Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases. The phenotypes often observed in NS are ambiguous and varied, including short stature, distinctive facial characteristics, learning problems, and a predisposition to leukemia4. Mutations in the SHP2, SOS, RAS, and RAF proteins have been identified in NS patients5, suggesting that NS is caused by the excessive activation of the RAS–MAPK pathway. Because this pathway plays crucial roles in various cellular phenomena8, different single mutations in one protein produce various phenotypes9, and the variation in phenotypes has hindered the development of medical treatments for NS. At the present time, only symptomatic treatments are available for NS patients. Clarification of the pathological mechanism of each NS mutant in hyperactivating the RAS–MAPK pathway is crucial to establishing techniques for the rational treatment of NS.Noonan syndrome (NS) is a congenital genetic disorder causing cardiac and developmental defects13. SOS is a guanine exchange factor (GEF) for the small GTPase RAS14. In the resting state of cells, SOS forms a complex with GRB2 in its C-terminal GRB2-binding domain and is located in the cytoplasm14. Upon stimulation of the receptor tyrosine kinases (RTKs) on the plasma membrane, including the epidermal growth factor (EGF) receptor (EGFR), the phosphorylation of tyrosine in the cytoplasmic domains of the RTKs is recognized by the SH2 domain of GRB2, resulting in the translocation of the GRB2–SOS complex from the cytoplasm to the plasma membrane14. On the plasma membrane, SOS interacts with the inactive GDP-bound form of RAS (RAS–GDP) to stimulate the exchange of RAS-bound GDP for cytoplasmic GTP, which activates RAS14.Mutations in SOS (SOS1) occur in 8–14% of all NS patients who have been genetically analyzed15. The N-terminal histone fold (H domain) and the third domain, the pleckstrin homology (PH) domain, bind acidic lipids. The second domain is the Dbl-homology (DH) domain. The fourth domain, the RAS-exchange motif (REM) domain, binds to RAS–GTP. The fifth domain, the Cdc25 domain, corresponds to the GEF activity and therefore binds to RAS–GDP. The last domain is the C-terminal GRB2-binding (G) domain. Therefore, SOS contains five direct and indirect membrane-associating domains  that are involved in its translocation to the membrane. A conformational change in the SOS structure and a positive feedback loop between RAS and SOS are important in the regulation of SOS activity, in addition to its association with multiple membrane components17. The intramolecular association between the H domain and the helical linker (HL) between the PH and REM domains, stabilizes the SOS structure in the closed form19. In this form, the interaction between the REM domain and RAS–GTP is prevented20. When SOS takes the open form and RAS–GTP binds to the REM domain, the GEF activity of SOS in the Cdc25 domain is strongly increased18, forming an SOS–RAS positive feedback loop.SOS consists of six domains21. Mutations in the Cdc25 domain may directly increase the GEF activity of SOS, inducing the hyperactivation of the RAS–MAPK pathway. However, mutations in the other domains must affect RAS–MAPK activation through indirect mechanisms. Some of these indirect mutations have been classified into two classes based on the location of the mutation21. Some mutations in the H and PH domains are thought to increase the affinity of SOS for the lipid components of the membrane, and others are thought to destabilize the closed conformation, as suggested from the crystal structure15. However, these possibilities have not been examined experimentally in the context of living cells, and the molecular mechanisms by which some mutations cause NS are largely unknown.NS mutations have been reported in all six domains and the HL of SOS22 is a useful way to examine SOS functions in living cells23. Although the translocation of SOS is infrequent in single living cells, its dynamics can be visualized clearly because the single-molecule imaging of SOS molecules conjugated with a fluorophore is highly sensitive. Single-molecule imaging also allows the analysis of the association and dissociation kinetics between SOS and the membrane components23. We found that the simultaneous association of the H and G domains to the plasma membrane is critical for generating an intermediate dissociation state of SOS on the plasma membrane. In the intermediate state, the SOS molecule seems to adopt an open conformation, which is stabilized by the function of the REM domain23. SOS activity simultaneously requires intact H, G, and REM domains, suggesting that in the intermediate state the autoinhibition of SOS in the closed state is released to complete the positive feedback loop with RAS23.A single-molecule kinetic analysis21  and is thought to maintain the autoinhibitory closed structure15. How R1131K causes NS is unknown. Because R1131 is located in the proline-rich region of the G domain21, which is used for the formation of the SOS–GRB2 complex, it may change the interaction with activated RTKs via GRB2. In addition to these molecules with single-point mutations and WT, we used the corresponding molecules with an additional defect in the REM domain 23) to assess the roles of the positive feedback reaction with RAS. Because the feedback reaction acts when SOS takes the open conformation23, comparisons with the REM(−) mutants should provide information on the SOS conformation. We found that the three NS mutations have unique effects on the basic processes of the membrane interaction.In this study, we analysed the single-molecule kinetics of the membrane interactions of three NS SOS mutants  and compared them with that of the wild type (WT). These three mutations occur in the DH, HL, and G domains, respectively21 Fig. . In the 23, single molecules of SOS were observed on the plasma membranes of the cells before and after stimulation with EGF , the densities of the NS SOS molecules on the cell surfaces  were increased  mutation  decreased the association rate constant of M269R to a level similar to that of WT/REM(−) Fig. . Our preion Fig. . The del23. The half-life of the membrane association (median dwell time) was taken as a simple indicator of the dissociation kinetics  mutation  was shorter than that of WT after EGF stimulation. The half-life of M269R/REM(−) was similar to that of WT/REM(−), indicating that the longer dwell time of M269R relative to that of WT was produced by the REM domain. The half-life of R552G/REM(−) was still longer than that of WT/REM(−) at every stage, indicating that this increase was independent of REM. R1131K/REM(−) had a half-life similar to that of WT/REM(−).Before cell stimulation, all the NS molecules displayed half-lives longer than that of WT Fig. . EGF stiion Fig. . As obse26, and its association with the basal amount of RAS–GTP. In cells expressing WT SOS, an increase in the density of RAF on the cell surface was observed after EGF stimulation for 8 min, and this increase persisted for 15 min  results from many molecular mechanisms. More than 30 different NS-related point mutations have been reported in the SOS molecule aloneHowever, it is possible that NS SOS activity is not simply determined by the density of SOS on the cell surface. Our results indicate that the mechanisms that induce excessive affinity for the cell surface are specific for each NS mutant. Membrane affinity is determined by the balance between association and dissociation. The observation of single SOS molecules revealed that M269R and R1131K have higher association rate constants with the plasma membrane components than WT, whereas R552G does not  mutation destabilizes the intermediate state probably because it prohibits the interaction of SOS with RAS–GTP, but does not reduce the association rate constant of WT with the plasma membrane23. However, in M269R SOS the REM domain contributes not only to the stability of the association state , but also to the initial association with the cell surface (large association rate constant with the membrane). In the crystal structure of the inactive form of WT SOS, the intramolecular interaction between the H domain and HL prevents the REM domain from interacting with the RAS molecule20. The REM domain in WT SOS seems to be exposed to RAS only after the molecule takes a specific membrane-associated conformation (i.e. the intermediate state) using the H and G domains simultaneously23.The changes in the interactions between each SOS mutant and the membrane components suggested by the single-molecule measurements are summarized in Fig. The REM-dependent increase in the association rate constant suggests that the REM domain of M269R in the cytoplasm is exposed to RAS before SOS associates with the cell surface, and binds directly to RAS on the membrane. The enhanced interaction between the REM domain and RAS–GTP amplifies the activity of RAS through the SOS–RAS positive feedback loop.15, which should be destabilized by the R552G mutation. However, the single-molecule kinetics suggest that unlike M269R, R552G adopts an active conformation only after its association with the membrane; i.e., R552G in the cytoplasm does not display an increased association rate constant for the plasma membrane. The reduced dissociation rate constant of R552G can be explained as a conformational preference that allows the simultaneous association of the H and G (and other) domains with the membrane components. In other words, R552G increases the probability of the open SOS conformation on the cell surface. In the open conformation, the probability of the interaction between the REM domain and RAS–GTP increases, thereby increasing the GEF activity. In WT and the other NS mutants investigated in this study, the REM/RAS–GTP interaction stabilized the membrane association state of SOS. However, in R552G the interaction between RAS and the REM domain did not determine the dissociation kinetics. A high probability of an open conformation will increase the REM/RAS–GTP interaction, inducing the hyperactivation of RAS by R552G, as suggested by the increased translocation of RAF in cells expressing R552G.R552 interacts with D140 and D169 in the H domain, maintaining the autoinhibitory structure of SOS27. S1134 and S1161 in the G domain are also phosphorylated by RSK, which is regulated by ERK, inducing the downregulation of ERK activity28. These serine residues are in the proline-rich region of the G domain, which forms the complex with GRB2. Because R1131 is located near these serine residues, it is possible that changes in the formation of the SOS–GRB2 complex explain the dysregulation caused by R1131K. The positive charge on lysine may neutralize the effect of the negative charges introduced by serine phosphorylation. In our experiment, the increased association rate constant of R1131K was largely explained by the effect of the G domain  are phosphorylated by ERK in the negative feedback to SOS activityain Fig. , consist17. We observed that the REM(−) mutation largely prevented the translocation of RAF, a typical effector of RAS, from the cytoplasm to the cell surface for activation23. Because the feedback response is nonlinear, involving the product of the SOS–RAS reaction (RAS–GTP), small differences in the reaction conditions will be amplified, resulting in large differences in the output. The differential properties of the three NS mutants in inducing the hyperassociation of SOS with the cell surface may cause mutant-specific dynamics of RAS activation, which cannot be explained simply by the cell-surface density of the SOS molecules. For example, although the density of SOS on the cell surface was higher for R552G than for M269R  and PrimeSTAR® Max DNA Polymerase (Takara). The plasmid encoding GFP–RAF was constructed as reported previously24.The construction of the cDNAs of Halo7-WT SOS and Halo7-REM(−) SOS has been described previously2. The cells on coverslips were transfected with cDNAs using Lipofectamine® LTX with Plus™ Reagent (Invitrogen) 2 days before the experiments. After transfection, the cells were starved for 16 h in minimal essential medium (MEM) without serum or phenol red, and supplemented with 1% bovine serum albumin (BSA). To observe the SOS protein, the cells were stained with 100 nM HaloTag® TMR Ligand (Promega) for 15 min immediately before the experiments and washed.All experiments were performed in HeLa cells (American Type Culture Collection). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37 °C under 5% CO−1. After 3 × 3 pixel averaging and background subtraction with MetaMorph (Molecular Devices) and Image J , single molecules were detected and tracked with in-house software29 and TrackMate30. The dissociation kinetic analysis and statistical analysis were performed with Matlab (The MathWorks) and Origin (Originlab) as described previously31. The photo-bleaching rate constant of TMR conjugated to SOS 23 was significantly smaller than the apparent values  of the dissociation rate constants of SOS from the plasma membrane components , based on an inverted microscope IX83, Olympus) equipped with an objective PlanApo 60× NA 1.49 (Olympus). A 555 nm solid-state laser  was used for illumination, and fluorescent movies were acquired with a CMOS camera  at a frame rate of 20 s, OlympusHeLa cells transfected with plasmids encoding the SOS molecules were incubated overnight in MEM, supplemented with 1% BSA. The cells were washed twice with Hank’s balanced salt solution (HBSS) and harvested in SDS solubilisation buffer. The proteins in the cell lysates were separated according to their molecular sizes on 10% or 8% polyacrylamide gel, and transferred to polyvinylidene difluoride membranes (BD Biosciences). The membranes were incubated in 5% skim milk with an anti-SOS1 antibody , anti-ERK antibody , or anti-pERK antibody . After the membranes were washed tree times with PBS, the membranes were incubated with a secondary antibody conjugated with alkaline phosphatase  or with horseradish peroxidase . Antibody bindings were visualized with BCIP/NBT Color Development Substrate (Promega) or with ECL Prime Western Blotting Detection Reagent Kit .Supplementary Information


