It is demonstrated that a Co capping layer essentially changes the domain structure and decreases the threshold of magnetization reversal, due to the formation of noncollinear magnetization in Co/Pt. It is shown that local irradiation with a focused He+ ion beam enables the formation of regions with decreased easy-axis anisotropy (magnetic bubbles) that have the inverse magnetization direction in the demagnetized state of Co/Pt. The experimental results demonstrate that the magnetic bubbles can be switched using a probe of a magnetic force microscope. The possible application of these effects for the development of magnetic logic and data storage systems is discussed.We present the results of magnetic force microscopy investigations of domain structures in multilayer [Co (0.5 nm)/Pt (1 nm)] The engineering of magnetic states in ferromagnetic nanosystems is one of the topical problems closely connected with the development of modern magnetic data processing systems and element base of spin electronics . One eff+ ion beam.In the present study, we investigate the possibilities of local modification of the domain structure in Co/Pt multilayer films with perpendicular anisotropy through the growth of additional patterned Co layers, and by local modification of the Co/Pt anisotropy parameter using a focused He5 were grown by DC magnetron sputtering on Si(100) substrate with Ta(10 nm)/Pt(10 nm) buffer sublayer. Additionally, we deposited Co capping layers with variable thickness. The Co capping layers were patterned using lithographic methods to locally change the domain structure. The Carl Zeiss “Supra 50VP” scanning electron microscope with “ELPHY Plus” exposure system  was used for the e-beam lithography hography ,16. Addiin Co/Pt . The magnetic properties of the samples were investigated by home-made magneto-optical Kerr effect (MOKE) technique. Domain structure was studied by magnetic force microscope (MFM) “Solver HV”  equipped by DC magnet with 500 Oe perpendicular (relatively to the sample) magnetic field. The MFM probes were standard NSG-11 cantilevers coated with Co with a thickness of 30 nm. Before the measurements, the probes were magnetized along the tip axes in a 1T external magnetic field. The MFM measurements were performed both in two-pass tapping mode and in noncontact constant-height mode. The phase shift of cantilever oscillations under the gradient of the sample magnetic force was registered as the MFM contrast. The MFM measurements were performed in a vacuum chamber with the rest gas pressure of 10−4 Torr that improved the MFM signal due to an increase of cantilever quality factor.M0. The coercive field is about 200 Oe. The MFM image of Co/Pt labyrinth domain structure in a demagnetized state (demagnetizing field was 200 Oe) is shown in The typical hysteresis loop  of the initial Co/Pt film is presented in M0 and the coercive field is 50 Oe) and a reduction in the size of the domains . In the case of the 1.3 nm Co capping layer, the remanent magnetization is 0.2 M0, the coercive field is 10 Oe and the typical domain width is 150 nm. The decrease of the domain structure scales with increasing thickness of the Co capping layer can be explained by effects of exchange interaction. The Co-Co/Pt exchange coupling leads to a decrease in the effective perpendicular anisotropy, and to an increase in the density of the domain walls in the Co/Pt subsystem. Similar effects of changes in the domain structure were mentioned in [As can be seen from ioned in , where CTo create laterally inhomogeneous domain structures in the Co/Pt sample, we fabricated spatially inhomogeneous Co coatings. As an example, It can clearly be seen that the deposition of a thin Co film substantially changes the lateral domain structure of the Co/Pt. From this point of view, the patterning of the upper Co layer enables the control of the domain structure of Co/Pt layers on small spatial scales.We investigated the change of magnetic domains caused by circular Co islands covering the Co/Pt. In Initially, the sample was uniformly magnetized in an external field of 500 Oe. The MFM image of the initial state is shown in + ion etching [We applied these effects in order to control the domain structure in planar Co/Pt nanowires (NWs). The NWs were fabricated by e-beam lithography and Ar etching . The latH1), and observed a partial remagnetization of the NWs in remanent state (H2) all NWs were remagnetized completely. The magnitude of the gap ΔH = H2 − H1 was equal to 50 Oe, which is quite enough for applications in a variety of devices based on NW remagnetization. The NW array was partly covered by a Co (1.3 nm) layer  near the boundary between the irradiated and unirradiated regions is shown in It can clearly be seen that periodic ion irradiation essentially changes the spatial structure of magnetization, and allows the realization of high-density lattices of inverted domains (bubbles). These objects can be used for application in data storage systems. The effects of magnetization reversal in the arrays of Co/Pt magnetic bubbles under an external magnetic field were investigated earlier in . Here, w+ ion beam. It was shown that the application of an additional Co layer allows a reduction in the characteristic scale of the domain structure and the magnitude of magnetization reversal fields. Therefore, the nanostructuring of Co/Pt films and covering Co layers enables the possibility to control domain wall nucleation and pinning/depinning processes for the development of magnetic devices. On the other hand, the application of local irradiation of Co/Pt films by ion beams opens up significant opportunities for creating highly dense arrays of controllable magnetic domains that can be used to develop novel and promising data storage systems.We have studied the peculiarities of domain structure in multilayer Co/Pt thin-films modified by patterned Co capping layers and by a focused He


Our results point to a role for Cdk5 in modulating TRPA1 activity.Transient receptor potential cation channel, subfamily A, member 1 (TRPA1), is activated by a broad range of noxious stimuli. Cdk5, a member of the Cdk family, has recently been identified as a modulator of pain signaling pathways. In the current study, we investigated the extent to which Cdk5 modulates TRPA1 activity. Cdk5 inhibition was found to attenuate TRPA1 response to agonist in mouse DRG sensory neurons. Additionally, the presence of active Cdk5 was associated with increased TRPA1 phosphorylation in transfected HEK293 cells that was roscovitine-sensitive and absent in the mouse mutant S449A full-length channel. Immunopurified Cdk5 was observed to phosphorylate human TRPA1 peptide substrate at S448A  Reactions were quenched with 70 µL 10% trichloroacetic acid. Samples were subsequently centrifuged for 5 minutes at 10,000x g, and 17 or 20 µL supernatant aliquots were spotted onto P81 phosphocellulose squares. Each reaction condition was performed in triplicate, and each replicate reaction was spotted onto phosphocellulose squares in triplicate . After air-drying in a fume hood, squares were individually washed in 6-well plates with 75 mM Phosphoric acid . A final 15 minute wash in acetone was followed by air-drying. Dried squares were placed in scintillation vials containing biosafe scintillation fluid,  and quantified in a Beckman LS-6500 scintillation counter. For inhibitor kinase assays, PD-98059 or Roscovitine , or vehicle alone  were added to immunoprecipitates, prior to addition of peptides and [32P] ATP.Cdk5 immunoprecipitates from rat brain were combined with TRPA1 peptides  and 5 µCi of i. However, in