Vibrio coralliilyticus cause the bleaching of corals due to decomposition of symbiotic microalgae. The V. coralliilyticus strain ATCC BAA-450 (Vc450) encodes a type III secretion system (T3SS). The gene cluster also encodes a protein (locus tag VIC_001052) with sequence homology to the T3SS-secreted nodulation proteins NopE1 and NopE2 of Bradyrhizobium japonicum (USDA110). VIC_001052 has been shown to undergo auto-cleavage in the presence of Ca2+ similar to the NopE proteins. We have studied the hitherto unknown secondary structure, Ca2+-binding affinity and stoichiometry of the “metal ion-inducible autocleavage” (MIIA) domain of VIC_001052 which does not possess a classical Ca2+-binding motif. CD and fluorescence spectroscopy revealed that the MIIA domain is largely intrinsically disordered. Binding of Ca2+ and other di- and trivalent cations induced secondary structure and hydrophobic packing after partial neutralization of the highly negatively charged MIIA domain. Mass spectrometry and isothermal titration calorimetry showed two Ca2+-binding sites which promote structure formation with a total binding enthalpy of −110 kJ mol−1 at a low micromolar Kd. Putative binding motifs were identified by sequence similarity to EF-hand domains and their structure analyzed by molecular dynamics simulations. The stoichiometric Ca2+-dependent induction of structure correlated with catalytic activity and may provide a “host-sensing” mechanism that is shared among pathogens that use a T3SS for efficient secretion of disordered proteins.Strains of the Gram-negative bacterium  The rise of intracellular calcium levels is also a typical response in both pathogenic and mutually beneficial plant/microbe interactions4. Recently, a potentially virulence-related gene of the prokaryotic coral pathogen Vibrio coralliilyticus  has been shown to code for a protein that carries a Ca2+-dependent “metal ion-inducible autocleavage” (MIIA) domain5. The gene is located in a cluster that encodes a type III secretion system (T3SS). The latter is expressed in a highly controlled manner in a large variety of Gram-negative plant and animal bacterial pathogens6. VIC_001052 was discovered by the sequence similarity of its MIIA domain with corresponding domains in NopE1 and NopE2 proteins from Bradyrhizobium japonicum USDA110  in one case but to “bleaching” of a coral (Pocillopora damicornis) in the other9, which might result from an attack on the algae10.The regulation of protein function by Ca110 Fig. , a benef2+-dependent autocatalytic activity in the protein domains of the distantly related bacteria raises the question how Ca2+ ions regulate the diverse biological functions by presumably similar structural transitions. However, neither the stoichiometry nor the structural basis of Ca2+ binding are known. At the molecular level, the most common Ca2+-binding protein domain is the EF-hand which exhibits a well-defined helix-turn-helix structure11 originally described for parvalbumin12. The canonical helix-turn-helix motif typically undergoes tertiary structural changes upon Ca2+ binding but maintains its characteristic secondary structure elements also in the metal-free state15. Related Ca2+-binding motifs have been described that are only partially structured16 or virtually unstructured17 in the absence of Ca2+ but fold into an EF-hand upon Ca2+ binding. The structural features of the MIIA domain from V. coralliilyticus are unexplored and the molecular mechanism of enzyme activity is unknown. With its large content of charged amino acids , the MIIA domain resembles intrinsically disordered proteins (IDPs) as revealed by IDP predictors  spectroscopy. In the absence of metal ions, the D116A-MIIA domain exhibited a negative CD signal at 198 nm, typical of a largely disordered peptide backbone . This reaction is inhibited by the D116A replacement (D116A-MIIA domain)one Fig. . In the ion Fig. . In addials Fig. .Figure 12+-induced structure formation implies an increase in secondary structure-specific H-bonds and possibly other intramolecular contacts which are not necessarily restricted to the actual metal ion-coordinating residues. We have used thermal denaturation to determine the free enthalpy that accompanies disruption of intramolecular contacts during unfolding in the metal-bound state of the D116A-MIIA domain. Figure 2+-bound protein between 18 °C and 92 °C. The transition from the folded to the unfolded state is clearly visible in the spectra and has been analyzed by fitting the integrated spectral changes by a two-state equilibrium unfolding model . The resulting sigmoidal could be well reproduced with enthalpic and entropic contributions of ΔH = 153 kJ mol−1 (±10 kJ) and ΔS = 452 J mol−1 (±30 J mol−1), respectively, with a transition midpoint temperature Tm = 66 °C . As shown in Fig. 2+-binding to the D116A-MIIA domain occurred exothermally with a molar ratio of two Ca2+-ions per protein. The data were modeled (VPITC-Origin Software) with two binding sites, exhibiting binding enthalpies between −57 and −54 kJ mol−1 and binding entropies between −38 and −59 J mol−1 K−1, corresponding to Kd ≤ 3.6 µM (±1.2 µM). The negative binding entropy agreed well with the demonstrated increase in secondary structure upon Ca2+.The spectroscopic data revealed the pronounced structural influence of Ca2+ or Eu3+. Figure 2+- and Eu3+-bound states. All three samples show the same base peak at m/z = 2981,1 representing the metal-free D116A-MIIA domain at the predominant +7 charge state. In the presence of both, Ca2+ and Eu3+, two additional peaks appear in the spectra found at m/z = 2986,5 and m/z = 2991,9 for the Ca2+ containing solution and at m/z = 3002,4 and m/z = 3023,9 for the Eu3+ containing solution, respectively. The observed mass shifts agree with the expected shift caused by one or two of the respective metal cations. The mass spectra clearly demonstrate the presence of two prominent metal-binding sites for Ca2+ in line with the ITC experiment. Moreover, the preservation of the stoichiometry upon substitution of Eu3+ for Ca2+ supports the formation of a structurally well-defined cation-binding site which, similar to other Ca2+-binding proteins, also accepts Eu3+ as a surrogate for Ca2+ 22. Given the consistency of the effects of Ca2+ and Eu3+ on the mass spectra and on the CD curves  which could be used as intrinsic fluorescent monitors of metal-ion-induced folding. The fluorescence emission maximum of both the wild type and the D116A-MIIA domain shifted from 357 to 349 nm when Ca2+ was added  after the spectroscopic experiments to reveal the appearance of the N- and C-terminal cleavage fragments SN and SC, respectively. In agreement with efficient cleavage at 100 µM Ca2+, also the 100 µM Ca2+-induced emission shift was of the same size as that evoked by 500 µM . In both cases, the metal ions induced hydrophobic clustering of the remaining single tryptophan Fig.  and exhides Fig. . The repnts Fig.  but suppnts Fig. .Figure 42+ induced hydrophobic packing less efficiently since 100 µM did not achieve full tryptophan emission shift in MIIA73-223  and 73 to 218 (MIIA73-218). Both truncated derivatives folded with the above studied metal ions as seen by their CD spectra Fig.  and both223 Fig.  and was 223 Fig. . The two223 Fig.  whereas 2+ was still able to support the typical spectral phenotype of the MIIA domain, it is unlikely that a Ca2+ binding site is present in the 25 C-terminal residues. The stoichiometry of only two cations binding to the MIIA domain despite the large excess of a negative charge hints at the presence of specific binding site structures, rather than non-specific electrostatic association of the investigated cations with disordered protein regions. The MIIA domain does not harbor a canonical Ca2+-binding motif. However, less restrictive sequences derived from the original 12 amino acid-long EF-hand Ca2+-binding loop .The above spectroscopic data show that the 25 C-terminal amino acids are not essential for folding but do regulate the efficiency of metal-ion-induced structure formation and catalysis. Since Ca2+-dependent folding potential may exist within the MIIA domain. Sequences with similarity to canonical Ca2+-binding loops are reasonable candidates for such a study. However, the simulations did not intend to prove or disprove binding at specific sites. Instead, we aimed at revealing the potential of short anionic stretches of amino acids in the wt-MIIA domain to reduce backbone flexibility upon Ca2+-coordination. The simulations were performed with a structure that was generated by homology modeling of the putative Ca2+-binding site 1 of the MIIA domain with human calmodulin EF-hand 3. MD simulations were restricted to the sixteen residues 176 to 191 (MIIA-domain numbering). Among these, the 178–189 segment exhibits the highest similarity to a canonical twelve amino acid-long Ca2+-binding loop. During 400 ns of total simulation time, Ca2+ stayed bound to the side chain carboxyls of four residues, namely D178, D180, E186, and E189 fluctuating between monodentate and bidentate coordination with Ca2+  exhibited a stable bidentate metal coordination as is typical of EF-hands.Due to the absence of experimentally determined structures of the MIIA domain or the related NopE proteins, we used MD simulations to investigate whether a local Caa2+ Fig. . At the ite Fig. , inset rvia the type III secretion system (T3SS) requires complete unfolding of the secreted proteins. Intrinsic disorder is over-represented in pathogenic proteins and may have evolved in favor of secretion efficiency but also as a mechanism to target multiple host proteins and to evade immune defense24. However,  folding may be required for biological function such as proteolytic activity of a bacterial pathogen in the host. Here, we have discovered intrinsic disorder in the ion-induced auto cleavage domain (MIIA) of the coral pathogen protein VIC_001052 from Vibrio coralliilyticus. The location of the VIC_protein in a cluster that contains a T3SS and the sequence similarity of the wt-MIIA domain with regions of the T3SS-secreted NopE proteins of Rhizobium sp. suggests that the VIC_protein is secreted in the same way. On a molecular level, it may act as a pathogenic protein in the coral cell or its endosymbiont by coupling metal-ion-binding structural transitions in a similar manner as the NopE proteins which are involved in symbiosis. However, the low secondary structure content of the MIIA domain was not expected, because the previously demonstrated autocatalytic activity implies structure at the catalytic and most likely also at the substrate site . Thus, the MIIA domain features properties that are increasingly recognized for intrinsically disordered regions in eukaryotic proteins that are involved in signaling, where structural substates are populated in diverse ligand protein interactions25 or upon cation binding26. Here, we have shown that the apparently conflicting features of disorder and catalytic activity can be reconciled by the Ca2+-induced folding of the MIIA domain. This differs from the more common role of Ca2+ as an allosteric regulator that repositions preformed secondary structure elements in Ca2+-regulated proteins, thereby promoting activity by more subtle tertiary structural changes27.Host infection by Gram-negative bacteria 2+ ions bind to the MIIA domain with enthalpies of ~−55 kJ mol−1 and a macroscopic Kd in the high nanomolar to low micromolar range. In contrast to canonical EF-hands, where Ca2+ binding is driven by entropy with only small enthalpic contributions30, the Ca2+-induced secondary structure formation in the MIIA domain is accompanied by an entropy decrease. The gain of favorable interactions within the metal ion-induced