The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain. C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are members of the CSK family of protein tyrosine kinases. These proteins suppress the activity of Src family kinases (SFKs) by selectively phosphorylating the conserved C-terminal tail regulatory tyrosine -phosphorylated peptide was studied. The reduced and oxidized forms of Csk can both bind with a Cbp peptide, with the reduced SH2 showing a slightly stronger affinity. Crystal structures of both oxidized SH2 and C122S SH2 were solved and refined. Comparison of the crystal structure of the different forms of SH2 suggests that only minor structural changes resulted from the disulfide bond. Analysis of biophysical data of the unusual disulfide bond provided insights into the role of the disulfide bond in SH2 domain.1H–15N HSQC spectrum of purified, uniform 15N-labeled reduced sample, such as the side chain peak of W82  was used in other studies  and without (green) Cbp peptide. Figure 1H–15N HSQC spectrum in the absence of ligand; therefore, data on chemical shift perturbation were not available for these two residues. In addition, βA–αA loop residues at the N-terminal underwent smaller chemical shift perturbation upon binding of the phosphopeptide. Comparison of the SH2 structures in putative active full length, inactive full length . Cbp was titrated with L-SH2 at a final concentration of 100 μmol/L. Well-resolved Y129 and G162 peaks were used to calculate To study the interaction between L-SH2 and Cbp, we used a 10-residue peptide with the sequence ISAMpYSSVNK derived from human Cbp protein . The Cbp peptide (ISAMpYSSVNK) was synthesized by GenScript  using standard solid-phase peptide synthesis methods and then resuspended in purified water before use.The human Csk SH2 domain genes containing the residues A73–A178 were amplified and cloned into the expression vector pTWIN1 (New England Biolabs) as described previously  NaN3, 5% D2O, and 0.1 mmol/L DSS . DTT (10 mmol/L) was added whenever necessary. In the 3D triple-resonance experiments, HNCA, HNCO, HNCACB, and CBCA(CO)NH were collected for backbone resonance assignment. The 1H chemical shifts were referenced to internal DSS. The 13C and 15N chemical shifts were referenced indirectly using the 1H/13C or 1H/15N frequency ratios of the zero point: 0.101329118 (15N) and 0.251449530 (13C) 2 + (0.154∆δN)2]1/2, where ∆δH and ∆δN correspond to the changes in amide proton and nitrogen chemical shift, respectively. The weight factor for 1H and 15N is determined from the ratio of the average variances of the amide nitrogen and proton chemical shifts observed for the 20 common amino acid residues in proteins deposited in the BioMagResBank , 1.0 mmol/L EDTA, 0.01%  was mixed with reservoir solution  and incubated in reservoir solution (1 mL) at 25 °C. Within one day, crystals typically grew as rod-shaped structures with dimensions 300 μm × 20 μm × 20 μm. X-ray diffraction data were collected, integrated, and scaled using HKL2000 /(IU − IN), where N and U stand for the fluorescence intensity of the native and fully unfolded species, respectively. Funf values were calculated from the linear extrapolation of the pre- and post-unfolding baselines. The unfolding free energy and m values in the relationship were obtained from the fitting of the denaturation data into a two-state model using the standard equation for all four L-SH2 protein samples. The apparent free energy of unfolding was determined according to the linear equation ΔGU,W0 = ΔGU,D0 + m[D],  where ΔGU,W0 is the apparent free energy of unfolding in water, ΔGU,D0 is the apparent free energy of unfolding at a denaturant concentration [D], and m is the coefficient expressing the dependence of the free energy of unfolding on the denaturant concentration.Fluorescence titrations were performed at 25 °C in 3 mL of 50 mmol/L Tris-HCl (pH 7.5) using excitation and emission wavelengths of 288 nm and 320 nm, respectively. The 10-residue synthetic phosphopeptide corresponding to the specific SH2-binding site in Cbp was titrated with the L-SH2 sample. The typically adopted protein concentration is 5 μmol/L. For the denaturation experiments, ultrapure guanidine hydrochloride (Sigma S0933) was used. Proteins were exposed to guanidine hydrochloride concentrations ranging from 0 to 6 mol/L at 0.1–0.2 mol/L steps. The concentrations of guanidine hydrochloride of the solutions were determined by measuring the index of refraction and by using the following equation: PDB number: 3EAC (oxidized) and 3EAZ (C122S mutant). The backbone chemical shifts of SH2 with and without Cbp peptide were deposited in BMRB with accession numbers 7141 and 7140, respectively.


In recent years reports from a number of countries, including Sweden, describe problems with diarrhoea in newborn piglets despite the use of previously effective preventive measures. This seemingly altered disease pattern of neonatal porcine diarrhoea (NPD) warrants investigations on the magnitude and manifestation of the problem. The aim of the present study was to investigate the herd-level prevalence of NPD in Sweden and to describe disease characteristics and intervention strategies used in affected herds. To obtain this information a questionnaire was developed and sent out to 170 randomly selected herds. The presence of NPD in the herds was specified as “Yes”, “No” or “Occasional cases” during the preceding year.Escherichia coli (ETEC). The most common treatment regimens used in affected herds included antimicrobials only  or antimicrobials in combination with supplementary fluids . Trimethoprim in combination with a sulphonamide was the drug of choice in 57% (24/42) of the affected herds whereas the remaining herds used a broad range of other antimicrobials . Furthermore, the risk of experiencing NPD was found to be higher in herds with >200 sows (OR = 4.0) compared to herds with <200 sows and in herds where more ambitious efforts  were made to save weak-born piglets (OR = 4.4).A response rate of 58% (98/170) was achieved. The total prevalence of farmer experienced NPD, including occasional cases was 79.6% (95% CI 70.6–86.4%). Most herds  employed maternal vaccination against enterotoxigenic The results of the present study indicate that Swedish farmers commonly experience NPD in their herds, often despite vaccination against ETEC. Considering the extent of this problem and its contribution to antimicrobial usage, improving alternative control strategies for NPD needs to be prioritized.The online version of this article (doi:10.1186/s13028-016-0261-0) contains supplementary material, which is available to authorized users. Neonatal porcine diarrhoea (NPD) is a common and serious problem causing significant production losses in pig farming worldwide –3. A numEscherichia coli (ETEC) and Clostridium perfringens type C, two agents that can be prevented by maternal vaccination combined with a high level of hygiene in the farrowing units [In Sweden, well-known causes of NPD include enterotoxigenic ng units , 15, 16.ng units –20. The ng units , 19, 20.This seemingly altered disease pattern of NPD warrants further investigations on the magnitude and manifestation of the problem. The aim of the present study was hence to investigate the prevalence of NPD in Sweden and to describe disease characteristics and intervention strategies used in affected herds. Furthermore, we aimed to identify herd characteristics and herd management practices that differentiate herds experiencing a problem with neonatal porcine diarrhoea from herds not experiencing NPD.A questionnaire was developed to obtain information on farm characteristics, management routines, and, in affected herds, disease manifestation and intervention strategies. The questionnaire was designed to be filled in by the farmers assisted by their veterinarian, and was pilot-tested in six piglet-producing herds to improve the comprehensiveness of the questions. These responses were not included in subsequent analysis.The study population was piglet-producing herds supervised by the Swedish Animal Health Service  or by Lunden’s Animal Health Service. In 2011, 740 piglet-producing herds  were supervised by the Swedish Animal Health Service, and 59 by Lunden’s Animal Health Service . This corresponded to approximately 86% of the total number of herds keeping sows in Sweden during 2011 . We hypoIn agreement with the 17 veterinarians participating in the study, an average of ten questionnaires was assigned per veterinarian. The sample size of 170 was hence indirectly decided by the study design. The random selection of herds  was however not stratified and resulted in four to 16 herds per veterinarian. Herds that had less than ten sows or had ceased their piglet production were replaced.The final questionnaire included 59 questions divided into two sections—“production and management routines”, and “disease characteristics and treatment of neonatal diarrhoea”. The section on production and management covered type of production, age of farrowing stables, number of sows in production, number of employees, recording of production data and production results, employment of all in-all out in the farrowing unit, washing and disinfection of the farrowing unit, vaccination routines against NPD, employment of nurse sows, entry of sows to the farrowing unit, treatment of post–partum dysgalactia syndrome (PPDS), monitoring of farrowing, and management of newborn piglets and their environment. The section on disease characteristics and treatment of NPD in affected herds included proportion of piglets affected, changes in prevalence of NPD, prevalence in litters from first parity sows, time point for the occurrence of diarrhoea, treatment of diarrhoea, efforts made to investigate the cause, and employment of prophylactic measures.The questionnaire design included multiple choice or dichotomous questions as well as a smaller number of open-ended or semi-open questions. Respondents were requested to base their answers on an average farrowing batch during the preceding year. The full questionnaire (in Swedish) is available upon request from the corresponding author.Questionnaires and a stamped, addressed return envelope, as well as a short missive with instructions to the veterinarians, were distributed by mail during February 2012 to the 16 veterinarians employed by the Swedish Animal Health Service. A reminder to the veterinarians was sent out in May. In November, a second reminder was sent out to individual veterinarians by e-mail together with a list of herds from which responses were missing. Three veterinarians withdrew from the study. Following an initial telephone contact, these questionnaires were instead distributed to the corresponding herds by mail. A similar procedure was employed for herds supervised by Lunden’s Animal Health Service (n = 11).In total, the 59 questions in the questionnaire corresponded to 63 different variables (including the outcome) as some questions included information on more than one parameter. Due to poor question design or low response rates (>25% missingness), 14 questions were discarded . See Fig. All