Mg2+ homeostasis is maintained through Mg2+ channels such as MgtE, a prokaryotic Mg2+ channel whose gating is regulated by intracellular Mg2+ levels. Our previous crystal structure of MgtE in the Mg2+-bound, closed state revealed the existence of seven crystallographically-independent Mg2+-binding sites, Mg1–Mg7. The role of Mg2+-binding to each site in channel closure remains unknown. Here, we investigated Mg2+-dependent changes in the structure and dynamics of MgtE using nuclear magnetic resonance spectroscopy. Mg2+-titration experiments, using wild-type and mutant forms of MgtE, revealed that the Mg2+ binding sites Mg1, Mg2, Mg3, and Mg6, exhibited cooperativity and a higher affinity for Mg2+, enabling the remaining Mg2+ binding sites, Mg4, Mg5, and Mg7, to play important roles in channel closure. This study revealed the role of each Mg2+-binding site in MgtE gating, underlying the mechanism of cellular Mg2+ homeostasis.Magnesium ions (Mg They bind to a number of proteins and nucleic acids, regulating a wide range of biological processes such as ATP utilization, enzyme activation, and maintenance of genomic stability  region (residues 1–263) and a C-terminal transmembrane (TM) region (residues 264–450)   (Hattori264–450) . The CP 264–450) . Thus, ttE state .2+-binding sites (Mg1–Mg7) can be identified, among which Mg1 is located on the 2-fold axis in the ion-conducting pore at the extracellular side of the TM region, whereas the other six sites are located in the CP region  contributes to the formation of the MgtE closed state  in both the Mg2+-free and -bound states  analysis of the MgtE CP region in the Mg2+-free state, conducted by our laboratory, revealed that the N domain tumbles widely in space, and that it transiently approaches the CBS domain  to which Mg2+-binding causes the changes in the structure and dynamics of each portion of MgtE, resulting in the closed MgtE state.Although the structure of Mgd states . A strucS domain . Based o2+-dependent changes in the structure and dynamics of full-length MgtE. Mg2+-titration experiments using the wild-type and mutant forms of MgtE revealed the functional roles of each Mg2+-binding site: Whereas the Mg1, Mg2, Mg3, and Mg6 sites exhibit a higher affinity for Mg2+ and stabilize the structure of the TM region, the Mg4, Mg5, and Mg7 sites play critical roles in changing the conformation and dynamics of the CP region including the plug helices, leading to channel closure. This study thus revealed the Mg2+-dependent gating mechanism of MgtE that underlies cellular Mg2+ homeostasis.In the present study, we used solution NMR spectroscopy to investigate MgEscherichia coli, solubilized in n-dodecyl-β-maltoside (DDM) micelles, and purified to homogeneity , which is sufficiently longer than the sum of the radii of a methyl group (1.6 Å) and a hydrated Mg2+ (4.8 Å).We next established the resonance assignments for twelve of the fifteen Ile δ1 methyl groups in the Mg mM Mg2+ . The rem2+ concentrations ranging from 0 to 5.0 mM. At 0 mM Mg2+  were observed, together with nine weak and/or broad signals . However, signals for I242CBS, I291TM, and I302TM were not observed. mM Mg2+ , three m2+ concentration increased  of the nine weak and/or broad signals became split in the Mg2+-bound state . It has been reported that at Mg2+ concentration of 4 mM, or above, the MgtE channel closes  of 0.8–2.5 mM, with Hill coefficient (n) values of 1.8–7.6. These results indicate that Mg2+ binding to multiple sites cooperatively affects the conformation and dynamics of the Ile residues at different locations in the MgtE molecule.In order to investigate Mgntration . The plo2+-free state to the Mg2+-bound state; the Ile residues in the TM and other regions are shown in the upper and lower panels, respectively. Clearly, three Ile residues in the TM region  and I260Plug experienced Mg2+-induced changes at Mg2+ concentrations of 0–2 mM, whereas the other Ile residues were affected at 2–3 mM Mg2+. The functionally more important Mg2+ concentrations are those at which the NMR spectral changes saturate , above which each Ile residue adopts the closed MgtE conformation. The normalized plots in 2+]sat values are 3 mM for the Ile residues in the plug helix and the TM region, and 4 mM for those in the N and CBS domains.2+-binding sites, Mg2, Mg3, Mg5 and Mg7 in the CP region contribute to the formation of the closed state, in addition to Mg1 in the TM region  was disabled by mutation of its Mg2+-coordinated acidic residue(s), and investigated the effect of the mutations on the Mg2+-dependent NMR spectral changes , superimposed on the spectra of the wild-type MgtE in the presence  differs from that of the wild-type in the presence of 4.0 mM Mg2+, as the signals for I190CBS, I201CBS, I242CBS, I291TM, I302TM, I338TM, I397TM, and I438TM did not appear in the former , were observed for the Mg2- (E258Q), Mg3- (D259N), and Mg6- (D95N) binding mutants , suggest2+ binding to the Mg1, Mg2, Mg3, and Mg6 sites is required for the Mg2+-induced changes in the conformation and dynamics of the whole MgtE molecule  identical to those seen in the wild-type protein, whereas the spectral changes for the Ile residues in the N domain (I28N and I84N), the CBS domain (I190CBS and I242CBS) and the plug helix (I260Plug) differed from the wild-type spectra  compared to 4 mM for the wild-type , the Mgobserved . The Mg2ild-type . The cha I190CBS , right.2+-induced changes in I190CBS and I242CBS were not observed , than the wild-type (4 mM) (Plug (n = 0.8) than that of the wild type (n = 1.8). These results suggest that Mg2+ binding to the Mg5 site elicits changes in the structure and dynamics of the N and CBS domains, as well as the plug helix, as shown by I28N, I84N, I190CBS, I242CBS, and I260Plug (For the Mg5-binding mutant (D226N/D250A), the Mgobserved . The Mg2e (4 mM) . In addiI260Plug , right.N, I84N and I260Plug, exhibited Mg2+-dependent changes that were identical to those in the wild-type , than the wild-type (4 mM) (Plug (n = 0.6) than that of the wild type (n = 1.8). These results suggest that Mg2+ binding to the Mg7 binding site elicits changes in the N domain and the plug helix, as shown by I28N, I84N, and I260Plug (For the Mg7-binding site mutant (E59A), the majority of the Ile signals, except for I28ild-type . The Mg2e (4 mM) . This muI260Plug , right.2+ binding to the Mg4-, Mg5-, and Mg7-binding sites does not affect Ile residues in the TM region, all affect the N domain Ile residues  were affected by Mg2+ binding to the Mg1, Mg2, Mg3, and Mg6 sites, whereas mutation of the Mg4-, Mg5-, or Mg7-binding sites did not affect the Mg2+-induced changes in the structure and dynamics of the TM region  cells. {u-2H, Ileδ1-[13CH3]}MgtE was expressed according to a previous study , 150 mM NaCl, 40 mM n-dodecyl -D-maltoside) (DDM), and 20 mM imidazole. After centrifugation at 14,000 × g for 30 min, the supernatant was applied to a TALON column . After washing the column with a buffer containing 50 mM HEPES-NaOH (pH 7.0), 150 mM NaCl, 1 mM DDM, and 20 mM imidazole, the protein was eluted with the same buffer supplemented with 150 mM imidazole. The N-terminal His × 6 tag was then cleaved using the HRV-3C protease. The cleaved His × 6 tag, undigested MgtE, and HRV-3C protease were removed by passing the sample through a HIS-select column .MgtE and its mutants were purified as follows. The harvested cells were suspended in a buffer containing 50 mM HEPES-NaOH (pH 7.0), 150 mM NaCl and 20 mM imidazole, supplemented with 1 mM phenylmethylsulfonyl fluoride and lysed by sonication followed by centrifugation at 1,750 × 2O). The pH value of the 100% D2O buffer was calibrated by adding 0.4 pH unit to the reading on the pH meter , 20 mM NaCl, 100% DpH meter .2+-titration experiments, small aliquots of the NMR buffer containing 20–200 mM MgCl2 were added. The MgtE concentration was 200 μM, whereas the DDM was approximately 10 mM, estimated from the signal intensity of the DDM methyl signal in 1H 1D spectra.NMR spectra were observed at 313 K on Bruker Avance 500, 600, or 800 MHz spectrometers equipped with a cryogenic probe. For Mg2+-free and bound states were obtained by site-directed mutagenesis. We observed methyl-TROSY spectra for fifteen individual Ile mutants, in which each Ile residues was mutated to Val . The spectra of these mutants were compared to those of the wild-type protein in the Mg2+-free and Mg2+-bound states, respectively. The missing signals in the spectra of the Ile mutants were assigned as the signals arising from the mutated Ile residues.The assignments of the Ile δ1 methyl-TROSY signals in the Mg1H and Δδ13C are the chemical shift differences in the 1H and 13C dimensions, respectively. The normalised factor (5.8) was determined from the ratio of the variance of methyl 1H and 13C chemical shifts, deposited in the Biological Magnetic Resonance Data Bank. The signal intensities of Ileδ1 methyl signals for a series of the titration spectra were normalised based on the intensity of the DDM methyl signal. The Mg2+ concentration reaching the half maximal values in the changes ([Mg2+]1/2) and Hill coefficients (n) were calculated using the equationsChemical shift differences, Δδ, were calculated using the equation:2+-auxotrophic E. coli strain (BW25113 ΔmgtA ΔcorA ΔyhiD DE3) was transformed with each MgtE expressing plasmid, and maintained in growth medium supplemented with 100 mM MgSO4. E. coli giant spheroplasts were prepared as described previously . Borosilicate pipettes , with a resistance of 5–8 MΩ, were filled with a pipette solution . After gigal seal formation, a patch of membrane was excised and the bath solution was exchanged with a batch solution containing 290 mM N-methyl-D-glucamine, 0.2 mM MgCl2, 300 mM glucose and 10 mM HEPES (pH 7.2). The membrane patch voltage was clamped and currents were recorded using an Axopatch 200B amplifier , coupled to an A/D converter  and controlled by the pclamp10 software . Currents were filtered at 2 kHz and sampled at 5 kHz.The Mgeviously . Spherop In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.2+ binding sites in ion-dependent gating of a Mg2+ channel, MgtE, revealed by solution NMR" for consideration by eLife. Your article has been favorably evaluated by Richard Aldrich (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Charles R Sanders (Reviewer #2).Thank you for submitting your article "Functional roles of MgThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision.This is an interesting paper on the role of the binding of seven Mg(II) ions to the microbial MgtE channel, providing the apparent Mg(II) binding affinity as well as Hill coefficients informing on binding cooperativity. This was a technically challenging project in which a combination of sophisticated isotopic labeling methods and methyl-TROSY methods allowed NMR spectra to be acquired for some 15 assigned isoleucine residues (spread rather evenly throughout the protein). This took quite a lot of work for a >150 kDa detergent/intact