folded state contributes to an overall enthalpy-driven and thus exothermic reaction. The thermal stability of the folded state of the MIIA domain is remarkably high and similar to that of the partially urea-destabilized 79 amino acids long N-terminal calmodulin domain (N-Cam) in the presence of 100–200 µM Ca2+ 29. The binding enthalpy of about −110 kJ for the two Ca2+ ions implies an unfolding enthalpy greater than +110 kJ. This agrees with the estimated value of 153 kJ from thermal unfolding which further indicates the additional thermal unfolding of residual structure outside the Ca2+-binding sites. However, the electrostatic interactions that underlie ion binding must be rather site-specific, because Ca2+ and Eu3+ bound with the same 2:1 stoichiometry despite their different charges. Eu3+ and Tb3+ also evoked secondary structural changes similar to Ca2+. Therefore, we believe that the lanthanides occupy the same two sites in the wt-MIIA domain as Ca2+ in analogy to proteins for which lanthanides have been used to probe Ca2+ binding sites22. It is remarkable that, despite the low structure-forming propensity of the MIIA domain, ion selectivity for Ca2+ and Mn2+ is clearly observed in both secondary structure formation and catalytic activity. The coupling of metal ion binding to folding in the MIIA domain may be responsible for efficient ion-discrimination of the MIIA domain as compared to the more subtle ion-specific structural differences in canonical EF-hand proteins. For example, Mg2+ recognizes and stabilizes the EF-hand in the N-terminal domain of human calmodulin (N-CaM), a process which has been proposed to confer a regulatory role to Mg2+ in Ca2+signaling29. In the case of the MIIA domain, Mg2+ does not stabilize any secondary structure. Nevertheless, there are intriguing similarities such as the interaction of the MIIA domain with Mn2+ which also binds to EF-hands29.We have shown that Ca2+ is similar to that of the intrinsically disordered protein prothymosin, an unstructured 110 amino acid-long protein that adopts secondary structure and undergoes compaction in the presence of Zn2+ but not Ca2+ or Mn2+ 31. Whereas prothymosin is entirely devoid of hydrophobic residues, the wt-MIIA domain folds in accordance with a hydrophobic collapse that leads to reduced solvent accessibility of two tryptophan residues. A similar Ca2+-induced lowering of the accessibility of hydrophobic residues has also been reported for the Nereis sarcoplasmic Ca2+-binding protein (NSCP)17, a non-canonical EF-hand protein with larger structural flexibility than calmodulins but lower intrinsic disorder than the MIIA domain. The hydrophobic clustering upon ion-induced folding of the wt-MIIA domain is probably crucial in preserving ion-regulated function This may explain the appearance of Zn2+-induced autocatalysis in the W223F and W172F mutants after prolonged incubation with the cation  was observed. Both criteria identified residues 178–189 where charge neutralization would reduce hydrophilicity near W172 and indirectly by loop formation which could bring W172 in the vicinity of conserved non-polar residues on the C-terminal side of E189 . This behavior may reflect a fundamental molecular property of the pathogenic or symbiosis-mediating proteins. They may harbor “close to canonical” Ca2+-binding motifs which are destabilized by the replacement of structure-promoting residues by amino acids that favor disorder. Thereby, only the additional cation binding energy promotes structural stability in these regions which is in line with recent views of the role of protein disorder in structure function relationships32. The MD result shows that such a replacement could well preserve cation binding but at the same time it would enhance translocation efficiency across membranes by lowering the unfolding energy.The proposed hydrophobicity-based structure formation in the Ca189 Fig. . Our MD 2+-induced secondary structure formation in the MIIA domain is likely to provide a sensor/signaling mechanism during early infection. The high sequence similarity with NopE proteins suggests that a common molecular “post secretion host-sensing” conformational switch may exist in both beneficial and pathogenic T3SS-dependently secreted proteins. Ca2+-dependent disorder-to-order transitions are typical of numerous proteins secreted by type 1 secretion systems34. It is thus remarkable that the MIIA domain of the coral pathogen resides in a T3SS gene cluster, but exhibits a similar Ca2+-dependent transition from a disordered secretion-adapted state to a more compact structure with hydrophobic interactions. This may provide the molecular mechanism for triggering enzyme activity and effector recognition under the influence of Ca2+ signals in the host(s) as sketched in Fig. Irrespective of the yet ambiguous assignment of the two metal-binding sites, the biological function of CaPlasmids and oligonucleotides used in this study are listed in Table Escherichia coli DH10B (Thermo Scientific). All plasmids derived from pVCD01935, which encodes a fusion protein consisting of MalE, the MIIA domain of the hypothetical protein VIC_001052 (amino acids 73–243 of the full-length protein) and a Strep-tag (Table Plasmids were propagated in ag Table . DerivatE. coli BL21 (DE3) (NEB). Cells were grown in lysogeny broth containing ampicillin (150 µg/ml) at 37 °C. At an OD600 of about 0.7, protein expression was induced by Isopropyl-β-D-thiogalactopyranosid  and cells were grown at 28 °C for 4 h. Cells were harvested and washed once with TKE buffer , pH 8,0). Cells were resuspended in TKE buffer containing lysozyme (0.1% w/v) and incubated for 1 h on ice. Cells were disrupted by sonication. Cell debris was removed by centrifugation and a final filtration step. The fusion protein was purified by affinity chromatography using a MBPTrapTM HP column  and eluted in TKE buffer containing 10 mM maltose. The MalE tag was removed by cleavage with thrombin over-night at 22 °C . The Strep-tag-carrying MIIA domain was purified using a StrepTrapTM HP column  and eluted in TNE buffer . Anti-DYKDDDDK (FLAG®) G1 Affinity Resin; GenScript  was used for purification of FLAG-tagged proteins according to the manufacturer’s protocol. For concentration of the protein and changes of the buffer, Amicon® Ultra Centrifugal filters  and Slide-A-Lyzer® Mini dialysis devices  were used. Protein samples were analyzed by SDS-PAGE36. Gels were stained with Coomassie Brilliant Blue G-250.For protein purification, the corresponding plasmids Table  were tra4, pH 8.0). The reaction was stopped with 10 mM EDTA and analyzed by SDS-PAGE.. For negative control, the metal ion solutions were replaced by Tris buffer. In addition to these standard conditions  in a volume of 10 µl were incubated for 30 min at room temperature with different metal salts  in Tris buffer . Thermal unfolding data were analyzed by a two-state unfolding model. The temperature-dependent mol-fraction fN of the native state was expressed as fN (T) = (1 + exp(ΔS·T-ΔH)/R·T)−1. The unfolding enthalpy ΔH and entropy ΔS were determined from this with the additional condition ΔH/ΔS = Tm, where Tm is the experimentally determined midpoint temperature of 50% denaturation.Circular dichroism (CD) spectra were recorded with a J-815 spectrometer  equipped with a continuously stirred temperature-controlled 1 cm cuvette. For thermal denaturation, the temperature increased at a rate of 1 °C/min from 18 to 92 °C. Two spectra were then accumulated between 185 and 250 nm  at constant temperature in intervals of 2 °C. Spectra were analyzed by DichrowebIntrinsic tryptophan fluorescence was measured at room temperature in a 100 µL cuvette using an LS55 spectrometer (PerkinElmer Life Sciences) with excitation at 285 nm and emission recorded from 290 to 450 nm (band width of 5 nm).2 (2 mM), 10 µL aliquots were further injected to 1.4 ml of MIIA-D116A domain (55 µM). Both solutions contained 20 mM Tris-SO4 at pH 8. The data were processed and analyzed using the MicroCal origin 7.5 software.Binding isotherms were measured with a MicroCal VP-ITC Instrument . After an initial injection of 5 µL of CaClMass spectrometry measurements were performed by using nano-ESI-FTMS. Samples were injected into nanospray capillaries from New Objective Inc.  with an inner tip diameter of 2 mm and analyzed with a Velos Pro Orbitrap Elite mass spectrometer . The temperature of the transfer capillary was fixed at 240 °C. All measurements were performed in the positive ionization mode with an applied voltage to the nanospray capillary of 3 kV. The voltages applied to the S-Lens and multipoles were automatically tuned to favour the transmission of high m/z values. Full MS spectra were recorded using the Orbitrap analyzer, by averaging at least 110 scans in the range of m/z 2000–4000. Acquisition and treatment of the data was done with the Xcalibur software.39. Additional parameters were employed for the bound Ca2+ ion40. The starting structure of a hypothetical Ca2+-binding site was generated by homology modelling using “SwissModel”41 with a 30 amino acid-long target sequence of the MIIA domain that contained the 12 amino acid-long subsequence (amino acid 178 to 189) which exhibited the highest similarity to the sequence pattern of Ca2+-binding loops in canonical EF-hands (PROSITE pattern PS00018). The Ca2+-binding loop of EF-hand 3 of human calmodulin  in the NMR structure (pdb-file 2kuh) served as the template;Molecular dynamics (MD) simulations and data analyses were performed using the AMBER 14 program package with the ff99SB force field applied to the protein domain2+-binding loop: FDKDGNGYISAAELRH,EF-hand 3 Ca173IEGIDSDQKGDLEVKEYNNINYSGGTVND202.MIIA-fragment: Y2+ ion was added at the position corresponding to that in the Ca2+-binding loop of calmodulin. The protonation state of the domain was adjusted to mimic the physiological pH. TIP3P waters were added with a minimum water layer thickness of 12 Å. Five hundred steps of steepest decent and five hundred steps of conjugate gradient with 500 kcal mol−1 Å−1 harmonic constraint on the protein were initially conducted after which another one thousand steps of steepest decent and 1500 steps of conjugate gradient were performed without constraints. The system was heated from 0 to 300 K with 10 kcal mol−1 Å−1. A harmonic constraint on the domain was performed for 40 ps followed by another 2 ns preconditioning run at 300 K without constraining the solute. Finally, a 400 ns MD run was performed with periodic boundary condition in the NPT ensemble. Simulations were terminated and restarted every 50 ns. The SHAKE algorithm, a 2 fs time integration step, 10 Å cutoff for non-bonded interactions and the particle mesh Ewald (PME) method were used.MD simulations were carried out with the modelled structure of amino acid 178 to 189 (underlined above) to which a single CaSupplementary Information