answers were treated as categorical variables. To facilitate the analyses, some answers were amalgamated into fewer categories. Answers on interval scale was categorised in quartiles (“sow batch size”). Other semi-open questions with numerical answers were often given in intervals, e.g. 2–3, and were categorised accordingly (“entry of sows to the farrowing unit” and “desired temperature in the farrowing unit”). The outcome “presence of neonatal diarrhoea”, was specified in the questionnaire as diarrhoea in piglets younger than seven days with the possible responses “Yes”, “No”, and “Occasional cases”. In the analyses, the outcome was treated as a binomial variable where “No” and “Occasional cases” were merged.Most variables Tables , 2, 3 arPrior to statistical analyses, missing values for explanatory variables in the section “production and management routines” were managed by multiple imputation (MI) using a nonparametric method in the CRAN package MissForest , 25. In 2 –test or Fisher’s exact tests in case of cell frequencies <5). The results were compared with equivalent analyses on the original non-imputed data with no considerable differences  was considered a better measure of herd size. Hence, a total of 12 explanatory variables were included . Forty-two herds reported experience of more than occasional cases of NPD during the previous year, 36 reported occasional cases and 20 reported no occurrence. Hence, the total prevalence of farmer experienced NPD, including occasional cases was 79.6% (95% CI 70.6–86.4%) whereas the prevalence of herds experiencing a recurrent problem with NPD was 43% (95% CI 33.5–52.7%).P = 0.68).Of the 98 herds included in the study, 43 had integrated or partially integrated production, 34 were specialised piglet producers, 18 were satellite herds in sow pool-systems, and three were specialised gilt-producing or nucleus herds. The majority of herds (n = 64) had less than 200 sows in production. Of the 63 herds that recorded production results with PigWin Sugg, 23 had production results that were classified as “good”, 10 had “intermediate” results, and 21 had production results that was lower than average (information was lacking from 9 herds). There was no statistical difference between NPD and non-NPD herds (C. perfringens type C and three in combination with C. perfringens type A and C (information was lacking from six herds). Eleven herds had changed vaccine during the last year. Most herds vaccinated sows and gilts at least three weeks prior to farrowing . However, in ten herds (four NPD and six non-NPD herds), gilts were only vaccinated once. Supplemental heating of the creep area was provided in all herds. However, in herds using heat lamps as the only heat source (n = 34), lamps with a low wattage (≤150 W) were more common in herds with NPD (P = 0.02).All in-all out was practiced in the farrowing unit in 86 of the 98 herds and 67 washed the farrowing units between each batch. Maternal vaccination against agents associated with NPD was routinely performed in 83 of the herds. All vaccinating herds used vaccines against ETEC, seven in combination with P < 0.2 in univariable tests are presented in Tables Herd characteristics and management routines with associations to NPD of P < 0.01). The estimated odds ratio for herds with >200 SIP was 4.0 (95% CI 1.7–10).The univariable logistic regression analysis on the effect of herd size  on the presence of NPD showed that herds with >200 SIP had a higher risk of having NPD compared with herds <200 SIP (P < 0.01). Herds where moderate to ambitious efforts were made to take care of weak piglets had a higher risk of having NPD compared with herds where none or only some efforts were made. The estimated odds ratio for herds where moderate to ambitious efforts were made was 4.4 (95% CI 1.9–11).The multivariable logistic regression analysis of management factors rendered a final model which only included the variable “efforts made to save weak-born piglets”  estimated that less than 25% of the piglets were affected by diarrhoea in an average farrowing batch. Diarrhoea was most common 1–3 days after birth (26 out of 42) and did often affect more than 50% of piglets in diarrhoeic litters (29 out of 42). The mortality due to NPD was estimated to be <10% in most herds (30 out of 42). In 29 of the 42 herds, the occurrence of diarrhoea in the herd was described as constant (18) or intermittent (11), whereas ten stated that the diarrhoea had decreased, and three that it had increased during the last year. Seasonal variations were only described in four herds (more during winter and spring). Diarrhoeic piglets were most commonly treated with antimicrobials only, or antimicrobials in combination with supplemental fluids . The drug of choice was trimethoprim/sulphonamide (24 out of 42) and 34 stated that treatment was successful in more than 75% of the cases. In case of poor response to initial treatment, 19 stated that they used fluoroquinolones as a second drug of choice. Efforts to investigate the cause of NPD had been undertaken in 13 out of 42 herds during the preceding year and three stated that the cause of diarrhoea had been established.The prophylactic measures used to prevent NPD were grouped as “changed vaccination routines” (5), “hygienic measures” (6), “vaccination” (2), “vaccination and hygienic measures” (2), “faecal feedback” (6), and “no specific measures” (20). Hygienic measures were often not specified, and although most herds with diarrhoea vaccinated against NPD, only two respondents mentioned vaccination alone as a specific prophylactic measure. Among the 21 respondents reporting that specific prophylactic measures were employed, the effect was appreciated to be poor by two, moderate by 12, and successful by five (three missing answers). Measures with successful effect were hygienic measures and/or vaccination. Faecal back feeding was estimated to have poor (2) or moderate effect (4). An overview of disease characteristics and treatment of NPD in affected herds are presented in Table C. perfringens . Moreover, a broad range of different antimicrobials were used for treatment . Taken together, these results suggest that NPD is a substantial problem and that prophylaxis and treatment regimens recommended against ETEC are not always efficient. Further, the results indicated that larger herd size (>200 SIP) and making more efforts to save weak-born piglets could be potential risk factors for experiencing NPD on herd-level.The total herd-level prevalence of farmer experienced NPD  indicates that diarrhoea in neonatal piglets is common among Swedish piglet-producing herds. Interestingly, many herds experienced more than occasional NPD cases, despite maternal vaccination against ETEC or ETEC and Previous studies on the herd-level prevalence of NPD are lacking and epidemiological studies on pre-weaning diarrhoea have hitherto focused on incidence rates on litter level , 28, 29.To investigate whether presence of NPD on herd level could be associated with certain herd characteristics or management routines we wanted to compare herds experiencing a more recurrent problem with NPD from herds with less prominent or no problems. Thus, for these analyses the categories “occasional cases” and “no” were merged.Before statistical analyses, missing data was handled by MI as MI analyses generally produce less biased and more precise  results than complete case analyses .P < 0.2  were found to be significantly associated with experiencing NPD (OR = 4.0). The categorisation of herds size as <200 or >200 SIP was based on the average Swedish herd size of 186 sows . Associa00 SIP weP < 0.01). Interestingly, making more ambitious efforts to save weak piglets was associated with a higher risk of experiencing diarrhoea (OR = 4.4). One explanation for this may be that an increased handling of piglets could lead to an increased spread of pathogens between litters. However, it could also be that more ambitious animal caretakers were more likely to report NPD, or that newborn piglets were given extra attention because the herd had a NPD-problem. Lastly, it cannot be excluded that promoting survival of weak piglets indirectly could lead to an increased number of NPD-cases as these pigs may be more prone to develop diarrhoea.By multivariate analysis of other herd characteristics and management routines (n = 11), efforts made to save week-born piglets were shown to have a significant effect on the likelihood of experiencing NPD  were more common in herds experiencing more than occasional cases of NPD. These results hence follow the same trend as “efforts made to save weak-born piglets” suggesting that an increased use of zootechnical interventions could be associated with NPD. Again, it is however not possible to make any causal inferences from the present investigation. Nonetheless, a high level of intervention measures has previously been suggested as a contributing factor to recurrent NPD problems in high-performance herds [Other factors significantly associated with NPD in the univariable tests were maternal vaccination against NPD, age of the farrowing building(s), recording of production results, washing of the farrowing unit, and employment of nurse sows  or combined with supplementary fluids . A smaller proportion  had only stated “by injection” which likely could be interpreted as injection with antimicrobials. Notably, microbiological investigations of NPD had only been undertaken in 31% (13/42) of the affected herds during the preceding year. In addition, one respondent stated that all piglets were treated with antimicrobials after birth to prevent NPD. This frequent use of antimicrobials against NPD makes the development of alternative control strategies a necessity. Highlighting this need, a study of antimicrobial usage in Swedish pig herds recently showed that the highest treatment incidence occurs in suckling piglets . TargeteE. coli (e.g. the use of penicillin or tylosin). Since many herds also reported more than occasional cases of NPD despite ETEC vaccination, the results suggest that prophylaxis and treatment regimens recommended against ETEC not always are efficient. This corresponds to recent pathological and microbiological studies, demonstrating occurrence of NPD unrelated to the presence of previously well-known enteropathogens in Sweden and Denmark [The drug of choice in the present investigation was trimethoprim in combination with sulfadiazine/sulfadoxine , which is the recommended treatment against ETEC. Interestingly, however, 40% 17/42) of the herds used other antimicrobials (one missing answer). This could suggest an unsatisfactory effect by trimethoprim/sulphonamide treatment due to resistant ETEC, or that the treatment targeted bacteria other than /42 of th Denmark . PrevaleThe distribution of the questionnaire via the herd veterinarians was chosen as we hypothesised that this would yield a higher response rate as compared to distribution by mail. However, some veterinarians (4 out of 17) were not able to complete the study and thus, the corresponding questionnaires were instead distributed directly to the herds by mail. Due to the small number of questionnaires per veterinarian, differences between veterinarians and distribution method were however not included in the analysis and hence, these potential confounders should be kept in mind when interpreting the results. Furthermore, we do not know if responder and non-responder herds differed in terms of size, production results etc. since no data from non-responders were available. A nationwide, up-to-date register of pig herds in Sweden including information on herd size would greatly facilitate this type of studies. However, in the 63 herds using PigWin Sugg, production data was comparable to the average results reported by Swedish PigWin users during 2012.Some information was not obtained due to inadequately designed questions n = 10). Unfortunately, this included treatment frequencies of post-partum dysgalactia syndrome (PPDS) that is a known risk factor for NPD . Unfortu, 28, 39.The results of the present study indicate that Swedish farmers commonly experience NPD in their herds, often despite vaccination against ETEC. Considering that antimicrobials are frequently used as treatment, preventive measures against NPD need to be improved. To find rational means for intervention, identification of factors potentially related to the disease is important. In the present study, larger herd size and making more ambitious efforts to save weak-born piglets were suggested as potential risk factors and further studies of the underlying biological reasons for these relationships are warranted.


