Mycobacterium avium subsp. paratuberculosis (MAP) infections in cattle are generally challenging to detect and cost-effective test strategies are consequently difficult to identify. MAP-specific antibody ELISAs for milk and serum are relatively inexpensive, but their utility is influenced by a number of factors such as herd size, herd composition and diagnostic sensitivity. The sensitivity of the test increases with the age of the tested animal, and therefore the general, or “mean effective sensitivity” , for detecting MAP within a herd is dependent upon the age distribution of the herd. For this study we used a dataset of cattle from 4,259 dairy herds and 4,078 non-dairy herds. The aim was to investigate the MES for groups of cattle considered to be reasonable entities for MAP surveillance and control, in order to assist the decision-makers in planning and optimizing these programs economically. We compared six different groups of cattle (three dairy and three non-dairy) in Denmark by calculating the MES for each herd in each group.The distribution of MES showed a large variation within and between groups, and in some groups we found a bimodal distribution of MES. Dairy herds generally showed higher MES than non-dairy herds. Dairy herds in a control programme for paratuberculosis showed a MES similar to all other dairy herds from which animals > 2.0 years were tested (both groups had a median MES = 0.60). For the non-dairy groups, the sensitivity became much higher when animals < 2.0 years and herds with less than 25 cattle were excluded, resulting in a median MES of 0.65.The results showed that MES could indicate the effectivity of testing different cattle groups for MAP, given that the data used are unbiased. Mycobacterium avium subsp. paratuberculosis (MAP) is a cause of financial loss to cattle farmers [ farmers , and con farmers –4. Nonet farmers .The diagnostic test sensitivity of ELISA for detection of infectious animals has been estimated at approximately 30 % , althougSurveillance programs for MAP can be focused on estimation of the prevalence on regional or national level, or on testing freedom of disease in an area. These estimates depend on the diagnostic sensitivity, which may depend on the sampling strategy, in particular if there is huge variation in the age-distribution. Design of sampling strategies is thus dependent on the expected test sensitivity in the study population, which can be measured with MES. Furthermore, targeted surveillance of specific subpopulations can be a cost-effective element in surveillance programs, while optimizing the coverage simultaneously with reducing the costs. MES can thus be a useful tool to describe the strength of studying a subpopulation, using for instance simulation modelling, so that the subpopulation with the highest MES can be chosen for surveillance. The objective of this paper was to estimate the distribution of MES in six different groups of cattle in Denmark. We stratified the cattle population into groups by the herd size and type (dairy and non-dairy), the age of each animal and by enrolment in the current MAP control programme. We also wanted to investigate the within-group as well as the between-group variation in MES and discuss the challenges in estimating the MES for these groups from available databases.A total of 1,586,725 cattle were present in Denmark on the sampling date, 1,165,004 of which were from 4,295 herds recorded as dairy herds, 344,259 cattle were from 4,078 herds recorded as non-dairy herds with more than 25 animals, and the remaining 77,462 cattle were from non-dairy herds not assessed in this study. The age distributions for the six populations are shown in Table This study is the first to provide distributions of mean herd sensitivity estimates for MAP infection in different populations. The MES calculated for a herd can be interpreted as the average sensitivity for a herd with a corresponding age-profile. The results are useful for optimizing MAP surveillance programs with regards to sensitivity, cost and number of samples to take from different groups. For example, a herd with a low MES may indicate that more samples should be collected from that herd in order to have the same information as would be obtained using fewer samples in a herd with a high MES. MES is also a useful way to measure the strength of a surveillance strategy. For example, we found that a relatively high MES (median 0.60) was obtained when testing animals above 2.0 years of age in dairy herds but a higher MES (median 0.65) was obtained when testing animals above 2.0 years in non-dairy herds with more than 25 animals. This means that large non-dairy herds are easier to detect MAP infection in, and the sample size can consequently be reduced slightly compared to dairy herds. However, in general we can adapt the sample size to achieve a comparable herd-level sensitivity even if the age-distribution differs. MES is also useful to show the potential of testing subpopulations, which could be further tested with simulation models.In all groups except Groups 2 and 3, the variation in the sensitivity distribution was fairly large. In Groups 1, 2 and 4 we found a bimodal distribution. We ascribe this (at least for the two first groups) to a bias in the database registration, resulting in some heifer-rearing herds being included in Group 2, adding to the within-group variation. Therefore, we suggest that MES should be used with caution, as it may not reflect the sensitivity of the intended group. This should also be considered when selecting herds for estimation of within-herd prevalences of MAP or other studies which rely on the sensitivity of the test . When deWe found that the MES was slightly higher for dairy herds than for non-dairy herds (Group 1 vs Groups 4 and 5), partly due to a bimodal distribution of the MES for non-dairy herds caused by a large proportion of young cattle intended for beef production purposes. This proportion of young cattle may differ seasonally, and consequently the sensitivity distribution also differs seasonally. This is not the case for dairy herds, where in Denmark calving occurs throughout the year. When comparing only animals > 2.0 years from large beef cattle herds and dairy herds (Groups 2 and 6), the MES in the beef herds was generally higher, but with more variation  to describe the herd-level sensitivity when testing for MAP. We calculated the MES for different subpopulations of cattle herds in Denmark and found a large variation between the investigated groups. Some groups had a bimodal distribution, but groups including young stock had low MES, whereas groups excluding young stock had on average high MES. However, the variation within a group was so large that it might be worthwhile to estimate the MES in a specific herd before embarking on a test scheme. The study confirms the general opinion that testing young stock is not beneficial. This updated information can be used to improve modelling of the effective sensitivity in mathematical and simulation models in order to optimize surveillance and control programs.The animals recorded in the Danish Cattle Database on 28 July 2014 were used to assess the within-herd age distribution in the six populations in question. By law, the birth date of all cattle born in Denmark must be recorded along with the date of death, and these recordings were used to establish the number and age of live animals. The database also recorded whether or not the herd was producing milk, and we were therefore able to divide herds into dairy and non-dairy groups. As a consequence, the latter group consisted of herds rearing calves or young stock smallholders (“hobby” farmers), as well as regular beef herds. We defined six groups of interest to explore in this study: 1) all cattle in Danish dairy herds; 2) all cattle > 2.0 years in Danish dairy herds; 3) all cattle > 2.0 years in dairy herds enrolled in the Danish control programme on paratuberculosis ; 4) all Testing young stock (< 2.0 years) by ELISA is generally discouraged due to low sensitivity . Their inclusion in some of the groups in the present study was mainly motivated by a desire to determine the impact on the mean herd sensitivity compared to testing of adult cattle alone. We used all dairy cattle herds irrespective of herd size, since in Denmark they generally consist of > 25 animals, with the mean herd size in Group 2 being 149 adult cattle > 2.0 years in each herd.Data for the MAP-specific ID Screen®  antibody ELISA used for milk samples was used, as relevant sensitivity and specificity estimates were available for this test. The effective test specificity has been estimated to 0.9866 –17, and The function for age-specific effective sensitivity used here has been described by Nielsen et al. (2013) and is defined as:Se(agei) is the sensitivity depending on the age of animal i. The herd mean effective sensitivity, MES, was then estimated by calculating the sensitivity for each cow in the herd using the function described above, and then taking the mean of the estimated sensitivities for all animals belonging to each group in each herd:where MESjk is the mean effective sensitivity for group j within herd k, where Njk is the total number of cows in group j within herd k, and Se(ageijk) is the sensitivity for animal i belonging to group j within herd k. Note that one herd may have cows belonging to multiple groups, and may therefore contribute several times to the results.So This study is theoretical and did not need to comply with any guidelines.