We demonstrate that, in Thermus thermophilus HB8, the uncharacterized protein TTHB144 is a cA4-activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease.Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN  active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA  •TTHB144 is a cyclic tetra-adenylate activated ribonuclease.•TTHB144 degrades cyclic tetra-adenylate at its CARF domain.•Self-limiting enzymes like TTHB144 may regulate anti-viral signaling in bacteria. Sulfolobus solfataricus (Sso) encodes dedicated ring nucleases, which degrade the cyclic tetra-adenylate (cA4) activator and deactivate Csx1 Thermus thermophilus HB8 (TTHB) has been investigated 4T. thermophilus also encodes an uncharacterized CARF domain-containing protein, TTHB144, which was reported to be Csm6-like 4. Furthermore, the enzyme degrades cA4 using its CARF domain. This enzyme therefore represents the first known example of a cOA dependent enzyme that degrades its own activator.The CRISPR system provides prokaryotes with adaptive immunity against mobile genetic elements  4-activated RNA cleavage by TTHB144 in a continuous assay  (2 > P) 4 degradation products with oligoadenylate standards generated using the E. coli MazF toxin, as described previously S. solfataricus ring nucleases, TTHB144 degraded cA4 to yield an A4 > P intermediate and A2 > P product (To investigate whether TTHB144 degraded cAs (PAGE) d, which  product e.4 degradation by TTHB144 CARF and HEPN domain variants. TTHB144 H368A, which has no ribonuclease activity, degraded cA4 similarly to wild-type protein, ruling out a role for the HEPN domain in cA4 degradation. However, cA4 degradation was abolished in the K94A variant and impaired in the T10A/T11A variant  in cA4 cleavage rate compared to the wild-type protein. The rate of RNA degradation (~ 5-10 min−1) thus appeared to exceed the rate of cA4 cleavage by approximately 3 orders of magnitude. Consequently, we investigated whether RNA binding at the HEPN domain stimulated cA4 degradation by the CARF domain by including unlabeled RNA in a cA4 degradation assay. TTHB144 degraded cA4 at a rate (0.010 ± 0.042 min−1) similar to cA4 degradation in the absence of RNA, suggesting that RNA binding at the HEPN domain does not affect cA4 degradation at the CARF domain.Subsequently, we evaluated cA variant d, suggesectively  allowingnuclease a, exhibi4 binding by the wild-type and variant enzymes, we carried out gel electrophoretic mobility shift assays with 32P-labeled cA4  cA4 at a reduced level. Hence, the TTHB144 cA4 binding and cleavage mechanism may be similar to that of the crenarchaeal ring nuclease Sso2081, where the equivalent residues, S11 and K106, have been shown to be important for cA4 binding and/or cleavage To examine cAeled cA4 c. The wi4 degradation activity and ribonuclease activity. The single-turnover rate of cA4 degradation by TTHB144 at 70 °C is slow: comparable to that of the less active S. solfataricus dedicated ring nuclease, Sso1393 4 degradation may function as a built-in control mechanism to limit the extent of ribonuclease activity. Faster rates of cA4 degradation could disable this arm of type III CRISPR-mediated immunity. Streptococcus epidermidis Csm6 activity during type III immunity has been shown to cause cell growth arrest TTHB144 is the first CARF family protein identified to harbor both cABiotechnology and Biological Sciences Research Council (Grant REF BB/S000313/1 to M.F.W.).This work was funded by a grant from the Author Contribution Statement: J.S.A. carried out the investigation and analysis and wrote the original draft of the paper; S.Grü. and S.Gra. contributed to the investigation and analysis; M.F.W. conceptualized and supervised the work. All authors contributed to the review and editing.


Most studies have evaluated poverty in terms of income status, but this approach cannot capture the diverse and complex aspects of poverty. To develop commodity-based relative deprivation indicators and evaluate their associations with mortality, we conducted a 6-year follow-up of participants in the Japan Gerontological Evaluation Study (JAGES), a population-based cohort of Japanese adults aged 65 and older. We analyzed mortality for 7614 respondents from 2010 to 2016. Cox regression models with multiple imputation were used to estimate hazard ratios (HRs) for mortality. Seven indicators were significantly associated with mortality: no refrigerator, no air conditioner, cut-off of essential services in the past year for economic reasons, and so on. Among participants, 12.0% met one item, and 3.3% met two items or more. The HRs after adjusting for relative poverty and some confounders were 1.71 (95%CI: 1.18–2.48) for relative deprivation, and 1.87 (95%CI: 1.14–3.09) for a combination of relative poverty and deprivation. Relative deprivation was attributable to around 27,000 premature deaths (2.3%) annually for the older Japanese. Measurement of relative deprivation among older adults might be worthwhile in public health as an important factor to address for healthy aging. Several studies have shown that relative poverty based on low income is significantly associated with poor health . HoweverThe association between relative deprivation and mortality was examined by some ecological studies. The use of social indicator approaches such as the Townsend deprivation index or Carstairs deprivation score has found that people in relatively deprived areas have a higher risk for standardized mortality rates ,7, canceTherefore, we examined the association between relative deprivation as one aspect of poverty and mortality among older Japanese adults after controlling for relative income poverty and other confounding factors.We used prospective cohort data from the Japan Gerontological Evaluation Study (JAGES), a large-scale population-based study of Japanese people aged 65 or older who were physically and cognitively independent. Baseline data were collected from August 2010 to January 2012, with a response rate of 66.3%. Of these, the present analyses used data on 7614 participants who answered a relative deprivation questionnaire, after excluding participants with missing information on sex and age. The average age of the respondents was 73.5 years (standard deviation (SD) = 5.6), and 53.0% were women. This study was performed on the basis of a collaborative research agreement with the municipality. Ethical permission (No. 13–14) was provided by the Ethics Board at Nihon Fukushi University.We retrieved information on death records from 2010 to 2016 from the government database of public long-term care insurance. This government database covers all respondents. Among these records, there were 514 (6.75%) deaths identified in the analysis sample.Commodity-based relative deprivation indicators have been developed using a consensual approach based on public opinion. These indicators were drawn from a review of daily necessities and basic needs in society ,14,15,16Relative poverty was defined as an income of less than half of the median annual equivalent income in the government statistics ; the thrDemographic variables included sex, age, years of education, and marital status at the baseline survey. In order to account for the health status at the baseline, presence of medical treatment, self-recognition of forgetfulness, and depressive symptoms were also considered. Medical treatment was determined by asking, “Are you currently receiving any medical treatment?”. Self-recognition of forgetfulness was measured by asking, “Do people around you notice your forgetfulness, for example, by telling you that you often ask the same thing?”. Depressive symptoms were assessed using the short version of the Geriatric Depression Scale (GDS-15), which was developed for self-administration in the community, using a simple yes/no format .First, we extracted the relative deprivation index that was associated with premature death among older Japanese adults by calculating crude hazard ratios (HRs) for mortality using Cox regression analysis. Second, we applied Cox regression analysis, starting with assessing the relationship between relative poverty (monetary poverty) and mortality, adjusting for the above covariates (Model 1). In Model 2, we assessed the association between relative deprivation and mortality, adjusting for relative poverty and covariates. The relative deprivation index was divided to ternary  in Model 2. In addition, we examined the binary category (none or anyone) in Model 2a, and the quaternion category  in Model 2b, respectively. We also examined the combination of relative deprivation and poverty in Model 3. To mitigate potential biases caused by missing information in predictors and covariates, we adopted the multiple imputation approach, under the missing at random assumption. We generated 20 imputed data sets using the multiple imputation by chained equations procedure. Finally, we calculated population attributable risk percentage (PAR%) in an older Japanese population. This estimation assumed that the adjusted HRs truly reflected causal impact, and that our results represented the entire older Japanese population. Data on annual mortality were obtained from governmental reports [The crude HR showed that the seven relative deprivation indicators were significantly associated with a higher risk for death, respectively . In partAfter adjustment for individual attributes and relative poverty , among rThe estimation of PAR% showed that about 27,000 premature deaths  or about 15,000 premature deaths  could be avoided annually if there was less severe relative deprivation in Japan .Relative deprivation is an important element in poverty, although it might be unsuitable for an international comparative study because the standard decent life varies by nation, culture, and period. To the best of our knowledge, this is the first study to examine the effect and impact of relative deprivation and relative poverty on mortality among older Japanese adults. Our results suggest the effectiveness of our seven relative deprivation indicators as social determinants of healthy ageing. Our findings are consistent with previous findings that analyzed poor social support ,21 and sAt the same time, our study added new evidence that relative deprivation has a stronger association with mortality than relative poverty if subjects experience relative deprivation in two or more items. There are several possible reasons for this finding. First, a relative deprivation index might capture severe poverty conditions better than a relative income approach. Whelan et al. revealed that people living in relatively deprived conditions experienced long-term and severe poverty throughout their life course . Some stAlthough the Europe 2020 strategy has adopted a strong material deprivation index as a goal for social inclusion in the next decade, there are few discussions regarding specific policy in Japan. Our results suggest that relative deprivation indicators could more accurately represent severe or absolute poverty in society that relative poverty indicators cannot address. The income approach has some limitations in the discussion of the poverty line, because the relationship between income and consumption behavior is complex. Relative deprivation indicators, which are composed of specific primary goods and resources, might be relevant to capture their part of “capabilities” . Our resThis study has several limitations. First, although we included indicators used in previous studies, our relative deprivation indicators did not cover the full range of daily resources among older people in Japan. Second, our analysis was limited to all-cause mortality. Third, our findings may be underestimated because people living in serious poverty and deprivation may have been less likely to participate in our survey. Finally, our data were not representative of the whole country. On the other hand, it is important to note that we did perform a large-scale survey concerning non-monetary poverty among older people in more than one municipality. Only a few studies have focused on relative deprivation among Asian older adults ,11,28.It is well-known that poverty is one of the social determinants of health. One important implication of our findings is that measurement of relative deprivation, along with relative poverty (monetary poverty), might be worthwhile in public health as an important factor for healthy aging. From a life course perspective, the impact of relative deprivation on health should be evaluated in older people as one of the cumulative disadvantages.