All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the “coordination shell volume,” also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76  I.Cyclophilin 40  is a two-domain protein member of the immunophilin family. In common with most of the other 20 cyclophilins in the human genome, Cyp40 acts as a peptidyl-prolyl-isomerase (PPIase) enzyme which speeds up rotation around the peptidyl prolyl amide bond resulting in more efficient folding of peptide chains.The two domain structure of Cyp40 PDB code 1IHG)IHG1. TheThe TPR domain comprises 7 helices. The first two helices, P (V216-K235) and Q (W239-A259), form the first of three TPR-motifs and are 9 residues longer than the eponymous 34 residue-long tetratricopeptide motif. In the second TPR motif formed by helix R (D262-K285) and S (W289-L300), helix R is again extended by an additional helical turn of four residues compared to a canonical TPR motif. An insertion in this location is a feature that distinguishes the immunophilin TPR domains from TPR domains present in the larger group of chaperone proteins that compete for Hsp70 or Hsp90 with Cyp40. The third TPR motif formed by helices T (T307-G319) and U (Y323-I336) conforms to the canonical repeat length of 34 amino acids. The 7th helix V (K341-K362) caps the domain. A well-defined positively charged groove formed by residues on helices P, R, and T provides the binding site for the C-terminal Hsp90 peptide. Site point mutations identified five residues  lining the groove as essential for Hsp90 binding.In an earlier paper,The TPR domain protein carboxy-terminus of Hsc70 interacting protein μs of all-atom MD simulations were performed for the (i) wild type (WT), (ii) K227A mutant, (iii) K308A mutant, and (iv) K227A + K308A double mutant.The observation that allosteric effects can be caused by site-point mutations in a TPR domain remote from the active site suggests that similar allosteric effects on the enzyme activity of the PPIase domain of Cyp40 could be engineered by means of site-point mutations of its TPR domain. Of particular interest are the carboxylate clamp residues Lys227 and Lys308 which form salt bridges with the MEEVD C-terminal residues of Hsp90 Fig. . In thisII.A.−1 for Langevin dynamics was used to maintain isothermal conditions. The Langevin Nosé-Hoover method was performed using an oscillation period of 100 fs and a damping time scale of 50 fs to maintain the pressure constant. First, 10 000 steps of minimization followed by 2 ns of equilibration was performed by keeping the protein fixed. Subsequently, the complete system was minimized for additional 10 000 steps without any constrains/restrains on the protein. The resulting conformer was equilibrated for 6 ns; where, during the first 2 ns of simulations, harmonic constraints having force constants of k = 2 kcal mol−1 Å−2 were applied to the backbone atoms. Each system was subsequently simulated for 720 ns  structure having PDB ID: 1IHGB.α atoms. The 4 residues at the N-terminal and C-terminal ends were not included in the alignment due to their high degree of fluctuations. After alignment/superposition, the covariance matrix C was constructedR is the 3 × 364-dimensional configuration vector composed of the instantaneous Cα-atom coordinates of residues S2-Y365 and R. Principal components (PCs) are constructed by performing eigenvalue decomposition of the covariance matrix,ith PC having a variance of All conformers in the WT, K227A mutant, K308A mutant, and K227A + K308A double mutant were aligned with the crystal structure using the CC.ΔRi)2⟩. The crystallographic temperature factor, or B-factor, is related to the MSF by ⟨(ΔRi)2⟩ = (3/8π2)Bi.Correlations between the motions of subsets of atoms can be extracted from the elements of the covariance matrix shown in Eq. D.α atom coordinates of a helix. For each helix PC1 vectors taken, Principal component analysis (PCA) is performed on all CE.HHHHHHDYDIPTTENLYFQG-Cyp40) (GeneArt™). Standard GATEWAY® methodology was used to generate an expression vector in pDEST™14 . Lysine to alanine mutants K227A, K308A and the double mutant K227A + K308A were introduced using the GeneArt Site-Directed Mutagenesis System (Thermo Fisher). Proteins were overexpressed in One Shot® BL21 Star™ (DE3) Chemically Competent cells (Thermo Fisher) as described in the work of Wear et al.The open reading frame for human Cyp40 was synthesized with a six histidine tag and a protease cleavage site, TEV, added at the N-terminus . Cyp40 was heated from 5 to 85 °C at a scan rate of 60 °C/h after a 5 min pre-scan equilibration. All samples were collected in triplicate. Data were normalized for concentration and baseline corrected by subtracting a buffer scan collected under the same conditions as the protein samples. Averaged data were analyzed to determine the mid-point melting temperature, Tm, and the calorimetric enthalpy, ΔH, using the software provided by the manufacturer .Differential scanning calorimetry experiments were performed on a MicroCal VP-capillary DSC system . Proteins were exchanged into 50 mM HEPES, pH8, 150 mM sodium chloride, 1 mM DTT to a final concentration of 13.7 G.cis to trans conversion of the substrate Suc-Ala-Leu-Pro-Phe-p-NA (Sigma) at 5 °C, as previously described by Wear et al.−1 α-chymotrypsin; 120 μM Suc-Ala-Leu-Pro-Phe-p-nitroaniline; 50 mM HEPES, pH8; 100 mM sodium chloride 14 μM lithium chloride; 1 mM DTT; 0.5 mM EDTA; and 3% 2,2,2-trifluoroethanol (v/v). The turnover was followed by measuring absorbance of the solution at 400 nM; this was recorded every 0.1 s for 120 s on a JascoV550 Ultraviolet Visible (UV/VIS) spectrophotometer . Protein concentration was determined from absorbance at 280 nm (ε280nm = 32 890 M−1 cm−1). Each experiment was repeated at least 6 times and results were reported as mean and standard errors.The PPIase activities of WT Cyp40, K227A, K308A, and their combinative mutant K227A + K308A were compared by determining the rate of the III.A.μs (2 × 0.72 μs), the MD simulations show significant deviations from this value, Fig. The interdomain distance between the centres of the TPR and cyclophilin domains as measured in the crystal structure is 41 Å. Over the course of 1.44 linker–Lys245helixQ, Asp200linker–Lys248helixQ, and Asp204linker–Lys248helixQ. These salt bridges have been monitored over the length of the simulation by measuring the distance between the center of mass of the oxygens in the acidic side chain and the basic nitrogens .The different conformations have also been analyzed by monitoring salt bridge distances of residues in the linker region L184-L213) that stabilizes and regulates the relative movements of the cyclophilin and TPR domains. In the crystal structure, the linker region  on all conformers of the WT, K227A mutant, K308A mutant, and K227A + K308A double mutant MD simulations. The principal component analysis is an effective and proven method to reveal the most prominent motions a protein exhibits along its MD trajectory, details of which are explained in Sec. Subsequent to the PCA, MD snapshots were projected onto the principal components (PCs). The distributions of the projections, 2.helixP-Ser274helixQ. Hydrophobic interactions between helices, particularly the last 5 amino acids E361KAAY365 on one TPR and the groove of another, provide stability for the fold in the crystal structure. When Cyp40 is in complex with Hsp90, additional non-covalent interactions are provided by binding MEEVD. Mutating K227A uncouples the two halves of the TPR domain.The TPR domain has very few direct H-bonds between residues maintaining the fold after Lys227As shown in Fig. 3.The distance between residues i and j is calculated from the trajectory as follows: the distance between the alpha carbons of residues i and j is calculated for all residue pairs at each time station of the trajectory and the average is obtained by summing up these values and dividing by the number of time stations.dij, between two residues i and j upon mutation are presented in Fig. dij upon mutation is denoted by a red point in the figure, and an increase is denoted by a black point. Red regions show decrease in distance, between −10 and −4 Å. Black regions show increase between 4 and 10 Å. This suggests that K227A Cyp40 has an average conformation that brings the last four helices  of the TPR closer to the Cyp40 domain, while the K308A mutation has the opposite effect of keeping domains apart. Interestingly this divergence involves the residues of the PPIase domain, 50-160, that are not in close proximity to the linker. In the double mutant, the situation is more complex; the active site face of the cyclophilin domain twists away from the TPR domain but the anterior face moves closer. This is shown in the red and black stripes for the double mutant in Fig. Changes in the distance, 4.Correlation matrices for each of the mutants were calculated see Sec. . Figure PPIase and 180-210linker, while red stripes highlight a decreased anticorrelation compared to WT Cyp40.The pattern of increased anticorrelations (black points) of the C-terminal helices T, U, V, W (residues 285-380) fits an exaggerated wagging motion of the last four helices which will be both enhanced negative and positive correlations with different groups of residues on the rotating cyclophilin Fig. . This ex5.m) have been measured for WT Cyp40 and each of the mutants as described in Sec. The MD simulations have been used to identify and quantify two different measures of conformational compactness each of which correlates with the melting temperature. Mid-point melting temperatures  to 6.72 Å (Dmut). They show an interesting correlation with melting temperature as determined using differential scanning calorimetry .As a measure of the compaction effect of the mutations, we developed a parameter defined as “coordination shell volume.” The radius of coordination shell volume is estimated as follows: The Ctry Fig. . The dif7.m values, Fig. As described above, the MD simulation identifies extended and compact conformations of Cyp40 which are measured by the distance between the centroids of the TPR and Cyp domains. The most extended state is adopted by K308A which has an interdomain distance of 43.3 Å (0% folded) and the most compact adopted all other forms at close to 34.0 Å; the size of this population varies. The “foldedness values” (or molecular compactness) also correlate with the measured TC.