and provided valuable input to the revisions of the manuscript. All authors read and approved the final manuscript.


Our data are consistent with a model in which 3B copy number expansion within the FMDV genome has allowed evolution of separate cis and trans acting functions, providing selective pressure to maintain multiple copies of 3B.For gene duplication to be maintained, particularly in the small genomes of RNA viruses, this should offer some advantages. We have investigated the functions of a small protein termed VPg or 3B, which acts as a primer in the replication of foot‐and‐mouth disease virus (FMDV). Many related picornaviruses encode a single copy but uniquely the FMDV genome includes three  copies of the 3B coding region. Using sub‐genomic replicons incorporating nonfunctional 3Bs and 3B fusion products in competition and complementation assays, we investigated the contributions of individual 3Bs to replication and the structural requirements for functionality. We showed that a free N‐terminus is required for 3B to function as a primer and although a single 3B can support genome replication, additional copies provide a competitive advantage. However, a fourth copy confers no further advantage. Furthermore, we find that a minimum of two 3Bs is necessary for  ATCCAmerican type culture collectionBHK‐21baby hamster kidney cell‐lot 21cDNAcomplementary deoxyribonucleic acidDMEMDulbecco’s minimum essential mediaEV71enterovirus‐71FBSfetal bovine serumFMDVfoot‐and‐mouth disease virush.p.t.hours post‐transfectionkbkilobaseMUSCLEMultiple Sequence Comparison by Log‐ExpectationNCBINational Center for Biotechnology InformationNEBNew England BiolabsORFopen reading framePBSphosphate‐buffered salinepolpolymerasepRepplasmid repliconproproteasePVpoliovirusRdRpRNA‐dependent RNA polymeraseRNAribonucleic acidS.D.standard deviationS.E.M.standard error of the meanSDS‐PAGEsodium dodecyl sulfate poly‐acrylamide gel electrophoresisNTTtranscription and translationVPggenome‐linked viral proteinWTwild‐typeY3Ftyrosine to phenylalanine substitution of the third residue in the protein1The evolution of small RNA viruses is largely driven by the error‐prone RNA‐dependent RNA polymerase (RdRp), which is responsible for replicating the viral genome. Hence, RNA viruses exist as populations of related quasispecies.Aphthovirus genus of the Picornaviridae family. The FMDV genome comprises an open reading frame (ORF) flanked at the 5ʹ and 3ʹ ends by untranslated regions. The single ORF encodes both the structural and nonstructural proteins starting with the leader protease followed by a capsid coding P1‐2A region. P1 is processed into the four structural proteins VP4, VP2, VP3, and VP1. Downstream of the P1‐2A region lies the nonstructural protein‐coding region, required for RNA replication and organized as two sub‐regions, P2 and P3. The P2 region encodes the 2B and 2C proteins, 2A being a C‐terminal extension of P1. The P3 region encodes 3A, triplicate copies of the replication primer, 3B , the viral protease 3Cpro and the RdRp termed 3Dpol.Foot‐and‐mouth disease (FMD) is a highly contagious disease affecting cloven‐hoofed animals, which imposes a global economic burden of between US$ 6.5 and 21 billion in endemic regions, and over US$ 1.5 billion annually in FMD‐free countries and regions.pol, the 3CD precursor protein and a viral RNA structure termed the cis‐active replicative element or cre.Most picornaviruses, such as the well‐studied poliovirus (PV), encode a single copy of 3B and although there is evidence for duplication of 3B in some, FMDV is the only picornavirus that has been demonstrated to have three  copies of the 3B coding region. This feature is almost 100% conserved among naturally occurring FMDV isolates to date.Earlier studies, using sub‐genomic replicons and infectious virions, have shown that although multiple copies of 3B may influence host specificity and virulence,22.1Baby hamster kidney cells‐lot 21 (BHK‐21) and HeLa cells were obtained from ATCC  and propagated according to standard methods.2.2pro proteolysis, and hence, produce a series of defined fusion proteins. An FMDV construct was also designed to incorporate four copies of 3B by duplicating the 3B1 sequence using alternative codons . The duplication was inserted between 3A and 3B1, while retaining the 3A‐3B1 and 3B1‐3B2 junctions. The replicon constructs used in this study are listed in Table Two previously described picornaviral sub‐genomic replicons in which the capsid protein coding regions were replaced with fluorescent reporter genes mCherry2.3AscI . RNA transcripts were generated from sub‐genomic replicon cDNAs using T7 RNA polymerase  in the presence of nucleoside triphosphates  and MnCl2 according to the manufacturer’s protocol and our established methods.Replicon plasmid DNA was linearized with 2.4BHK‐21 cells were propagated in duplicate wells of a 12‐well plate according to standard methods. Cells were transfected with 1 µg of purified mCherry or GFP replicon RNA transcripts using Lipofectin reagents  according to the manufacturer’s protocol. Replication was monitored hourly for red and/or green fluorescence within an