Myxococcus xanthus CarD and CdnL, have distinct functions whose molecular basis remain elusive. CarD, a global regulator linked to the action of several extracytoplasmic function (ECF) σ-factors, binds to the RNAP β subunit (RNAP-β) and to protein CarG via an N-terminal domain, CarDNt, and to DNA via an intrinsically unfolded C-terminal domain resembling eukaryotic high-mobility-group A (HMGA) proteins. CdnL, a CarDNt-like protein that is essential for cell viability, is implicated in σA-dependent rRNA promoter activation and interacts with RNAP-β but not with CarG. While the HMGA-like domain of CarD by itself is inactive, we find that CarDNt has low but observable ability to activate ECF σ-dependent promoters in vivo, indicating that the C-terminal DNA-binding domain is required to maximize activity. Our structure-function dissection of CarDNt reveals an N-terminal, five-stranded β -sheet Tudor-like domain, CarD1–72, whose structure and contacts with RNAP-β mimic those of CdnL. Intriguingly, and in marked contrast to CdnL, CarD mutations that disrupt its interaction with RNAP-β did not annul activity. Our data suggest that the CarDNt C-terminal segment, CarD61–179, may be structurally distinct from its CdnL counterpart, and that it houses at least two distinct and crucial function determinants: (a) CarG-binding, which is specific to CarD; and (b) a basic residue stretch, which is also conserved and functionally required in CdnL. This study highlights the evolution of shared and divergent interactions in similar protein modules that enable the distinct activities of two related members of a functionally important and widespread bacterial protein family.Two prototypes of the large CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-binding proteins,  Myxococcus xanthus, a 316-residue global regulatory protein, CarD, is involved in the control of light-induced carotenogenesis, starvation-induced development of multicellular fruiting bodies, and other processes -labeled proteins, in MOPS minimal medium containing 2.5 g/l 13C6-glucose and 1 g/l 15NH4Cl as the sole carbon and nitrogen sources 222 = 895 for 0% helix and (1–3/Nr) for 100% helix, where Nr is the number of residues residues .0.05% NaN3in 9:1 v/v H2O/D2O or pure D2O. Probe temperatures were set using a methanol sample. Standard triple resonance NMR methods were used for data acquisition, processing, and 1H/15N/13C NMR chemical shift assignments, which were deposited at BioMagResBank . Distance constraints, obtained from a 3D NOESY [1H-13C]-HSQC and two 2D [1H-1H]-NOESY spectra  in H2O/D2O and/or D2O, and  torsion angle constraints, from TALOS, were used as input in structure calculations using a standard iterative protocol of the program CYANA 2.1 [NMR data were acquired in a Bruker AV-600 or AV-800 US2 spectrometer equipped with a z-gradient triple resonance cryoprobe using 0.5 or 0.2 mL samples of 0.5–1 mM protein in 100 mM NaCl/50 mM sodium phosphate buffer /YANA 2.1 ,31,32. OYANA 2.1 . MOLMOL YANA 2.1  was usedin vivo [in vivo remained to be tested and was examined here. In the M. xanthus response to light, CarD is implicated in the activation of the carQRS regulatory operon from its promoter, PQRS, which is recognized by RNAP holoenzyme containing the ECF-σ factor CarQ [+ phenotype; carD-deleted (ΔcarD) strain does not become red in the light  exhibited a Car- phenotype . In marked contrast to the ΔcarD or ΔcarDNt strains, the strain expressing carDNt acquired the red color associated with carotenogenesis, albeit at a slower rate than the wild-type strain  that aligns with CdnLNt, can on its own interact with RNAP-β. A striking difference between CarDNt and CdnL is that the former also interacts with CarG [1–72 or by the C-terminal part of CarDNt spanning residues 61 to 179, CarD61–179  estimated for CdnL. Hence, even though sequence-based analysis predicts similar secondary structures for CarDNt and CdnL, far-UV CD spectroscopy suggests differences between the two.Sequence-based secondary structure predictions suggest close correspondence between CarDNt and CdnL, with five β-strands and a short helix within the N-terminal stretch, and five helices in the C-terminal part . These pl module . Using f1–72 could be stably expressed and its NMR spectra exhibited good overall quality and signal dispersion  being (0.7± 0.2) Å for the structurally ordered backbone segments between residues 9 and 67  the equivalent modules in CdnL and its homologs [E. coli TRCF-RID, and to a lower degree (Z-scores <4.5), various other proteins with Tudor-like domains [1–72 and CdnLNt suggests that the different secondary structures for CarDNt and CdnL based on far-UV CD likely stem from differences between CarD61–179 and CdnLCt.The CarD4 and β5 . Most ofing DALI  yielded homologs ,20–22 an domains ,37. The 1–72 also mirrors CdnLNt in specific contacts with RNAP-β. In CdnL, mutating F36, M49, or P51 to alanine did not affect its protein stability or folding but abrogated interaction with RNAP-β [19–148  V123 or E125, but not D122 or K124, impaired the interaction with CdnL [19–148 with CarDNt was impaired on mutating V123 or E125, but not D122 or K124 , which does not interact with CarG, is also indispensable [in vitro and mutation of the highly conserved W or of the basic R128/K129 residues did not have a drastic effect; only the F125A and the R90A/R91A/R93A mutations produced noticeable effects, the triple mutant being almost equivalent to lack of CdnL in vitro and in vivo [Elimination of CarDNt results in the same phenotype as that caused by the complete deletion. That disrupting its interaction with RNAP, which occurs via CarDpensable . Severalpensable ,19,21. B in vivo .M. xanthus strains in which the wild-type gene was replaced by each of these mutant carD alleles , K130/R132 (R128/K129 in CdnL), and K93/R95/R97 in CarD (corresponding to R90/R91/R93 in CdnL) . The Car alleles  and theyexpected . Hence, was Car- . MoreoveD strain . BACTH aD strain , implyinQRS-lacZ activity was considerably diminished in the R95A mutant, albeit somewhat less than with the K93A/R95A/R97A mutant  by RNAP holenzyme with the primary housekeeping σA; and that this activity of CdnL is impaired by mutations disrupting the interaction with RNAP-β [in vivo at PQRS, the alternative ECF-σ CarQ-dependent promoter whose activation requires CarD, suggesting absence of direct CdnL action at this promoter [QRS but not on rRNA promoters [QRS expression on disrupting CarD/CarDNt binding to RNAP-β therefore suggests that this interaction, unlike with CdnL, is not as critical in transcriptional activation mediated by CarD. Other CarD interactions should then be more decisive determinants of its function.Mutations that impair CdnL interactions with RNAP-β were very adverse to cell growth and survival . It is th RNAP-β ,12,18,19promoter . By contromoters ,10. The 61–179 segment, but the exact molecular mechanism by which CarG acts together with CarD in ECF-σ promoter activation remains enigmatic. In the HMGA-driven assembly of the large transcriptionally competent complex in eukaryotes known as the enhanceosome, a key role is played by transcriptional factors that do not bind DNA but rather provide a protein scaffold for interaction with various other regulatory factors [One interaction indispensable in every known CarD-dependent process is that with CarG ,5,8. We  factors ,39. CarG factors , but thein vitro that we have thus far never detected with either CarDNt or CdnL [in vivo nucleoid localization of CarD [in vivo of mycobacterial CdnL and RNAP σA and its absence from non-promoter regions also suggested that CdnL is targeted to the genome through its interaction with RNAP and not through direct binding to DNA [in vivo [in vivo on mutating an AT-rich site lying upstream of the ECF-σ promoter PQRS to which CarD exhibits minor groove binding via its HMGA-like domain [A second characteristic CarD interaction is that mediated by its HMGA-like C-terminal domain. This domain confers an intrinsic DNA binding capability to CarD  or CdnL . Hence,  of CarD , whereas of CarD . The colg to DNA . The Car[in vivo ,7,29. The domain ,7,29,41 e domain ,39,42. TO formation at rRNA promoters was abolished on mutating the basic segment [O complex and whether it has a similar role in CarD function, however, remain open. This basic segment and an adjacent highly conserved tryptophan have been linked to DNA binding based on the weak, nonspecific binding to double-stranded DNA observed in vitro with the mycobacterial CdnL homolog, and single mutations of the basic residues or the tryptophan were detrimental for M. tuberculosis growth and viability [M. xanthus, mutation of the basic segment, but not of the tryptophan, drastically impaired CarD function in vivo, just as was observed with CdnL. Thus, while the conserved tryptophan appears to play an important but unknown role in the function of the M. tuberculosis protein, it does not appear to do so in M. xanthus CarD or CdnL. On the other hand, the basic residues are functionally crucial in both M. xanthus proteins as well as in that of M. tuberculosis, even though in contrast to the latter neither CarDNt nor M. xanthus CdnL exhibited any intrinsic DNA binding in vitro. That CdnL with its intact basic segment is required in RPO formation hints that any crucial and specific contacts potentially involving this segment would more likely be with elements within RPO (not naked DNA), and structural models have been proposed for possible interaction in RPO with the transcription bubble or RNAP-σA [A promoters, but resolving these issues for CarD or CdnL would require further higher resolution analyses in future studies. Nevertheless, the structure-function dissection of CarDNt reported here has highlighted four distinct interactions associated with CarD and their relative functional importance: those involving CarG and a conserved basic residue segment in CarD61–179 are crucial for CarD function, that with RNAP is apparently dispensable, and that with DNA via the HMGA-like domain contributes synergistically to maximize activity.It is remarkable that, despite the divergences discussed above between CarDNt and CdnL, both have a conserved basic residue segment crucial for their respective functions. With CdnL, its ability to stabilize RP segment . The detiability ,19,21. I RNAP-σA ,22. It iM. xanthus genome suggested that gene duplication, divergence, horizontal gene transfer and domain fusion events may have occurred quite extensively in the evolution of the sensory complexity of this bacterium [In an early study, we speculated that CarD may have evolved from an interkingdom domain fusion of a preexisting bacterial domain akin to CarDNt or CdnL and an eukaryotic HMGA-like domain acquired by horizontal gene transfer . Subsequacterium . The sim1–72 are 2LT1 for structural coordinates deposited in the Protein Data Bank, and 18194 for NMR chemical shifts in BioMagResBank (http://www.bmrb.wisc.edu/).Accession codes for CarDS1 File(DOC)Click here for additional data file.