α atom in the cyclophilin and TPR domains of WT Cyp40, K227A Cyp40, K308A Cyp40, and the double mutant (Dmut)  shown in Fig. The intradomain correlation matrices were separately calculated from the average fluctuation vectors for each Csee Sec. . For exasupplementary material) domains. Domain average B-factors were calculated as n is the number of residues in each domain, and are show in Table 1 of the supplementary material. The domain average B-factor of the linker domain was significantly higher than those for the cyclophilin (Å) and TPR domains (Å). For the WT, the domain average B-factor of the linker was 3.1 and 3.3 times as much as the cyclophilin and TPR domains, respectively. Similarly, for the K227A mutant, the linker domain average B-factor was 2 and 2.5 times as much as the cyclophilin and TPR domains. For the Dmut simulations, average linker domain fluctuations were 2.4 and 2.1 times as much as the cyclophilin and TPR domains. For the K308A mutant, on the other hand, ratios were much smaller; the linker domain fluctuated on average only 1.7 and 1.3 times as much as the cyclophilin and TPR domains.B-factors were calculated separately for cyclophilin, TPR Fig. , and linμM s−1, Dmut 0.44 μM s−1, K227A 0.47 μM s−1, and K308A 0.52 μM s−1[Fig. Experimentally we have shown that all forms of Cyp40 were enzymatically active. The measured enzyme activities for the three mutants all show a small increase in turnover rate compared with WT Cyp40: WT, 0.31  s−1Fig. , which sAlthough we have not yet identified a specific mechanism explaining the enhanced enzyme activity caused by the lysine mutations in the remote TPR domain, it is interesting to note that the all three mutations do have a clear effect on the fluctuations of specific regions of the cyclophilin domain. The most prominent effect is the increased fluctuation in the divergent loop (60PTTGKPLH67) which may enhance substrate recognition. This effect is most pronounced for K308A Cyp40. Also interesting is an increase in fluctuation of the catalytic residues H141 and H146 observed in the K227A mutant.D.p endotherm. It is significantly different for each of the mutants. ΔH provides an absolute measurement of the heat energy uptake and it is notable that WT has a significantly higher value  compared to ΔH for Dmut . The two single K to A mutants have intermediate values. This suggests that unfolding energy is related to the number of free lysines which are likely to form a strong network of hydrogen-bonded interactions with solvent. The difference in the melting enthalpy between WT and the three mutants are ΔΔH K227A = 5.5 kcal/mol, ΔΔH K308A = 10.0 kcal/mol, and ΔΔH Dmut = 16.3 kcal/mol. To a first approximation it would seem that the individual contributions of the single point mutations are additive with a Dmut value of 16.3 kcal/mol close to 15.5 kcal/mol, the combined values of the individual mutants. Reducing the hydrogen bonding network for the double mutant Cyp40 lowers the overall stiffness of the protein thus increasing entropy. This is a feature of thermophilic proteins which enables them to function at higher temperatures:ΔH is the normalized calorimetric enthalpy of un-folding (ΔH), measured as the area under the curve of the C value 16 kcal/molIn Fig. IV.The long simulation times of the WT and the three mutants show clear differences in the domain movements caused by site point mutations. K308A favours the stretched form with 43 Å separation between the cyclophilin and TPR domains distinguish stretched and compressed forms, while K227 about 50% of the conformers adopt a compressed form with a separation of 34 Å. These two forms can be rationalised by the domain motions identified by principal component analysis  averages for each domain are provided. Data files providing the means and variances of internal distances and correlations between residues are given.See


By analyzing the different phenotypes of two Chinese DFNA9 families with the same mutation located in the intervening region between the LCCL and vWFA domains of cochlin and testing the functional changes in the mutant cochlin, we investigated the different pathogeneses for mutations in LCCL and vWFA domains.in vitro cell experiments.Targeted next-generation sequencing for deafness-related genes was used to identify the mutation in the proband in family #208. The probands of family #208 and family #32 with the same p.C162Y mutation were followed for more than 3 years to evaluate the progression of hearing loss and vestibular dysfunction using pure-tone audiometry, caloric testing, electrocochleogram, vestibular-evoked myogenic potential, and video head-impulse test. The disruption of normal cleavage to produce secreted LCCL domain fragments and the tendency to form aggregations of mutant cochlins were tested by The two families showed different clinical symptoms. Family #32 was identified as having early-onset, progressive sensorineural hearing loss, similar to the symptoms in DFNA9 patients with cochlin mutations in the vWFA domain. The proband of family #208 endured late-onset recurrent paroxysmal vertigo attacks and progressively deteriorating hearing, similar to symptoms in those with cochlin mutations in the LCCL domain. We therefore suggest that the disrupted cleavage of the LCCL domain fragment is likely to cause vestibular dysfunction, and aggregation of mutant cochlin caused by mutations in the vWFA domain is responsible for early-onset hearing loss. The p.C162Y mutation causes either disruption of LCCL domain fragment cleavage or aggregation of mutant cochlin, resulting in the different phenotypes in the two families.This study demonstrates that DFNA9 families with the same genotype may have significantly different phenotypes. The mutation site in cochlin is related to the pathological mechanism underlying the different phenotypes. The full length cochlin contains 550 amino acids, consisting of N-terminal secretary signal peptide, LCCL domain , ivd1, vWFA1 domain (von Willebrand factor A), ivd2 and vWFA2 domain [ragments . CurrentL domain , and sixL domain –16. OnlyL domain . These mfunction –21.COCH gene varies and correlates with the location of the mutation in cochlin [The phenotype of DFNA9 patients due to mutations in  cochlin , 23. Fam cochlin . Up to t cochlin , 24. Ves cochlin . The var>A (p.C162Y) mutation located in exon 7 encoding ivd1 in COCH gene, the same mutation we previously reported in a DFNA9 family (#32) [in vitro experiments, we also investigated the mechanism underlying the pathogenesis due to the p.C162Y mutation. The relationship between genotype and pathological mechanism was then explored in association with information on DFNA9 families previously reported by others.Recently, we identified a Chinese DFNA9 family (#208) carrying a c.889Gly (#32) . The proMedical history and pedigree investigation were taken from all members in family #208 in 2012. Audiological tests and vestibular assessment were performed in 2012 and repeated in 2015 for the proband in family #208. The clinical information for the proband in family #32 was collected in 1998 and was used for comparison of phenotype between the two families. Written informed consent was obtained from all participants. This study was approved by the Medical Ethics Committee of Peking University First Hospital.forward primer 5’-CAAGATCTCCATTTGGGAAGG and reverse primer 5’-ACCCACTTGGCCTCCAAAG were used for amplification of exon 7 of COCH gene in the family members of this pedigree. PCR products were directly sequenced using an ABI 3730 sequencer.A blood sample was collected from the proband in family #208. Genomic DNA was extracted using the Qiagen blood DNA extraction kit . The targeted exome sequencing included 136 nuclear genes and mitochondrial genes known to be responsible for nonsyndromic or syndromic hearing loss. After the construction of an enrichment library using the MyGenostics GenCap enrichment kit  , the samPure-tone audiometry (PTA) for sensorineural hearing loss diagnosis was performed by standard audiometry  in a sound-proofed room according to clinical standards. Tympanometry was performed using a Madsen Otoflex 100 immittance system. The transient evoked otoacoustic emissions (TEOAEs) and distortion product otoacoustic emission (DPOAE) recordings were conducted using a Madsen Capella OAE System.Eye movements were recorded via the ICS Medical VG-30 infrared camera goggles and subsequent eye movements were analyzed using ICS Medical CHARTR ENG version 4.1 software. Spontaneous nystagmus testing was performed, while Random Saccade and Smooth Pursuit Eye movements were recorded as per nystagmography. Monitored eye movements were analyzed by computer and compared to age-matched and in-house validated normative data in terms of gain, latencies and accuracy of eye movements.This was performed with the patient supine and head anteroflexed at 30°. Irrigation with hot (50°C) and cold (24°C) water was maintained for 30 seconds with a between-stimuli interval of 5 minutes. Symmetry of peak angular slow-phase velocity was analyzed and unilateral canal paresis (UCP) was assumed at a side difference >15%.This was performed using SmartEP 3.86 equipment from Intelligent Hearing Systems . The exam was conducted using a tympanic electrode . The electrode was positioned close to the posteroinferior quadrant of the tympanic membrane. The reference electrode was positioned on the mastoid bone of the contralateral ear with the ground electrode on the forehead. Click stimuli and tone bursts at frequencies of 1000 Hz were utilized. A reproducible SP/AP  ratio of greater than 37% was considered abnormal for the click stimulus.This was recorded from the contracted cervical muscles (cVEMP) via the sternocleidomastoid muscle through stimulation by short-tone bursts . Surface EMG activities were recorded from Ag/AgCl surface electrodes ipsilaterally placed over the middle part of the muscle. The reference electrode was placed on the upper sternum and the ground electrode was positioned between the eyebrows. Using the ICS ChartrEP 200  and an insertphone type headphone, the recorded VEMPs were measured by the mean latencies of peaks p13 and n23 and the amplitudes of peak p13 to peak n23.ICS Impulse  was used in this test which recorded the corrective saccades when rapidly turning the head to the side of vestibular weakness. Approximately 20 horizontal head impulses were manually applied to each side with random timing and direction. The same eye was recorded simultaneously with video-oculography. Two data sets were obtained for each recording session to show the reliability of the calculated gains. Video images were analyzed online to calculate eye position and velocity using a pupil detection method. The criterion for a normal vestibular ocular reflex (VOR) velocity gain was that it should be 0.68 or greater, based on head impulse test (HIT) data from 12 previously published healthy asymptomatic subjects.