channel complex. Binding was monitored examining the Mg(II)-concentration dependence of peak positions and/or intensity. Different peaks exhibited changes that led to different apparent Kd and Hill coefficients. Additional NMR studies conducted using Mg(II) ion binding site mutants, led to the realization that three of the Mg(II) sites form a distinct set of sites from the other 4 sites. In particular, the three sites are proposed to be responsible for stabilizing the open form of the channel, whereas the other 4 may help stabilize the channel but also exert other modulating effects, perhaps leading to channel closure at higher ion concentrations.The reviewers agreed that the work is interesting and important. However, they felt that relating Mg(II) binding results to function would be critical to establish the role of the bound ions. In particular, it was pointed out that the binding data and structural inferences that suggest highly cooperative ion binding should be reconciled with existing functional data ), which show no cooperativity in the channel response to increased Mg(II) concentration. Furthermore, similar functional data on mutants targeting Mg(II) binding sites should help resolve the role of the groups of the sites. For example, mutations of sites 4, 5, and 7 should lead to a channel that does not close at elevated Mg(II) concentrations.Reviewer #1:The manuscript by Osawa et al. describes an elegant study of a bacterial magnesium channel MgtE (homologous to eukaryotic SLC41), which is responsible for the maintenance of the intracellular Mg levels. The channel is tightly regulated by Mg, and closes at Mg concentrations above 5 mM. Previous crystal structures showed that the dimeric channel contains one central Mg ion in the permeation pathway (Mg1) and 6 additional sites per subunit in three cytoplasmic regions: N-domain, CBS domain and plug helix. To address the role of the Mg binding sites, the authors employed Ile Methyl-TROSY NMR. They found that in the absence of Mg, N-domain and plug residue I260 showed high mobility. Titrating in Mg, led to changes of Ile peaks characteristics throughout the protein with EC50 varying between 0.8 and 2.5 mM, and with high Hill coefficients from 2 to 7. The authors mutated out each Mg binding site one at a time, and found Mg binding to sites Mg1, 2, 3 and 6 was essential for all conformational changes. In contrast, binding to sites Mg4, 5, and 7 was not essential for changes in TM regions, but was required for changes in N-domain, CBS domain, and plug helix (Mg 5 and 7). The authors construct a model whereby at low, but physiologically relevant Mg concentrations, Mg binds cooperatively to sites 1, 2, 3 and 6 to produce a conducting state; at higher concentrations Mg binds cooperatively to sites 4, 5 and 7, increasing interactions between N-domain and CBS and leading to channel closing. I found the work compelling even if there were some unresolved questions.2+ binding at each site on the conformation and dynamics of MgtE”, first paragraph), the authors say that Mg binding to sites 1, 2, 3 and 6 affect Ile in TM region "cooperatively". The last term is ambiguous here. In particular, the TM Ile sites show Mg-dependent changes with different EC50s and Hill coefficients, such that, for example, I397 has EC50 of 0.8 (Nhill=2.8) while I338 has EC50 of 2 mM (Nhill=5.2). The latter residue shows sensitivity more similar to residues in the N- and CBS-domains. Therefore, while Mg binding to the sites might be cooperative, the structural changes in TM region may occur in two steps (with binding of Mg to the first and second sets of binding sites). This needs to be clarified.1) While the spectra of WT apo protein and Mg1 mutant in the presence of 4 mM Mg are similar as the authors state, they are not identical. The authors should discuss what are the differences and what might be their origin. In Discussion .2) According to Figure 5E, Mg4 mutations do not affect Mg-dependent changes of I260Plug, while both Mg7 and Mg5 mutations decrease cooperativity, but not affinity. Text in the sixth and seventh paragraphs of the subsection “Role of each Mg3) The statement about cooperative binding to sites 4, 5, 7 is confusing. Binding to site 4 does not seem to affect the structural changes in the plug, while binding to sites 5 and 7 does.4) Can the signal changes, such as the chemical shift changes and peaks' sharpening/appearance be interpreted in terms of changes of protein dynamics?5) What kind of cooperative coupling is expected between the subunits in the dimer?Reviewer #2:This is an interesting paper on the role of the binding of seven Mg(II) ions to the microbial MgtE channel, providing the apparent Mg(II) binding affinity as well as Hill coefficients informing on binding cooperativity. This was a technically challenging project in which a combination of sophisticated isotopic labeling methods and methyl-TROSY methods allowed NMR spectra to be acquired for some 15 assigned isoleucine residues (spread rather evenly throughout the protein). This took quite a lot of work for a >150 kDa detergent/intact channel complex. Binding was monitored examining the Mg(II)-concentration dependence of peak positions and/or intensity. Different peaks exhibited changes that led to different apparent Kd and Hill coefficients. Along with the results of additional NMR studies conducted using Mg(II) ion binding site mutant, this led to the realization that three of the Mg(II) sites can regarded a distinct set of site from the other 4 sites. In particular, the three sites are proposed to be responsible for stabilizing the gated form of the channel, whereas the other 4 may help stabilize the channel but also exert other structural and/or dynamic roles in modulating Mg(II) channel function. Exactly what these other roles is was a little vague. This is an admirable and difficult study that significantly advances our understanding of Mg(II) channel structure and function. I especially admire the care with which they quantitative the Mg(II)-dependence of the very modest changes in NMR peaks positions/intensities.eLife.This well written paper could be published essentially as is. However, the degree to which this NMR study established exactly what these two sets of Mg(II) binding events are doing in terms of channel function would be greatly enhanced by accompanying [Mg(II)]-dependent channel function measurements for both WT and the mutants they characterized by NMR. Such measurements are not reported in this paper. I would strongly encourage the authors to consider revising this paper to include the results of functional measurements (perhaps provided by expert collaborators). Without this, I view this paper as borderline in terms of providing enough new insight into MgtE channel function to justify publication in a journal with the stature of Reviewer #3:2+ in and out of compartments is important to understanding both the molecular mechanisms and function of this ion. The authors of this study use solution-NMR to study the dynamics of the prokaryotic Mg2+ channel, MgtE. The size and helix content make this a challenging NMR target. Addressing this, the group used methyl-TROSY with specific labeling of isoleucines as reporters of channel dynamics in detergent micelles. Coupled with site directed mutagenesis of residues responsible for binding Mg2+ residues , the authors initially titrate and then use a set Mg2+ concentration to study what role binding plays in the dynamics of MgtE.Magnesium is a ubiquitous ion necessary for many biological functions with several unique chemical properties that differentiate it from other divalent cations such as Calcium. These include a very high energy of dehydration, small ionic but very large hydrated radius. How ion channels and transporters move Mg2+ binding sites. They arrive at a model where 4 of 7 Mg2+ sites act on the transmembrane domains and Plug Helix, whereas the remaining 3 of 7 Mg2+ sites are principally focused on dynamics in the cytoplasmic domain. They propose a model based on the absence or presence of Mg2+ on channel function. This is a useful finding that advances our knowledge of how the structure, dynamics and function reveal the mechanisms of Mg2+ permeation in an ion channel.The authors have used NMR in previous studies to begin to address MgtE dynamics using PRE. Further, they've characterized the function of MgtE. Working from these previous studies, the authors combine this information to derive a model that asks how the dynamics of the transmembrane region, N-domain, CBS, and net cytoplasmic portion are related to the known MgQuestions/concerns:E. coli is Mg2+, and the estimated cellular concentrations are 30 mM, the free Mg2+ is estimated to be around 0.3 mM for E. coli. How does the model in Figure 7 account for this range and for the estimated binding affinities?1) Physiological range – While 0.05% of dry weight mass of 2+ binding behavior at the different Mg2+-binding sites of MgtE, and the know Mg2+ dependence of the channel open probability, as determined by electrophysiological methods .2) It is suggested that the authors establish an explicit correlation between the behavior of the NMR-derived Mg2+ binding sites in ion-dependent gating of a Mg2+ channel, MgtE, revealed by solution NMR" for further consideration at eLife. Your revised article has been favorably evaluated by Richard Aldrich (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "Functional roles of MgThe manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The main issue that the reviewers raised during the initial review was the lack of clear correspondence between the conformational responses of the protein to binding of Mg ions as detected by NMR and the functional behavior. The authors have addressed the criticism by providing functional data on mutations targeting every Mg binding site . The data are succinctly summarized in the figure in their point-by-point response. What is still missing is putting these data in perspective of the current NMR study. In other words, how do these data agree (or disagree) with Figure 7? This is clearly a very complicated system, and perhaps not all data are expected to be in perfect correspondence, but key aspects should be addressed. For example, what is the role of sites 2, 3 and 5 if their mutations are not detrimental for channel opening but are for channel closing? I would have liked the authors to include the figure into the paper and discuss clearly to what extent their proposed model is or is not fully consistent with functional data. The reviewers agreed that the work is interesting and important. However, they felt that relating Mg(II) binding results to function would be critical to establish the role of the bound ions. In particular, it was pointed out that the binding data and structural inferences that suggest highly cooperative ion binding should be reconciled with existing functional data ), which show no cooperativity in the channel response to