Proteins with highly similar tandem domains have shown an increased propensity for misfolding and aggregation. Several molecular explanations have been put forward, such as swapping of adjacent domains, but there is a lack of computational tools to systematically analyze them. We present the TAndem DOmain Swap Stability predictor (TADOSS), a method to computationally estimate the stability of tandem domain-swapped conformations from the structures of single domains, based on previous coarse-grained simulation studies. The tool is able to discriminate domains susceptible to domain swapping and to identify structural regions with high propensity to form hinge loops. TADOSS is a scalable method and suitable for large scale analyses.https://github.com/lafita/tadoss.Source code and documentation are freely available under an MIT license on GitHub at Bioinformatics online. Protein misfolding and aggregation is a major problem for cells and organisms, and the cause of severe human diseases like Alzheimer’s. Recent studies have shown an increased propensity of aggregation in proteins containing identical domains in tandem  model, as described by The running time for an example domain of 159 residues on a MacBook Pro 2.9 GHz Intel Core i5 with 16 GB RAM is about 4 s. The method scales quadratically with the number of residues in the input domain structure .This work was supported by the intramural research program of the National Institute of Diabetes and Digestive and Kidney Diseases (grant number ZIA DK075104-06) of the National Institutes of Health to P.T. and R.B.B. This work was funded by the European Molecular Biology Laboratory.Conflict of Interest: none declared.bty974_Supplementary_DataClick here for additional data file.


The role of these three Trp residues for domain folding, stability and binding of the RAGE ligand S100B was investigated through site-directed mutagenesis, UV/VIS, CD and fluorescence spectrometry, protein–protein interaction studies, and X-ray crystallography. The data show that the Trp triad stabilizes the folded V-domain by maintaining a short helix in the structure. Mutation of any Trp residue increases the structural plasticity of the domain. Residues Trp61 and Trp72 are involved in the binding of S100B, yet they are not strictly required for S100B binding. The crystal structure of the RAGE-derived peptide W72 in complex with S100B showed that Trp72 is deeply buried in a hydrophobic depression on the S100B surface. The studies suggest that multiple binding modes between RAGE and S100B exist and point toward a not previously recognized role of the Trp residues for RAGE-ligand binding. The Trp triad of the V-domain appears to be a suitable target for novel RAGE inhibitors, either in the form of monoclonal antibodies targeting this epitope, or small organic molecules.The receptor for advanced glycation end products (RAGE) recognizes damage-associated molecular patterns (DAMPs) and plays a critical role for the innate immune response and sterile tissue inflammation. RAGE overexpression is associated with diabetic complications, neurodegenerative diseases and certain cancers. Yet, the molecular mechanism of ligand recognition by RAGE is insufficiently understood to rationalize the binding of diverse ligands. The N-terminal V-type Ig-domain of RAGE contains a triad of tryptophan residue; Trp The receptor for advanced glycation end products  is a cell surface receptor that senses a group of molecules that are commonly summarized as damage-associated molecular patterns (DAMPs). DAMP ligands of RAGE are structurally diverse and include advanced glycation end products (AGEs), for which the receptor is named, members of the S100 protein family , nucleicThe mature human RAGE receptor is a 382-residue cell surface protein with three Ig-like extracellular domains, termed as the V-, C1- and C2-domains; a single transmembrane domain; and a short cytoplasmic tail. The intracellular, C-terminal 44 residue tail is required for RAGE signaling, but the exact mechanism of intracellular signaling activation has not been unambiguously established. Activation of RAGE is initiated by binding of DAMPs to the extracellular domains. Most ligands bind to the N-terminal V-domain , while a2+ weakly (>350 μM), and two typical-EF-hand motifs, which bind calcium with higher affinity (<50 μM) , where F0 is the fluorescence intensity (FI) in the absence of the quencher Q and F is the FI in the presence of the quencher at the concentration [Q].Acrylamide quenching of tryptophan fluorescence measurements was performed using a protein concentration of 1 μM in quartz cuvette with a 5 mm path length. Acrylamide concentrations were incrementally increased to 0.25 M and a correction factor of rylamide . The samSYPRO Orange (Sigma) was obtained as a 5000× solution and initially diluted to 100× in DMSO, then further diluted to 10× in PBS, pH 7.4. This solution was mixed 1:1 with 5 μM V-domain in 2 M ammonium sulfate. The ammonium sulfate was identified as a suitable additive for the thermofluor assays of the V-domains by screening with the Hampton Crystal Screen HR2-100 (Hampton Research). Thermal denaturation was done in 50 μl volumes on a Stratagene Mx3000P qPCR instrument with a customized excitation/emission (492 nm/610 nm) filter set-up. The temperature was increased in 1°C intervals at a rate of 2°C/min. Two independent experiments were performed with five samples of each V-domain variant. The obtained FI versus temperature (T) data were analyzed by calculating the relative change in fluorescence per °C as a function of temperature (ΔFI/ΔT vs. T).2 was titrated by incremental addition of V-domain stock solutions in a quartz cuvette with reduced path length of 5 mm. After each addition of V-domain, an equilibration time of 2 min was allowed so the increase in sample volume at the end point of the titration was less than 3%. Titrations were repeated three to five times and the best fit for the data was obtained using a 1:1 RAGE:S100B stoichiometry model as described previously . PDB entries used are listed in Click here for additional data file.


We propose a theoretical framework to predict the deformation mechanism of the γ-TiAl single crystal without lattice defects by combining the generalized stacking fault energy and the Schmid factor. Our theory is validated against an excellent testbed, the single crystal nanowire, by correctly predicting four major deformation mechanisms, namely, ordinary slip, super slip, twinning, and mixed slip/fracture observed during the tensile and compressive tests along 10 different orientations using molecular dynamics simulations. Interestingly, although lattice defects are not taken into account, the theoretical predictions match well with existing experiments on bulk specimen with only a few exceptions; the exceptions are discussed based on the size-dependent deformation mechanism in the presence of preexisting dislocation sources. We expect that the method in this paper can be generalized to study various ductile intermetallic crystals where conventional Schmid law does not hold well. Despite these excellent properties, industrial applications have been limited by its inherently low ductility. To develop the compound with enhanced ductility, the microscopic deformation mechanisms must be understood1.Over the past few decades, TiAl intermetallic compound has attracted much attention because of its use in applications such as LPT (low pressure turbine) blades in aircraft engines and turbine wheel in automobile engines enabled by its outstanding high temperature mechanical properties including good thermal and oxidation resistance2 phase and the ductile γ phase, and its plasticity is accommodated mostly by the ductile γ phase. Numerous studies have been performed to understand the mechanical behaviors of TiAl alloy and each of the constituent phases14. The brittleness of α2 has been explained by the limited number of active slip systems, based on the detailed analysis on its slip systems and dislocation structures14. Deformation mechanisms of single-crystal γ-TiAl, prepared either from TiAl alloy or by direct growth for various orientations have been investigated via transmission electron microscopy (TEM) or scanning electron microscopy (SEM) analyses13. The effects of different γ/γ interfaces and lamellae orientations of TiAl compound have also been studied12. In addition, the motion of existing dislocations and their effects on mechanical properties in γ-TiAl have been investigated17.TiAl alloy is composed of the brittle α19, a systematic prediction on the deformation behaviors of γ-TiAl is still lacking, thereby limiting the understanding of the mechanical properties of TiAl alloy such as orientation-dependent yield stress, elongation, and deformation mode of PST  crystals20. Although a few analyses based on the Schmid factor exist18, it is well known that the Schmid factor alone is not sufficient to predict the deformation mechanism of single-crystal metals23. For example, in an experimental study5, γ-TiAl single crystals under compression along seven loading directions at root temperature undergo plastic deformation mediated by ordinary dislocations except one direction, although the Schmid law predicts that only two loading directions prefer the ordinary dislocation while the superdislocation or the twinning mechanism has the highest Schmid factor for the other five directions. In addition, although both superdislocations and ordinary dislocations are decomposed into partial dislocations, existing studies consider the Schmid factors for full ordinary dislocations and full superdislocations without accounting for the partial slips18.However, although its unique slip system Fig.  includin24 with a few exceptions. We discuss the origin of the discrepancy by considering size-dependent deformation mechanism in the presence of preexisting dislocation sources.In the present study, in order to overcome the limited applicability of the Schmid law in the γ-TiAl crystal, we suggest a theory to predict the deformation mode of the crystal without lattice defects by combining generalized stacking fault energy (GSFE) surface and the Schmid factor accounting for the partial slips. We test the theory against an excellent testbed, the single crystal nanowire, by performing molecular dynamics (MD) simulations of both compressive and tensile tests along 10 different orientations. We reveal that four major deformation mechanisms of ordinary slip (slip by ordinary dislocation), super slip (slip by superdislocation), twinning, and mixed slip/fracture occur depending on the loading condition, and that the theoretical prediction matches with the deformation mode observed in MD simulations for all loading conditions tested in the study. Having established the validity of our theory, we predict the deformation mode of single crystal compression experiments by employing the GSFE obtained from first-principle density function theory (DFT) calculations, and show that the predictions match well with experimental results in the literatureThe remainder of this paper is organized as follows. In Section 2, we discuss the unique slip systems in detail and present a theoretical model to predict the preferred deformation mode for a given loading condition. We calculate the ideal critical resolved shear stress (ICRSS) of five distinct slip events  partial slip, twin, inverse super slip, and ordinary slip) and provide a systematic method to compute the critical stress for each to occur based on the GSFE surface and the Schmid factor. In Section 3, we provide a detailed description of the MD simulations and show that the observed deformation mechanisms for 20 different loading conditions match well with the theoretical predictions. We then discuss the origin of different deformation mechanisms between the theory and the existing bulk experiments for a few exceptional orientations by considering size-dependent deformation mechanism in the presence of preexisting dislocation sources. A summary and an outlook for future research are presented in Section 4.0 intermetallic structure, which is composed of alternating Ti and Al atomic layer along the [001] axis, as shown in Fig. abc] or {def) introduced by Hug et al.25. The perfect L10 crystal structure can also be described as the repeated stacking of three consecutive {111) atomic layers. A stacking fault is a planar defect, where these stacking sequences are interrupted by the relative displacement between two adjacent planes. This interruption involves excess energies compared to a perfect