isolated fura-2 loaded DRG sensory neurons undergoing the same protocol, roscovitine markedly attenuated propofol and AITC-induced increases in [Ca2+]i . Control DRG neuron ratios and inhibitor-exposed HEK293 cell ratios were not significantly different.Summarized data represent the ratio of peak heights post-exposure to vehicle or inhibitor compared to pre-exposure peak heights . Mean po3.2.nd exposure to propofol was performed to assess TRPA1 responses subsequent to Cdk5 inhibition. Following 20 minutes washout in HBSS, a final exposure to propofol demonstrated the reversible nature of the TRPA1 response attenuation. A representative trace is shown in Fura-2-loaded DRG neurons were exposed to the TRPA1 agonist, propofol, to establish baseline response levels, followed by a 20 minute superfusion with HBSS containing the Cdk5 inhibitor, roscovitine (50 µM). A 23.3.in vitro kinase assays were performed using both S448 wild-type and S448A mutant peptide substrates. Mean disintegrations per minute (DPMs) were 15.4-fold higher in wild-type peptide vs. S448A mutant peptide ,  peptide , indicatin vitro kinase assays using S448 wild-type peptide substrates were repeated in the presence of either roscovitine (Cdk5 inhibitor), PD-98059 (MAPK inhibitor), or DMSO (vehicle). Cdk5 inhibition resulted in a marked (highly significant) reduction in phosphorylation compared to vehicle-treated control. MAPK inhibition with PD-98059 had no significant effect on phosphorylation compared to vehicle-treated control. Radioactive counts were 17.3-fold higher in vehicle vs. roscovitine-treated peptides, p < 0.001, while PD-98059-treated peptides did not differ significantly from vehicle-treated , or doubly-transfected with myc-tagged TRPA1/RFP empty vector (control) 24 hrs after transfection with TRPV1. Myc-tagged mouse TRPA1 was immunoprecipitated from transfected HEK293 cells with anti-myc IgG, and subjected to western blot with anti-phosphoserine IgG. TRPA1 phosphoserine immunoreactivity was quantified and normalized to total TRPA1 after stripping and reprobing with anti-myc IgG, to correct for differences in transfection efficiency and loading. Baseline levels of TRPA1 phosphoserine immunoreactivity were observed in the control, non-Cdk5/p25-transfected state . Moreove4.in-vivo investigation indicated that Cdk5 inhibition attenuates the formalin-induced flinch response in rats i induced by TRPA1 agonists, while vehicle-treated DRG neurons failed to exhibit reduction in [Ca2+]i signal upon re-treatment with TRPA1 agonists. As an additional control, inhibition experiments were repeated using TRPA1-transfected HEK293 cells, rather than DRG neurons, as the model system. Cdk5 inhibition is expected to be without effect in this cell line, due to a lack of significant Cdk5 activity [2+]i in response to TRPA1 agonists in transfected HEK293 cells, providing evidence for the target-specific action of the Cdk5 inhibitor, and confirming that attenuation of rise in [Ca2+]i is not due to direct inhibitor interaction with TRPA1 itself.Incubation with the Cdk5 inhibitor, roscovitine, resulted in a significant attenuation of the rise in [Caactivity ,31. ResuAn intriguing aspect of these results concerns the context-dependence of the requirement for Cdk5 activity for TRPA1 function. When endogenously expressed in the ‘native’ nociceptor environment of DRG neurons (with the full coterie of regulatory and binding partners present), TRPA1 response to agonist demonstrates a dependence upon Cdk5 activity. When transfected into the ‘artificial,’ non-nociceptor context of HEK293 cells however, the presumably more ‘naked’ TRPA1 receptor (lacking in regulatory factors and binding partners present in nociceptive neurons) responds robustly to agonist stimulation, in a manner independent from Cdk5 activity. This is most likely due to the fact that HEK293 cells do not express the neuron specific p25 regulatory factor required for Cdk5 activation ,17,30. MThat a more layered and intricate regulatory machinery would have evolved to control and modulate TRPA1 nocisensor function within the highly-specialized environment of the nociceptive sensory neuron appears eminently logical. The placement of Cdk5 within such a regulatory framework is also far from surprising – as a factor principally active in post-mitotic neurons, which has previously been demonstrated to play key roles in multiple aspects of neuronal function – including pain signaling ,32. Cdk54.3.Kinase phosphorylation of substrates is generally a short-term modification unless actively maintained, due to ubiquitous protein phosphatase activity. Thus, incubation with kinase inhibitors leads to rapid de-phosphorylation of kinase substrates – and just as readily, washout of inhibitor, by returning the kinase to an active state, allows for rapid re-phosphorylation of substrates. Therefore, TRPA1 modulation by Cdk5 activity, whether by direct or indirect interaction, would be expected to be a reversible on/off process. Our results demonstrate that TRPA1 responses exhibit the anticipated reversibility under the influence of Cdk5 inhibition/washout, congruent with channel modulation by kinase action. Additionally, Cdk5 inhibition with roscovitine exhibited a dose-dependent effect on TRPA1 responses, as expected for a biological response to enzyme inhibition.4.4.in vitro Cdk5 kinase assays were performed using wild-type S448 and S448A mutant peptide substrates. Our findings indicate robust Cdk5 phosphorylation of the wild-type S448 peptide, while phosphorylation levels for the S448A mutant peptide were close to background levels – clearly demonstrating that Serine 448 can act as a Cdk5 substrate in vitro. These results were further bolstered by Cdk5 kinase assays performed in the presence of either PD-98059 (MAPK inhibitor) or roscovitine (Cdk5 inhibitor). Cdk5 specificity of the observed phosphorylation was reliably confirmed in these assays, as Cdk5 inhibition significantly reduced TRPA1 peptide phosphorylation, while MAPK inhibition had no significant effect on peptide phosphorylation relative to uninhibited control.In the interest of locating potential Cdk5 phosphorylation sites on TRPA1, bioinformatics searches were performed using the publically-available NetPhosK  and Scan4.5.19. To investigate the effect of Cdk5 activity on global serine phosphorylation of the TRPA1 ion channel, phosphoserine immunoreactivity was probed via western blot of TRPA1 immunoprecipitated from TRPA1/Cdk5/p25 co-transfected  and TRPA1/RFP empty vector co-transfected (control) HEK293 cells. Data from the present study indicate that the addition of Cdk5/p25 increases total phosphoserine immunoreactivity of TRPA1, consistent with kinase modulation of receptor activity by a phosphorylative mechanism. Moreover, incubation of the TRPA1/Cdk5/p25 transfected HEK293 with roscovitine results in a decrease in phosphoserine immunoreactivity indicating Cdk5 is responsible for increasing phosphoserine phosphorylation. Finally, we observed no phosphoserine immunoreactivity in HEK293 cells that were transfected with the TRPA1 mutant S449A in combination with Cdk5/p25. These data indicate that S449 is the determinant of Cdk5 phosphorylation in the full length TRPA1 channel. We conclude that in addition to T673 serving as a phosphorylation site for Cdk5 regulation of TRPA1 function [In light of the altered TRPA1 physiological responses observed with Cdk5 inhibition in DRG neurons, attempting to ascertain the mechanism(s) by which Cdk5 influences TRPA1 activation is a logical next goal. Phosphorylation of channel residues as a means of regulation has