increased Mg(II) concentration.2+ level. This is consistent with our NMR results that detect the Mg2+-dependent changes in the population of Mg2+-bound conformation of I260Plug , which serves as a good probe for the conformational change of the gate region (N424TM and L263Plug).The patch clamp analysis in our recent paper  indicated that open probability decreases in response to the elevation of the MgPlug is affected by the Mg2+-binding to Mg1, 2, 3, 5, 6 and 7 . The Mg2+-binding to these sites differentially affects the conformation of I260Plug, resulting in the small Hill coefficient value of 1.8 ± 0.3. This weak cooperativity might not be observed in the patch clamp analysis, due to the differences in the experimental conditions, such as high salt concentration, existence of the Mg2+-concentration gradient and membrane potential across the lipid bilayer for the patch clamp analysis, whereas low ion strength of 20 mM NaCl is suitable to detect the Mg2+-binding effects by NMR with high sensitivity.The conformation of I260Furthermore, similar functional data on mutants targeting Mg(II) binding sites should help resolve the role of the groups of the sites. For example, mutations of sites 4, 5, and 7 should lead to a channel that does not close at elevated Mg(II) concentrations.Thank you very much for the suggestion. The functional data for the sites, 1, 2, 3, 5, and 7 were previously reported , as follows:Mg1-binding mutant, D432A, exhibited no channel activity.2+.Mg2-binding mutant, E258Q, did not close at 20 mM Mg2+.Mg3-binding mutant, D259N, did not close at 20 mM Mg2+.Mg5-binding mutant, D226N/D250A, did not close at 20 mM Mg2+, but closed at 20 mM Mg2+.Mg7-binding mutant, E59A, did not close at 10 mM Mg2+. We described this as follows, “We then examined the role of the rest of Mg2+-binding sites, Mg4 and Mg6, in the MgtE channel activity. Although wild-type closed at 10 mM Mg2+ on the periplasmic side, D91A/D247A mutant for Mg4 and D95A mutant for Mg6 did not close even at 20 mM Mg2+, which is similar to D226N/D250A mutant for Mg5 analysed previously . […] In other words, every Mg2+-binding site, Mg2-Mg7, is required for the Mg2+-dependent closure of MgtE channel.”Here, we have performed the functional experiments for the binding mutants for the sites 4 and 6 , indicating that these mutants do not close at 20 mM MgReviewer #1:[…] 1) While the spectra of WT apo protein and Mg1 mutant in the presence of 4 mM Mg are similar as the authors state, they are not identical. The authors should discuss what are the differences and what might be their origin.CBS, I338TM, I397TM, and I438TM were observed for the WT apo protein , whereas they were not observed for the Mg1 mutant in the presence of 4 mM Mg2+. As the signals for these residues were observed at different chemical shifts for the WT protein in the presence of 4 mM Mg2+ , missing of these signals in the spectrum for the Mg1-binding mutant in the presence of 4 mM Mg2+ suggests that these signals were broadened due to the chemical exchange among multiple conformations, caused by the partial and inhomogeneous binding of Mg2+ to the sites Mg2-Mg7.As the reviewer pointed out, there are differences in these two spectra: the signals for I168CBS, I338TM, I397TM, and I438TM, which were observed for the wild-type at 0 mM Mg2+. The missing of these signals is presumably caused by the exchange broadening between Mg2+-bound and unbound states for the sites Mg2-7 of the Mg1-binding mutant.”The description regarding the differences has been added as follows, “The only differences are the missing of the signals for I1682+ binding at each site on the conformation and dynamics of MgtE”, first paragraph), the authors say that Mg binding to sites 1, 2, 3 and 6 affect Ile in TM region "cooperatively". The last term is ambiguous here. In particular, the TM Ile sites show Mg-dependent changes with different EC50s and Hill coefficients, such that, for example, I397 has EC50 of 0.8 (Nhill=2.8) while I338 has EC50 of 2 mM (Nhill=5.2). The latter residue shows sensitivity more similar to residues in the N- and CBS-domains. Therefore, while Mg binding to the sites might be cooperative, the structural changes in TM region may occur in two steps (with binding of Mg to the first and second sets of binding sites). This needs to be clarified.In Discussion .Thank you for the comment. The mutations at either site of Mg1, 2, 3, or 6 caused the similar changes in the NMR signals for the identical set of the Ile residues , suggesting that the MgTM, I397TM, and I438TM, which lie in the proximity of Mg1 at the extracellular side of the TM region, change at lower Mg2+ concentrations, the residues I291TM and I338TM, which locate at the intracellular side of the TM region, change at the Mg2+ concentrations similar to the changes of the Ile residues in the N and CBS domains. The structural changes in two steps seem to reflect that the latter residues at the intracellular side of the TM region are also affected by the Mg2+-binding to the intracellular domains.Whereas the residues I302Accordingly, we revised the Discussion, as follows,“cooperatively” has been removed.We have removed “appears to be cooperative and”.2+-binding to the Mg2+ sites, Mg2, Mg3 and/or Mg6 in the intracellular domains.”We have added a paragraph, “It should be noted that the structural changes in the TM region seem to occur in two steps . […] The structural changes in two steps seem to reflect that the latter residues at the intracellular side of the TM region are also affected by the Mg2+ binding site in the Mg2+-induced conformational change in MgtE” contradicts the figure .2) According to Figure 5E, Mg4 mutations do not affect Mg-dependent changes of I260Plug, while both Mg7 and Mg5 mutations decrease cooperativity, but not affinity. Text in the sixth and seventh paragraphs of the subsection “Role of each MgPlug. We revised the ninth paragraph of the subsection “Role of each Mg2+ binding site in the Mg2+-induced conformational change in MgtE” to state that Mg5 and Mg7 decreased the cooperativity of I260Plug, and line 274 to state that Mg7 also affects the plug helix. In addition, we revised the right panel of Figure 6B.We are grateful for the reviewer’s comment. As the reviewer pointed out, the Mg5- and Mg7-binding mutants decreased the cooperativity of I2603) The statement about cooperative binding to sites 4, 5, 7 is confusing. Binding to site 4 does not seem to affect the structural changes in the plug, while binding to sites 5 and 7 does.Plug is mostly suppressed at 3 mM , Mg2+ binding to Mg5 and Mg7, which affects I260Plug, saturates at 4 mM .”We agree with the reviewer’s comment and have revised the text as follows, “Although the fast motion of I2604) Can the signal changes, such as the chemical shift changes and peaks' sharpening/appearance be interpreted in terms of changes of protein dynamics?Yes, they can be. Both signal line width and the way of the chemical shift changes reflect protein dynamics. Basically, a nuclear spin with a fast tumbling motion gives a sharp NMR signal, whereas a spin tumbling slowly gives a broad signal. The rate of the chemical exchange between conformations A and B as compared to difference in the resonance frequencies for the conformations A and B also affects the appearance of the signals  Principles and Practice ISBN: 978-0-12-164491-8).5) What kind of cooperative coupling is expected between the subunits in the dimer?2+ at the site 1 bridges the TM regions of the two subunits, forming the Mg2+-permeation pathway at the interface of the two subunits in the TM region. As described in the last paragraph of the subsection “The effects of Mg2+ binding at each site on the conformation and dynamics of MgtE”, the N domains tumble fast in the apo state, and they interact with the two CBS domains in the Mg2+-bound state. Mg2+ at the site 5 and 7 bridges the CBS domain in one subunit with the plug helix and the N domain of another subunit, respectively, which are assisted by Mg2+ at the site 4 and 6 that bridge the N and CBS domains in the same subunit. Thus, cooperative coupling is expected between the subunits in the dimer, which is achieved by the inter-subunit bridging by the Mg2+ at the sites 1, 5, and 7 directly .The MgReviewer #2:[…] This well written paper could be published essentially as is. However, the degree to which this NMR study established exactly what these two sets of Mg(II) binding events are doing in terms of channel function would be greatly enhanced by accompanying [Mg(II)]-dependent channel function measurements for both WT and the mutants they characterized by NMR. Such measurements are not reported in this paper. I would strongly encourage the authors to consider revising this paper to include the results of functional measurements (perhaps provided by expert collaborators). Without this, I view this paper as borderline in terms of providing enough new insight into MgtE channel function to justify publication in a journal with the stature of eLife.2+]-dependent channel function measurements for the wild type and the Mg2+-binding mutants for the sites, 1, 2, 3, 5 and 7 . The functions are summarized as follows:We appreciate time and effort by the reviewer #2, as well as the positive response regarding our manuscript. As stated in the manuscript, we previously reported the -dependent channel function for the rest of the binding mutants for the site 4 (D91A/D247A) and 6 (D95N), both of which have not closed at 20mM Mg2+ . These data are consistent with our NMR results that the binding mutants for the site 4 and 6 exhibited incomplete spectral changes at 4 mM Mg2+ .Here, we have added the data for the  Questions/concerns:2+, and the estimated cellular concentrations are 30 mM, the free Mg2+ is estimated to be around 0.3 mM for E. coli. How does the model in Figure 7 account for this range and for the estimated binding affinities?1) Physiological range – While 0.05% of dry weight mass of E. coli is Mg2+ concentration for the MgtE channel closure is proposed to be 4 mM, which is 10-fold higher than the cellular free Mg2+ level. Recently, we reported that ATP binding to the CBS domain of MgtE decreases the Mg2+-concentration for the MgtE gating , suggesting that the gating seems to occur at the Mg2+ concentration as low as 2-3 mM Mg2+ in the presence of ATP. However, the Mg2+ concentration is still much higher than the cellular free Mg2+ level.Thank you very much for the comment on the important point. The Mg2+ level at 0.3 mM, the population of the Mg2+-bound conformation of I397TM, which lie in the proximity of the site Mg1, is about 0.1 , suggesting that MgtE exhibits Mg2+ conductivity at the Mg2+ level. When extracellular Mg2+ level is elevated and the excessive amount of Mg2+ enters into E. coli cells, the gate of MgtE is supposed to close.Under the cellular Mg2+ binding behavior at the different Mg2+-binding sites of MgtE, and the know Mg2+ dependence of the channel open probability, as determined by