>A (p.C162Y) mutation in COCH cDNA in the plasmid. The sequences in the wild-type and mutant COCH plasmids were confirmed by sequencing. Human kidney cell line HEK29 cells were divided into two groups: one was transfected with wild-type COCH plasmid, and the other was transfected with the mutant COCH plasmid.The mammalian expression plasmid containing the open reading frame of mouse COCH cDNA (NM_001135058) with a DDK-myc tag at the 3’ end was obtained from Origene . We also used a site-directed mutagenesis kit  to produce the c.889Ghttp://www.uniprot.org/).After 48 hours, the cultured cells were collected in the lysis buffer  supplemented with protease inhibitor cocktail  to produce cell lysate. After separating the lysate in SDS–PAGE, the gels were silver-stained and digested for LC-MS/MS analysis. The sample was separated by a 120-min gradient elution at a flow rate of 0.300 μL/min with the Dionex 3000 nano-HPLC system. The Q Exactive mass spectrometer was operated in the data-dependent acquisition mode using Xcalibur 2.1.3 software and a single full-scan mass spectrum was collected with an Orbitrap mass spectrometer  followed by 20 data-dependent MS/MS scans. The MS/MS spectra were searched against the Uniprot database  diluted in 5% non-fat milk/Tris-based saline/0.05% Tween 20 solution. The blotted antibody was recognized by 1:10,000 peroxidase-conjugated mouse anti-rat IgG antibody, and developed with the enhanced chemiluminescence method .The transfected cells were fixed in 4% paraformaldehyde. After washing three times, they were incubated in a blocking solution (10% goat serum/PBS/0.25% Triton X-100) for 1 h. The cells were then incubated with 1:400 mouse anti-FLAG antibody  in the blocking solution at 4°C overnight. After washing, the cells were incubated with 1:500 Alexa Fluor 488-conjugated goat anti-mouse IgG  in PBS at room temperature for 1 h. After washing three times and staining with DAPI, the slides were observed with a confocal microscope.Family #208 is a Chinese family with DFNA9 of autosomal dominant inheritance spanning four generations . Six famCOCH gene  initially presented with bilateral sensorineural hearing loss of a decreasing pattern involving low frequencies  associatCOCH gene, the affected members presented with early-onset hearing loss in the 2nd decade of life [In our hearing loss family #32 with the same mutation of c.889G>A (p.C162Y) in  of life . The proNormal videonystagmography in the neurotologic examination excluded the central causes of her vertigo. Caloric testing showed unilateral canal paresis on the left side with a side difference of 30% and the abnormalities returned to normal in the follow-up study, consistent with the self-reported degree of vertigo. For the electrocochleogram, the SP/AP ratios were 51% and 38% for left and right ears, respectively, and increased to 62% and 50%, respectively, on follow-up study , indicatMutations in cochlin are gain-of-function mutations resulting in DFNA9. Mutant cochlins may have either abnormal formation of an intramolecular disulfide bond or impaired post-translational cleavage to produce two shorter fragments –26. We tHEK293 cells were transfected with wild-type or c.889G>A (p.C162Y) mutant cochlin cDNA to examine the functional changes of the mutant cochlin. After transfection for 48 hours, the 10x concentrated cultured medium was subjected to SDS-PAGE and silver staining . The banThe cell lysate of transfected cells was harvested and separated in non-denatured polyacrylamide gel to conduct western blot using rat anti-cochlin antibody as the primary antibody . More diCochlin mutations and clinical findings in this study and in previous reports are summarized in COCH were followed up to observe the disease course of DFNA9. Based on the mutation sites in cochlin and their correlation with molecular defects in cochlin, we made the assumptions that vestibular dysfunction may be caused by disruption of normal cleavage to produce secreted LCCL domain fragments and that the cochlear symptoms may be closely related to aggregation of mutant cochlins. More DFNA9 families with different genotypes and phenotypes are needed to confirm the assumptions.In this study, the probands in the two families with the same mutation c.889G>A (p.C162Y) in Although the variation of genotype-phenotype correlation in DFNA9 is only limited to cochlear and vestibular dysfunctions, this is of clinical importance for differential diagnosis. The affected members in family #32 had the phenotype of deafness without any vestibular symptoms, quite different from the phenotype in family #208. Genetic background or environmental factors may be the factors that determine the presence of disrupted cleavage of secreted LCCL domain fragments or aggregation of the p.C162Y mutant cochlin. For example, the severe vertigo attacks and sudden deafness in family #208 always appeared in situations of feeling tired or working for a long period.Some DFNA9 cases with cochlin mutations in the LCCL domain had a phenotype similar to Ménière’s disease , 28. TheECochG is an electrophysiological test that can reflect elevation of inner ear pressure and distention of the basilar membrane. ECochG measures the ratio of summating potential to nerve action potential (SP/AP) in response to auditory stimuli. A SP/AP ratio greater than 37% is thought to be a sign of endolymphatic hydrops seen in Ménière’s disease , 30. TheThe otolithic input to the sternocleidomastoid principally arises from the saccule , 32, andS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI‐anchored, is highly N‐glycosylated and carries two O‐glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino‐proximal fasciclin 1 domain and was unaffected by removal of the GPI‐modification signal, a highly conserved N‐glycan or the deletion of predicted O‐glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)‐exit and plasma membrane localization of FLA4, with N‐glycosylation acting at the level of ER‐exit and O‐glycosylation influencing post‐secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy‐proximal fasciclin 1 domain and that its amino‐proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy‐proximal Fas1 domain and its normal cellular trafficking depends on N‐ and O‐glycosylation.Fasciclin‐like arabinogalactan proteins ( FLA4, a protein involved in root growth, salt tolerance and seed coat mucilage development consists of two fasciclin domains, multiple predicted glycosylation sites and a GPI‐anchoring motif. The functional significance of these features is elusive. Our structural functional dissection shows that the carboxy terminal fasciclin domain is crucial for FLA4 function while the amino‐terminal fasciclin domain, GPI‐anchoring N‐linked and O‐linked glycosylation are important for intracellular trafficking but not for function. In the 145 residues of the Fas1‐1 domain the 1001 genomes database yielded 11 non‐synonymous DNA polymorphisms giving rise to eight divergent aminoacid substitutions in the FLA4 protein sequence, while in the Fas1‐2 domain there were only two highly conserved polymorphisms  domain , the construct UBQ:F4C∆Fas1‐1∆PR1.5 in which four more residues including the predicted N‐glycosylation site N207 were deleted, was functional in only 50% of tested transgenic lines (n = 10). Removal of seven more aminoacid residues in the F4C∆Fas1‐1∆PR1.6 construct completely abolished genetic function  residues in oligomannosidic but not in complex N‐glycans. Treatment of F4C extract with Endo‐H induced a molecular weight shift of a minor fraction of F4C resulting in a weak sharp band of approximately 66 kDa  cleaves the glycosidic links between the innermost GlcNAc residue and asparagine of N‐glycoproteins in both oligomannosidic and complex N‐glycans, however, the enzyme is blocked by α(1–3) fucosylation at the innermost GlcNAc of complex N‐glycans. Treatment of F4C with PNGase F resulted in an electrophoretic pattern identical to the one seen after Endo‐H digestion. This suggested that most complex N‐glycans on F4C might be α(1–3) fucosylated. To test this hypothesis we crossed FLA4:F4C with the GDP‐L‐fucose biosynthesis defective mur1‐2 mutant  fucosylated complex N‐glycans contributed around 13 kDa to the major fraction of F4C. Variably sized additional post‐translational modifications contributing 7–17 kDa were only present on the Endo‐H insensitive F4C fraction. We next tested the N‐glycosylation of two amino‐proximally truncated versions of F4C namely F4C∆Fas1‐1 and F4C∆Fas1‐1∆PR1.6. Like full‐length F4C, a small fraction of F4C∆Fas1‐1 experienced a downward shift in apparent molecular weight after digestion with Endo‐H and PNGaseF  or H2 domains  the most conserved position for N‐glycosylation in Fas1 domains was 31–39 residues upstream the H1 domain. We detected this predicted glycosylation site in every single Fas1 domain FLA except FLA19 and in every tandem Fas1 domain FLA except FLA15 to FLA18, FLA20 and Fas1‐2 in FLA2. (Table N‐glycosylation at residue N207 in the Fas1‐2 domain by replacing it by glutamine (Q) in the full‐length fusion protein to generate the transgene UBQ:F4C_N207Q. The relative abundance of the resulting protein was comparable to the wild‐type, suggesting that this post‐translational modification was not crucial for either protein stability or turnover in vivo  proline hydroxylation and O‐galactosylation. Classical AGPs contain (XO)≥2 repeats in their peptide sequence with X typically a Ser, Thr or Ala residue preceding hydroxyproline  , only the heterogeneous fraction but not the discrete 66 kDa band reacted with LM14 and JIM13  contains two proline doublets (APP and SPP). Comparing putative s Figure . In the A Figure . On westA Figure a. ConsisA Figure . The redA Figure b. SimilaA Figure c. Co‐locA Figure e. LM14 rA Figure e. In ours Figure  indicatisos5 root growth and salt tolerance phenotype and previously its seed coat mucilage phenotype  core fucosylated complex N‐glycans and to a small extent with oligomannosidic glycans and that it carries AGP‐type O‐glycan epitopes. The most highly conserved N‐glycosylation site in the FLA family is at the amino‐proximal margin of the Fas1 domains and might be a key determinant for the subcellular trafficking within this group of proteins. Deletion of this glycosylation site in FLA4 led to predominant retention of the protein in the ER which is an example how an individual N‐glycan in