been previously reported in multiple other members of the TRP superfamily, including TRPV1, TRPV4 and TRPV6 ,35–37, a4.6.The mechanism(s) by which Cdk5 affects TRPA1 ion channel activity is an important question to be answered. In the case of modulation of TRPA1 by Cdk5, a ‘phosphoryl only’ mechanism would appear unlikely, as the channel is functional when transfected into cells which lack Cdk5 activity, but is dysfunctional in DRG neurons, in the absence of Cdk5 activity. Thus, Cdk5 influence on TRPA1 channel function (and of TRPV1 function ), whetheA simple model consistent with the present data would be one in which Cdk5 phosphorylation of TRPA1 prevents binding of an allosteric inhibitory factor  to the receptor. Such an inhibitory factor might be postulated to act by sequestering de-phosphorylated TRPA1 away from the plasma membrane, and/or might help place the plasma membrane-localized ion channel into an inactive/less active conformation or complex. Notably, either of these mechanisms have the potential to be easily reversible – which is consistent with the results observed in the present investigation.As improved understanding of TRPA1 regulation and functional interactions can potentially inform the development of rationally-designed therapeutics targeting the receptor, the novel findings described in this study have potential utilitarian implications, in addition to enhancing our knowledge of basic nociceptor biology. Further investigations into the mechanistic interactions underlying Cdk5 regulation of TRPA1 (and TRPV1) promise to offer additional insight in this regard.


Canine atopic dermatitis is a common pruritic skin disease often treated with allergen immunotherapy (AIT). AIT in dogs traditionally begins with attempting to identify clinically relevant environmental allergens. Current allergen testing methodologies and immunotherapy techniques in dogs are not standardized. Immunotherapy with a mixture of allergenic extracts selected based on regional aerobiology rather than intradermal tests or serum IgE assays has been described. The objective of this study was to evaluate the effectiveness of regionally-specific immunotherapy in dogs with atopic dermatitis. The medical records of a veterinary dermatology referral clinic were searched for dogs with atopic dermatitis that began regionally-specific subcutaneous immunotherapy from June, 2010 to May, 2013. An overall assessment of treatment effectiveness  was assigned based upon changes in pruritus severity, lesion severity, and the reduction in concurrent medication(s) during a follow-up period of at least 270 days. Baseline characteristics that might predict treatment success were analyzed with the Spearman’s correlation and the Kruskal-Wallis tests.r = 0.72, p < 0.001) and lesion severity , but not with the dogs’ baseline characteristics. Adverse reactions were reported in 7/286 (2.4%) of treated dogs.Of the 286 dogs that began regionally-specific immunotherapy (RESPIT) during a 3 year period, 103 met the inclusion criteria. The overall response to RESPIT was classified as excellent in 19%, good in 38%, fair in 25%, and poor in 18% of dogs. The response classification correlated significantly with a reduction in pruritus severity (Under the conditions of this study, RESPIT was safe and effective for the treatment of atopic dermatitis in dogs. Malassezia dermatitis frequently develop in atopic dogs.Canine atopic dermatitis (AD) is a common inflammatory and pruritic skin disease that is frequently associated with sensitization to environmental allergens . AffecteCanine AD often requires long-term management and therapy . There iAllergen immunotherapy prescriptions are customized for each dog. An optimal allergenic extract mixture would contain only allergens that elicit clinical signs upon natural exposure. However, customizing an allergenic extract involves multiple subjective variables. Veterinarians choose which allergens to test for, whether to test with IDT or SIA, which laboratory’s SIA to use, how to interpret borderline reactions, which positive reactions are deemed clinically relevant, what dose of each allergen to include in the ASIT prescription, and by what schedule and route it will be administered. These variables are not trivial. Within a geographic region, the allergens veterinary dermatologists evaluate with IDT vary substantially , as do tAlthough subject to the variables described above, AIT has been found to be effective for the management of canine AD in one placebo-controlled and multiple retrospective studies . ResultsAn alternative to AIT is RESPIT, allergenic extract mixtures that are formulated based on a dog’s geographic location rather than individual allergy test findings. Reports on the use of non-specific AIT mixtures in dogs or humans are limited –12. The The electronic medical records  of a veterinary dermatology clinic were searched for dogs with AD for which RESPIT  was prescribed on the same date as an initial examination between June 1, 2010 and May 31, 2013. The diagnosis of AD was made based on identifying characteristic clinical features and ruling out alternative diagnoses [The following history and examination findings from the initial encounter on day 0 (D0) were exported to a spreadsheet: patient identification, date of birth, gender, breed, weight, encounter date, pruritus visual analogue scale (PVAS) , seasonaOn the basis of the changes in the dogs’ PVAS, LSI, and those concomitant systemic medications with substantial evidence of efficacy  between D0 and D270+, an overall assessment score was assigned by the primary investigator as follows: 1 (poor) = no clinical change or a deterioration, 2 (fair) = improvement, but concurrent medications could not be substantially decreased, 3 (good) = greater than 50% improvement in clinical signs and reduction in medications, 4 (excellent) = complete remission without concurrent medications . The perP-value of <0.05 was considered statistically significant. Trends in bivariate relationships were highlighted by local regression trend lines [All statistical analyses were carried out in the statistical software R: A Language and Environment for Statistical Computing  version 3.1.3. Continuous and categorical characteristics were analyzed with Spearman’s correlation and the Kruskal-Wallis test, respectively. A nd lines .n = 103) and excluded (n = 183) dogs were not significantly different (p > 0.05).During a 3 year period, 286 dogs with AD began RESPIT on the day of an initial examination during which the pruritus severity was recorded. Of these dogs,103 (36%) returned for an examination after 270 days while still receiving RESPIT, thereby meeting the inclusion criteria. Most commonly, dogs were excluded due to poor compliance. Eighty-five dogs received only the initial prescription of RESPIT, an insufficient volume to last 270 days. Three of these dogs transitioned to an oromucosal formulation of RESPIT. Fifty-five dogs had their prescriptions renewed for the last time before day 200 and likely did not receive a volume sufficient to reach D270+. These 55 dogs were not examined after D270 while receiving subcutaneous RESPIT. Two of these dogs also transitioned to the oromucosal formulation of RESPIT. D270+ examinations while receiving RESPIT were not performed for 39 dogs that did have RESPIT prescriptions renewed after day 200. Four additional dogs had lengthy (7 month to 4 year) lapses in therapy before the D270+ examinations. The D0 baseline characteristics  of the included , weight , gender (p = 0.2), pruritus severity , lesion severity , seasonal history (p = 0.2), and