electrophysiological methods .2) It is suggested that the authors establish an explicit correlation between the behavior of the NMR-derived Mg2+ concentrations of 0, 5, 20 mM Mg2+, respectively . In addition, the Mg2+ binding mutant Mg1 does not show the conductance, whereas the Mg2+ binding mutants, Mg2, 3, 5, and 7 do not close at 20 mM Mg2+. Here, we have executed patch clamp analyses for the Mg2+ binding mutants, Mg4 and 6, which also do not close at 20 mM Mg2+. The open probabilities of the wild type and mutants are plotted .We are grateful for the reviewer’s suggestion. The open probability of the wild type MgtE is about 0.4, 0.2, and 0 at the Mg2+-binding mutants, Mg2, 3, and 5 in the presence of 4-5 mM Mg2+ exhibit high similarity to the spectrum of the wild type in the absence of Mg2+, rather than that of the wild type in the presence of 5 mM Mg2+, indicating that these mutants do not form a structure in the closed state, in the presence of 4-5 mM Mg2+.As shown in Figure 5 and Figure 5—figure supplement 3, the NMR spectra for the Mg2+-binding mutants, Mg4, 6, and 7 in the presence of 4-5 mM Mg2+ provided NMR spectra, which are similar to that of the wild type protein in the presence of 5 mM Mg2+. However, the NMR signals from the CBS domain, particularly I190CBS, which lie at the interface between two CBS domains in the dimer, were not observed. This NMR data suggest that the relative orientation of the two CBS domains of these mutants are not fixed even in the presence of 4-5 mM Mg2+. Since the gate exists at the top of the plug helices on the CBS domains, the fluctuation of the relative orientation of the two CBS domains cannot stabilize the closed gate.Other Mg2+-binding mutants, Mg2-7, in the presence of saturating amount of Mg2+, is consistent with the electrophysiological data, in which the mutants Mg2-7 do not close in the presence of 20 mM Mg2+.Therefore, the NMR-derived structural information on the Mg2+ concentrations for the NMR-derived conformational change (4-5 mM) and the electrophysiological experiments (20 mM) are due to the differences in the experimental conditions, such as low ion strength of 20 mM NaCl for the high sensitivity of the NMR spectra; high salt concentration, existence of the Mg2+-concentration gradient and membrane potential across the lipid bilayer for the patch clamp analysis.The differences in the MgThe manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:The main issue that the reviewers raised during the initial review was the lack of clear correspondence between the conformational responses of the protein to binding of Mg ions as detected by NMR and the functional behavior. The authors have addressed the criticism by providing functional data on mutations targeting every Mg binding site . The data are succinctly summarized in the in their point-by-point response. What is still missing is putting these data in perspective of the current NMR study. In other words, how do these data agree (or disagree) with Figure 7? This is clearly a very complicated system, and perhaps not all data are expected to be in perfect correspondence, but key aspects should be addressed. For example, what is the role of sites 2, 3 and 5 if their mutations are not detrimental for channel opening but are for channel closing? I would have liked the authors to include the into the paper and discuss clearly to what extent their proposed model is or is not fully consistent with functional data.Thank you very much for raising the important point again, which should have been appropriately addressed in the previous revision. Accordingly, we have added the figure from the previous response to the manuscript as Figure 5—figure supplement 1C.2+-binding mutants shown in Figure 5—figure supplement 1 and other reported data , as follows:We have addressed the correspondence between the conformational responses of the protein to binding of Mg ions as detected by NMR  and the functional behavior of the Mg2+ conductivity, whereas a mutation at either of the sites Mg2, Mg3, or Mg6 does not . […] The functional roles of each of these Mg2+-binding sites therefore underlie the intracellular Mg2+ homeostasis mediated by MgtE.”“Electrophysiological results indicated that a mutation at Mg1 abolishes Mg


Root resorptions are assessed and diagnosed using different radiographical techniques. A comparison of the ability to assess resorptions on two-dimensional (2D) and three-dimensional (3D) radiographs is, hitherto, lacking. The aims of this study were to evaluate the accuracy of 2D  and 3D  radiographic techniques in measuring slanted root resorptions compared to the true resorptions, a histological gold standard, in addition to a comparison of all the radiographic techniques to each other.Radiographs , in addition to histological sections, of extracted deciduous canines from thirty-four patients were analyzed. Linear measurements of the most and least resorbed side of the root, i.e., “slanted” resorptions, were measured using an analyzing software (Facad ®). For classification of slanted root resorptions, a modified Malmgren index was used.PAN underestimated the root length on both the least and most resorbed side. Small resorptions, i.e., low modified Malmgren scores, were more difficult to record and were only assessed accurately using CBCT. The root resorption scores were underestimated using PA and PAN. In assessment of linear measures, PAN differed significantly from both CBCT and PA.CBCT is the most accurate technique when measuring and scoring slanted root resorptions. Accurate diagnosis and assessment of the extent of root resorption is important in orthodontic treatment and treatment planning. The diagnosis of root resorptions mainly depends on radiographic examinations. Conventional two-dimensional 2D) radiographical techniques are the most commonly used methods for diagnosis of root resorption. These methods, however, have limitations such as superimposition of structures onto the two-dimensional plane, distortion projection errors, and blurred images –5. FurthD radiogrDuring the 1990s, cone beam computed tomography (CBCT) was introduced and is now widely used. Studies have shown that CBCT imaging is a more reliable tool for detection of small resorptions compared to conventional panoramic (PAN) radiograph , 9.When periapical (PA) radiographs were compared to CBCT images, root lengths were underestimated by an average of 2.6 mm compared to 0.3 mm underestimation in CBCT images. In a comparison of the accuracy of CBCT and PA radiographs in detecting small periapical resorptions, CBCT showed poor accuracy in detecting simulated resorptions smaller than 0.8 mm in diameter and excellent when the lesions were larger than 1.4 mm in diameter. On the other hand, PA radiographs demonstrated poor accuracy for all simulated resorption sizes. A higher accuracy was reported with PA radiographs when lesions were larger (2 to 5 mm) in diameter –13. LineMalmgren root resorption scoring system with index scores from 0 to 4 is commonly used to classify the severity of horizontal root resorption. In order to assess slanted root resorptions, a modified Malmgren index is needed . A thoroThe patient in this study is from a previous prospective randomized trial on interceptive treatment of palatally displaced canines (PDCs) by extracting the deciduous canine. Details about the patients, their recruitment, inclusion, and exclusion criteria are described in this study . Sixty-sThe study was approved by the research ethics committee of Sahlgrenska Academy at the University of Gothenburg, Sweden, and by the radiation protection committee. Parents and patients have received verbal and written information, in addition to informed consent provided by the patient or the parent in accordance with the Declaration of Helsinki.All patients had a set of radiographic images before extraction of deciduous canines. These radiographs included, at least, two PA, one PAN, and one CBCT. The PAN and CBCT were taken on the same day as the extraction of the deciduous canine, while the PA radiographs were taken not more than 3 months before the extraction.PA radiographs were taken by the general practitioners in the public dental clinics, in bisecting angle and parallel techniques. At least two radiographs on each tooth in a different angle to detect the position of the permanent canine were taken. All PA radiographs were scanned in high resolution (2000dcm). PAN were taken at the Department of Oral and Maxillofacial Radiology at the Institute of Odontology at Sahlgrenska Academy, with 1.7-degree magnification . CBCT was performed at the Department of Oral and Maxillofacial Radiology at the Institute of Odontology at Sahlgrenska Academy, with 3D Accuitomo FPD  with a 360-degree rotation. The volume used was 60 × 60 mm. Primary data reconstructions were made by acquisition software  at the Accuitomo workstation, providing axial, frontal, and sagittal views. Secondary reconstruction was then made using the i-Dixel software. A sagittal radiographical view of the deciduous canine was performed with a Sectra Imtec IDSS multiplanar reconstruction program with a reference line placed at the middle of the pulp chamber and another intersecting line with a 45-degree angle. This was made in order to obtain 3D CBCT images comparable to 2D periapical images.Extracted deciduous teeth were stored in 5% buffered formaldehyde. Before embedding, the teeth were washed several times in 70% ethanol, with a final wash in absolute ethanol. Then, the teeth were embedded in epoxy-resin , and each tooth was cut sagittally, in a bucco-lingual direction into halves in a Leitz low-speed saw microtome. From the central part of the teeth, three sections were cut with a thickness of 110 μm. All sections were photographed with fiber-optic surface illumination in a Nikon SMZ800 microscope equipped with a Nikon Digital Sight DS-F1 camera. The slanted root resorption evident on the most central cut of the tooth was used as gold standard Fig. .Fig. 2Miwww.random.org. The radiographical and histological images were imported into an analyzing software  and were calibrated regarding magnification. Assessment of the most resorbed and the least resorbed root side was made on each image by two calibrated examiners (EA and HAL). Two linear measurements were done on each image. The most resorbed (MR) side of the root was defined as the distance from cemento-enamel junction to the most resorbed point on the root. The less resorbed (LR) side of the root was defined as the distance from the cemento-enamel junction to the least resorbed point on the root surface. This was considered a “slanted” resorption  and histological images of 34 patients, in total 136 images, were coded with a numeric sign, and the order of the images was randomized in ion Fig. . The oriA first set of measurements was blinded; thus, root resorption measurements were performed by each operator on each radiographic image without comparing the image to the gold