Ephs are transmembrane receptors that mediate cell-cell signaling. The N-terminal ectodomain binds ligands and enables receptor clustering, which activates the intracellular kinase. Relatively little is known about the function of the membrane-proximal fibronectin domain 2 (FN2) of the ectodomain. Multiscale molecular dynamics simulations reveal that FN2 interacts with lipid bilayers via a site comprising K441, R443, R465, Q462, S464, S491, W467, F490, and P459–461. FN2 preferentially binds anionic lipids, a preference that is reduced in the mutant K441E + R443E. We confirm these results by measuring the binding of wild-type and mutant FN2 domains to lipid vesicles. In simulations of the complete EphA2 ectodomain plus the transmembrane region, we show that FN2 anchors the otherwise flexible ectodomain at the surface of the bilayer. Altogether, our data suggest that FN2 serves a dual function of interacting with anionic lipids and constraining the structure of the EphA2 ectodomain to adopt membrane-proximal configurations.  •EphA2 receptor ectodomain mainly interacts with lipid bilayers via its FN2 domain•Anionic lipids are recruited by positively charged residues in FN2•Mutations K441E and R443E abolish the specific binding of FN2 to anionic lipids•The FN2-bilayer interaction orients the EphA2 ectodomain relative to a membrane Chavent et al. combine multiscale molecular dynamics simulations with a biochemical assay to describe the interactions of the EphA2 receptor FN2 domain with the anionic lipids of a model membrane. This provides insights into the dynamic behavior of the receptor at the cell surface. The ephrin receptors (Ephs) are the largest group within the family of receptor tyrosine kinases (RTKs). Ephs play critical roles in many developmental processes  and are Although detailed structural data are now available for all intra- and extracellular Eph domains , and modIII9 domain of fibronectin on  surfaces (We have explored the interaction of the EphA2 FN2 domain with model lipid bilayers. Molecular dynamics (MD) simulations provide a powerful computational tool to explore the interactions between proteins and membranes . This apsurfaces . Here, wWe performed coarse-grained (CG) dynamics simulations  for the first peak changes from 7.5 for the wild-type  with the membrane, thus integrating structures from both crystallography  and nuclWe first constructed an atomistic model of the FN2 domain attached to the TM helix corresponding to the NMR structure of the TM dimer  see Exp. We perf2X11; but without the ephrin ligand) and on the unliganded (PDB: 2X10) conformation . We generated models based on the liganded conformation  resumed a stable position in which both the FN2 domain and the LBD domain interact with the membrane. The LBD residues involved in this interaction were largely aromatic. Both the FN2 and LBD domain interactions attract anionic lipids , the flexible FN1-FN2 linker allowed for movement of the EphA2 ectodomain, although the receptor remained in a generally upright orientation domains (sometimes denoted as “lipid rafts”) are densely packed regions that are enriched in signaling proteins and play important roles in membrane signaling events . The lipcis interaction with ephrins, also found in lipid nanodomains  (We used two different protocols. We coarse grained the FN2 domain A using a2K9Y)  to gener2K9Y) . We then2K9Y)  to smoot2X10 or 2X11 PDB entries. In the latter case, we removed the ephrin ligand. For each structure we used Modeller (http://www.ks.uiuc.edu/Research/vmd/plugins/mutator/) on the atomistic model of the liganded monomer to construct the K441E + R443 mutant, then coarse grained it as described above.We constructed two different systems, corresponding to the ectodomain monomers in an unliganded or liganded state using, respectively, one monomer from the Modeller  to constBased on the atomistic monomer models, we constructed each dimer by superimposing each of two ectodomain monomers onto the PDB structures of the ectodomain + TM dimers, then placing the full dimer in the membrane. We coarse grained the membrane and the proteins (making sure that we did not link the monomers together by the elastic network) as described above. We then applied the Alchembed methodology  to removwww.gromacs.org)   and the acs.org) . For theacs.org)  composedrecently . The intembranes . The intAtomistic simulations were carried out using the GROMOS96 43a1 force field . Water aVMD  was usedP29317) was cloned into the AgeI-KpnI cloning site of a pHLsec vector  transiently in HEK293T cells, in the presence of kiffunensine . Proteing for 10 min. We repeated the experiments seven times. For each set the bound protein fractions were visualized by western blot using mouse anti-pentaHis antibody (Qiagen). Data analysis was done with ImageJ (We prepared lipid vesicles by drying dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), or a 1:1 (w/w) mixture of DOPG and DOPC under constant argon flow to avoid oxidation. We re-suspended the resultant lipid film in buffer  and sonicated the mixture for 15 min to generate vesicles. Final lipid concentrations were 2 mg/ml. We mixed 100 μl of vesicle suspension with equal amounts of wild-type or mutant EphA2 FN domain 2 protein, incubated these at room temperature for 10 min, and centrifuged them at >20,000 × h ImageJ . In eachM.C. performed and analyzed simulations. E.S. performed experiments. M.C., E.S., E.Y.J., and M.S.P.S. designed the research and together wrote the manuscript.


Domain patterns of perpendicularly magnetized ultra-thin ferromagnetic films are often determined by the competition of the short range but strong exchange interaction favouring ferromagnetic alignment of magnetic moments and the long range but weak antiferromagnetic dipolar interaction. Detailed phase diagrams of the resulting stripe domain patterns have been evaluated in recent years; however, the domain fluctuations in these pattern forming systems have not been studied in great detail so far. Here we show that domain fluctuations can be observed in ultra-thin two-dimensional ferromagnetic Fe/Ni/Cu(001) films with perpendicular magnetization in the stripe domain phase. Non-stroboscopic time-resolved threshold photoemission electron microscopy with high temporal resolution allows analysing the dynamic fingerprint of the topological excitations in the nematic domain phase. Furthermore, proliferation of domain ending defects in the vicinity of the spin reorientation transition is witnessed. The walls between domains in magnetic thin films close to a spin reorientation transition fluctuate owing to thermal excitation. Here Kronseder et al. image these fluctuations in iron/nickel/copper films using time-resolved threshold photoemission electron microscopy. Competition between attractive short range and repulsive long range interactions may lead to intricate pattern formation that can be observed in diverse complex systems12356789101112In the prototypical systems Fe/Cu(001) and Fe/Ni/Cu(001), many modulated phases such as stripe phases, labyrinthian phases and bubble phases have been discovered and have been classified in appropriate phase diagrams314151718192021222426In the following, we study perpendicular domain phases of the 2D ferromagnetic system Fe/Ni/Cu(001) with high real-time temporal (<1 ms) and spatial resolution (<100 nm)An overview of the observed domain pattern in the vicinity of the spin reorientation transition (SRT) in Fe/Ni/Cu(001) for a typical wedge-type thin-film sample (for sketch see dSRT indicated in dSRT=dFe=1.8±0.3 ML (monolayers). The minimum domain width at the SRT is around 300 nm. Note that we do not focus on the details of the SRT that may be spiral-like14In this system, a well-studied SRT occurs at a thickness et al.et al.et al.From these types of experimental data, one can in principle determine the predicted domain phases in this system. Similar to the seminal experiments by Vaterlaus According to theory, on raising the thickness (temperature), the smectic phase eventually transforms to the nematic phase mediated by the two types of fluctuations. When the stripe bending energy is smaller than the stripe orientation energy, meandering of the domain walls can be observed that decreases positional order and causes the smectic crystal to lose its orientational rigidity827In most of our experiments, an orientationally ordered phase  is followed by a completely disordered phase before the SRT occur, as shown for instance in In the following, we will demonstrate that a non-stroboscopic imaging technique with high temporal resolution can be used efficiently to search for the dynamical fingerprint of the stripe domain phase of this 2D magnetic model system. This has previously only been possible by monitoring average quantities such as the ac-susceptibility24314The concept of the approach is straight forward. In a perfect smectic stripe phase, for example, it is expected