the calendar month of the D270+ examination . D0 age , a reduction in lesion severity , the duration of therapy to D270+  and the total duration of therapy as of the date of the data retrieval .The response classification at D270+ significantly correlated with a reduction in pruritus severity  were suspected by pet owners to have experienced adverse reactions to RESPIT, including three with increased pruritus, and one each with vomiting, blepharitis, restlessness, or urticaria. In 5/7 dogs the dose was decreased and RESPIT was continued. In one dog with increased pruritus during the induction phase, RESPIT was temporarily discontinued then resumed 1 month later following the induction schedule up to a lower maintenance dose. In the dog that reportedly developed hives after two injections, RESPIT was discontinued by the owner and the dog was lost to follow up.In this study, 59/103 dogs (57%) had good or excellent responses to RESPIT. Similar rates have been reported in studies evaluating AIT effectiveness , 19, 20.The efficacy of immunotherapy using uniform allergen mixtures has been evaluated in two randomized controlled trials in atopic dogs, both reported only in abstract form with limited details or analysis , 11. In Beneficial effects of immunotherapy with imperfectly matched or unrelated allergens have also been reported in cats and humans , 21, 22.Whereas perfectly matching an atopic dog’s clinical sensitivity is the objective of AIT, the mechanism of action of RESPIT may be both allergen-specific and non-specific. Phylogenetically related allergens frequently cross react on IDT in atopic dogs . About 3In the present study, the median duration of therapy at the time of the D270+ evaluation was 12 months and the majority of D270+ evaluations (74/103) occurred between 9 and 15 months after beginning RESPIT  was at the low end of the wide range reported for AIT 5–50%) [% was at –50% % w.H2 to a TH1 allergen response [In children with rhinitis or asthma who are sensitive to house dust mite, AIT may have a tolerogenic effect, preventing the sensitization to additional allergens by inducing a shift from a Tresponse . Irrelevresponse .A limitation of this study was the open, retrospective design, similar to most studies on the effectiveness of AIT . A placeThis retrospective study suggests that subcutaneous RESPIT is a safe and effective alternative to AIT in atopic dogs with the advantage of avoiding the subjectivity involved in allergy testing and AIT formulation.


Traditionally, the left hemisphere has been thought to extract mainly verbal patterns of information, but recent evidence has shown that the left Inferior Frontal Gyrus (IFG) is active during inductive reasoning in both the verbal and spatial domains. We aimed to understand whether the left IFG supports inductive reasoning in a domain-specific or domain-general fashion. To do this we used Multi-Voxel Pattern Analysis to decode the representation of domain during a rule search task. Thirteen participants were asked to extract the rule underlying streams of letters presented in different spatial locations. Each rule was either verbal (letters forming words) or spatial (positions forming geometric figures). Our results show that domain was decodable in the left prefrontal cortex, suggesting that this region represents domain-specific information, rather than processes common to the two domains. A replication study with the same participants tested two years later confirmed these findings, though the individual representations changed, providing evidence for the flexible nature of representations. This study extends our knowledge on the neural basis of goal-directed behaviors and on how information relevant for rule extraction is flexibly mapped in the prefrontal cortex. A recent body of evidence suggests that executive functions might be subject to a degree of functional fractionation and that they can be mapped along the axes of the prefrontal cortex (PFC) –5. This On one side, some studies suggest that the fractionation along the left-right dimension is mostly domain-based. Evidence from neuroimaging studies shows that verbal information is preferentially processed in the left ventrolateral PFC (vlPFC), while spatial information is preferentially processed in the right vlPFC ,8, suggeHowever, some recent models ,10 propoInductive reasoning was used recently to address the question of whether the gradient along the left-right dimension is domain-based by manipulating rule domain within different types of rule search . BabcockThe latter evidence is particularly intriguing, and supports at least two possible explanations. The first is that the left IFG supports a domain-independent cognitive process required for the general task, such as rule extraction. The second possible account is that the left IFG holds representations of domain-specific information required for the task. These two possibilities could not be disentangled using the univariate analyses presented in Babcock and Vallesi (2015). Multivariate analyses, however, offer an opportunity to support one or the other explanation as these analyses are intended to specify the type of information processed in a region. Indeed, Multi-Voxel Pattern Analysis (MVPA), a type of multivariate analysis, is sensitive to information contained in fine-grained patterns, which goes undetected by univariate analysis ,18.These fine-grained patterns can separate the two posited explanations. For the first, we expect that the left IFG is employed in the same manner for both domains, thus there would be no difference in the neural patterns associated with the verbal and the spatial domains in this region. For the second explanation, we expect that the left IFG holds independent representations for the verbal and the spatial domains. These representations should be decodable in this region as separate neural patterns.To tease apart these two explanations we employed a support vector machine (SVM)-based MVPA to examine whether rule domain can be decoded in the left IFG. Specifically, we examined the representations of the verbal and spatial domains in the regions involved in the rule discrimination phase as reported by Babcock and Vallesi . The abips ≥ .112). The two sessions used the same experimental procedure and parallel forms of same task . All participants were right-handed native Italian speakers with no known neurological or psychiatric conditions. All of them had normal color vision as measured by the Ishihara Color Test ..48].In conclusion, the present data support the hypothesis that frontally-based hemispheric specialization could be process-based . In partThe reason for this result could be that the left IFG reaches its full processing capacity, an explanation which is consistent with previous evidence suggesting that a load-sensitive bilateral fronto-parietal network may be engaged whenever a hemisphere has reached its limit in learning and executing new rules ,50. The In fact, searching for both verbal and spatial domains requires participants to evaluate verbal patterns as well as spatial patterns. In order to optimize the search process, the right IFG could constrain the possible number of patterns to evaluate.This inter-hemispheric asymmetry could arise particularly when the cognitive demands imposed by the inductive reasoning task are fairly low, such as when the rule domain to attend to is known . In fact, when the cognitive demands are low, the computational capacity of a single hemisphere could suffice to successfully execute the task at hand . HoweverS1 Appendix(PDF)Click here for additional data file.