standard. In a second not blinded measurement set, the root resorption level in each radiographic image was compared to gold standard. The data from the blinded and not blinded measurement sets was analyzed by each operator in a 2 months’ interval period.t tests were used to compare the blinded and the not blinded data sets. p values < 0.05 was considered statistically significant. The inter-examiner reliability was assessed with Cohen’s kappa with less than 5% deviation from the gold standard. The Malmgren classification of the gold standard sections was compared to the radiographic sections using Mann-Whitney test. All statistical tests were performed using the statistical package in Excel.Two root resorption measurements were performed for each tooth; one measured by examiner EA and the other by examiner HAL. The mean of the two measurements was used for the statistical analysis. Paired For both blinded and not blinded measurements, PAN underestimated the root length on the least and most resorbed side. However, measurements with CBCT and PA were similar to gold standard Fig. .Fig. 4BlThere was a statistically significant difference between the root scores for the PAN and PA images compared to the gold standard, while CBCT values did not differ. Also, blinded and not blinded recordings did not differ statistically. Small resorptions were more difficult to record than large resorptions, except for middle resorptions, i.e., grade 6. Resorption grade 1 was recorded accurately only with CBCT. On the contrary, resorption grade 5 was accurately recorded even on PAN radiographs. Resorption grade 6 was severely underestimated by all techniques. Most of grade 6 resorptions were detected on CBCT. Grade 6 resorptions in the apical third were misdiagnosed on the PA and PAN radiographic images as grade 1 or grade 2 (Fig. In assessments of linear measures, there was no difference between CBCT and PA in either blinded or not blinded measurements. PAN, however, differed statistically significant from both CBCT and PA, both blinded and not blinded, in this sense Fig. .Fig. 6CoA statistical significant difference was detected for not blinded PA images compared to CBCT and for both blinded and not blinded PAN compared to CBCT Fig. .Fig. 7MoThe inter-examiner reliability, as assessed by Cohen’s kappa, differed for the radiographic techniques. For PAN, the kappa value was 1.0, i.e., a perfect agreement. For PA images, the kappa value was 0.71, i.e., a substantial agreement, and, finally, for CBCT, the kappa value was 0.44 demonstrating a moderate agreement. The inter-examiner agreement for the modified Malmgren root index was 0.88, demonstrating a good agreement between the two examiners.This study shows that CBCT is the most accurate radiographical technique when measuring root length and root resorptions, using a modified Malmgren score, in deciduous maxillary canines. In this study, for the first time, the most common routine radiographs, i.e., PAN and PA radiographs and CBCT, were compared to gold standard histology of true resorptions on extracted teeth.Orthodontic diagnosis and treatment planning is essentially influenced by radiographic interpretations of the site and severity of root resorptions. Periapical radiographs and PAN are the most commonly used conventional radiographic techniques in orthodontics although they have limitations –5. CBCT,To determine the degree of root resorption, different approaches have been employed, being linear measurements, scoring of horizontal root resorption degree, or using scales to describe mesial, distal, buccal, and lingual resorptions , 20. In A statistically significant difference was found in modified Malmgren scores for PA images and PAN compared to gold standard. On the other hand, CBCT did not differ from gold standard. It was evident that grade 6 resorptions with intact apex were the most difficult to detect, especially on 2D x-rays due to superimposition of structures. Large resorptions were easily interpreted with all techniques, while low resorption grades, especially grade 1 could be interpreted only on CBCT images. Other studies have, as well, showed that CBCT is a superior diagnostic tool in determining location and dimension of small root resorptions compared to PA radiographs , 22, 23.Furthermore, when linear measurements were assessed and compared to histology, CBCT was once again found to be the most accurate technique. Interestingly, linear root length measurements of the most resorbed side made on PA images were close to gold standard, while the least resorbed side was somewhat overestimated. PAN, however, underestimated the root length in both blinded and non-blinded measurements. Several other studies have also reported the limits with PAN for assessing root resorptions , 25, 26.To overcome the difficulties in identifying tooth structures due to superimposition of 3D structures onto a 2D plane, distortion projection errors, and blurred images, blinded and not blinded assessments of root length and resorptions were performed. This has important clinical relevance since the root resorption detection on the radiograph is considered blinded. Interestingly, the linear root length measurements and index scoring did not differ regardless if it was blinded or not. Even when the assessments were done not blinded, PAN underestimated compared to histology but also when it was compared to CBCT. This clearly demonstrates that PAN is not an accurate method to detect root resorption in the canine region. Similarly, PA differed in assessing resorptions with Malmgren scores; however, in linear measurements, it did not differ. When the different radiographic techniques were compared to each other, PAN was found least accurate in blinded as well as not blinded measurements compared to CBCT and PA , 27. HowThe inter-examiner reliability test in the present study showed better agreement with the least accurate radiographic techniques, i.e., PA images and PAN, as it showed lower kappa values with the CBCT which was the most accurate technique. In the literature, other results, with better inter-examiner reliability for CBCT than for both PA and PAN, have been presented. A reason for this difference might be the use of a modified Malmgren score in the present study. This shows that it is easier to have a high agreement between two observers using a less accurate technique , 28, 29.CBCT is the most accurate technique, both blinded and not blinded, when measuring root length and detecting root resorptions using a modified Malmgren score. PA images were comparable with CBCT for blinded and not blinded root length measurements but were not accurate when assessing root resorptions using the modified Malmgren score. PAN is not a good diagnostic tool for measuring blinded and not blinded root length and root resorptions. A better kappa agreement was seen for the least accurate technique, i.e., PAN.


CPFE) is an important risk factor for lung cancer (LC), because most patients with CPFE are smokers. However, the histological characteristics of LC in patients with CPFE (LC‐CPFE) remain unclear. We conducted this study to explore the clinicopathological characteristics of LC‐CPFE. We retrospectively reviewed data from 985 patients who underwent resection for primary LC, and compared the clinicopathological characteristics of patients with LC‐CPFE and non‐CPFE LC. We identified 72 cases of LC‐CPFE, which were significantly associated with squamous cell carcinoma (SqCC) histology  and higher tumor grade , compared to non‐CPFE LC. Most LC‐CPFE lesions were contiguous with fibrotic areas around the tumor , and this association was independent of tumor location. Furthermore, dysplastic epithelium was identified in the fibrotic area for 31 (52.5%) LC‐CPFE lesions. Moreover, compared to patients with pulmonary fibrosis alone in the non‐CPFE group (n = 31), patients with CPFE were predominantly male (P = 0.008) and smokers (P < 0.001), with LC‐CPFE predominantly exhibiting SqCC histology (P = 0.010) and being contiguous with the tumor‐associated fibrotic areas (P < 0.001). Multivariate analysis revealed that CPFE was an independent predictor of overall survival . Our results indicate that LC‐CPFE has a distinct histological phenotype, can arise from the dysplastic epithelium in the fibrotic area around the tumor, and is associated with poor survival outcomes.Combined pulmonary fibrosis and emphysema ( Combined pulmonary fibrosis and emphysema (CPFE) is a clinical syndrome that is characterized by the following findings: a history of smoking; the presence of dyspnea, pulmonary hypertension, and hypoxemia; relatively normal spirometry and lung volumes in the context of severely impaired gas exchange; radiographically confirmed upper‐lobe emphysema; lower‐lobe fibrosis; and poor survival We retrospectively evaluated cases of lung resection for pulmonary masses, which were treated at Shinshu University Hospital between December 1995 and December 2013, using the hospital's Thoracic Surgery and Pathology database. We used the patients' electronic medical records to retrieve their clinical data, which included age, sex, smoking status, tumor location , history of connective tissue disease and drug treatment, and follow‐up data. Our research protocol was reviewed and approved by our institutional ethics review committee.To evaluate the clinicopathological significance of lung cancer in patients with CPFE (LC‐CPFE) after complete tumor resection, we divided patients into LC‐CPFE and non‐CPFE lung cancer groups based on their chest high‐resolution computed tomography (HRCT) findings We also divided the non‐CPFE group into three groups to evaluate the prognostic significance of LC‐CPFE based on their HRCT images : lung cancer in patients with only pulmonary fibrosis (LC‐PF), patients with only emphysema (LC‐Emp), and patients with normal lungs (LC‐Norm). The radiographical criteria for CPFE were used to define the LC‐Emp group (fulfilled criterion 1 only), the LC‐PF group (fulfilled criterion two only), and the LC‐Norm group (fulfilled neither criteria). To account for interobserver variation, we omitted the LC‐Norm group, and the LC‐CPFE, LC‐PF, and LC‐Emp groups were independently determined by MZ, AY, a radiologist (SK), and two pulmonologists . All reviewers were blinded to the patients' information, and consensus was used to resolve discrepancies in the group assignments. At this point, we also excluded patients with pulmonary fibrosis that was induced by connective tissue disease and drug treatment from the LC‐CPFE and LC‐PF groups, based on their medical records.To explore the histological characteristics of LC‐CPFE, all histological slides from that group were reviewed under a multiheaded microscope by MZ and two pathologists , who were blinded to the clinical and radiological findings. In addition, the histological slides from the LC‐PF group were reviewed in a similar manner, and their clinicopathological characteristics were compared to