that fluctuations are suppressed due to geometric constraints. In a fully disordered phase or a smectic/nematic phase with topological defects, on the other hand, the domain patterns are less constrained and one might expect fluctuations to be more pronounced. Our unique experimental method allows us to record images with exposure times as short as 450 μs enabling the analysis of non-stroboscopic real-time movies of the fluctuating domains as a function of effective temperature.2, images recorded at 200 frames per second) in the nematic stripe domain phase with pronounced defects marked in colour. The Fe-wedge/(9 ML)Ni/Cu(001) sample is the same as shown in wD≈2,060 nm and wD≈800 nm are rescaled to the case of wD≈400 nm. This figure reveals that the relative maximum of the fluctuating domain area in fact increases slightly with decreasing domain width, whereas the absolute maximum fluctuating domain area decreases as shown in To evaluate the observed fluctuations, we calculate the local change of the domain area and register the time the fluctuating area remains in the new state (dwell time) before it jumps back into its original configuration or into a new state defined by an area change of at least 5–10 pixels depending on the field of view and corresponding resolution. We first apply this evaluation method to the nematic domain phase with varying domain width. Typical domain images with decreasing domain width are shown in et al.Next, we take a closer look at the dynamical fingerprint of topological defects such as the ones marked in In conclusion, we have imaged fluctuations of the domain patterns in the vicinity of a spin reorientation transition for the first time with high temporal and spatial resolution. We identify the relevant topological defects and evaluate their dynamical signatures. We conclude that in the particular striped domain phase investigated here, A-type defects drive the phase transition into a less ordered phase. The presented data may prove to be of importance for the analysis of phase transitions in 2D systems. Furthermore, the recorded movies can be viewed as experimental realizations of extremely large scale Monte Carlo simulations of a truly 2D system and may allow to shed light on finite size effect problems often encountered in these simulations.−1 in ultra-high vacuum with a base pressure better than 2 × 10−10 mbar and a pressure during evaporation <3 × 10−10 mbar. During evaporation reflection high-energy electron diffraction oscillations are monitored confirming layer-by-layer growth and serving as an accurate thickness calibration. No contamination of the films is visible in X-ray photoemission spectroscopy.The Fe/Ni/Cu(001) samples are prepared using a well-established protocol. We first prepare the Cu(001) single crystal by cycles of soft sputtering and annealing until sharp reflection high-energy electron diffraction spots are visible. The magnetic films are evaporated at a rate of 0.4 ML minTo be able to perform fast non-stroboscopic imaging with large magnetic contrast and high spatial resolution, we revert to the recently discovered contrast mechanism of magnetic circular dichroism in threshold photoemission applied to a photoemission electron microscope. The illumination unit comprises a 405 nm (3.06 eV) laser diode with up to 600 mW, typically set to ∼(100−200) mW, a linear polarizer and a rotatable quarter-wave plate. Since the work function of Fe and Ni is around ∼5 eV, Cs adatoms were deposited before PEEM operation to lower the work function and, hence, to increase the photocurrent.C.H.B. and M.K. planned the experiment. M.K. performed the experiments. C.H.B. and M.K. analysed the data. T.N.G.M., M.B., M.V. and M.Z. helped with the data analysis. C.H.B and M.K. wrote the manuscript.How to cite this article: Kronseder, M. et al. Real-time observation of domain fluctuations in a two-dimensional magnetic model system. Nat. Commun. 6:6832 doi: 10.1038/ncomms7832 (2015).Supplementary Figures 1-6, Supplementary Notes 1-5 and Supplementary ReferencesThe Supplementary Movie 1 shows the fluctuating domains of the domain configuration shown in Fig. 2 of the main text and fluctuations in the vicinity of topological defects. Also shown is the evolution of the A-type defect discussed in the manuscript, and the decay of a C-type defect driven by thermal fluctuations and leading to the proliferation of A-type defects.


H2 domain. The oligosaccharide not only orients the CH2 domains but plays an important role in influencing IgG effector function, and engineering the IgG-Fc oligosaccharide moiety is an important aspect in the design of therapeutic monoclonal IgG antibodies. Recently we reported the crystal structure of glycosylated IgG4-Fc, revealing structural features that could explain the anti-inflammatory biological properties of IgG4 compared with IgG1. We now report the crystal structure of enzymatically deglycosylated IgG4-Fc, derived from human serum, at 2.7 Å resolution. Intermolecular CH2-CH2 domain interactions partially bury the CH2 domain surface that would otherwise be exposed by the absence of oligosaccharide, and two Fc molecules are interlocked in a symmetric, open conformation. The conformation of the CH2 domain DE loop, to which oligosaccharide is attached, is altered in the absence of carbohydrate. Furthermore, the CH2 domain FG loop, important for Fcγ receptor and C1q binding, adopts two different conformations. One loop conformation is unique to IgG4 and would disrupt binding, consistent with IgG4's anti-inflammatory properties. The second is similar to the conserved conformation found in IgG1, suggesting that in contrast to IgG1, the IgG4 CH2 FG loop is dynamic. Finally, crystal packing reveals a hexameric arrangement of IgG4-Fc molecules, providing further clues about the interaction between C1q and IgG.The Fc region of IgG antibodies, important for effector functions such as antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis and complement activation, contains an oligosaccharide moiety covalently attached to each C The typical pattern of glycosylation is that of a heptasaccharide bi-antennary core, which can additionally contain fucose, galactose and sialic acid residues *, derived from the Rea IgG4 myeloma protein , was enz2.2xia2 package  are referred to in accordance with the C1-type immunoglobulin domain strand definition of Data were collected at beamline I04-1 at the Diamond Light Source . Integration was performed with XDS within the  package , and fur package . The str package  using prge (4C54 . Refinemge (4C54  and manu54 (Coot . TLS gro54 (Coot  and POLY54 (Coot  within P54 (Coot . Data pr33.1The asymmetric unit of the deglycosylated IgG4-Fc (degly-Fc)* structure contains two interlocked Fc molecules related to one another by a pseudo-symmetric two-fold rotation A. No intH2 and CH3 domains is essentially identical for all four chains, which could be superposed with r.m.s. deviations of 0.39–0.90 Å. While there are local differences at the interfaces between the four chains of the degly-Fc asymmetric unit, some due to side chain disorder, the general features can be described as follows. The CH2 domain from chain A simultaneously contacts the CH2 domain from chain C and the CH3 domain from chain D. The overall molecular packing is such that CH2-CH2 and CH2-CH3 domain interactions for chain B are with chains D and C, those for chain C are with chains A and B, and those for chain D are with chains B and A, respectively, with an average buried surface area of 1470 Å2.The overall orientation of CH2-CH2 and CH2-CH3 interfaces is presented from the perspective of chain B , flanked by others forming van der Waals interactions (Phe243 and Phe296). The CH2-CH3 domain interface between chains B and C is formed predominantly from van der Waals interactions. This interface comprises CH2 domain FG loop residues Pro329 and Ser330 (chain B), and Lys340, Tyr373, Leu398 and Phe404 (chain C)  B.With the exception of conversion of Asn297 to Asp297 through the activity of PNGase F, and conformational differences in loop regions (described below), some due to the absence of oligosaccharide, there were no significant differences between the overall structure of deglycosylated IgG4-Fc and glycosylated IgG4-Fc .3.2IgG typically contains a heptasaccharide bi-antennary core, with additional fucose, galactose and sialic acid residues . The serH2 domain, burying a total area of ∼1000 Å2. The patch exposed by the absence of carbohydrate in the degly-Fc structure is partially covered by the intermolecular CH2-CH2 domain interface, burying a total surface area of ∼750 Å2. While the surface buried by the CH2-CH2 domain interface is not identical to that buried by carbohydrate, residues Phe243 and Arg301 participate in both CH2-CH2 domain and CH2–carbohydrate interactions.In glycosylated IgG4-Fc, the heptasaccharide core covers the surface of the C3.32* moiety, to a tetrasaccharide (MN2F)2* moiety, causes the CH2 domains to approach one another, and the distance between Pro329 Cα atoms, located at the top of the CH2 domains (E. coli) revealed an even more closed CH2-CH2 conformation, with Pro329-Pro329 Cα distances of 18.9 Å and 19.6 Å for the two Fc molecules of the asymmetric unit  domains , decreas domains . The cryric unit , while tric unit    B and C. tructure B and C, tructure .3.4H2 domain DE loop, which includes Asn297 to which the oligosaccharide is attached, displays a conserved conformation in glycosylated IgG-Fc structures. It is altered, however, in enzymatically deglycosylated  or Phe296 (IgG2 and IgG4) from the DE loop adopt a range of positions, pointing towards or away from the oligosaccharide, independent of the presence or absence of a fucose residue linked to the first N-acetylglucosamine. However, in degly-Fc, Phe296 faces towards the inner face of the Cric unit A.3.5H3 domain plays a key role in controlling Fab-arm exchange (FAE) in IgG4  is important for both Fcγ receptor and C1q binding  differs by ∼1.6 Å, which would have the effect of moving the CH2 FG loop closer towards the Fcγ receptor , suggesting a role for Ala327 in positioning Pro329 for the IgG1/Fcγ interaction.A conserved, but shifted, Calso Gly . Much sm3.71HZH, 4NHH; P622), although the same space group was reported for intact IgG4 crystals  Fc receptor, rheumatoid factor and TRIM21 (tripartite motif 21) binding  . The IgGcontacts B, perpen binding . The sam binding , and the1HZH, The degly-Fc structure and a structure of an intact IgG antibody, both solved at 2.7 Å resolution (PDB accession number H3-CH3 domain contact (shown in A (chain A) and Pro387B (chain B), van der Waals interactions between Tyr436A and Tyr436B, and hydrogen bonds between Asn434A, Glu380B and Tyr436B, and between Gln438A and Gln438B (main chain). Ile253A from the CH2 domain protrudes into a pocket created by Leu251B, Ile253B, His310B, Gln311B, Leu314B (CH2 domain) and His435B (CH3 domain). Additionally, His435A from the CH3 domain forms van der Waals interactions with Ile253B and Ser254B from the CH2 domain. Met252 from the CH2 domain adopts different side chain positions, but forms van der Waals interactions with nearby residues, including Met428, Asn434 and His435.Residues involved in the IgG1 and IgG4 hexamer interfaces are identical. Cshown in B include3.8H2 domains adopt a range of orientations in relation to the CH3 domains  adopt an open conformation. Superposition of a number of IgG-Fc structures available from the Protein Data Bank onto the degly-Fc hexamer (formed by chains A and B) using CH3 domain Cα atoms revealed clashes between residues from the CH2 domain AB loop. Even superposition of chains C and D from the degly-Fc structure revealed clashes, implying that a structural change within the CH2 domain is required to form the hexamer.In IgG, the C domains . In the H2 domains onto the degly-Fc hexamer, revealed close contacts mediated by Ile253 from the CH2 AB loop in the vast majority of structures analysed, including close contacts between Ile253A and Ile253B and Ile253A and His310B, where A and B refer to interfacing chains  and 1.21 Å (IgG4)  .Fig. 5CH3.9H2 domain BC and FG loops can adopt a different conformation, disrupting the binding site (H2 domain, although the degly-Fc structure presented here does reveal a dynamic FG loop with the ability to adopt the conserved, IgG1-like conformation.In human IgG1, residues Asp270, Lys322, Pro329 and Pro331 are important for C1q binding  (indicating site . FormatiH2 domain.The effect of a recently described Glu345Arg mutation in enhancing complement activation  can now While residues at the IgG1 and IgG4 hexamer interfaces are identical, the interactions formed are not. For example, in both IgG1 and IgG4, variation in the Met252 side chain position is observed. Furthermore, while similar, the IgG1 and IgG4 hexamers are not identical when superposed. In a number of IgG3 allotypes, Arg435 and Phe436 replace His and Tyr, found in IgG1 and IgG4, respectively. Since these residues are both at the interface, allotypic sequence differences and variation in side chain position could affect the orientation of Fc molecules within the hexamer, which in turn might alter the disposition of the C1q binding sites, and the molecular surface presented to C1.4H2 DE loop in the absence of carbohydrate. Furthermore, the CH2 FG loop adopts two different conformations, one unique to IgG4, and one more akin to the conserved conformation found in IgG1. Finally, crystal packing reveals a hexameric arrangement of Fc molecules, similar to the IgG1 hexamer involved in C1 binding. The deglycosylated IgG4-Fc structure reported here thus extends our understanding of this structurally less well characterised IgG subclass.In summary, we solved the structure of enzymatically deglycosylated, serum-derived human IgG4-Fc at 2.7 Å resolution. The structure comprises a novel, interlocked arrangement of two Fc molecules and reveals structural alteration of the C


We incorporate the chiral centres into one of the organic ligands for the synthesis of the chiral COFs. We subsequently in situ prepare the COF-bound capillary columns. The prepared chiral COFs and their bound capillary columns give high resolution for the separation of enantiomers with excellent repeatability and reproducibility. The proposed strategy provides a promising platform for the synthesis of chiral COFs and their chiral separation application.Covalent organic frameworks (COFs) are a novel class of porous materials, and offer great potential for various applications. However, the applications of COFs in chiral separation and chiral catalysis are largely underexplored due to the very limited chiral COFs available and their challenging synthesis. Here we show a bottom-up strategy to construct chiral COFs and an  Porous materials can be used in columns for gas separation applications. Here, Yan and co-workers from a bottom-up method have produced chiral covalent organic frameworks and from an in situ strategy formed capillary columns for chiral gas chromatography. Covalent organic frameworks (COFs) are a novel type of crystalline porous materials with highly ordered structures solely constructed from organic building units via strong covalent bonds123441679121315161819Resolution of enantiomers is of great significant either in pharmacology or in biology, since pure enantiomers may profoundly differ in biological interactions, pharmacology and toxicity23S-pyrrolidine was utilized to functionalize the pore wall of a prepared COF via the subsequent coordinative incorporation to introduce the chiral centres and catalytically active sites in the COF frameworks. The synthesized chiral functionalized COF was then employed as a metal-free heterogeneous catalyst for chiral organocatalysts with high activity, enantioselectivity and recyclability.Although lots of COFs with multi-functions have been reported, the preparation of COFs with chiral functions has been rarely reported. Synthesis of a stable crystalline chiral COFs remains a major challenge that prevents their practical application in chiral catalysis and separation. The general strategies for introducing the chiral functional moieties into the COF networks can be summarized as post-synthesis and bottom-up strategiesin situ growth approach for the fabrication of chiral COF-bound capillary columns for chiral separation. In this work, the 1,3,5-triformylphloroglucinol (Tp) is first functionalized with chiral (+)-diacetyl-L-tartaric anhydride ((+)-Ac-L-Ta) to form the chiral functionalized monomer CTp. 1,4-phenylenediamine (Pa-1), 2,5-dimethyl-p-phenylenediamine (Pa-2) and benzidine (BD) are then condensed with CTp to obtain chiral COFs CTpPa-1, CTpPa-2 and CTpBD, respectively. The prepared chiral COFs show high thermal stability. Furthermore, the fabricated chiral COF-bound capillary columns give high resolution for the separation of enantiomers. The results reveal the promising aspects for the fabrication of chiral COFs via the bottom-up strategy and the great potential of chiral COFs as a platform for chiral separation.Herein, we report a bottom-up strategy for the synthesis of chiral COFs and show an et al. reported a series of COFs TpPa-1, TpPa-2 and TpBD with remarkable stability28L-Ta  reveals the successful formation of imine bonds after the condensation of the aldehyde groups on CTp and the amino groups on Pa-1 (−1 (C=O of ester) and 1,735 cm−1 (C=O of carboxyl) imply the successful introduction of (+)-Ac-L-Ta into the structure. In addition, the disappearance of N–H stretching peaks at 3,300–3,400 cm−1 of Pa-1  characteristic peak of CTpPa-1 at 1,664 cm13C nuclear magnetic resonance (NMR) spectroscopy  and pore size (12 Å) than CTpPa-1 due to the additional methyl group of Pa-2 (2 g−1) and pore size (18 Å) than CTpPa-1 due to the extended phenyl of BD , and 1,604 cm−1, 1,583 cm−1 and 1,520 cm−1 (phenyl