Human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)—a critical enzyme of the viral life cycle—undergoes a complex maturation process, required so that a pair of p66 precursor proteins can develop conformationally along different pathways, one evolving to form active polymerase and ribonuclease H (RH) domains, while the second forms a non-functional polymerase and a proteolyzed RH domain. These parallel maturation pathways rely on the structural ambiguity of a metamorphic polymerase domain, for which the sequence–structure relationship is not unique. Recent nuclear magnetic resonance (NMR) studies utilizing selective labeling techniques, and structural characterization of the p66 monomer precursor have provided important insights into the details of this maturation pathway, revealing many aspects of the three major steps involved: (1) domain rearrangement; (2) dimerization; and (3) subunit-selective RH domain proteolysis. This review summarizes the major structural changes that occur during the maturation process. We also highlight how mutations, often viewed within the context of the mature RT heterodimer, can exert a major influence on maturation and dimerization. It is further suggested that several steps in the RT maturation pathway may provide attractive targets for drug development. The accessory proteins negative regulatory factor (Nef) p66 have been interpreted to indicate the formation of a stable, symmetric p66/p66 homodimer containing two structurally and magnetically equivalent, stable RH domains -labeled p66 in our lab obtained under conditions similar to those of Sharaf et al. [Importantly, all of the NMR spectra that have been obtained for the Ile-labeled p66/p66’ homodimer reveal many subunit-specific chemical shift differences, consistent with the conclusion that the p66/p66’ homodimer exists as a structural heterodimer, and consistent with the above conformational selection process in which two conformationally distinct p66 monomers form an initial homodimer. Fortuitously, despite their close structural similarity, the RH and RH’ domains initially present in the p66/p66’ homodimer yield many separate resonances, allowing their separate conformational evolution to be individually tracked . In cont domains . Howeverf et al.  are domiM , many of these factors can exert their effects by influencing the earlier domain rearrangement process, rather than by directly influencing the monomer association step. Even perturbations that interfere with the subsequent subunit-specific RH domain unfolding step can influence the reversibility of the entire maturation pathway, and hence the fraction of p66 that is dimeric. Therefore, dimer stability provides a readout for multiple steps in the RT maturation pathway.extended conformation, and the second corresponding to dimerization of polymerase molecules in the E and M conformations p51MM and p51C conformations , this additional enhancement of the E/C ratio will enhance dimer formation  perturbations of the structure of the dimer interface that are transmitted from the NNRTI binding pocket ,83; and extended structure of the polymerase domain [In addition to the NNRTIs, other active site ligands, including dsDNA, hybrid RNA–DNA, and metal–nucleotide complexes are each expected to stabilize the dimer as a result of selective binding to the e domain ,88,89. Te domain ,90). In As in the case of symmetric multimers such as HIV protease, the metamorphic RT polymerase domain must accommodate a pair of mutations for each mutational variation in the p66 precursor. This constraint can be mitigated in protein regions with differential sensitivity to mutations. For example, the β7’–β8’ loop on p66’ (or p51) plays an important role at the dimer interface , while tNumerous studies have shown that RT mutations can influence dimer stability ,95,96,97E/M conformer ratio [M, Leu289 is solvent exposed, while in the structure of apo RT which serves as a model for p66E, Leu289 is located in a small fingers:thumb interface and makes hydrophobic contacts with fingers residues Phe61, Ile63, and Leu74 in the predominant closed conformation . Structural analysis indicates that the buried Asp110 and Asp186 residues in p51 make multiple H-bond and salt bridge contacts with other residues, while the buried Trp402 and Trp406 stabilize the compact conformation due to hydrophobic interactions. Trp406 Nε-H is also involved in stabilizing H-bond interactions in the compact p51 subunit. The role of the aspartyl residues of the YMDD motif in stabilizing the compact conformation of p51 has also been demonstrated by studies of Mulky et al. [extended conformation as neutral or slightly destabilizing with the additional possibility that W402R and W406R may be capable of dimer stabilization due to the formation of inter-subunit H-bond interactions. Consequently, while the L234A mutation is expected to lower the E/C conformational ratio of p66, the rescue mutations are expected to increase this ratio, primarily due to destabilization of the compact conformation. In summary, the effects of the dimerization inhibiting and rescue mutations studied by Tachedjian et al. [Tachedjian et al.  further am VADAR  for resiy et al. . Alternan et al.  appear tn et al.  and illuM and p66C, introduction of the larger side chains in the compact conformer will lead to local structural disruption that may be transmitted further into the structure, perturbing the dimer interface. The corresponding region in p66 in the apo structure is somewhat less crowded, but more importantly, exhibits substantial intrinsic flexibility that allows the accommodation of large NNRTIs. Movement of side chains in the NNRTI site of the E conformation can thus allow the accommodation of mutations requiring substantially greater side chain volume. It was determined that the most extreme side chain expansion (corresponding to a G190W mutation) was able to enhance dimer stability nearly as effectively as the NNRTI efavirenz (EFV) [E/C conformer ratio, similar to the mechanism discussed above for NNRTIs  . Consistnz (EFV)  shows thr NNRTIs . This exAs outlined above, subunit-specific RH domain unfolding is the final step in the maturation of the p66/p66’ homodimer. Mutations that significantly destabilize the RH domain can result in premature unfolding and proteolysis that effectively uncouples this step from dimer formation. Precursors containing mutations at the Phe440–Tyr441 cleavage site or in the segment between the domain N-terminal Tyr427 residue and the cleavage site have been reported to result in excessive formation of p51 and low or even absent levels of the RT heterodimer ,53,112. M illustrated in Abram et al. identified T477A as a rescue mutation for F440V and several other mutations that destabilize the RH domain and lead to premature processing ,113. BasThe RT maturation processes outlined above are mostly accomplished within the virion after it has separated from the host cell. Various lines of evidence indicate that the viral polyproteins exist primarily as a linear assembly of folded proteins and protein domains ,115, adoThe structures of the RT heterodimer and its p66/p66’ homodimer precursor A are arrThe polyprotein structure of pro-Pol results in cooperative dimerization, in which formation of an initial homodimer facilitates