multiple‐site glycosylated proteins can crucially affect protein localization. Individual N‐glycans can function in roles such as ligand binding, protein folding or ER‐exit. Similar to our results the substitution of two conserved N‐glycosylation sites N43 and N122 of the S‐locus receptor kinase (SRKb) led to ER‐retention without affecting genetic function  that was released by PNGase F treatment and that appeared 7–17 kDa larger than the 66 kDa fragment, corresponded to the O‐glycosylated and de‐N‐glycosylated fraction of F4C. The relatively homogeneous molecular weight of F4C around 91 kDa suggests that mature native FLA4 might have a molecular weight of around 62 kDa including N‐ and O‐glycosylation. This number is in good agreement with data for poplar PtFLA6  and Joseph Kieber , respectively. Growth conditions and root measurement and salt treatment were as previously described  , followed by most of the FLA4 coding region (At3g46550: residues 29–420) and the HSP18.2 (At5g59720) terminator  we transformed sos5‐1 plants by floral dip  was crossed with transformant lines to generate a homozygous sos5‐2−/− (Col‐0/Col gl) background to assess the potential genetic interactions of the endogenous sos5‐1 mutant protein with the recombinant FLA4‐citrin (F4C) protein or its variants.The FLA4:F4C construct was based on pGREEN179 . Before electrophoresis the samples were heated at 95°C for 5 min in the presence of loading buffer . The proteins were separated on an 8.5% SDS‐PAGE according to  followed by horseradish peroxidase‐conjugated anti‐rabbit secondary antibody  and Amersham ECL Prime Western Blotting Detection Reagent . Blots were exposed to X‐ray film  and recorded on a ChemiDoc XRS Imager (Bio‐Rad). Endo H (New England BioLabs) and PNGase F (New England BioLabs) digestions were performed as previously described  for 2 min at 30 minLaemmli,  and elecm Tris–HCl pH 8.0, 400 mm sucrose, 20 mm EDTA, 1 mm DTT and 1% proteinase inhibitor cocktail . The insoluble debris was removed by centrifugation at 300 g for 10 min at 4°C. The 300 g cell wall pellet is washed once. Microsomal membranes were separated from soluble proteins by centrifugation at 100 000 g for 1 h at 4°C. Two‐phase partitioning using Triton X‐114 and GPI‐PLC digest was performed as previously described  in a μMACS magnetic separator. We eluted the protein from the column with 80 μl of SDS‐PAGE loading buffer  to purify citrin tagged protein according to manufacturer's instruction with the following modifications. Binding of 400 μl extract to 50 μl anti‐GFP‐tagged magnetic beads was done at 4°C for 20 h. Washing was done with 6 × 200 μl of extraction buffer including 0.1% SDS and a final rinse with 100 μl wash buffer . We analysed the peptide mixture as described ‐galactanase as described  (1:10 000) followed by chemiluminescence detection . In this study the AGP antibodies LM14  freeware and mounted with Adobe Photoshop. Unless otherwise indicated imaging settings and contrast operations were identical between compared specimens.We mounted seedlings in dHhttp://chibba.agtec.uga.edu/duplication/). Before sequence alignment, using Clustal Omega     suppresses F4C abundance.Click here for additional data file.Figure S13. The clustered proline residues in the two PR domains are not required for FLA4 function in root growth and NaCl tolerance.Click here for additional data file.Table S1. Conserved N‐glycosylation sites at N‐terminal margin of Fas1 domains in Arabidopsis FLAs.Click here for additional data file.Table S2. Oligonucleotide primers used in this study.Click here for additional data file. Click here for additional data file.


Mitochondria are dynamic organelles that continually adapt their morphology by fusion and fission events. An imbalance between fusion and fission has been linked to major neurodegenerative diseases, including Huntington’s, Alzheimer’s, and Parkinson’s diseases. A member of the Dynamin superfamily, dynamin-related protein 1 (DRP1), a dynamin-related GTPase, is required for mitochondrial membrane fission. Self-assembly of DRP1 into oligomers in a GTP-dependent manner likely drives the division process. We show here that DRP1 self-assembles in two ways: i) in the presence of the non-hydrolysable GTP analog GMP-PNP into spiral-like structures of ~36 nm diameter; and ii) in the presence of GTP into rings composed of 13−18 monomers. The most abundant rings were composed of 16 monomers and had an outer and inner ring diameter of ~30 nm and ~20 nm, respectively. Three-dimensional analysis was performed with rings containing 16 monomers. The single-particle cryo-electron microscopy map of the 16 monomer DRP1 rings suggests a side-by-side assembly of the monomer with the membrane in a parallel fashion. The inner ring diameter of 20 nm is insufficient to allow four membranes to exist as separate entities. Furthermore, we observed that mitochondria were tubulated upon incubation with DRP1 protein in vitro. The tubes had a diameter of ~ 30nm and were decorated with protein densities. These findings suggest DRP1 tubulates mitochondria, and that additional steps may be required for final mitochondrial fission. Mitochondria are dynamic organelles that frequently divide and fuse . MitochoMembers of the large GTPase dynamin superfamily, mechano-enzymes are responsible for mitochondrial fusion and fission . DespiteArabidopsis thaliana DRP1 indicates the N and C termini of the GTPase domain form a BSE domain . Th. Th67]. The difference between human DRP1 and Yeast Dnm1 is particularly striking given their high amino acid similarity. In the absence of lipids, human DRP1 helices form a 1-start turn similar to dynamin 1, while Yeast Dnm1 assembles into 2-start helices in the presence of lipids . In the DRP1 is the main mediator of mitochondrial fission. Similar to earlier studies, DRP1 like dynamin is composed of a mechanochemical core of GTPase, a middle, and GTPase effector domain regions. In place of the pleckstrin homology domain in dynamin however, DRP1 contains an unstructured variable domain whose function is not yet fully resolved . StudiesOne striking difference between the molecular arrangement of human DRP1 and dynamin 1 is the arrangement of the domains with respect to the center of the oligomers, presumably reflecting a ~90° rotation of the monomer towards the membrane. For Yeast Dnm1, the GTPase domain is located on the outside, the PH domain closer to the membrane and the GED/MID domain and stalk, in the middle , 22, 42.DRP1 and Yeast Dnm1 lack the predicted PH lipid-binding domain of dynamin 1. In its place, DRP1 and Yeast Dnm1 harbor an uncharacterized B-insert or VD domain. Similar to the previous study of Yeast Dnm1, we were unable to resolve the position of the B-insert in our 3D structure . There iUpregulation of DRP1 plays a critical role in mitochondria on a string (MOAS) and tubulation phenotype, which are often found in pathological conditions such as stroke, brain trauma and altered brain metabolism associated with progression of Alzheimer’s disease (AD) –73. SimiIn the current model of membrane constriction by dynamin, Yeast Dnm1, and DRP1, GTP hydrolysis mediates constriction of the oligomers and brings the membranes that are meant to be fused closer together , 42, 54.In the presence of GTP or GNP-PNP in vitro, DRP1 tubulates mitochondria by forming rings or short helices around the mitochondria, to mediate fission . Such obOur model proposes multiple sites on the mitochondria where DRP1 binds, forming helices to tubulate the organelle. The final constriction for fission may, however, require a GTP-dependent oligomerization of a ring-like assembly . DRP1 meS1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 FigCurrent model of dynamin self-assembly. Dynamin dimers are formed through the interactions of their stalk domains (shown in blue). Two dimers assemble by the interaction of the GTPase domains (shown in green). The PH domain (shown in pink) binds and inserts into the lipid membrane . B) Our proposed model for DRP1 self-assembly. The DRP1 monomers assemble in a parallel fashion with respect to the membrane, stalk and GTPase domains, which are equidistant from the membrane. The interaction of DRP1 with the lipid membrane is shown in plan-view (left cartoon) and side-view (right cartoon).A) (DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


A decline ratio map of flow stresses was proposed and divided into five deformation domains, in which the flow stress behavior was correlated with different microstructures and dynamic softening mechanisms. The results reveal that the dynamic recovery is the sole softening mechanism at temperatures of 300 to 400 °C with various strain rates and at temperatures of 400 to 450 °C with strain rates between 1 and 10 s−1. The level of dynamic recovery increases with increasing temperature and with decreasing strain rate. At the high deformation temperature of 450 °C with strain rates of 0.001 to 0.1 s−1, a partially recrystallized microstructure was observed, and the dynamic recrystallization (DRX) provided an alternative softening mechanism. Two kinds of DRX might operate at the high temperature, in which discontinuous dynamic recrystallization was involved at higher strain rates and continuous dynamic recrystallization was implied at lower strain rates.The hot deformation behavior and microstructural evolution of an Al-Zn-Mg-Cu (7150) alloy was studied during hot compression at various temperatures (300 to 450 °C) and strain rates (0.001 to 10 s A good Dynamic recovery (DRV) and dynamic recrystallization (DRX) are the typical softening mechanisms in metals and alloys during deformation at elevated temperatures ,4. AlumiMoreover, during the plastic deformation process, most of the deformation work is converted to heat . Plasticet al. [To date, the previous studies have been mainly on determining the Zener–Hollomon parameter (Z), the activation energy for hot deformation and various material constants in the constitutive equations for describing the hot deformation behavior of 7xxx aluminum alloys ,26. The et al.  measuredet al.  proposedIn the present paper, the hot deformation behavior of an Al-Zn-Mg-Cu alloy (7150) is studied using uniaxial compression tests performed at various temperatures and strain rates. The microstructural evolution during the hot deformation is investigated in order to understand the various dynamic softening mechanisms at different deformation conditions. Based on the different decline levels in flow stresses with respect to peak stresses, a decline ratio map of flow stresses is proposed to correlate