groups) in the CTpPa-1-bound capillary column reveals the successful bonding of CTpPa-1 in capillary column -1-phenylethanol, (±)-1-phenyl-1-propanol, (±)-limonene and (±)-methyl lactate on chiral COF-bound capillary columns within 5 min . HoweverL-tartaric anhydride-functionalized capillary column  and entropy change (ΔS), and the chiral part of enthalpy change (ΔΔH) and entropy change (ΔΔS) of the enantiomer–selector phase transfer were measured -enantiomers have much larger negative entropy change than the (−)-enantiomers, indicating the (+)-enantiomers become more ordered in the microenvironment of chiral COFs. Meanwhile, such configuration fit is more favourable for the (+)-enantiomers to interact with the chiral COFs than the (−)-enantiomers, resulting in more negative values of ΔH for the (+)-enantiomers. The relatively smaller absolute values of ΔΔH for (±)-limonene and (±)-1-phenyl-1-propanol CTpPa-1- or CTpPa-2-bound capillary columns show the chiral discrimination of (±)-limonene and (±)-1-phenyl-1-propanol is due to steric hindrance with additional weak π–π interaction or/and hydrogen bond between (+)-enantiomers and the chiral COFs. In contrast, (±)-1-phenylethanol and (±)-methyl lactate give much larger absolute values of ΔΔH, suggesting chiral discrimination seems to be the result of an additional strong π–π interaction or/and hydrogen bond for the most retarded (+)-enantiomersTo further understand the retention and chiral discrimination of enantiomers, the enthalpy change (Δmeasured . The detn=7), day to day (n=5) and column to column (n=3) were 0.15–0.30%, 1.11–1.89% and 2.35–3.41%, respectively, which demonstrates the excellent repeatability and reproducibility of the prepared chiral COF-bound capillary columns.The repeatability and reproducibility of the chiral COF-bound capillary columns were investigated with CTpPa-1 as an example . The relin situ growth approach to fabricate chiral COF-bound capillary columns for chiral gas chromatographic separation. The prepared chiral COFs CTpPa-1, CTpPa-2 and CTpBD exhibit two-dimensional eclipsed layered sheet structure with high thermal stability, while their bound capillary columns show high resolution for the separation of enantiomers with excellent repeatability and reproducibility. We find that the (+)-enantiomers show more ordered in the microenvironment of the chiral COFs with stronger interaction than the (−)-enantiomers, and that the high-resolution chiral separation is driven by enthalpy. The proposed strategy is easily to be extended to the preparation of other chiral COFs and their bound capillary columns. We believe that our work will promote the synthesis of chiral COFs and their wide applications in chiral separation and catalysis. Further attempt will be made to construct new chiral COFs by introducing the chiral functional groups on the diamine for catalysis or other applications.We have reported a bottom-up strategy to prepare chiral COFs, and an L-tartaric anhydride (1.2 mmol) were separately dispersed in anhydrous THF (10 ml) under ultrasonication . The Tp dispersion was dropwise added into the solution of (+)-diacetyl-L-tartaric anhydride at room temperature under Ar atmosphere. The mixture was refluxed at 60 °C for 24 h, then cooled down to room temperature. Water (10 ml) was added to the mixture to suspend the reaction. After the solvent was evaporated, the yellow-green residue  was washed with water and dried under vacuum. 1H-NMR ): 10.03 , 5.60 , 5.56 , 2.12 , 2.10 . FT-IR (powder): 3,451, 2,958, 1,756, 1,722, 1,644, 1,605, 1,433, 1,254, 1,071, 967, 607 cm−1. MALDI-TOF-MS for C33H30O27: 857.141 [M-H]− .1,3,5-Triformylphloroglucinol  and (+)-diacetyl-14H12NO8)n: C 52.17; H 3.74; N 4.35. Found: C 50.92; H 3.88; N 4.15. FT-IR (powder): 1,744, 1,735, 1,664, 1,605, 1,583, 1,518, 1,452, 1,256, 822, 612 cm−1. PXRD (2 theta): 4.6°, 8.1°, 25.8°.Typically, a pre-reaction mixture containing CTp (0.1 mmol), Pa-1 (0.15 mmol), absolute ethanol (18 ml) and anhydrous THF (2 ml) was mixed under ultrasonication  to obtain a homogeneous dispersion and then transferred into a three-necked flask equipped with a condenser. The mixture was refluxed at 80 °C for 4 h with Ar protection. The obtained dark-red-coloured precipitate was collected and washed with absolute THF and ethanol three times, then dried at 120 °C under vacuum for 24 h to get the CTpPa-1 (77.5 mg) in the isolated yield of 76%. Analysis Calculated for  was treated sequentially with 1 M NaOH for 2 h, water for 30 min, 0.1 M HCl for 2 h, water again until the outflow reached pH 7.0, and methanol for 30 min. The pretreated capillary was filled with a methanolic solution of 3-aminopropyltriethoxysilane , and incubated in a 40 °C water bath overnight after both ends of the capillary were sealed with rubbers to obtain an amino-modified capillary. The amino-modified capillary was rinsed with methanol to flush out the residuals, and dried with a stream of nitrogen at 120 °C overnight for further use.2 for 2 h to remove the solvent. Finally, the prepared CTpPa-1-bound capillary column was conditioned with a temperature program: 80 °C for 30 °C min, ramp from 80 to 200 °C at a rate of 2 °C min−1, and 200 °C for 1 h.Solution A was obtained by suspending CTp (0.1 mmol) in ethanol (9 ml) and THF (1 ml), while solution B was obtained by dissolving Pa-1 (0.15 mmol) in ethanol (9 ml) and THF (1 ml). The above two solutions were cooled in an ice bath, and then mixed to obtain the pre-polymerization solution of CTpPa-1. The amino-modified capillary was quickly filled with the pre-polymerization solution with a syringe and incubated in an 80 °C water bath for 4 h after both ends of the capillary were sealed with rubbers. The prepared capillary column was rinsed with THF and ethanol to remove residuals, then flushed with NThe authors declare that the data supporting the findings of this study are available within the article and its How to cite this article: Qian, H. L. et al. Bottom-up synthesis of chiral covalent organic frameworks and their bound capillaries for chiral separation. Nat. Commun. 7:12104 doi: 10.1038/ncomms12104 (2016).Supplementary Figures 1-55, Supplementary Tables 1-8, Supplementary Methods and Supplementary References


Restorer of fertility (Rf) gene encoded by the nuclear genome. An Rf gene of Lead rice type CMS, Rf2, encodes a small mitochondrial glycine-rich protein. RF2 is expected to function by interacting with other proteins, because RF2 has no motifs except for glycine-rich domain.The pollen function of cytoplasmic male sterile (CMS) plants is often recovered by the RF2-interacting candidate factors (RIF1 to RIF4). A study of subcellular localization demonstrated that only RIF2 was targeted to mitochondria. A pull-down assay using E. coli-produced recombinant GST-tagged RF2 and His-tagged RIF2 confirmed that RF2 interacted with RIF2. RIF2 encodes ubiquitin domain-containing protein.To elucidate the protein that interacts with RF2, we performed yeast two-hybrid screening. We identified four genes and named These results suggest that RIF2 is a candidate factor of a fertility restoration complex of RF2.The online version of this article (doi:10.1186/s12284-014-0021-6) contains supplementary material, which is available to authorized users. Restorer of fertility (Rf) gene encoded by the nuclear genome. Many Rf genes are reported to encode pentatricopeptide repeat (PPR) protein, which is involved in the processing of mitochondrial RNA  type CMS, encodes a mitochondria-targeted 152-amino acid protein containing glycine-rich domain , which is caused by an aberrant mitochondrial gene, is unable to produce functional pollen. Pollen function is often recovered by a Toriyama  for a rei et al. ). Rf2 isi et al. ).6 clones) made from the mature anther RNA of rice  using MA fertility restoration factor is expected to function in mitochondria. We therefore investigated the subcellular localization of RIF1, RIF2, RIF3 and RIF4. Coding sequences of RIF proteins were PCR-amplified using primers listed in Additional file Each of the resulting plasmids that express GFP fusion proteins was introduced into rice protoplasts with a plasmid containing CaMV35S promoter-F1F0 ATPase mitochondrial targeting signal-RFP (Arimura and Tsutsumi ) using tE. coli-produced recombinant GST-tagged RF2 (GST-RF2) and His-tagged RIF2 (His-RIF2). The coding sequence of mature RF2 that lacks a predicted mitochondrial targeting signal sequence was PCR-amplified using primers Rf2_CDS_CACC217F and Rf2_CDS_BamHI456R . RF2 ofKT and TK conceived and designed the experiments. SF performed the experiments and drafted the manuscript. KT and YI revised the manuscript. YI and SK supervised Y2H experiments. All authors read and approved the final manuscript.Additional file 1: Table S1.: Primers used in this study. (XLS 10 KB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Below are the links to the authors’ original submitted files for images.