more extensive dimer formation . Since i20 lowers the equilibrium dissociation constant for p66 by about 30-fold. Formation of a complex with double-stranded RNA (dsRNA) formed from vRNA annealed with tRNA(Lys3) requires an extended conformation of the polymerase domain, which will stabilize the dimer and presumably accelerate the maturation of the p66’ subunit shown in In addition to the higher polyprotein concentration in the virion, other interactions are likely to facilitate RT dimerization. There is evidence that a pre-initiation reverse transcription complex (RTC) that includes viral RNA (vRNA), tRNA(Lys3), and RT is formed within the virion ,126. In In addition to supporting dimer formation, the high concentration of p66 within the virion can compensate for many p66 mutations that reduce but do not prevent dimer formation. Thus, Wapling et al.  observedThe virion pH is not well regulated, making the intra-virion pH subject to variations as a result of changes in the viral medium . Since Reverse transcriptases derived from HIV-1 and HIV-2 show only ~60% sequence identity ; howeverThe coding economy that results from reliance on a metamorphic RT comes at the cost of constraints on mutational variability and reliance on a complex maturation pathway. Recent NMR, crystallographic, and modeling studies have provided initial insights into this pathway, and suggest that maturation may be an attractive target for therapeutic intervention. In contrast, nucleoside drugs that target mature RT can also interfere with activities of host polymerases, and in some cases have been shown to inhibit the mitochondrial polymerase, leading to associated HIV lipodystrophy . In the The feasibility of targeting the final, subunit-specific RH domain unfolding step using an RH-active site-directed ligand has recently been demonstrated . As discThe RH:thumb’ interface—which is proposed to be absent in the initial homodimer structure A—represe


Antibodies are central to the growing sector of protein therapeutics, and increasingly they are being manipulated as fragments and combinations. An improved understanding of the properties of antibody domains in isolation would aid in their engineering. We have conducted an analysis of sequence and domain interactions for IgG antibodies and Fab fragments in the structural database. Of sequence-related properties studied, relative lysine to arginine content was found to be higher in CH1 and CL than in variable domains. As earlier work shows that lysine is favoured over arginine in more soluble proteins, this suggests that individual domains may not be optimised for greater solubility, giving scope for fragment engineering. Across other sequence-based features, CH1 is anomalous. A sequence-based scheme predicts CH1 to be folded, although it is known that CH1 folding is linked to IgG assembly and secretion. Calculations indicate that charge interactions in CH1 domains contribute less to folded state stability than in other Fab domains. Expanding to the immunoglobulin superfamily reveals that a subset of non-antibody domains shares sequence composition properties with CH1, leading us to suggest that some of these may also couple folding, assembly and secretion. As a tool they have proved to be invaluable for clinical2 and research purposes3, and are the backbone of the burgeoning protein therapeutics (biotherapeutics) portfolio4. This latter area in particular is leading to an increased interest in the properties that determine the stability and interactions of antibodies. Compared to other biotherapeutics, antibodies are a favoured affinity platform because of their characteristic high affinity and target specificity, with applications in many different disease sectors5.Antibodies are key components of the immune system, present in vertebrates from the evolution of jawed fish onwards7. A major constraint on biotherapeutics is the high concentration required for storage and delivery in the home-use injection format8, facilitating high binding efficacy with wide-spread  use. Antibodies occur at naturally high concentrations in the blood9, thus providing a good starting point for stable formulations. The detailed behaviour of antibodies, particularly at high concentrations, is dependent on solution conditions. Research into how sequence and structure influence antibody stability and the formation of soluble and insoluble aggregates is therefore required. Improved understanding in this area would impact on several areas of pharmaceutical drug development, including production, storage and delivery. Identification and engineering of solubility enhancing features in the immunoglobulin framework could improve next generation protein therapeutics. Nevertheless, variations in complementarity determining regions (CDRs)10 and framework regions11 can lead to significant challenges, for example in minimising aggregation at high concentrations.There can, however, be limitations to the use of therapeutic antibodies in the clinic, where producing suitable pharmaceutical formulations has proved to be difficult12. CDRs are located within the variable Ig domains of the heavy and light chains, with conserved Ig domains forming the framework of the antibody. CH1 domains illustrate the diversity that is possible on a conserved Ig domain fold. The CH1 domain alone is not stable in folded form13, requiring proline isomerisation and interaction with the CL domain for folded state stability. This process forms part of the quality control for antibody passage through the secretory pathway14. Antibodies must be assembled prior to secretion and function. Where an antibody is not correctly assembled, heavy chains are retained in the endoplasmic reticulum (ER), but isolated light chains can be secreted15. Heavy chain retention is due to binding between the CH1 domain and a molecular chaperone 16 that recognises the incompletely folded CH1. Addition of light chains can disrupt the heavy chain complex with BiP, leading to assembly and secretion17. The importance of CH1 in retaining heavy chains within the ER is demonstrated in studies where CH1 deletion allows the heavy chain to be secreted18. Interestingly, despite its intrinsically disordered protein (IDP) nature in isolation, the CH1 domain has been reported to not contain the sequence characteristics of IDPs14.CDRs are contained within the variable domains of the heavy and light chains, contributing to fundamental differences in the properties of immunoglobulin (Ig) domains that are formed around the immunoglobulin fold. These differences are of increasing interest as candidate protein therapeutics are constructed from engineered fragments of antibodies. Antibody protein sequence is generally well conserved within a class, except for the CDRs, that determine antigen binding specificity within the variable region of each chain19, with the interaction between CH1 and CL domains in the Fab fragment being maintained even in the presence of sufficient GuHCl to denature individual domains. More recently, it has been suggested that immunoglobulin G (IgG) unfolds in two major steps, the order of which is dependent on the degree to which CDRs destabilise the variable domains20. Within the Fc fragment, the CH2 domain has been shown to unfold before the CH3 domain21.Differences between Ig