those of the LC‐CPFE group. The histological subtype was determined according to the 2015 WHO classification for lung tumors The chi‐square test and Fisher's exact test were used for categorical data, as appropriate. Mann–Whitney U tests were used for continuous data. The survival rates were calculated using the Kaplan–Meier method, and were compared using the log‐rank test. Multivariate analysis was performed using Cox's proportional hazards model. All statistical tests were two‐sided and used a 5% level of significance. All data analyses and graphing were performed using JMP software .LCO, 50.9 ± 16.5%), and there were no patients with CPFE and pulmonary hypertension, as patients with pulmonary hypertension are excluded from lung cancer resection at our institution. Compared to patients with non‐CPFE lung cancer, patients with LC‐CPFE were predominantly men  and older . Furthermore, LC‐CPFE was associated with a larger tumor size . The numbers of LC‐CPFE cases at each pathological stage were 20 (27.7%) at stage IA, 22 (30.5%) at stage IB, 14 (19.4%) at stage IIA, 2 (2.7%) at stage IIB, 10 (13.9%) at stage IIIA, and 4 (5.6%) at stage IV; LC‐CPFE was associated with a significantly higher stage, compared to the other groups (P < 0.001). The most common histological subtype of LC‐CPFE was squamous cell carcinoma , which was followed by adenocarcinoma , large cell carcinoma , small cell carcinoma , and large cell neuroendocrine carcinoma . The tumor grade of the LC‐CPFE group was significantly higher than those in the other groups (P < 0.001).This study evaluated records from 1647 patients; patients without CT images or sufficient clinical and histological information, patients with metastatic tumors, and patients who had undergone chemo‐radiotherapy before surgery were excluded. Therefore, we included 985 patients in this study, and discovered that this group included 72 patients with LC‐CPFE, 82 patients with LC‐Emp, 31 patients with LC‐PF, and 800 patients in the LC‐Norm group. The clinicopathological characteristics of the patients with LC‐CPFE and non‐CPFE lung cancer are summarized in Table P < 0.001) and LC‐Emp (P = 0.001) groups . The LC‐CPFE group exhibited significantly poorer OS compared to the LC‐Norm (P < 0.001) and LC‐Emp (P = 0.002) groups . Patients with CPFE adenocarcinoma or squamous cell carcinoma exhibited poorer survival outcomes compared to patients with non‐CPFE adenocarcinoma  or non‐CPFE squamous cell carcinoma .The curves for overall survival (OS) and disease‐free survival (DFS) in the four groups are shown in Figure ups Fig. A. Howeveups Fig. B. SimilaP < 0.001) and mortality  compared to the patients without CPFE. The multivariate analysis using the Cox proportional hazards model revealed that CPFE was a significant and independent predictor of OS , although it was not an independent predictor of DFS .Table n = 28, 38.8%), which was followed by the right upper lobe , the left lower lobe , the left upper lobe , and the right middle lobe . Approximately half of the LC‐CPFEs were located in the lower lobes . Most cases of LC‐CPFE exhibited fibrotic changes around the tumor , although seven cases of LC‐CPFE exhibited emphysematous changes around the tumor, and six cases of LC‐CPFE did not exhibit fibrotic or emphysematous changes around the tumor. We observed metaplastic epithelium in the fibrotic area around the tumor in all cases , atypical metaplastic epithelium in 33 cases , and dysplastic epithelium in 31 cases . Most cases with the squamous cell carcinoma subtype were located contiguous with the fibrotic area , and dysplastic squamous epithelium in the fibrotic area was identified in approximately half of these cases . Most cases with the adenocarcinoma subtype were also located contiguous with the fibrotic area , and dysplastic glandular epithelium in the fibrotic area was identified in more than half of these cases .Table n = 19), the most common subclass was solid adenocarcinoma , which was followed by papillary adenocarcinoma , acinar adenocarcinoma , invasive mucinous adenocarcinoma , and lepidic adenocarcinoma  .Among the adenocarcinoma cases in the LC‐CPFE group  and smokers . Furthermore, LC‐CPFE was associated with a significantly smaller tumor size . The most common histological subtype of LC‐CPFE was squamous cell carcinoma , and the most common subtype of LC‐PF was adenocarcinoma . Among the adenocarcinoma cases in the LC‐CPFE group, the most common subclass was solid adenocarcinoma , and only one case (3.2%) of solid adenocarcinoma was observed in the LC‐PF group. Compared to the LC‐PF lesions, the LC‐CPFE lesions were significantly more likely to be located along the fibrotic area (P < 0.001). The lung cancers along the fibrotic area in both groups exhibited dysplastic epithelium in the surrounding fibrotic area, although there was no significant difference between the two groups (P = 0.187). Five patients in the LC‐CPFE group experienced acute exacerbation and subsequently died, while three patients died because of acute exacerbation in the LC‐PF group; this difference was not statistically significant (P = 0.634).The clinicopathological characteristics of the patients with LC‐CPFE and LC‐PF group are summarized in Table Since the concept of CPFE was proposed by Cottin et al. n = 59, 81.9%) were associated with the fibrotic area around the tumor in the patients with CPFE, and this relationship was independent of the tumor's location. Furthermore, a majority of our cases  exhibited dysplastic epithelium in the fibrotic area. Thus, we hypothesize that pulmonary fibrosis is strongly associated with lung cancer development in patients with CPFE. To the best of our knowledge, this is the first report regarding this relationship in patients with CPFE, although a few researchers have reported similar findings for lung cancer in patients with pulmonary fibrosis. For example, Kawasaki et al. analyzed the relationship between cancer location and regions of idiopathic pulmonary fibrosis (IPF), and reported that 50% of the cancers occurred in the fibrotic parenchyma and that 29% of the cancers occurred in the marginal area of the fibrosis We found that 55.5% of the lung cancers in patients with CPFE were located in the lower lobes, which is compatible with the findings of the previous studiesn = 31, 5.5% exhibn = 7, 36.8%) as the most frequent subclass, no cases of adenocarcinoma in situ or minimally invasive adenocarcinoma, and only one case of lepidic adenocarcinoma. Moreover, tumors without a lepidic component were observed in a majority of the cases . These findings may indicate that lung adenocarcinomas in the CPFE group were different from those in the non‐CPFE group. After the IASLC/ATS/ERS multidisciplinary classifications of lung adenocarcinoma were published in 2011 Nineteen of the 72 LC‐CPFE cases exhibited adenocarcinoma histology, with solid adenocarcinoma (In conclusion, the findings of this study indicate that LC‐CPFE may arise from dysplastic epithelium in the fibrotic area around the tumor, and that the process of developing lung cancer may be similar to that of LC‐PF. However, as our patients with LC‐CPFE exhibited significantly poorer outcomes, we suggest that CPFE should be considered as an important background disease for patients who have undergone resection for lung cancer.There are no conflicts of interest to declare.


Allium tuberosum is a well-known spice as well as a herb in traditional Chinese medicine, used for increasing libido and treating erectile dysfunction. However, not many studies have been done to evaluate the sexual enhancing properties of A. tuberosum. The aim of this study was to evaluate the aphrodisiac and vasorelaxant properties of A. tuberosum on corpus cavernosum smooth muscle (CCSM) as well as checking the effect on enhancing male rat sexual behavior, libido, potency as well as its spermatogenic properties.n-BuOH extracts (ATB) of A. tuberosum  and Viagra was used as the positive control drug. The extract/drug was administered by gastric probe once daily for 45 days and the sexual behavior was analyzed by exposing the male rats to female rats in the estrus period.The seeds were powdered and sequentially extracted with hexane, ethyl acetate and butanol. Male Wistar rats were administered with graded doses of the ATB relaxed corpus cavernosum smooth muscle (68.9%) at a concentration of 200 μg/ml. The results obtained from the animal studies indicated that ATB significantly increased mount frequency (MF), intromission frequency (IF), ejaculation frequency (EF), ejaculation latency (EL) and markedly reduced post ejaculatory interval (PEI), mount latency (ML), and intromission latency (IL). Furthermore, a remarkable increase in the test for potency was observed as witnessed by marked increase in erections, quick flips, long flips and total reflex. In addition, ATB significantly improved the sperm viability and count as well as increased the concentrations of testosterone, follicle stimulating hormone (FSH), and phosphatases in the treated animals.A. tuberosum could stimulate sexual arousal and enhance sexual execution in male rats, thus providing valuable experimental evidence that A. tuberosum possesses sexual enhancing properties.Thus our results suggest that  Sexuality is a major fundamental principle of reproduction involving conjugation, conception and procreation. Sexual dysfunction is the inability to attain normal sexual activity, including loss or partial erection, inability to keep erection, premature ejaculation, reduced libido, orgasm, arousal disorder and lack of detumescence. Varying degrees of sexual dysfunction is estimated to occur in approximately 30 million men globally , 2. SubsThe role of testicular phosphatases like acid phosphate (ACP) and alkaline phosphate (ALP) are considered vital as they are regarded as functional indicators of spermatogenesis. ACP are found in spermatogenic cells and the role of these enzymes are intensified during the differentiation of germ cells from spermatogonia into spermatocytes and spermatids. Lysosomal ACP is also involved in the digestion of phagocytosed exogenous compounds containing phosphate residues and they assist in the penetration of the spermatozoon through the egg. ALP on the other hand play a significant role as regards the motility and survival of sperm, testicular development as well as in the process of spermatogenesis , 7. AlteAllanblackia floribunda Oliver, Fadogia agrestis, Afromomum melegueta, Lepidium meyenii, Kaempferia parviflora, Terminalia catappa, Turnera diffusa, Cnestis ferruginea, and Allium tuberosum, -β-D-galactopyranoside (18), gitogenin 3-O-{O-β-D-glucopyranosyl- (1---2)-O-[β-D-xylopyranosyl-(1---3)]-O-β-D-glucopyranosyl-(1---4)-β-D-galactopyranoside (19) [Mimaki et al., [18 and 19 from A. tuberosum.An extensive chemical isolation and chemical purification of ATB extract using various chromatographic techniques led to the isolation of 19 compounds Fig. . The comnine (2) , uracil mine (4) , 