crystal, called the generalized stacking fault energy (GSFE). The GSFE surface is calculated by moving upper half of the perfect crystal along the directions parallel to the {111) plane (or gamma surface) and calculating the energy difference between the initial state and the deformed state. The GSFE landscape of the γ-TiAl single crystal is different from that of the FCC structure, as shown in Fig. 26 and Zope & Mishin27), ab initio density functional theory (DFT) calculations and experiments are summarized in Table γ-TiAl single crystal has an L113, plastic deformation of bulk single-crystal γ-TiAl involves twinning, ordinary dislocation and superdislocation on the {111) plane. Among these, the formation of superdislocation is a unique feature that distinguishes γ-TiAl from ordinary FCC structures. Two types of superdislocations with burgers vectors of 〈011] and 1/2〈112] are often considered, as shown in Fig. ab initio GSFE surface19, fully dissociated superdislocations involve four partials and three stacking faults, as follows:Because of the aforementioned unique GSFE, the γ-TiAl crystals deform differently from the FCC crystals. According to previous experimental studies28 or two partials and SISF30. Nevertheless, in the present theoretical study, we considered the fully dissociated superdislocations with four partials and ordinary dislocation with two partials.Because of the limited experimental resolution, many experimental studies reported that superdislocations dissociate into three partial dislocations and two stacking faultsThe slip on the {111) plane is initiated either by one SISF partial or two CSF partials, as shown in Fig. 31. Because the pre-strain originated from the surface stress of nanowire is non-negligible and the applied strain at the yield point is even higher, it may be necessary to incorporate the strain-induced change of GSFE surface to enhance the accuracy of deformation mode prediction. However, as shown in the later part, although we use the GSFE obtained at zero strain for the prediction of the deformation mechanism, we find that our theoretical predictions match very well with MD simulations for all 20 loading conditions considered in the study. Indeed, the previous study on FCC crystals23 also shows that the deformation mode can be predicted accurately without accounting for the strain-induced GSFE change.We note that the GSFE surface moderately changes with the strainσICRSS of each partial slip from the maximum slope of the corresponding section of GSFE curve, as summarized in Table σICRSS calculation in this work assumes the sliding of the perfect half space sliding instead of dislocation nucleation from a surface, it will determine the preference between different slip events; a similar method has been applied to predict the deformation mode of FCC crystals by Cai & Weinberger23.For each partial dislocation involving the aforementioned five categorized events, we obtain the GSFE curve by projecting the GSFE surface along the partial Burgers vector directions constituting each event, as shown in Fig. 0 crystal and FCC crystal is not significant because the dislocation core structure of both crystals involves stacking faults and thus is planar. Although the core structure of the 1/2〈110] ordinary screw dislocation in TiAl gamma crystal is known to be non-planar exceptionally, the lattice friction stress (the Peierls stress) at 0 K is estimated by a DFT method to be relatively small value of around 0.01C44 (C44 = 68 GPa)32. The lattice friction stress at 300 K is expected to be significantly smaller due to thermal fluctuation33. Hence, although the lattice friction resistance is not taken into account, the deformation mode prediction based on the minimum crystal stress criterion works well for FCC crystals23 and also for L10 crystal of the TiAl alloy in this study. We note that our method is not expected to work at a very low temperature where the lattice resistance of the ordinary screw dislocation becomes significant.We extended the methodology to the intermetallic system involving more complex GSFE surface with more deformation modes. We note that the lattice friction resistance of both L1S of each partial slip and calculate the critical axial stress by σc = σICRSS/S. We first compare the critical axial stresses of one SISF partial and two CSF partials for all available {111) slip planes and choose the preferred slip direction with lower critical stress. We note that, because we only consider three directions toward saddle points of the GSFE surface in the Step-1 deformation  loading, we can obtain the Schmid factor ion Fig. , negativion Fig. , which i34 to investigate the deformation mode of a single-crystal nanowire by MD simulations. First, we construct γ-Ti50Al50 nanowires with 3:1 aspect ratio to have 18 nm height and 6 nm diameter with given orientation. The periodic boundary condition is imposed only along the loading direction (x-axis). Prior to the loading tests, we equilibrate the specimen at 300 K by using NPT ensemble simulation for 30 ps, during which the equilibrium length is obtained. We further equilibrate the nanowire by 30 ps of NVT ensemble simulation at the equilibrium length. Next, we perform uniaxial tension and compression tests of nanowires along the x-axis with 108s−1 strain rate up to 30% strain by imposing incremental strain of 0.1% every 10 ps with 300 K NVT ensemble. To understand the orientation dependent deformation mechanism, we perform the uniaxial compression and tension tests for 10 different crystallographic orientations: 〈001], 〈010], 〈011], 〈102], 〈110], 〈111], 〈112], 〈120], 〈201], and 〈211]. The simulation results are visualized using OVITO 35 and POV-Ray36.We use LAMMPS  software26 and Zope & Mishin27. A key difference between two EAM interatomic potentials is the relative magnitude of the APB and CSF energies in the relaxed GSFE map. As depicted in Fig. The choice of a reasonable interatomic interaction model is crucial for the classical MD simulations. We test two embedded-atom method (EAM) interatomic potentials developed by Farkas & JonesAb initio Simulation Package)37. The exchange-correlation functional was described within the Generalized gradient approximation (GGA) and parameterized by Perdew, Burke, and Ernzerhof 38. K-point meshes were constructed using the Monkhorst-Pack scheme39. We used a 11 × 5× 19 k-point mesh and a plane-wave cutoff energy of 320 eV. To calculate the stacking fault energy, we constructed 6 atomic layers along (111) plane, having spacing of 13.8 Å, 4.9 Å and 2.8 Å along the (111) slip plane normal and We also explain the details about the GSFE landscape in Fig. In a series of MD simulations, we found that γ-TiAl nanowires deform by four types of mechanisms: ordinary slip, twin, super slip and mixed slip/fracture mode. In order to identify four deformation modes, we calculated relative displacement vectors of the atoms above and below of the slip planes. This vector was calculated by the position of the atoms for each step when they are moved. Stacking faults were identified by common-neighbor analysis. Four representative deformation mechanisms are visualized in Fig. For all loading conditions considered in the study, we find a good match between the theoretical prediction and simulation results, as shown in Table T, 9.43 GPa for 〈011]T, 21.96 GPa for 〈001]T, 11.01 GPa for 〈102]T) overestimates the MD results. Nevertheless, the yield stress in MD simulations reflects the critical stress prediction to some extent, although the order of yield stress can be slightly different because of the nonlinear stress-strain curve and the detailed dislocation nucleation mechanism of each.Our analysis also provides a qualitative understanding of the stress-strain curves of different loading conditions, as shown in Fig. 13 in a wide range of temperature. To make a more appropriate comparison with experiments, we predicted the preferred deformation modes based on the ICRSS from DFT calculations  is considerably larger than the nanowires used in MD simulation; therefore, the specimen generally contains defects that can act as the Frank-Read source, pre-existing statistically stored dislocations, and etc. In comparison, the nanowire in our MD simulation is a lattice-defect-free specimen whose deformation is governed by dislocation nucleation. The critical stress criterion based on the Schmid factor and GSFE are relevant to predict the nucleation of dislocation or twin in defect-free nanocrystals. However, as the sample size increases, it becomes progressively important to consider the stress to form superdislocation and twin in the entire domain in the presence of pre-existing defects, as discussed in the literature44.The typical size of the specimen in the experimentτs is the critical resolved shear stress to emit a bs is the magnitude of the Burgers vector of the superdislocation, μs is the effective shear modulus for superdislocation, and d is the sample size . Figure τp is the critical resolve shear stress to emit a 1/6 bp is the magnitude of the Burgers vector of the twinning partial, μp is the effective shear modulus for the twinning partial, and γSISF is the stacking fault energy. Details of the derivation of (4) can be found in the Supplementary Note dc) can be obtained by equating  and 18.8 nm from the first principle calculation . The parameters required for the calculation are summarized in Table 45. Hence, we could suspect that the deformation twinning is unlikely to occur in the experimentally-tested millimeter scale single crystal samples which are orders of magnitude larger than the critical size predicted from theoretical calculations.The critical size is predicted to be 48.9 nm from the Farkas & Jones EAM potential (In summary, we suggest a theoretical framework to analyze the deformation behavior of a γ-TiAl single crystal without lattice defects and benchmarked the theory against MD simulations. MD simulations revealed that the γ-TiAl single-crystal was deformed by four types of deformation behaviors: ordinary slip, twinning, super slip, and mixed slip/fracture. We predict the deformation mode based on the critical stress calculation obtained from the Schmid factor and the GSFE curve and find a good match with the simulation results. Hence, we show that the limited applicability of the Schmid law on the γ-TiAl can be resolved when GSFE curve is also taken into account. Interestingly, our theoretical predictions on the deformation mechanism matched well with existing experiments on bulk specimen with only a few exceptions, although the bulk specimen inherently have preexisting lattice defects. We suggest size-dependent deformation mechanism originated from the preexisting dislocation source as a plausible cause of the discrepancy, in addition to the inaccuracy of the DFT calculations and different chemical composition. We note that the suggested analysis method in the present study can be applied to other intermetallic systems involving the formation of superdislocations.Supplementary Information