flow stress behavior with dynamic softening mechanisms at various deformation conditions.2.3. The cast ingots were homogenized at 465 °C for 24 h, followed by a direct water quenching. Cylindrical samples 10 mm in diameter and 15 mm long were machined from the homogenized ingots. Uniaxial compression tests were conducted on a Gleeble 3800 thermomechanical simulation unit at strain rates of 0.001, 0.01, 0.1, 1 and 10 s−1 and deformation temperatures of 300, 350, 400 and 450 °C, respectively. During the tests on the Gleeble unit, the samples were heated to the desirable deformation temperature at a heating rate of 10 °C/s and held for 3 min to ensure a homogenous temperature distribution through the samples. The samples were deformed to a total true strain of 0.8 and then immediately water-quenched to maintain the microstructure at the deformation temperature. Some selected samples were also deformed to different true strains of 0.1, 0.3 and 0.5, followed by water quenching, in order to investigate the microstructural evolution during the deformation process.The experiments were conducted on an Al-Zn-Mg-Cu alloy. The chemical composition of the experimental alloy analyzed by an optical emission spectrometer is given in 2 with a scanning step size of 3.0 μm was selected. In addition, EBSD analysis was performed to measure the grain size of the as-homogenized sample using the linear intercept method [2 with a scanning step size of 5.0 μm was selected for the sample. Samples for TEM observation were mechanically ground to the thicknesses of 35–60 μm followed by electropolish in a twin-jet polishing unit, which was operated at 15 V and −20 °C using a 30% nitric acid and 70% methanol solution. The disks were observed in a transmission electron microscope , operated at 200 kV.The microstructures of the as-cast and as-homogenized materials were examined prior to hot deformation under an optical microscope. The intermetallic phases were identified using the electron backscattered diffraction (EBSD) technique and the energy dispersive spectroscope (EDS) with a scanning electron microscope . All deformed samples were sectioned parallel to the compression axis along the centerline and then polished and etched in Keller solution for optical microscope observation. Additionally, some deformed and as-homogenized samples were selected for EBSD analysis. In the EBSD analysis, the boundaries of both the grains and subgrains are defined as low angle boundaries (LABs), medium angle boundaries (MABs) and high angle boundaries (HABs) for which the misorientation angles of boundaries occur in the ranges of 1°−5°, 5°−15° and greater than 15°, respectively . The stet method ; a surfa3.3.1.2(η), Al6(FeCu), Mg2Si, Al2CuMg(S) and Al2Cu(θ) intermetallic phases, distributed in the interdendritic boundaries. The Al2CuMg phase was observed to be attached with the MgZn2 phase (lamellar eutectic morphology) and Al2Cu phase (isolated droplet morphology) in the aluminum matrix. These phases have been commonly observed in as-cast 7xxx aluminum alloys [2 and Al2Cu phases were completely dissolved into the aluminum matrix, while the coarse particles of Al6(FeCu), Mg2Si and Al2CuMg were only dissolved partially, and some of them were retained in the matrix  and σp is the value of peak stress.where σ−1, the decline ratio map of flow stresses at various deformation conditions for the 7150 alloy is presented in −1, the decline ratio value again decreased. This map can be divided into five deformation domains:According to the experimental data obtained under deformation at temperatures of 300 to 450 °C and at strain rates of 0.001 to 10 sdR between −13% and −6%, when the deformation was performed at a high strain rate of 10 s−1.Domain I illustrates a significant decrease in flow stress after the peak stress with the values of −1, as well as at higher temperatures of 400 to 450 °C with a strain rate of 1 s−1. Either a slight decrease or a continuous increase in flow stress after the peak stress is observed, and the values of dR vary between −8% and 17%.Domain II represents the deformation conditions at temperatures of 300 to 400 °C with strain rates of 0.1 to 1 s−1. A significant decline in flow stress is observed with the values of dR between −28% and −10%.Domain III represents the deformation performed at temperatures of 300 to 400 °C with low strain rates of 0.001 to 0.01 sdR approximately −12%, during the deformation at a high temperature of 450 °C with strain rates of 0.01 to 0.1 s−1. It is of interest to notice that only a single peak following a steady-state flow was observed in the flow stress curves of Domains III and IV . −1, the original grains were severely torn and broke into irregular deformation bands , the boundaries with misorientation angles between one and 15° decreased dramatically and were associated with a rapid increase in high angle boundaries. This result suggests that DRX occurred accompanied with an increasing fraction of high angle boundaries, as a result of the formation of recrystallized grains. This is in good agreement with the microstructure observation in Based on the microstructure observation of samples deformed at various deformation conditions, DRV generally occurred during hot deformation in Domains I, II and III, and DRX took place when the deformation was conducted in Domains IV and V. For a further investigation of the dynamic softening mechanisms during the hot deformation processes, a quantitative measurement of misorientation angle distributions of boundaries for samples deformed at different deformation conditions was carried out using the EBSD technique, and the results are illustrated in 4.The decline ratio map of flow stress as a function of temperature and strain rate is divided into five domains, which can be utilized to study the relationship between flow stress behavior and various dynamic softening mechanisms in the Al-Zn-Mg-Cu alloy (7150) during hot deformation at different deformation temperatures and strain rates.−1 (Domain I), the sample temperature rose continuously with increasing strain at each target deformation temperature. The maximum overheating temperature reached 22 °C at the target temperature of 450 °C and increased as high as 35 °C at the target temperature of 300 °C. Those results suggest that the heat release during deformation at the high strain rate of 10 s−1 plays an important role in the decrease of flow stress, leading to a significant thermal softening, as confirmed by other researchers [In Domain I, the microstructure observation confirms that DRV is the main dynamic softening mechanism during hot deformation . Althougearchers –18.−1 and 0.1 s−1, the sample temperatures kept close to the target temperatures (within 2 °C). Thus, it is reasonable to consider that the deformation was under an isothermal process. Even at the intermediate strain rate of 1 s−1, the temperature increase was generally limited within several degrees (4 to 8 °C). Therefore, it is believed that the thermal softening effect in this case plays a minor role in the flow softening.However, in other domains, when the deformation was carried out at low strain rates between 0.001 s−1) or a slight decrease at a lower strain rate (0.1 s−1). It is evident that the effect of work hardening was dominant during hot deformation at the strain rate of 1 s−1, whereas the dynamic softening slightly overcame the work hardening at the lower strain rate of 0.1 s−1.Domain II represents a typical recovered structure , indicat−1, a large number of spherical and rod-shaped precipitates with an average size of 60 nm appeared  micrograph gives an overview of the recrystallized grain and its surroundings, and four bright-field TEM images show the enlarged views in the corresponding areas. The misorientation angles of the boundary were determined by measuring the shift of the Kikuchi line intersections on the crossing boundary under the TEM mode [Moreover, in Domain IV, the deformed microstructure reveals a partially recrystallized microstructure at the serrated grain boundaries , which pTEM mode ,23.It can be seen that the recrystallized grain C is comprised of a partially bulged original grain boundary  and a br−1 in Domain V, the bulging of grain boundaries and the formation of recrystallized grains by DDRX were not observed in −1. At a true strain of 0.3, subgrains were well developed inside the original grains  during hot deformation. Recovered structures are generally observed in the samples deformed in Domains I, II and III. In these cases, DRV is the sole dynamic softening mechanism during hot deformation, which is commonly reported in 7xxx aluminum alloys ,22,46,51−1, where DRX occurs during hot deformation, leading to softening and the reconstituting of the microstructure [Recently, several works have been done to optimize the hot working processes of 7xxx aluminum alloys using the processing maps –55. Corrtructure . This istructure –55. On ttructure ,54,55. T5.−1. The level of flow stress decreases with an increasing deformation temperature and with a decreasing strain rate.The hot deformation behavior of an Al-Zn-Mg-Cu alloy (7150) is systematically investigated at temperatures of 300 to 450 °C and strain rates of 0.001 to 10 sThe decline ratio map of flow stresses is proposed and further divided into five deformation domains, in which the flow stress behavior is correlated with different microstructures and dynamic softening mechanisms at various deformation conditions.−1. The level of dynamic recovery increases with increasing temperature and with decreasing strain rate.The dynamic recovery is the sole softening mechanism of the alloy when the hot deformation is performed at temperatures of 300 to 400 °C with various strain rates, as well as at temperatures of 400 to 450°C with strain rates between one and 10 s−1, the heat release during deformation plays an important role in the decrease of flow stress, leading to a significant thermal softening. At low strain rates of 0.001 to 0.01 s−1 and temperatures of 300 to 400 °C, the coalescence of dynamic precipitation, accompanied by enhanced dynamic recovery, results in a considerable decline in flow stress.At the high strain rate of 10 s−1, providing an alternative softening mechanism. The results reveal that two kinds of DRX may operate at the high temperature, in which DDRX is involved at the higher strain rates and CDRX is implied at the lower strain rates.The dynamic recrystallization takes place when the deformation is conducted at the high deformation temperature of 450 °C with strain rates of 0.001 to 0.1 s