domain contributions to antibody stability are illustrated by measurements of unfolding rates, in which Fab unfolding is slow compared with that of Fc22. Domains within the IgSF superfamily can be divided according to whether they more closely resemble the variable (V) or constant (C) domain in the immunoglobulin. Many representatives of IgG domains, particularly in Fab fragments are available in the protein structural database 23. Structural analysis of protein domains and their interactions can be accomplished at a simple level through studies of charge interactions and shape complementarity. Methods for predicting charge interactions in proteins have been developed based on continuum electrostatics models. Most ionisable groups in proteins are sited at the water accessible surface, and in this case a Debye-Hückel model with the dielectric value of water is effective24. The pH-dependent contribution to folding energy is then calculated from the interactions between ionisable sites using a Monte Carlo scheme for sampling protonation sites26. These methods give the predicted contribution of ionisable groups to folded state stability of an isolated domain, or of the interaction between domains.Antibody Ig domains are part of the immunoglobulin superfamily (IgSF), a large family of proteins containing Ig domains, with many acting as cell surface receptorsThe current work carries out a sequence and structural analysis of IgSF domains, commencing with Fab fragments, as these are abundant in the PDB, studying their variation and confirming that CH1 is an outlier in terms of certain properties. These properties are then analysed for domains in the wider IgSF, which demonstrates a spectrum of similarity to CH1 domains that is orthogonal to sequence identity. It is suggested that a subset of domains within the IgSF may be part of assembly and secretion quality control that is analogous to that mediated for IgG by CH1 domains.27. Here, we plot z-score deviation for the property, R percentage composition subtracted from K percentage composition (K-R), relative to the average K-R over the high throughout solubility dataset28. Domain median values are higher than the reference value for all domains, with K-R values for constant domains exceeding those of the variable domains in the Fab fragment, and CDRs have the lowest K-R  adaptation, and is balanced with a higher value in the constant domains, reminiscent of engineered solubility fusion tags. It is not clear why arginine may be less favourable than lysine for protein solubility. Discussion has centred on its strength of interaction (compared with lysine) in salt-bridges and cation-pi pairings27. Charge interactions generally play a role in antibody stability, and are thought to modulate the resistance of variable domains to aggregation29. Overall charge is reflected in the pI  content is, on average, enriched relative to arginine (R) for more soluble proteinsK-R Fig. . Thus, i pI Fig. , with CL30. Differences in the compositions of other amino acids could give insight to the biophysical features that yield an atypical IDP-like character for the CH1 domain. For example, a smaller number of the bulky and aromatic hydrophobic F and Y for CH1 could indicate that non-polar packing incorporates more smaller hydrophobic groups (e.g. V), with a concomitant increase in the torsional degrees of freedom that require freezing on packing into the folded domain. Non-polar packing would still form the folding core, but with a higher thermodynamic entropy cost for CH1 than for other Fab domains, due to immobilising a greater number of torsional degrees of freedom. Under-representation of charged residues  could give a lower contribution of charge interactions to stabilisation of the folded domain.Sequence entropy is a measure of the degree to which amino acid composition is enriched in some amino acids, with higher values reflecting a more uniform composition across amino acids. This information entropy is calculated from the amino acid compositions of a sequence, and the absolute value ranges between extremes of zero (where the entire sequence is constituted by a single amino acid type) and 4.32 . The CH1 domain and CDRs have relatively low sequence entropy  content was investigated further. Figure ins Fig. . The pre14 can be associated with its sequence composition. Within that composition, the lack of folding stability for CH1 appears to be the result of more subtle effects than the simple balance of hydrophobicity and charge used in a standard measure of folding potential32.Taken together, these results suggest that, in part, the unconventional IDP-like character of the CH1 domainInteractions between ionisable groups at neutral pH have been predicted for the Fab domains, using continuum electrostatics methods. These are structure-based calculations with results given as absolute values, rather than the deviations to population averages shown for sequence-based calculations. Ionisable groups contribute less to folded state stability for the CH1 domain than the other Fab domains Fig. . This ob33 were scanned for similarity of sequence composition to CH1 domains. This method used a vector comparison, based on amino acid compositions, to probe the difference between each IgSF domain and a representative CL domain, against the equivalent difference for representative CH1 and CL domains. The aim of the scan was to emphasise amino composition features (vector components) that differentiate CH1 from CL. As a low sequence entropy also differentiates CH1 from other antibody domains, this property is added to form a double threshold of similarity of IgSF domains to CH1. Figure 34 than is the CL domain from the same IgG.Human representatives of the IgSF35. Although ICAM3:D1 can fold independently of other domains36, there are a number of interesting features. The closely related ICAM1:D1  to folding stability for these D1 domains are low at −6.5 kJ/mole for ICAM3 , and −7.8 kJ/mole for ICAM1 , similar to the CH1 domain values , we looked for evidence of 3D domain structures being determined only in the context of domain-domain interactions. Taking thresholds for the cosine similarity parameter (>0.3) and sequence entropy  has a 3D structure reported. Although ICAM3-D1 folds as an independent monomer, it is extensively glycosylated which may increase its folded state stability35. A close neighbour, ICAM1-D1 (just below the threshold that selects 11 CH1-like domains), is not extensively glycosylated, and is not stable as an independent domain. We suggest that there may be other domains within the IgSF that possess similar IDP-like character to the CH1domain, and that mediate assembly and secretion in a parallel manner to IgG antibodies. Experience with ICAM1-D1 suggests that such domains could be engineered to acquire folded state stability39. Further work could alter CH1 and other IgSF domains to determine key elements (beyond proline content) that determine IDP-like character, and shed light on a set of atypical intrinsically disordered proteins. It will also be of interest to establish the degree to which glycosylation of some IgSF domains contributes to folded state stability. Since protein structural analysis is a convenient indicator of intrinsic stability, this work will advance as the structures