Activities & Participation” were examined. Results showed that there were subgroups of patients whose functioning improved over time. The number of problems in “Activities & Participation” decreased in acute patients over time, whereas in chronic patients, an increase was found. The significant difference in relative variability for the environmental factor “support and relationships” reflects the increase in facilitators in acute patients, whereas the number of facilitators in chronic patients remained unchanged over time. Age at event, time from event, and relative variability in “Environmental Factors” were significant predictors of relative variability in “Activities & Participation”. It is of clinical relevance that patients with disorders of consciousness are kept in a supportive and facilitative environment, in order to prevent a decline in their functioning. Moreover, caregivers should receive tailored support in order to enhance and facilitate appropriate care of patients with disorders of consciousness.Disorders of consciousness are neurological conditions associated with low levels of functioning which pose a serious challenge to public health systems. The current study aimed to examine longitudinal changes in functioning in patients with disorders of consciousness and to identify associated biopsychosocial factors using the International Classification of Functioning, Disability, and Health. An Italian sample of 248 patients was assessed longitudinally. Differences in relative variability (an index of change that controls for baseline levels) between acute and chronic patients and predictors of relative variability in “ Evaluation of assumptions showed that relative variability in Activities & Participation, relative variability in Body Functions, and time from event were not normally distributed. Therefore, a logarithmical transformation log 10 [(max + 1) – p-values < 0.05 were considered to indicate statistical significance. Data were analyzed using SPSS version 18.0 . With respect to the Mann Whitney analyses, the effect size Cohen’s d was calculated using G*Power software and interpreted as ≤ 0.19 = marginal, 0.20–0.49 = small, 0.50–0.79 = medium, and ≥ 80 = large. In all analyses, hypothesis testing was two-tailed and A flow chart of patient inclusion is shown in The socio-demographic characteristics of the sample are presented in The numbers and percentages of patients whose functioning improved, remained stable, or deteriorated with respect to the extension and severity indexes of the four ICF domains are presented in A > 20% improvement was found for the chapters Structures of the cardiovascular, immunological and respiratory systems (s4) and Structures related to movement (s7) (extension index) and s7 (severity index). Within the Activities & Participation domain (a > 20% improvement was found for the chapters Mobility (d4), Self care (d5), Domestic life (d6), and Community, social and civil life (d9) (extension index) and d4 and d6 (severity index). With respect to capacity, a > 20% improvement was found for the chapter d6 (severity index). Lastly, for the Environmental Factors domain (a > 20% improvement was observed for chapter Support and relationships (e3) (severity index). In relation to the barriers, there were no improvements >20% for any of the chapters. For the Body Functions domain , the extn domain , in relas domain , the extImprovement and deterioration in extension and severity of problems in the total scores on the four ICF domains are presented in Z = − 2.75, p = 0.006, Cohen’s d = 0.78), reflecting the relatively large increase in facilitators pertaining to this chapter in acute patients (relative variability = 34.13), whereas the number of facilitators in chronic patients remained stable (relative variability = 1.30). With regards to the other chapters within the Environmental Factors domain, a decrease in the number of facilitators was observed in acute patients, with the largest decrease found in the chapter Natural environment and human-made changes to environment (e2) (relative variability = −23.08), whereas chronic patients showed a decrease in the number of facilitators in the chapters Products and technology (e1) and e2 and small increases in the chapters Attitudes (e4) and Services, systems and policies (e5). A comparison of the relative variability in the various aspects of functioning and disability as defined by ICF between T0 and T1 is presented in t = −2.080, p = 0.040) indicates that the older a patient is at the time of the acute event, the lower his relative variability is in the number of problems in Activities & Participation performance domain between T0 and T1. Results of the regression analysis, in which relative variability in Activities & Participation performance was regressed on age at the time of an acute event, time from event (at T0), and relative variability in the Body Functions domain and the chapters of the Environmental Factors domain, are presented in t = −2.532, p = 0.013) shows that the longer the time from the acute event is, the lower is the patient’s relative variability in the number of problems with respect to Activities & Participation performance between T0 and T1. Similarly, the negative relationship between time from event (assessed at T0) and relative variability in Activities & Participation performance  suggests that the more relative variability a patient has in the Environmental Factors domain, the lower his relative variability is in the number of problems within the Activities & Participation performance domain. Overall, the regression model explained 17.7% of the variance . Finally, the negative association between relative variability in the Environmental Factors domain and the relative variability in the Activities & Participation performance domain  to systematically monitor biopsychosocial functioning and disability in patients with disorders of consciousness. Routine reexamination of functioning in these patients is fundamental to evaluate the effects of interventions  and to iPatients with disorders of consciousness are by definition completely dependent upon their environment. In this study, we conclude that it is important that the environment of these patients is maintained and adapted to support and facilitate them, especially in the long term, in order to prevent an inevitable decrease in their functioning and, subsequently, an increase in their disability. It remains an important public health responsibility of policy makers to ensure tailored support to caregivers over time in order to continue to provide appropriate care to patients with disorders of consciousness who show low levels of functioning and high needs of environmental facilitators.


The hydrophobic core, when subjected to analysis based on the fuzzy oil drop model, appears to be a universal structural component of proteins irrespective of their secondary, supersecondary, and tertiary conformations. A study has been performed on a set of nonhomologous proteins representing a variety of CATH categories. The presence of a well-ordered hydrophobic core has been confirmed in each case, regardless of the protein’s biological function, chain length or source organism. In light of fuzzy oil drop (FOD) analysis, various supersecondary forms seem to share a common structural factor in the form of a hydrophobic core, emerging either as part of the whole protein or a specific domain. The variable status of individual folds with respect to the FOD model reflects their propensity for conformational changes, frequently associated with biological function. Such flexibility is expressed as variable stability of the hydrophobic core, along with specific encoding of potential conformational changes which depend on the properties of helices and β-folds. This α/β classification varies with respect to the participation of both folds in proteins, as well as their orientation. Purely helical  or purely β-shaped (immunoglobulins) structures, as well as proteins characterized by near-equal participation of both types (lactate dehydrogenase or carboxypeptidase), can be identified in protein databases . The supersecondary structure, which determines the mutual arrangement of secondary folds can be expressed as follows (the following list is derived from a commonly used biochemistry textbook ):βαβ – hhttp://www.cathdb.info/ 27 Apr 2016) database, which currently (as of 27 April 2016) contains 26 million domains arranged into 2738 families. Structural classification is performed automatically upon inclusion of a new protein in PDB, via an exhaustive search for proteins homologous to the new entrant [Attempts to introduce a detailed structural classification of domain units in large proteins have resulted in the creation of the CATH/Gene3D , the work also discusses proteins capable of binding specific ligands (cytochrome) as well as antigen-binding proteins (immunoglobulin).We establish that each of the presented molecules (or domains) contains a “seed” (of variable size) representing its hydrophobic core, which, according to textbook knowledge, is responsible for tertiary structural stabilization. As shown in our other papers, local deviations from the “idealized” hydrophobic core structure  are often linked to the protein’s biological activity. The presence of cavities or exposure of hydrophobic residues on the surface creates suitable conditions for ligand binding and protein complexation respectively. Eliminating such discrepancies from analysis of the hydrophobic core structure invariably leads to identification of parts of the molecule for which the idealized distribution of hydrophobicity closely corresponds to observed values. Such parts are thought to mediate structural stabilization of the protein (or domain) as a whole –9.In this work we show that, regardless of secondary and supersecondary conformational properties, the presence of a more or less prominent hydrophobic core is a common phenomenon. We identify proteins where the core is highly consistent with theoretical values (3D Gaussian) as well as those which exhibit local deviations from the theoretical model, typically associated with the capability to bind ligands or attract complexation partners. The study set is very diverse, yet exhibits common characteristics which point to the necessary presence of water in their environment — this applies to many different molecules encountered in the cell, but particularly to proteins.Restricting protein structure analysis to topological aspects would disregard the most important factor — the influence of the water environment, which plays an active and often decisive role in the folding process. Hence, our study of the hydrophobic core — a product of the environment, which determines the biological activity of proteins.Water environment is treated as the external force field which together with the internal force field (non-bonding interaction) participates in structuralization process and ensures the biological activity. The presented paper is an attempt to show the possible interpretation of structural effects which may express the role of water.Our analysis is based on a set of proteins derived from a popular biochemistry textbook . Table 1An in-depth presentation of the fuzzy oil drop model can be found in an Open Access publication , 28. In The fuzzy oil drop model is a modification of the oil drop model originally introduced by Kauzmann . The oriThe actual (observed) hydrophobicity distribution depends on the location of each residue in the protein body . Each rBoth hydrophobicity distribution profiles — expected (T) and observed (O) — can be compared quantitatively. Quantitative expression of the differences between the expected (T) and observed (O) distribution is possible by using the Kullback-Leibler divergence entropy formula :\documenDKL expresses the distance between the observed (p) and target (p0) distributions, the latter of which is given by the 3D Gaussian (T). The observed distribution (p) is referred to as O.The value of For the sake of simplicity, we introduce the following notation:DKL is a measure of entropy it must be compared to a reference value. In order to facilitate meaningful