3-formydine (7) , adenosid B (10) , tuberosd B (12) , tuberos et al.,  neogitog et al., ; Kim et  et al., , using sA. tuberosum, a widely consumed vegetable in China with renal protective activity as well as for the treatment of impotence and nocturnal emissions [A. tuberosum treatment enhanced stimulatory effects on male sexual behavior. Indices of male sexual efficiency such as mount and intromission frequencies are parameters that are usually measured in the determination sexual motivation, penile erection efficiency and ejaculatory reflexes activation [A. tuberosum on vital indicators of sexual vigour and potency thus enhancing sexual performance.Erectile dysfunction (ED) is a health condition which leads to the inability of a man to maintain an erection of his penis sufficiently for a period of time for normal sexual intercourse or for achieving sexual satisfaction. The currently available treatment for ED have several unpleasant side effects as well as limited efficiency . Therefomissions . This prtivation , 43. TheA. tuberosum may facilitate erectile function and the highest adminstered dose could produce similar effect as viagra in the male rat sexual parameters. In addition, the effect of ATB extract on potency was also investigated. The effect of ATB on the frequencies of erections (E), quick flips (QF) and long flips (LF). ATB cause a marked increase in the tested parameters of penile reflexes  in all the treated rat groups when compared to control group.It is generally accepted that mount latency (ML) and intromission latency (IL) are parameters that are considered to be a mark of sexual motivation. These parameters are inversely proportional to sexual carving. An increase in these parameters suggests lack or loss of sexual appetite/drive . TreatmeA. tuberosum extracts could stimulate increase in the level of testosterone thus improving sexual motivation and activity of the rats. The relaxation of the smooth muscle has been proposed to be associated with erection [Previous studies have suggested that the level of testosterone in the blood is essential for arousing sexual desire or stimulation. This sex hormone plays important modulatory role of male sexual behavior in the CNS. It is also noteworthy to indicate that the activity of these sex hormones has a direct correlation with erectile function in the peripheral nervous system (PNS) . The Leyerection . Nitric erection . Furthererection . This hyA. tuberosum which are steroidal saponins and nucleosides may have played a critical role in the aphrodisiac activity. These constituents present in ATB extract may have caused a stimulation of the body’s testosterone levels which in turn is expressed in the enhancement of the male rat’s sexual capacity as indicated in this study. Saponins have been previously suggested to have the ability to bind to certain hormone receptors/enzymes involved in the synthesis of these hormones, thus enhancing the physiological function of the hormone [The effect of plant bioactives on sexual enhancement is thought to be mediated through different mechanisms of action such as induction of the relaxation of smooth muscle corpus cavernosum through the L-arginine/nitric oxide pathway , enhance hormone .Spermatogenesis, the process that produces spermatozoa from stem cells of the spermatogonia. The number of sperm in the testes is increased during this process. The results from this present study indicated a significant dose dependent increase in the sperm count, sperm motility and sperm viability of the rats administered with ATB suggesting the possibility of the spermatogenic properties of the extract. The effect of plant or its bioactive on the spermatozoa characteristics is thought to be mediated through the hypothalamic-pituitary-testicular axis of the animal. The increase in sperm count and motility in the treated rats might due to the effect of the extract on the maturation of the spermatozoa as well as the androgen enhancing effect of ATB. The extract may also have facilitated the spermatogenesis as well as aiding the functioning of epididymis .Alkaline phosphatase (ALP) is involved in the mitosis of spermatogenic cells as well as glucose transport that is necessary by the spermatozoa in the seminal fluid and for steroidogenesis. Acid phosphatase (ACP) are found in the in lysosome of leydig cells in the testis and are involved in the synthesis of protein by abduction of sex hormones and sperm physiology . AlteratA. tuberosum significantly enhances sexual behavior parameters, thus indicating that the plant possesses potent aphrodisiac activity.Taken together, our findings clearly demonstrate that the butanol extract from


The Mg2+ ions in seawater played an important role in lowering the electron transfer of the rust layer in the Cr-containing steel. The corrosion resistance of the Cr-containing steel is superior to that of blank steel in Mg2+ ions containing seawater. XPS and XRD results indicated that the formation of MgFe2O4 and a mixed layer (Cr oxide + FeCr2O4 + MgCr2O4) improved the corrosion resistance of the low-alloy steel in the seawater.This study examined the synergic effect of alloying the element Cr and the environmental element Mg The water ballast tank (WBT) is a structure that is used for controlling vessel weight with seawater to provide vessel balance and stability. The repetition of the seawater inflow and outflow induces the exposure of the WBT to various marine environments, and the seawater acts as a corrosion factor, due to the various aggressive ions and organisms ,2,3,4,5.In recent years, numerous studies have attempted to improve the service life of the WBT. The primary focus of these studies comprises the following two parts: coating technologies and corrosion-resistant alloy designs. A zinc (Zn) epoxy-primer coating is mainly used in the ballast tank for the prevention of corrosion; however, this coating is not an ideal protector of the ballast tank because of existing defects, and therefore high-corrosion-resistant steel is required ,9,10,11.α-CrxFe1-xOOH, called Cr-goethite, and this compact rust layer protects the matrix from chloride anions by changing the surface property under the cation-selectivity condition.The typical alloying elements that are used to increase the corrosion resistance are not only chromium (Cr), aluminum (Al), and nickel (Ni), but a combination of the three elements is also used. In particular, regarding the effect of Cr in the high-strength low-alloy (HSLA) steels that are reported by Blekkenhorst et al. , it was 3) and magnesium hydroxide (Mg(OH)2). CaCO3 and Mg(OH)2 are attributed to the cathodic reduction of oxygen (O). The deposits impede the transport of dissolved O to the metal surface and eventually decrease the corrosion rate. The precipitate formations are derived according to the following reactions [The corrosion of carbon steel is influenced by various seawater constituents. The chloride and sulfate ions are representative anions that cause serious corrosive problems. The major cations that contribute to the corrosion property are calcium (Ca) and magnesium (Mg) ions, as they produce local alkaline surface conditions, due to the precipitative effects of calcium carbonate . 2+ ions, in seawater.In regard to the seawater cation, Ca is distributed in the rust layers on both the blank- and 0.7-wt.%-Cr steel rust layers. Mg, however, showed a different trend in both of the specimens, as follows: It is distributed throughout the rust layer on the blank steel, but it is condensed in the 0.7-wt.%-Cr steel; furthermore, the concentrated-Mg region is included in the Cr-enrichment layer. From this result, it was expected that both the Mg and the Cr would contribute to the corrosion resistance. Thus, to identify the Cr–Mg synergic effect, the experiment was conducted in both the presence and the absence of the Cr-alloying element and the MgpR) can be obtained from the EIS and LPR data. Then, the pR is transferred to the corrosion-current density, using the following equation [corri is the corrosion current density (μA/cm2). The impedance parameters of each specimen, including the pR, are listed in corri through Faraday’s law [M is the molar mass of the metal (g/mol), z is the number of transferred electrons per metal atom, F is Faraday’s constant, and ρ is the metal density (g/cm3). The corrosion rates of the specimens after 30 days of immersion were calculated using electrochemical impedance spectroscopy (EIS) and the linear polarization resistance (LPR) test. By fitting the EIS data and using the slopes of the potential and the current, the polarization resistance (equation :(3)Rp=βaay’s law , as follW is the weight loss (g), A is the exposure area (cm2), t is the immersion time (h), and ρ is the density (g/cm3). 2+ ions. The corrosion rates calculated by EIS, LPR, and weight loss tests showed the similar trend in all three experiments: The average corrosion rates of the steels decreased in the following order: 0.7-wt.%-Cr steel without the Mg2+ ions ≈ blank steel without the Mg2+ ions > blank steel with the Mg2+ ions > 0.7-wt.%-Cr steel with the Mg2+ ions. This result shows that the Cr-alloying element only improved the corrosion resistance in the seawater containing the Mg2+ ions. The difference in the corrosion rates of the blank steel, in the presence and absence of the Mg2+-containing seawater, is due to the Mg(OH)2 that acts as a physical barrier. In the 0.7-wt.%-Cr steel, the difference in corrosion rate between the Mg2+-containing and Mg2+-free seawater solutions is larger than that of the blank steel, which can be explained by not only the Mg2+-containing effect, but also the barrier effect between the Mg2+, Fe2+, and Cr3+ ions. As shown in 2+, Fe2+, and Cr3+ ions that are concentrated near the substrate will form a compound and act as a barrier against corrosion.Also, the weight loss-calculated corrosion rate was determined using the following equation :(5)CorroThe Nyquist and Bode plots of the specimens after 1 day of immersion are shown in s represents the solution resistance, Cdl is the capacitance that is generated by the electric double layer, and Rct is the charge-transfer resistance. The RE is the reference electrode , and WE is the working electrode (specimens). The experiment results show that the pR values are similar, regardless of the presence of the Cr-alloying element and the Mg2+ ions in the seawater.The equivalent circuit consists of the following elements: Rs represents the solution resistance, Crust is the rust capacitance, Rrust is the rust resistance, Cdl is the capacitance that is generated by the electric double layer, and Rct is the charge-transfer resistance. The RE is the reference electrode , and WE is the working electrode (specimens). Also, CCr is the capacitance that is formed by the Cr-enrichment layer and RCr is the Cr-enrichment-layer resistance. In this instance, the capacitors were replaced with the constant phase elements (CPEs) for more accurate EIS-data fitting, where not only a double layer capacitance (C) but also a phenomenological coefficient (n) have been included. The CPE impedance takes the following form:Q is an effective CPE coefficient, ω is the sine-wave-modulation angular frequency, j is an imaginary number, and n is the phenomenological coefficient that characterizes the phase shift [rust, CCr, and Cdl were replaced with CPE1, CPE2, and CPE3, respectively.Also, the Nyquist plot impedance spectra after the 30 days of immersion are shown in se shift ,30,31,32α). The depression angle is an empirical factor that represents the deviation from the ideal capacity [n) [In the Nyquist plot, the degree of depression of the semicircle, with a center below the real axis, determines the depression angle  ,32,33:(7pR) that is represented in rust + Rct for the blank steel and Rrust + RCr + Rct for the 0.7-wt.