IncuCyte Zoom  as previously described.2.53H]‐U (uridine) per well at 1‐hour post‐transfection (h.p.t.). At 8 h.p.t., total RNA was harvested using guanidinium thiocyanate‐phenol‐chloroform methodsg using an SW 55 Ti rotor for 50 minutes at room temperature. Gradient fractions were counted by scintillation for [3H] in a scintillation cocktail according to standard protocols.BHK‐21 cells seeded in duplicate wells of a 6‐well plate were pretreated for 1 hour with 10 µg/mL of actinomycin D at 37°C. Pretreated cells were transfected with 4 µg of replicon RNAs, before radiolabeling with 18 µCi of  labeled methionine according to the manufacturer’s protocol. Following a 40‐minute incubation, reactions were chased with excess unlabeled methionine/cysteine with sample collection at 30‐minute intervals. Reactions were stopped by addition of 2x Laemmli buffer and boiling. Proteins were separated by 12% SDS‐PAGE and detected by autoradiography.Coupled transcription/translation reactions  were carried out using expression plasmid constructs of FMDV WT or mutated P3 polyproteins in the presence of ‐U. To maximize the labeling of replicon RNA, BHK‐21 cells were pretreated with actinomycin‐D to reduce incorporation of [3H]‐U into cellular RNA. After the addition of [3H]‐U at 1 h.p.t, total cellular RNA was harvested at 8 h.p.t., separated by sucrose gradient centrifugation and analyzed by scintillation counting  and the replicon with a single functional 3B3  had slightly reduced levels of [3H]‐U incorporation, although the differences were not statistically different from WT. The replicons containing only a functional 3B1 or 3B2  had nine‐ and eightfold reduction in [3H]‐U incorporation compared to WT, respectively. Incorporation of [3H]‐U from a replicon with a replication‐defective 3D‐GNN substitution was used to measure levels of input translation and the levels recorded here were similar to cells alone or to the construct lacking all three 3Bs (3B123Y3F).To address the question of why FMDV maintains three copies of 3B, we first determined the efficacy of each 3B protein in priming genome replication using a sub‐genomic replicon Figures  and S3. Y3F, 3B2Y3F, or 3B3Y3F) and these all replicated similarly to WT . However, constructs lacking a functional 3B3  appeared to replicate less well, although this was only statistically significant for the former. Reporter expression from a construct with a replication‐defective 3D‐GNN substitution was used to measure levels of input translation. Although levels of reporter expression recorded were low as expected, it is interesting to note that for the construct lacking any active 3Bs , expression was slightly higher than for the 3D‐GNN control for unknown reasons.We also monitored replication over time by measuring mCherry fluorescence using our established protocols.T Figure . FurtherThese data are broadly consistent with those shown in Figure 3.2pro enzyme which recognizes defined boundary sequences, the natural 3A‐3B1 and 3B3‐3C junction sequences were maintained in all cases to minimize effects due to differential processing  when mCherry replicons were co‐transfected with ptGFP replicons containing a single functional 3B, suggesting that they competed equally with each other, thus, reducing the level of expression of each. These data Figure , togethe3.3pro. Replicons were generated incorporating alanine substitution to disrupt cleavage boundaries between individual 3B proteins, between 3A and 3B1, and between 3B3 and 3C in different combinations  or C‐terminal 3B (Δ3B1 3B2/3B3Y3F) or to both as a control (Δ3B1 3B2Y3F/3B3Y3F). Replicon RNA was then generated and transfected into BHK‐21 cells and monitored for replication as described above. In agreement with the data in Figure Further constructs were generated to investigate whether the position of the 3B molecule within a fusion product was important. Replicons were made where 3B1 was deleted (Δ3B1). The 3B2 and 3B3 proteins were then fused together by substitution of the cleavage boundary. Y3F substitutions were introduced to either the N‐terminal 3B .Having demonstrated the importance of multiple 3Bs in replication and the requirement for a free N‐terminus, we asked the question whether additional copies of 3B might be tolerated and if so, whether these would offer a further replicative advantage. A mutant construct (termed 3Bx) was designed that encoded an additional copy of 3B1 at the C‐terminus of 3A, while retaining the natural 3A‐3B1 and 3B1‐3B2 cleavage motifs at the new 3A‐3Bx and 3Bx‐3B1 junctions Figure . Fluores Figures  and S7. 