Aptamers are short, single-stranded oligonucleotides that bind to specific target molecules. The shape-forming feature of single-stranded oligonucleotides provides high affinity and excellent specificity toward targets. Hence, aptamers can be used as analogs of antibodies. In December 2004, the US Food and Drug Administration approved the first aptamer-based therapeutic, pegaptanib (Macugen), targeting vascular endothelial growth factor, for the treatment of age-related macular degeneration. Since then, however, no aptamer medication for public health has appeared. During these relatively silent years, many trials and improvements of aptamer therapeutics have been performed, opening multiple novel directions for the therapeutic application of aptamers. This review summarizes the basic characteristics of aptamers and the chemical modifications available for aptamer therapeutics. The idea of using single-stranded oligonucleotides as affinity molecules for various target compounds was initially proposed in 1990 . The conSingle-stranded RNA fold into a vast set of tertiary structures depending on their different primary structures. Therefore, RNA aptamers have a high potential to act as molecular mimics of proteins. This concept arose in our previous investigations of the translation machinery, in which we found that a polypeptide release factor encodes a tripeptide anticodon as a molecular mimic of a tRNA anticodon ,9,10. ThIn this review, we present an overview of the structural basis of aptamers and chemical modifications of aptamers for therapeutic application.Aptamers prefer to interact with positively charged surfaces of the target proteins due to the negatively charged nature of backbone linkages. For example, the crystal structures of aptamers in complex with target proteins, such as those of nuclear factor (NF)-κB, bacteriophage MS2 capsid, and thrombin showed electrostatic interactions  11,12,1,112,13. We previously reported a 23-nucleotide (nt) RNA aptamer targeting the Fc domain of human IgG1 (hFc1) . Since tThe conformational diversity and targeting specificity of RNA aptamers provide a promising potential for their therapeutic applications as inhibitors of protein–protein interactions. To achieve this goal, several features of the aptamer are required. First, therapeutic aptamers should be highly stable in the body. Since natural RNA and DNA are susceptible to endogenous nucleases, chemical modifications on the sugar and/or phosphodiester backbone should be adequately included . Second,O-methyl) are widely used to confer nuclease resistance. After the identification of a candidate aptamer sequence by SELEX, these modifications could be incorporated in the course of chemical synthesis. However, introducing modifications at all nucleotides is hardly tolerated, because a sugar modification affects the aptamer activity pyridine (Ds)–2-nitro-4-propynylpyrrole (Px) base pair ,65. TheyUnlike artificial bases, ribose-block replacement has many more limitations because of its incompatibility with enzymatic manipulation. Although 4′-thioribonucleotides are successfully incorporated by T7 RNA polymerase , other tTaken together, artificial nucleotides are expected to be the next generation of aptamers. Effective polymerization method and sequence strategy must be developed. Furthermore, their biological safety must be elucidated for clinical application.Computational analysis has been used to assist aptamer discovery. For example, secondary structure prediction software such as MFold  or CentrThe recent development of next-generation sequencing, also known as deep sequencing, provides vast sequence information. Hence, a bioinformatical approach for aptamer generation is worth considering. In the classical SELEX method, frequently appeared sequences after selection would be estimated as candidate aptamers. Instead of a frequency-based picking-up strategy, several algorithms for high-throughput SELEX data have been reported. MPBind  exploits the Meta-Z-Score and ranking candidate sequences . FASTAptCollectively, in silico methods will accelerate aptamer development. Moreover, since computational procedures will reduce the selection rounds, it would be beneficial to predict an aptamer with artificial nucleotides that is difficult to make with standard SELEX. Hence, these approaches support the creation of aptamers with new chemistry.The emergence of artificial nucleotides and computational methods might lead us to the next generation of aptamers. The following sections discuss the future field of therapeutic aptamers. Conventional aptamer targets are mainly focused on extracellular proteins. There is a lot of room for identifying intracellular targets. Actually, there have already been inhibitory nucleotides for transcription factors, the so-called decoy oligonucleotides. Decoy oligonucleotides are designed from the consensus sequence of DNA/RNA binding proteins and inhibit their function. Aptamers for other cellular proteins could be produced in principle. Another possible future direction is multi-functionalization of aptamers by assembling molecules. It takes advantage of the convenient chemical conjugation of aptamers.Although intracellular delivery is one of the major concerns, few strategies have been proposed. Single-stranded phosphorothioate ASO have been known to be delivered into organs without special carriers. Denichenko et al. recently developed single-stranded RNA decoy oligonucleotides targeting splicing factors . The autPlasmid or viral vector-based expression is another way to implement intracellular aptamers. There are some reports that develop anti-NF-κB aptamers, which include the p50 aptamer ,89 and tGenerating a multi-functionalized aptamer is another interesting issue. Since an aptamer drug is usually produced by chemical synthesis, conjugating an aptamer to another molecule is easy to design. For example, aptamers engaged with chelating agents have already been reported as imaging tools in magnetic resonance imaging (MRI)  and posiAptamer multimerization is an alternative way to create new functions. Tandem two-OX40 receptor aptamers activate anti-tumor immunity . Since tIn addition to multivalent aptamers, connecting different aptamers is also feasible. For example, Muller et al. reported that a chimera aptamer composed of two aptamers targeting different sites of thrombin had better inhibitory activity than the monovalent version . ConjugaAnother fascinating example of the use of bifunctional aptamers is the delivery of a therapeutic compound to the central nervous system. It is well known that the transferrin receptor (TfR)-binding molecule has the potential to cross the blood–brain barrier. The chimera aptamer consisting of the TfR aptamer and the EpCAM aptamer is reported to be delivered into the brain . There aWe have dedicated over one and a half decade to therapeutic aptamer discovery. Although aptamers act in a similar way to antibodies, superior characteristics such as low immunogenicity, high affinity, and chemical synthesis prompted us to develop therapeutic aptamers. Conformational plasticity of RNA is an important basis for the discovery of good therapeutic aptamers, and recent advances in SELEX technologies and chemical modifications could accelerate aptamer development. Moreover, computational approaches and artificial nucleotides described in this article further expand the possibilities to create next-generation aptamers. Once combined with the concept of functionalized aptamers, multiple successes in aptamer therapeutics could be achieved in the future.


Sipunculus nudus on the prokaryotic community composition in a tidal flat, an onsite investigation was conducted in Suixi in the Beibu Gulf  in the burrow sediments, non‐burrow sediments and the sediments without peanut worm disturbance (control). The16S rRNA gene Illumina MiSeq sequencing was used to investigate the microbial communities and their response to bioturbation by S. nudus in a sandy tidal flat. A total of 18 bacteria phyla were detected, and Proteobacteria and Cyanobacteria constituted the majority of the prokaryotic community in the samples. The distribution of the relative abundances of genera showed that approximately 6.99%–17% of the reads in the samples were classified into 25 known genera. Sulfate‐reducing bacteria (Desulfococcus and Desulfosarcina) were the most abundant taxa, followed by Thermodesulfovibrionaceae LCP‐6, indicating that sulfate reduction is the main process in the sandy tidal flat. The abundances of Desulfococcus, LCP‐6 and Cyanobacterium in the non‐burrow sediment were greater than in the burrow sediment, suggesting that the anoxic condition is more suitable for Desulfococcus and LCP‐6 when the activity of S. nudus is absent. The biomass of Cyanobacterium was decreased by the feeding bioturbation of S. nudus. Meanwhile, the relative abundance of the Bacteroidetes Luteimonas in the burrow sediments was significantly greater than in the non‐burrow sediment, and there was a strong relationship between S. nudus bioturbation and increased in oxygen contents and oxidation‐reduction potentials in the burrow sediment. The abundances of Desulfococcus and LCP‐6 were greater in the middle layer (20–30 cm) than in the top layer in the non‐burrow sediment. However, the middle and bottom layers  had higher abundances of these genera than did the upper layers  in the burrow sediments. The abundances of the Fusobacteria Propionigenium and the Spirochaetes Spirochaeta were greater in the middle and bottom layers  than in the top layers  in the burrow sediment, but this phenomenon was not found in the non‐burrow sediment. This study demonstrates that bioturbation by S. nudus plays an important role in reshaping the bacterial community composition in intertidal regions.To understand the impacts of peanut worms  We aim to reveal the dominant bacterial community composition, diversity, and microbial functions along the sandy tidal flat in Beibu Gulf through high‐throughput 16S rRNA gene sequencing. Meanwhile, we investigated the response of microbial communities to the bioturbation of 22.1S. nudus farming, and the area belonged to middle and low tidal zones. The period of low tide was less than 6 hr. The surface sediment of the tidal flats primarily consisted of quartz sand (as high as 93%), and most of the sand grain size dimensions were less than 0.85 mm  was in the tidal flat in the eastern region of Beibu Gulf  where the same management method was applied. The sediments were derivived from three distinctive types  with three replicates of each type. The burrow sediment was from the area where the worms were farmed and worm burrow activities occurred. The non‐borrow sediment was from the area where worms were distributed, but there was no burrow activity. The control zone sediment (Control) was collected from the area where no S. nudus was found. The samples from burrow and non‐burrow zones at the depths of 0–10, 10–20, 20–30, and 30–40 cm were labled as burrow 1, 2, 3, and 4, and sediments 1, 2, 3, and 4, respectively. The sediment cores in the control zone were not distinguished by layers (0–40 cm). All the samples were then divided into two parts and were stored into an insulated incubator with dry ice and were then immediately transported to the laboratory. One part of the samples was used for physiochemical analyses, such as particle size composition, moisture, organic matter, sandy content, and oxidation‐reduction potential (ORP). Particle size composition was obtained through a fine sieve filtration method. Organic matter content was measured using the combustion method at 550°C. ORP was measured by using an oxidation reduction potentiometer  after the pore water from different layers was filtered. The other samples were used to determine the bacterial community composition according to 16S rRNA gene.Three cores were drilled in parallel in each of the burrow, sediment, and control areas in the sandy tidal flat Figure . The dis2.3® DNA Isolation Kit according to the SDS method  pipeline was used to process the raw sequences and generate overlapped tags . The differences between the same layer in the sediment and burrow groups were analyzed, and differences were considered significant at p < 0.05.The microbial alpha diversity was analyzed using the number of observed species, the Chao I Chao, , Shannon33.1p < 0.05, Table p < 0.05, Table The environmental parameters from the sampling site are shown in Table 3.2In total, 984,268 raw reads were generated from the samples. After filtering, 901,334  were used in the analysis. A total of 448,398 pairwise reads were generated from the nine sediment samples, and the number of effective tags generated from the nine sediment samples ranged from 11,483 to 17,272 with a total of 130,965. All the valid sequences were used for further analysis of bacterial composition and diversity analysis.p < 0.05), and the burrow sediment 3 had the highest number of OTUs and bacterial species. However, non‐burrow sediment 3 (20–30 cm) had the highest Shannon index and Simpson's index  than in the upper layers (0–20 cm). In the present study, the average Shannon index was lower in the non‐burrow sediments (6.35) than in the burrow sediments (6.75). In addition, the microbial diversity was evenly distributed in the four layers of the S. nudus burrows . However, this index varied among the four layers of non‐burrow sediments.The alpha diversity indicators the Chao1 estimator, the number of observed species, and the Shannon and Simpson's indexes are shown in Table diment 3 0–30 cm h3.3p < 0.05), and the abundance of Cyanobacteria (43.54%) was greater in the surface of non‐burrow sediments than that in each layer of the burrow sediments . In addition, the Nitrospirae in non‐burrow sediment 3 was significantly higher than the burrow sediments (p < 0.05).During the study period, 18 bacteria phyla were detected in the nine samples and all the reads were classified into bacterial phyla. Proteobacteria and Cyanobacteria comprised the majority of the microbial communities in the samples, and