comparisons, we have introduced another opposite boundary distribution (referred to as “uniform” or R) which corresponds to a situation where each effective atom possesses the same hydrophobicity density . This distribution is deprived of any form of hydrophobicity concentration at any point in the protein body:Since Comparing O|T and O|R tells us whether the given protein (O) more closely approximates the theoretical (T) or uniform (R) distribution. Proteins for which O|T > O|R are regarded as lacking a prominent hydrophobic core. To further simplify matters we introduce the following relative distance (RD) criterion:RD < 0.5 is understood to indicate the presence of a hydrophobic core. Figure DKL  may be calculated for specific structural units  In such cases the bounding ellipsoid is restricted to the selected fragment of the protein. It is also possible to determine the status of polypeptide chain fragments within the context of a given ellipsoid. This procedure requires prior normalization of O and R distributions describing the analyzed fragment, whose length is denoted as N. Note that any selected fragment must be of a reasonable length — calculations cannot be performed for an individual residue.Generally the idealized distribution (T) is treated as the target for O|T calculation, while the unified distribution (R) is used when computing O|R.The above procedure will be applied in the analysis of proteins described in this paper. By restricting our analysis to individual fragments, we can determine whether a given fragment participates in the formation of a hydrophobic core. In particular, fragments representing well-defined secondary folds which satisfy RD < 0.5 are thought to contribute to structural stabilization, while fragments for which RD > = 0.5 are less stable. Such fragments, if present on the surface of the protein, may potentially form complexation sites. Specific fragments are derived by analyzing the protein’s secondary conformation. Identification of secondary folds and the composition of protein domains follows the CATH  and PDBsA graphical presentation of RD interpretation is shown in Fig. This category is represented by the nonhomologous domain 1 of lactate dehydrogenase (1A5Z – domain 1) and phosphoglycerate kinase (1FW8 – domain 2). The α,β-superfold can be described as a central twisted β-sheet surrounded by a ring of helices. RD values for both domains and their individual secondary folds are listed in Table From the structural point of view both proteins provide examples of the so-called flavodoxin fold  with a cThermotoga maritima and folds near the boiling point of water. Thermostable proteins are the focus of a separate study based on the fuzzy oil drop model (publication currently in preparation).Lactate dehydrogenase (1A5Z) is derived from the hyperthermophilic bacterium The catalytic residues in 1A5Z, as well as its disulfide bonds, are all located in domain 2 — thus, we will not consider them in the presented analysis.Domain 2 of phospoglycerate kinase (1FW8-D2) exhibits RD > 0.5, which means that its hydrophobic core is deformed. The domain contains two β-sheets — a parallel sheet  which also potentially affects interaction.It furthermore appears that the α,β-superfold — particularly the twisted β-sheet — contains a hydrophobic core and therefore improves the stability of its parent domain.The all-β-sheet superfold is represented by Cu, Zn superoxidase dismutase and concanavalin A. This type of all-β domain has the shape of a β-barrel. Both proteins appear to contain hydrophobic cores (RD < 0.5 in each case).2+. Similarly, in 1CON the cadmium ion (Cd2+) is bound by a locally discordant β-fragment.In 1B4L enzymatic and heavy metal ion binding residues are all located in loops. Table Cu, Zn superoxide dismutase adheres to the theoretical hydrophobicity distribution model as a whole, although some secondary folds diverge from the model. The helical fragment at 57–61 is an example (RD > 0.5). Similarly, in 1CON, despite the overall adherence of the molecule and its β-sheet, four individual β-folds are found to be discordant.Both proteins Fig.  exhibit This category is represented by triose phosphate isomerase and pyruvate kinase domain 1. The core structure comprises a centrally located β-barrel where individual β-folds are interconnected by α-helices located outside of the fold. The sample proteins differ with respect to their composition and biological activity.Elimination of catalytic residues Table  lowers tLocal deviations from the theoretical model caused by a ligand are commonplace. The ligand — by virtue of its presence — enforces a conformation which represents a balance between the protein’s own folding tendencies and the altered conditions introduced by the ligand’s polarized atoms. The end result of this process depends strongly on the ligand’s hydrophobicity .Residues which diverge from the model, despite not taking part in ligand binding, may represent structurally encoded “readiness” for structural changes in the α/β domain.Despite the observed differences, both domains share certain similarities. The helical fold at 95–100 in 1AMK exhibits a similar deviation from theoretical values when compared with the folds at 319–321 and 362–364 in 4DRS D2. It seems that local instability in all these areas may promote structural rearrangement.Three proteins representative of the β-barrel domain are characterized in Table Each of the above listed proteins represents a different type of β-barrel. The assessment of hydrophobic core structure is, likewise, different in each case. It should be noted that the protein exists to fulfill a specific biological role and that its structure is a means to this end.1RBP is a retinol binding protein. The presented domain contains three disulfide bonds which promote tertiary structural stability. Two of these bonds stabilize the domain in a “discordant” state, which is very likely their principal role.In order to facilitate comparisons with other types of β-dominated supersecondary structures, we have included triole phosphate isomerase (1TIM) in our study. The corresponding form is an α/β barrel consisting of eight pairs of alternating β-strands and α-helices rolled up to form an inner barrel of eight parallel β-strands surrounded by an outer barrel of eight parallel helices. Analysis based on the fuzzy oil drop model indicates the presence of a well ordered hydrophobic core. Stabilization is provided by the arrangement of β-folds (RD = 0.493), while catalytic residues deform the domain (their elimination results in RD = 0.478).Discordant helixes include the fragments at 95–103 (RD = 0.617), 130–137 (RD = 0.725), 138–154 (RD = 0.527), 168–170 (RD = 0.671), 177–197 (RD = 0.551), 198–204 (RD = 0.590). Catalytic residues are located in direct proximity to the active site. Experience with the fuzzy oil drop model indicates that enzymatic active sites are, in most cases, deficient in terms of hydrophobicity and diverge significantly from theoretical values. In the presented cases catalytic residues belong to the helices at 95–103, 168–170, 177–197, and 198–204, with RD values in excess of 0.5 in all cases.E. coli. Topologically, this protein is a sequence of antiparallel helical fragments described as an up-down-up-down 4-helix bundle.The arrangement of helical and β-folds in higher-order structures varies greatly both with respect to the quantity of each and their conformation. An example of a purely α protein is provided by the b562 cytochrome (1APT) from In contrast, the Fab IgG immunoglobulin domain (7FAB) is comprised almost entirely of β-folds, with its structure characterized as a sandwich of 3- and 4-stranded antiparallel β-sheets.A combination of helical and β-motifs is found in the 163-residual domain of dogfish lactate dehydrogenase (6LDH), which contains a 6-stranded parallel β-sheet with crossovers between β-strands and α-helices forming a right-handed helical turn with flanking β-strands.The cytochrome is characterized by RD = 0.426, suggesting the presence of a well-ordered hydrophobic core. All its helical fragments also satisfy RD < 0.5 . It seems that this protein depends strongly on the stabilizing influence of hydrophobic interactions. Given that the protein’s biological role is to bind heme, its structure provides suitable conditions for firm anchoring of the ligand at its center, with the polypeptide chain forming an “envelope” around the heme molecule.The Fab IgG domain fragment consists of two chains: the light chain (L) and the heavy chain (H), each of which is further composed of two domains (labeled V and C). All these structures exhibit a similar conformation, often referred to as an immunoglobulin-like domain: a sandwich comprising two β-sheets which we will refer to as the upper core and lower core respectively.Under the CATH classification the Fab fragment is characterized as 2.60.40.10 mainly β-sandwich. Both cores are linked by a centrally placed disulfide bond. The biological role of the Fab fragment is to recognize antigens via so-called CDRs — short variable loops which interact with the antigen, triggering a process called immunological signal transduction. This produces structural changes in the Fc fragment of immunoglobulin, priming it for complexation of the C1q complement, which is a first step in a cascade of reactions resulting in destruction of the cell which originally presented the antigen.The role of the Fab fragment is therefore to bind the antigen and trigger a signaling pathway. Both processes require a flexible structure, capable of accommodating the antigen and producing structural changes elsewhere in the molecule. The flexibility must, however, remain selective so as to ensure that the correct ligand is recognized and the correct signal sent.This situation is evidenced by the RD status of individual β-folds in all four domains: VH, CH1, VL, and CL  is stable as a whole and includes a stable β-I sheet, while all other domains are characterized as relatively unstable and therefore flexible. The stabilizing influence of SS bonds likewise varies: only in the CL domain does the affected fragment remain consistent with theoretical predictions.Immunoglobulins work in an unpredictable environment — the antigen is never known a priori. Thus, a system characterized by variable local stability creates suitable conditions for targeted signaling while preserving the immunoglobulins ability to interact with a variety of antigens.A review of immunoglobulin-like domains  can be found in . The autThe dogfish lactate dehydrogenase N-terminal 163-aa fragment is an example of a 6-strand parallel β-structure linked by a right-handed helical turn. The domain as a whole is characterized by RD = 0.489, while elimination of its N-terminal fragment (1–19 aa), which significantly disrupts the globular form, further reduces RD to 0.428. Evidently, the domain contains a prominent hydrophobic core. Table The author of  also addTable Cytochromes are proteins which act as “envelopes” for their ligand  . It seemIt is up to each researcher to select fragments of interest, depending on the problem being studied. In our experience, fragments which diverge from theoretical predictions produced by the FOD model are typically associated with biological function. The status of particular secondary fragments in supersecondary structures may quantitatively reveal their contribution to overall supersecondary stabilization.In summary, we can state that the targeted stability required in many biological processes can be supplied by a combination of nonbinding interactions, structural stabilization mediated by hydrophobic forces, and the presence of disulfide bonds. As shown in , SS-bondDiscussions concerning the fundamental role of the hydrophobic core have a long history  with botIn the context of these observations and the analysis presented in this paper we can conclude that the answer to the question posed in the title is “yes”. We can furthermore state that the fuzzy oil drop model is able to describe the hydrophobic core structure in both qualitative and quantitative terms including the substantial and specific difference of hydrophobicity distribution observed in amyloids .