3.5trans to support replication, as for the enteroviruses PV and enterovirus 71 (EV71).pol active site could be rescued in trans by co‐transfection with a second replicon containing a functional 3Dpol. However, not all substitutions within the 3Dpol could be rescued, demonstrating unique cis and trans activities.Y3F substitutions were introduced into WT or mutant ptGFP replicons. RNAs from these constructs were co‐transfected with RNA from WT, 3D‐GNN, or 3BY3F mCherry replicon constructs (or with yeast tRNA as a transfection control). Replication was monitored by both ptGFP and mCherry expression and data shown at 8 h.p.t.  but had WT 3B3, replicated to higher levels to those relying solely on WT 3B1 or 3B2, consistent with some of the studies above. However, the effects were subtle and there were slight differences between replication measured via fluorescent reporter expression or via radiolabel incorporation into RNA, possibly due to the different sensitivities of the assays. We also showed that the addition of a fourth 3B coding region  offered no replicative advantage. However, importantly, our studies using combinations of 3Bs, rendered inactive by substitution or by deletion (while maintaining boundary sequences) in competition experiments indicated that a competitive advantage was conferred by possession of more than one 3B.NT assays demonstrated the expected overall changes in polyprotein boundary cleavage in that substitution of the boundary increased the abundance of the expected precursor. However, in some cases it appears that reducing processing at one site may interfere with the normal cleavage cascade, resulting in enhanced proteolysis at others. It should also be noted that it was not possible to ascertain some small size changes, for example, the fusion of two 3Bs, and some proteolysis of the mutated boundaries was still observed as may be expected from the modest alterations introduced. More dramatic boundary substitutions would have resulted in greater inhibition of proteolysis but this may have resulted in structural changes, which we chose to avoid. However, when taken overall, the data showed that 3Bs with C‐terminal fusions were replication‐competent, whereas N‐terminal fusions were not tolerated, suggesting that the location of the uridylylated tyrosine at position three in 3B is essential for it to function as a primer for RNA synthesis.Constructs were also generated with N‐ or C‐terminal fusions, that is, between 3B molecules and 3A or 3C. By leaving only one 3B functional, it was possible to ascertain whether the fusion could be tolerated for replication. The Tpol uncovered a tight network of interactions securing the N‐terminus (and Y3 residue) of 3B within the active site cavity of 3Dpol.pol to reach the active site. This potentially explains why the N‐terminal fusions were not tolerated; the 3A‐3B1 fusion would require “threading” the full‐length of 3A through the 3Dpol cavity before Y3 could reach its site of action, which is implausible given the narrow dimensions of the cavity ; we would argue that this is plausible and may itself explain why Y3 is located so close to the N‐terminus of 3B. This result is also compatible with the suggestion that during the optimal functioning of the replication complex, the active 3B is derived from the N‐terminus of a 3B‐3C(‐3D) precursor and not from the C‐terminus of a 3A‐3B precursor.Interestingly, previous structural studies of 3B in complex with 3Dy Figure . While 3Sub‐genomic replicons offer the advantage of being able to separate replication from other parts of the virus lifecycle. In this way, we have been able to tease apart the relatively subtle advantages that multiple copies of 3B provide. However, although we have clearly demonstrated that two copies are better than one, the role for a third copy is less clear. It is possible that further competitive passages would be needed for the advantage conferred by three copies to become apparent. It is also possible that the possession of three copies of 3B is important for other aspects of the viral lifecycle, such as immune evasion or virion assembly.trans complementation required a helper with a minimum of two functional 3Bs. Our previous studiesin trans to support replication. Together, this suggests FMDV may have evolved two distinct mechanisms of replication, where the extra copies of 3B are nonessential but preferable for optimal cis active replication through active 3B‐3C(‐3D) precursors, but the extra copies are necessary to facilitate trans complementation via 3A‐3B delivery. This may offer a replicative advantage, thus, providing the selective pressure to maintain multiple copies of the 3B coding region in the genome.In conclusion, although the single 3B present in the enterovirus replicons used here could rescue a replicon with a defective 3B, rescue of a defective FMDV replicon lacking any functional 3Bs by The authors declare no conflict of interests.O.O. Adeyemi, J.C. Ward, M.R. Herod, D.J. Rowlands, and N.J. Stonehouse designed the research; O.O. Adeyemi, J.C. Ward, and M.R. Herod designed and conducted the experiments; O.O. Adeyemi, J.C. Ward, J.S. Snowden, and M.R. Herod analyzed data; O.O. Adeyemi, J.C. Ward, and N.J. Stonehouse wrote the manuscript; M.R. Herod, D.J. Rowlands, and N.J. Stonehouse edited the manuscript; D.J. Rowlands, M.R. Herod, and N.J. Stonehouse supervised the project; D.J. Rowlands, M.R. Herod, and N.J. Stonehouse are the guarantors of this work and, as such, had full access to all the data and take responsibility for the integrity of the data and the accuracy of the data analysis.Fig S1‐S8Click here for additional data file.Supplementary MaterialClick here for additional data file.