Bacteroidetes, Planctomycetes, Nitrospirae, Actinobacteria, Acidobacteria, Chloroflexi, Gemmatimonadetes, Verrucomicrobia, Chlorobi, Spirochaetes, Lentisphaerae, Calditrichaeota, Fusobacteria, Firmicutes, and Fibrobacteres were also found in the samples Figure a. In the3.4The distribution of relative abundances at the family level showed that approximately 20%–43% of the reads in the samples were classified into 25 known families  > LCP‐6 (3.55%) > Cyanobacterium (0.93%) > Desulfosarcina (0.37%) > Luteimonas (0.34%) > Propionigenium (0.27%) > LCP‐26 (0.14%) > Robiginitalea (0.11%) > Sulfurimonas (0.109%). Desulfococcus was dominant in the sandy sediment, and its abundance was higher in the layer of 20–30 cm than in the other layers. The abundance of LCP‐6 was greater in the two lowest layers than in the top two layers, but the abundance of Cyanobacterium was higher in the top layers than in the bottom layers. In the burrow sediments, the sequence of abundance was Desulfococcus (3.03%) > Luteimonas (2.92%)> LCP‐6 (1.56%) > Cyanobacterium (0.46%) > Desulfosarcina (0.36%) > Robiginitalea (0.28%) > Propionigenium (0.26%) > Planctomycete (0.15%) > LCP‐26 (0.13%) > Spirochaeta (0.127%). Some other genera were also found in the samples, including Loktanella, Fluviicola, Planctomyces, Helicobacter, Clostridium, and Marinobacterium. The abundances of Desulfococcus, LCP‐6, Propionigenium and Spirochaeta were higher in the bottom layers  than in the surface layers . In addition, the abundances of Luteimonas, Cyanobacterium, Loktanella and Fluviicola were greater in the top layers  than in the bottom layers  in burrow sediments, and the species of bacteria that were dominant in the upper layers  were more abundant in the burrow sediments than in the non‐burrow sediments. Notably, the abundance of Luteimonas was greater in the burrow sediments than in the non‐burrow sediments and was significantly greater in the top two layers  than in the other samples.The distribution of the relative abundances at the genus level showed that approximately 6.99%–17% of the reads in the samples were classified into 25 known genera Figure b. In the3.5The 25 most abundant bacteria were analyzed, and the results are shown in Figure 3.6In this study, PCoA1 and PCoA2 represent the factors that affect the dispersal of groups, and they explain the difference of the 83.15% and 10.48%, respectively Figure . The sam44.1S. nudus than in the other intertidal sediments , and plays an important role in the transfer of organic matter and bacterial communities in the sediment. Previous studies found that the relative abundances of the genera Propionigenium of Fusobacteria, Fusibacter of Firmicutes, Spirochaeta of Spirochaetes, Desulfococcus of Deltaproteobacteria, and Nitrospirae LCP‐6 are increased by the bioturbation of the macrobenthos in mudflat sediments  of the non‐burrow sediments than in the top layers, and a similar situation was found in the burrow sediments. Meanwhile, the abundances of the Fusobacteria Propionigenium and the Spirochaetes Spirochaeta were greater in the bottom layers  than in the surface layers  in burrows. Propionigenium can ferment succinate and organics to form propionate under oxygen‐deficient conditions, and Spirochaeta is the genus of free‐living saccharolytic spirochetes . This characteristic would contribute to the fast process of biogeochemical cycles in sandy tidal flats.The abundances of 4.3Desulfococcus and Desulfosarcina) were the most abundant microbial organisms in the present study. The Thermodesulfovibrionaceae LCP‐6 in the Nitrospirae phylum also had a high relative abundance in the genus’ distribution, indicating that sulfate reduction was the main cycling process in the sandy tidal flat. The Desulfobacteraceae is a family of Proteobacteria, and most species of Desulfobacteraceae reduce sulfates to sulfides to obtain energy and are strictly anaerobic  than in the surface layers  in burrows, which showed that the bioturbation of S. nudus can create better conditions for the growth of bacteria.Sulfate‐reducing bacteria , and the sulfide accumulation could be a reason for their continuous migration, causing growth decline of S. nudus in the Beibu Gulf.The SRB were mainly found in the layer of 0–20 cm in Erhai, and they were mainly found in the layer of 0–7 cm in Hongfenghu. The main reason for their abundant distribution in these locations was that there was more sulfate matter accumulated in the sediment in Erhai but no large amounts of organic matter and sulfate accumulation in the sediment in Hongfenghu (Liang et al., S. nudus. Proteobacteria and Cyanobacteria constitute the majority of the prokaryotic community in the sandy flat in Beibu Gulf. Sulfate‐reducing bacteria were the main group in the sandy tidal flat, while the Desulfococcus and Nitrospirae Thermodesulfovibrionaceae LCP‐6 varied significantly at the depth of 20–30 cm between burrows and sediments. The anoxic condition and rich organic matter in the deep layer might be responsible for the variation of SRB community, especially for LCP‐6. The accumulation of sulfide can lead to slow growth of S. nudus and low biodiversity in a short time. The variation of sulfate metabolism in the sediment and burrows need further study in the future.In conclusion, the bacterial community composition can be affected from the surface to the bottom layer by the bioturbation of The authors declare no conflict of interest.L.J.W., H.R.P., G.Y.J., C.S.W., X.X.Y, Q.J.G., M.Z.H., and Z.C.B. contributed to the experiment design and sampling. L.J.W. collected the data and wrote the manuscript. M.Z.H., and Z.C.B. revised the final version of the article, P.S.R. processed the data.None required.https://figshare.com/s/2d74a80aeb46ca8874db.The OTU data is available in the Figshare repository at


N = 54) show that women can escape this vicious cycle through the meaningful reconstruction of adversity. Women can put a halt on the on-going negative chain reaction through focusing on the meaning and purpose of adversity. We name such life stories narratives of meaningful endurance, which are characterized by structure, authorship and meaningful reconstruction, in opposition to its counterpart, narratives of non-directional distress. The three respective components of SOC - comprehensibility, manageability and meaningfulness - enable the attainment of a narrative of meaningful endurance and individuals with a stronger SOC are more likely to tell narratives of meaningful endurance. Theoretical and policy implications of our findings are discussed.Migrant women in Europe have a higher incidence of health problems and have disproportionately high unemployment rates. We examine how Dutch and Turkish, Moroccan and Surinamese first and second generation migrant women escape the vicious cycle between health problems and unemployment by using the theory of the Sense of Coherence (SOC). We study how SOC works and whether SOC is also applicable outside the domain of health. Our findings from life story interviews ( SOC is a general orientation to life which represents the extent to which individuals (1) perceive arising issues as structured, predictable and explicable, 2) feel able to deal with arising issues, and (3) are willing and motivated to deal with these arising issues . We first discuss the literature on the mechanisms through which SOC operates and how the literature on coping through narratives could offer a new perspective on how SOC operates. After discussing the background of the empirical research and elaborating how we use narrative analysis, we present the results in three main sections; We firstly go into the structural analysis of the narratives looking specifically at how the reinforcing relation between health and employment is narrated. Secondly, we examine how women escape these vicious cycles by discussing three types of narratives; narratives of meaningful endurance, narratives of non-directional distress and narratives in transition. Thirdly, we discuss the relation between these types of narratives and SOC. Lastly, we will discuss how our results may contribute to our understanding of and policies promoting migrant women to escape the vicious cycle between health problems and unemployment.In this study, we set out to examine the mutually reinforcing relation between health problemsbiographical disruption which can be repaired through what researchers variably refer to as biographical work to achieve biographical reinforcement   provide convincing causal explanations for the self, (2) reflect the richness of lived experience, and (3) advance socially-valued living action” , Moroccan (N = 19), Turkish (N = 19) and Surinamese (N = 8) descent. We included women from Moroccan, Turkish and Surinamese descent as these women have a disproportionately high incidence of health problems  living in the four largest cities of the Netherlands . The respondents were between 26 and 55 years old, with an average age of 39.5 years old. Besides the regular headaches and/or neck, shoulder and back aches, the extent of further psychological and physical health problems varied greatly. A fairly high percentage of 37% of the respondents indicated they are, or were, suffering from depression at some point during their life course. Lastly, 32% of the women in the sample were currently employed.We used purposive sampling to select respondents  due to their limited proficiency in Dutch. The interviews lasted from about 1 to 3 h, were audio-recorded, transcribed ad verbatim, made anonymous by using fictitious names and coded using Atlas.ti. After completing the interviews, we asked all participants to fill in the standardized and validated 13-item Sense of Coherence Questionnairenarratives of meaningful endurance (N = 32) and narratives of non-directional distress (N = 16), which differ in structure, positioning of the narrator and meaningful reconstruction. This typology represents ideal types, an abstraction and simplification from reality, in which some narratives may incorporate elements of both ideal types but in which the majority more or less closely reflected one of these ideal types. Some life story interviews (N = 6) seemed to be positioned in between these two ideal types. We labelled them narratives in transition, reflecting the ongoing transformation of these narratives. In the third step of the analysis we compare the SOC scores between groups of individuals with different types of narratives using ANCOVA analysis in SPSS.The analyses consisted out of three steps. Firstly, we identify boundaries of different segments in the transcripts to analyse how segments function strategically in the narrative using structural analysis  reconstruction of the life story is set in motion. By retelling the past, these women develop new perspectives on the present and the future.These two types of narratives are so-called ideal-types placed on a continuum. Narratives that were more difficult to categorize show how women are actively engaged in reconstructing and reshaping their life stories. These narratives seem to be “In the beginning, I was really afraid to make mistakes, I didn’t even dare to pick up the phone, pfff, I thought I am so not going to do that, even though I was doing that for over 15 years before in my previous job and I used to be great at that type of work. […] Yes, actually, not very long ago huh? You know, well, if I look back I really think I have grown a lot in the last months. I am not so insecure anymore. And also, you know, all these women that come here and are going through like this debt restructuring program or have psychological issues. I see where they are, where I was before, and now I can help them a little, and also see how far I got, do something valuable instead of sitting at home. When you say like, it was only 6 months ago, then I think by myself, wow! I don’t reflect on that enough. I have seen the lowest point, now I’m on my way back up. Maybe it was not for nothing, all of this, maybe, I don’t know”. In the previous part of this interview Lisa is mostly telling about her burnout and her debt issues. While telling her life story she reflects on the progress she made in this period, it creates a shift in the way she narrates. Instead of focusing on her own hardship and suffering, she starts focusing on the valuable work she is doing and the progress she made, and hesitantly - “maybe, I don’t know”- starts the meaningful reconstruction of her past experiences. This excerpt highlights how meaningful reconstruction is an on-going process and how meaning can change over time.Lisa, a 39-year-old Dutch woman, for example tells