%-Cr steel. In the 0.7-wt.%-Cr steel, the calculated depression-angle value of the Mg2+-containing seawater specimen is 17.433° and that of the Mg2+-free specimen is 21.186°. This result means that the characteristics of the Cr-enrichment layers that formed on both specimens are different, as follows: The 0.7-wt.%-Cr steel with the Mg2+ ions comprises a dense layer compared with that without the Mg2+ ions. Also, the depression-angle values of the blank steel are 21.213° and 22.833°, with and without the Mg2+ ions, respectively. This porosity difference, regarding the rust layer, is due to the physical barrier that is caused by the Mg2+ ions in the seawater, which also causes the corrosion rate difference for the blank steel.2+ ions, for the 30-day immersion in the synthetic seawater at 60 °C. The splitting of the phase angle frequency curve indicates the presence of other phases.2+-free seawater, meaning that the capacitance values of the Mg2+-containing seawater are larger, as identified in rust value is larger in the Mg2+-containing seawater. Because the capacitance is inversely proportional to the thickness, the rust layer in the Mg2+-free seawater is thicker, due to the greater corrosion. Also, the Cdl value, which is formed as the ions are absorbed onto the electrode surface, is larger in the Mg2+-containing seawater. Thus, the different values are reasonable because the Mg2+-containing seawater comprises a higher quantity of adsorbable ions. In the 0.7-wt.%-Cr steel, the capacitance values—Crust, CCr, and Cdl—are larger in the Mg2+-containing seawater. The Crust that represents the rust layer thickness shows the formation of a thin rust layer, due to the lower level of corrosion. The Cdl difference can be associated with ion absorption due to the Mg2+ ions in the seawater. The Cdl values between the blank and 0.7-wt.%-Cr steels, however, indicate a significant difference. This phenomenon means that the Cr-enrichment layer interrupts ionic absorption on the metal surface. Here, the CCr, the thickness of the Cr-enrichment layer, represents the production of a thin Cr-enrichment layer in the Mg2+-containing seawater that is identified by the detected Cr element in ct is a resistance value that prevents electron transfer from the metal layer to the rust layer, thereby indicating its relation to the metal corrosion rate. The larger Rct values of the blank and 0.7-wt.%-Cr steels in the Mg2+-containing seawater indicate the suppression of the electron transfer compared with the Mg2+-free seawater.In 2+-free seawater during the 30-day immersion. The tendencies, distributions, and concentrations of the Fe, oxygen (O), and Cr between the rust layer and the substrate are similar to those of the Mg2+-containing seawater, as shown in 2+ ions, represents the Cr-enrichment layer, and the average thicknesses are approximately 25 μm and 45 μm, respectively. The Cr-enrichment layers are produced by the corrosion of the specimens, and the Cr3+ ions are redeposited onto the steel surface through reduction reactions [2+ environment means that more corrosion occurred on the steel surface. This result means that the corrosion rate is more affected by the porosity than the thickness of the Cr-enrichment layer. The pR values that were measured at the 1- and 30-day immersions, which are shown in 2+-containing seawater environment, both the blank and 0.7-wt.%-Cr steels increased the pR over time, whereas the pR decreased for both of the specimens in the Mg2+-free-seawater environment; this is because the base steel corrosion continues through the porous region of the rust layer.eactions . Thus, t3O4), iron(III) oxide (Fe2O3), and iron hydroxide (FeOOH), and the corrosion resistance. The increase in the amount of the Fe oxide products resulted in an enhanced corrosion resistance because the Fe oxide products act as the corrosion protection layer of the steels [2, chromium(III) oxide (Cr2O3), chromium hydroxide (CrOOH), and iron(II) chromite (FeCr2O4), but it is also owing to the formation of Mg and the Cr-containing product, magnesium(II) chromite (MgCr2O4). MgCr2O4, the structure of which is spinel, is known for its corrosion resistance improvement property [2O4 increases the corrosion resistance of the 0.7-wt.%-Cr steel in seawater.To determine the chemical composition of the corrosion product after the 30-day immersion test, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) measurements were carried out. The local area near the Cr-enrichment layer was detected using XPS with the cross-section specimen, and the whole area was detected by XRD with a top-view specimen. e steels ,40. Simiproperty ,42,43,44property . Thus, t3O4) and magnesium iron oxide (MgFe2O4), which are not shown in the XPS data, were detected. Wang et al. [2O4 in the rust layer exerts a protective effect on weathering steel. Also, since the structure of MgFe2O4 is spinel, like that of MgCr2O4, this improves the corrosion resistance. The MgFe2O4 acts as an n-type-semiconductor corrosive film that works as a cation-selective rust layer [3O4, the MgFe2O4 with Fe oxide, Cr oxide, FeCr2O4, and Mg(OH)2, the corrosion resistance of the 0.7-wt.%-Cr steel is the highest in seawater. In particular, for the 0.7-wt.%-Cr steel in seawater, the Cr oxide, FeCr2O4, and MgCr2O4 mixed layers contributed to the enhancement of the corrosion resistance. The corrosion resistance of the blank steel in seawater is the second highest, due to the MgFe3O4, the MgFe2O4 with Fe oxide, and Mg(OH)2. The corrosion resistance of the blank steel in the Mg2+-free seawater, which only comprises an Fe oxide corrosion barrier, is lower. Although the 0.7-wt.%-Cr steel in the Mg2+-free seawater consists of Fe oxide, Cr oxide, and FeCr2O4 barriers, the corrosion resistance is similar to that of the blank steel under the same seawater conditions. This phenomenon is due to the less-protective Cr oxide and FeCr2O4 layers and the galvanic-corrosion effect. On the steel surface, the Cr oxide and FeCr2O4 layers without the MgCr2O4 comprise the porosity property, as shown in the EIS results. Also, since the Cr oxide and the FeCr2O4 do not cover the steel surface perfectly, the Cr oxide behaves as the large cathode and the bare-steel surface behaves as the small anode. Thus, the large ratio of the cathode-to-anode surface area accelerated the galvanic-reaction corrosion of the 0.7-wt.%-Cr steel [The XRD spectra of the specimens are shown in g et al.  reportedst layer ,47,48. BCr steel .2) was excluded from the D1141 standard for the preparation of the Mg2+-free seawater, and the chlorine (Cl) concentration was adjusted with sodium chloride (NaCl).The EIS analysis and the LPR test were performed using the EG&G PAR VMP2 potentiostat/galvanostat . To perform the electrochemical tests, the three-electrode electrochemical system consists of a low-alloy steel, two pure graphites, and a saturated calomel electrode (SCE) that served as the working, counter, and reference electrodes, respectively. The EIS analysis was carried out to calculate the corrosion rate and to observe the change in the rust layer with an amplitude of 10 mV, at frequencies ranging from 100 kHz to 1 mHz (or 10 mHz in the 1-day case). With the use of the Zsimpwin software , the impedance plots were interpreted on the basis of the equivalent circuit, according to a suitable fitting procedure. The LPR tests were carried out at a potential sweep of 0.166 mV/s, from an initial potential of −20 mVocp, to a final potential of 20 mVocp. The polarization resistance was obtained from the slope of the potential versus the current-density curve.6H12N4), 500 mL of hydrogen chloride (HCl), and balanced distilled water. These specimens were also degreased for 10 min in an ultrasonic cleaner with ethanol, followed by cleaning with distilled water and drying with N2, and then their final mass was measured to four decimal places. To improve the reliability, this experiment was performed repetitively in triplicate.Weight-loss measurements were performed according to the ASTM standard, G31 . The lenα energy source and the D/Max 2500 V PC XRD instrument  with a scan rate of 3°/min from 5–100°.To investigate the effect of Cr and the relationship between Cr and the seawater cation, a surface analysis was carried out. The corroded surface features after the 30-day immersion in the 60 °C synthetic seawater, depending on the solution states, were observed using the JXA-8900R EPMA . The chemical composition of the rust layer was identified using the ESCALAB 250 XPS instrument  with a monochromatic Al-K2+, on the corrosion of low-alloy steels in synthetic seawater at 60 °C, using EIS, LPR tests, weight-loss tests, and a surface analysis. Based on the previously presented results, the following conclusions can be drawn:2+ ions in the seawater are also concentrated on the Cr-enrichment layer.From the EPMA results in the seawater, the 0.7-wt.%-Cr steel represents the Cr-enrichment layer, and the Mg2+-containing seawater, revealed the following order for the average corrosion rates of the steels: 0.7-wt.%-Cr steel without the Mg2+ ions ≈ blank steel without the Mg2+ ions > blank steel with the Mg2+ ions > 0.7-wt.%-Cr steel with the Mg2+ ions. The Cr-alloying element only improved corrosion resistance in the Mg2+-containing seawater, and the reason is not only the Mg2+-containing effect, but also the barrier effect between the Mg2+, Fe2+, and Cr3+ ions.The EIS analysis, LPR tests, and weight-loss tests, with and without the Cr-alloying element and the MgThe EIS interpretation of the blank and 0.7-wt.%-Cr steels suggests that both of the steels under the seawater condition show a rust layer, with a relatively nonporous corrosion on the steel surface.3O4, MgFe2O4, MgCr2O4 with Fe oxide, Cr oxide, FeCr2O4, and Mg(OH)2. Also, the high-porosity condition of the Cr oxide layer was changed to a low-porosity condition.The XPS and XRD analyses showed that the rust layer of the 0.7-wt.%-Cr steel in the seawater comprises effective protection barriers, such as MgFeThis study investigated the synergic effect of the alloying element, Cr, and the environment element, Mg