about how she started volunteering at a community center after suffering from a burnout. p < .001). The covariate, age, is not significantly related to level of SOC,  = .763, p = .387). The covariates for ethnic background were also not significantly related to SOC, being from Surinamese  = .031, p = .861), Turkish  = .355, p = .554) or Moroccan  = .058, p = .811) background was not significantly related to the level of SOC (with Dutch women as a reference group). Planned contrasts show that individuals with a narrative of meaningful endurance have a significantly higher level of SOC than individuals with narratives of non-directional distress , and than individuals with narratives in transition . However, there is no significant difference between individuals with a narrative of non-directional distress and individuals with a narrative in transition . It appears that indeed individuals with a stronger SOC are more likely to narrate their life stories in a narrative of meaningful endurance.We have argued that narratives are a potential mechanism through which SOC contributes to escaping the vicious cycle between health problems and unemployment. The life story interviews highlight how, through narratives of meaningful endurance, the vicious cycle between health problems, unemployment and other negative developments can be broken, through the meaningful reconstruction of previous hardship. Hence, we would expect that individuals with a strong SOC are more inclined to tell narratives of meaningful endurance. The analysis of covariance shows that there is a significant difference in level of SOC across individuals with different types of narratives after controlling for age and ethnic background  = 20.228, The life history interviews show that not only are individuals with a stronger SOC more likely to narrate their life stories in a narrative of meaningful endurance, the different components of SOC appear to be related to the subsequent characteristics of this type of narrative. It appears that the three components of SOC, comprehensibility, manageability and meaningfulness, play a role in enabling individuals to achieve structure, authorship and meaningful reconstruction in their life narratives  (Riessman, narrative reconstruction (Williams, The process of meaningful reconstruction which we described using our own terms, could also be described as on Bury, , knittinThe second aim of this study was to expand the use of SOC outside the domain of health and to examine the mechanisms through which SOC operates. Our findings highlight the potential of using SOC theory outside the domain of health research. SOC, as a general orientation to life, does not only protect individuals’ health from risk factors. SOC also enables individuals to meaningfully reconstruct previous hardship and deal with arising issues so as to limit their negative impact on, for example, employment. SOC theory, with its encompassing character and focus on success factors, is a promising theory to apply on new issues and fields of study in future research. Secondly, we looked into whether SOC might operate through enabling individuals to narrate their life story in such a way that it prevents the negative reinforcing relation between health problems and unemployment to continue. Our findings (see Table enables individuals to narrate their life story in a certain way which makes strong SOC individuals more likely to have a narrative of meaningful endurance. To illustrate, individuals who score high on the meaningfulness component of SOC perceive that life has a purpose and is meaningful. However, when a relative passes away, they lose their job or suffer from severe health problems, they may not perceive this adversity as serving some meaningful purpose. If you generally perceive life as meaningful, you are better able to narrate adversity as serving a meaningful purpose, but this is not a given.It could also be argued that the types of narratives we found are not a mechanism through which SOC operates, but a direct reflection of SOC, a way to ‘measure’ SOC in qualitative studies. Yet, having a narrative of meaningful endurance is not the same as having a strong SOC. The Sense of Coherence is a general orientation to life which stabilizes in early adulthood (Antonovsky, how women achieved these elements of their life narratives, the narratives could all be placed on the continuum between narratives of meaningful endurance and narratives of non-directional distress.A potential limitation of this study may be the diverse nature of the sample with respect to ethnic background. However, despite the ethnic diversity in the sample, we were able to place all narratives of the women in our sample on the continuum between these two types of narratives. Therefore, we believe that the diverse nature of the sample strengthens our findings. Another potential limitation of this study is that some women were not native Dutch speakers. We aimed to limit the effect of language difficulties on the results by offering the use of an interpreter. Three respondents decided to make use of an interpreter during the interview and the subsequent filling in of the Orientation to Life Questionnaire. However, non-native speakers who did not make use of an interpreter did not differ in how they narrated their life stories. Despite the obstacles non-native speakers experienced in speaking Dutch, they managed, through body language, tone, occasionally using translation tools and meaningful silences to narrate their life story in all its richness. Here it is also important to note that the ways in which women created structure, causal order, authorship and applied meaningful reconstruction varied greatly. However, irrespective of narratives of non-directional distress and narratives of meaningful endurance. These findings may also be of interest for interventions directed at promoting health and/or employment. The results of several previous studies show that various types of interventions, of varying length and among different populations, are able to increase the level of SOC (Delbar & Benor, In this study we explore the mechanisms through which SOC operates and the use of SOC outside the realm of health. We find support for two ‘grand’ narratives through which SOC operates:


The hereditary spastic paraplegias (HSPs) are genetic motor neuron diseases characterized by progressive degeneration of corticospinal tract axons. Mutations in SPAST, encoding the microtubule-severing ATPase spastin, are the most common causes of HSP. The broad SPAST mutational spectrum indicates a haploinsufficiency pathogenic mechanism in most cases. Most missense mutations cluster in the ATPase domain, where they disrupt the protein's ability to sever microtubules. However, several putative missense mutations in the protein's microtubule interacting and trafficking (MIT) domain have also been described, but the pathogenicity of these mutations has not been verified with functional studies. Spastin promotes endosomal tubule fission, and defects in this lead to lysosomal enzyme mistrafficking and downstream lysosomal abnormalities. We investigated the function of three disease-associated spastin MIT mutants and found that none was able to promote normal endosomal tubule fission, lysosomal enzyme receptor trafficking, or lysosomal morphology. One of the mutations affected recruitment of spastin to endosomes, a property that requires the canonical function of the MIT domain in binding endosomal sorting complex required for transport (ESCRT)-III proteins. However, the other mutants did not affect spastin's endosomal recruitment, raising the possibility of pathologically important non-canonical roles for the MIT domain. In conclusion, we demonstrate that spastin MIT mutants cause functional abnormalities related to the pathogenesis of HSP. These mutations do not directly affect spastin's microtubule-severing capacity, and so we identify a new molecular pathological mechanism by which spastin mutations may cause disease. The hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative disorders characterized by spastic paralysis of the legs. This is caused by progressive degeneration of the longest axons of the corticospinal tract, the main motor pathway that connects the brain to the spinal cord Harding, . To dateSpastin is a microtubule-severing ATPase that uses energy from ATP hydrolysis to sever microtubules. In microtubule severing, spastin's ATPase domains associate to form a hexameric ring structure with a central pore, within which basic residues interact with the C-terminal acidic tail of tubulin   domain with two non-canonical members of the endosomal sorting complex required for transport (ESCRT)-III, CHMP1B and IST1  (raised against a glutathione S-transferase fusion protein that incorporated residues 86–340 of M1-spastin) antibodies were produced as previously described  in 3.8% (vol/vol) formaldehyde in phosphate buffered saline (PBS) and permeabilized in PBS containing 0.1% (vol/vol) saponin (Sigma-Aldrich) or 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich). Coverslips were labeled with primary and secondary antibodies as previously described  or repetitive freeze-thaw cycles with liquid nitrogen (glass). Ultrathin (50–70-nm) conventional sections were cut using a diamond knife mounted to a Reichart ultracut S ultramicrotome. Sections were collected onto copper grids stained using lead citrate. Sections were viewed on a FEI Tecnai transmission electron microscope at a working voltage of 80 kV.Yeast two hybrid (Y2H) assays were carried out using the Clontech Matchmaker system as previously described  were cloned into the BamHI-XhoI sites of pGEX-4T2 . The MIM domains of IST1, residues 319–364, and the single MIM of CHMP1B, residues 174–199, with the addition of a myc and a 6His tag were cloned into the BamHI-NotI sites of pGEX-6P-1 .Escherichia coli and subsequently purified on GST sepharose as described in Collins et al. (Protein was expressed in BL21(DE3)pLysS s et al. . Spastins et al. , whereasGST pull-down assays were carried out as follows: 2 nmole of both bait (MIT domain) and prey (MIM) protein were bound to 20 μl packed volume of glutathione sepharose beads in 1 ml pull-down buffer (PBS + 0.1% NP-40 + 1 mM DTT) overnight at 4°C with rotation. Pull-downs were then washed three times in ice-cold pull-down buffer and resuspended in 30 μl of 3x protein sample buffer. Samples were then separated on an 18% Tris-Tricine gel at 120 V for 3 h in duplicate, half of each pull-down Coomassie stained to assess total protein, and the other half subject to Western blotting and subsequent detection of prey protein bands with anti-myc antibody (Millipore).t-tests or one-way ANOVA with Bonferroni's (when multiple treatments were compared against each other) or Dunnett's  post hoc tests for multiple comparisons tests, using GraphPad Prism version 5.01 for Windows, GraphPad Software, La Jolla California, USA (www.graphpad.com).Statistical analyses were performed by paired two-tailed Three putative mutations in the MIT domain of spastin were identified from the existing literature, E112K, N184T, and L195V  or the disease-associated K388R mutation (which blocks ATP hydrolysis), failed to rescue the increased tubulation of sorting nexin 1  seen in cells lacking endogenous spastin . VPS4 normally acts to remove ESCRT-III proteins from endosomes, while the dominant negative form traps ESCRT-III proteins and associated proteins, including spastin, on the endosomal membrane, so revealing transient endosomal localizations  in the endosomal recruitment of spastin, as compared to the wild-type protein. Thus, in the case of this mutation, defects in endosomal tubule fission, with consequent downstream defects on lysosome function, are probably caused by insufficient endosomal spastin. However, although structural studies of the binding of the MIT domain to CHMP1B show that this mutation lies within the MIM binding site and directly participates in MIM binding, surprisingly, it did not appear to affect MIT-MIM binding when assayed by GST pull-down  for the publication of any potentially identifiable images or data included in this article.An additional description of a family with an N184T spastin mutation was recently published (Kadnikova et al., RA performed experiments, analyzed the data, helped direct the research, and helped write the article. JE performed experiments, analyzed the data, and helped write the article. ER directed the research, analyzed the data, and wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.