of more IgSF domains are mapped.Since sequence properties are related to protein solubilityFab structures were obtained from the PDB using the text search query ‘Fab’ and excluding similarly named structures. Only the heavy (‘H’) and light (‘L’) chain components of structures were retained, missing some occurrences with non-standard naming conventions, but providing a convenient filter for a single copy of the Fab fragment within a larger PDB file. For the dataset of 1119 structures, a degree of heterogeneity in chain length was observed. In order to avoid scanning of all structures individually, a further filter was employed, based on heavy and light chain lengths. Lower and upper quartiles of chain length were derived for both chains, and structures with chain lengths between these values selected (between 219 and 228 amino acids for heavy chain fragments and between 213 and 218 amino acids for the light chain). This reduced the dataset to 387 Fab structures.40. Interdomain regions were found between residues 110 and 130 for the heavy chain fragment, and residues 100 and 115 of the light chain. In order to determine break points between domains more precisely, the amino acid sequences of heavy chain fragments and light chains were aligned using MUSCLE41, and the interdomain regions searched for conserved sequence interdomain motifs. Of the 387 heavy chain fragment sequences, 375 were found to contain a VS motif between residues 110 and 130, followed by either an S or A. For the light chain, two search motifs were used. The majority followed a E[LIV]KR pattern (311/387). To increase this number, the search pattern TVL[GSA] was also identified, present in 44 light chain interdomain regions. One or other of these motifs could be found in 355 of the 387 light chain interdomain regions between residues 100 and 115.The interdomain regions between the constant and variable domains of heavy chain fragment and light chain were identified by studying a small number of the models visually using the molecular graphics software Visual Molecular Dynamics (VMD)42, the CDR regions for 247 of the Fabs were identified, and the resulting amino acid sequences formed a CDR dataset.Combining motif filtering for heavy and light chains gave 350 Fab structures from 387, for which domains were obtained from splitting at the interdomain motifs. A further filter was applied, requiring that all domains had a length within 10 amino acids for the mean for that domain in the 350 subset, with a decrease to 333 Fab fragments and associated domains , available as structures and sequences. Structures were also extracted for VL:VH and CL:CH1 domain dimers. The dataset used for calculations therefore consisted of 333 Fabs, 666 VL:VH/CL:CH1 dimers, and 1332 individual immunoglobulin domains. Using the SAbDab database32. Additionally, lysine and arginine composition are compared, as previous work indicates that lysine may be preferred over arginine in more soluble proteins27. In analysis of sequence-based properties, calculated values are given as deviations from population averages in a high-throughput dataset of Escherichia coli protein solubility (derived from quantification of protein expressed in soluble and insoluble forms)28. This allows comparison both between the subsets being studied (e.g. CH1 and other IgG domains), and of these subsets to a proteome average, albeit the E. coli proteome. The deviation value is the z-score measure of how many population standard deviations a property is away from the population average (positive or negative).Sequences were processed using software developed in our group that calculates amino acid compositions and other properties, such as pI, sequence entropy (a measure of the extent to which a protein is enriched in a subset of amino acids), and folding propensity 43. A Debye-Hückel model24, was used to generate the pH-dependent component of folding energy. This method uses a partitioning of ionisable group energy25, together with Monte Carlo sampling of ionisation states26, to predict pKas and pH-dependent energy. The value of ionisable group interactions at pH 7.0 was then extracted, as an estimate of the contribution of ionisable group interactions to folded state stability. Calculation of SASA was made with in-house code, partitioning according to contribution by polar or non-polar atoms. Contributions to dimerisation  were calculated, either for electrostatic energy or SASA, by subtraction of the individual subunit contributions from that of the dimer.Structures were investigated using continuum electrostatics for predicting the contribution of ionisable groups to structural stability44, and Biopython45 used in the processing.IgSF domain sequences were aligned with clustal33, and corresponding sequences retrieved from UniProt46. The 477 identifiers were cross-referenced with UniProt and 1229 immunoglobulin domain amino acid sequences extracted. The human IgSF sequence dataset was also processed to give amino acid compositions. To compare amino acid composition between CH1 and other IgSF domains, code was written to create a 20-dimensional vector of amino acid composition values. A representative CL domain composition (CLREF) was subtracted from a representative CH1 domain (CH1REF) and IgSF domain composition vectors, to increase sensitivity to the differences between CH1 and CL domains. For an IgSF domain X, the vector dot product, (X − CLREF). (CH1REF − CLREF), was calculated and used to derive the cosine of the angle between these differenced vectors. A cosine close to zero relates to little similarity, whilst close to 1 is highly similar i.e. the closer the cosine to 1, the more similar is the deviation of domain X from CLREF to the deviation of CH1REF from CLREF. In this way, it is possible to assess the degree to which an IgSF domain recapitulates the features that separate CH1 from CL domains. This measure of similarity was combined with sequence entropy to visualise in a 2D plot those IgSF domains most similar to CH1. Visualisation of sequence alignment for the entire human IgSF using COBALT47 was made with iTOL48.To make wider comparisons, identifiers for other members of the immunoglobulin superfamily (IgSF) in humans were obtainedThe datasets analysed during this study are available from the corresponding author on reasonable request.


In conjunction with slight barrier lowering, these two effects are sufficient to explain the observed emission current distribution. These results suggest that different electronic properties at domain walls are not necessary to observe localized enhancement in domain wall currents.Ferroelectric domain walls have continued to attract widespread attention due to both the novelty of the phenomena observed and the ability to reliably pattern them in nanoscale dimensions. However, the conductivity mechanisms remain in debate, particularly around nominally uncharged walls. Here, we posit a conduction mechanism relying on field-modification effect from polarization re-orientation and the structure of the reverse-domain nucleus. Through conductive atomic force microscopy measurements on an ultra-thin (001) BiFeO 3 film resulting from the formation of a field-induced meta-stable twisted domain nucleus.Understanding the conductivity at the nominally uncharged domain walls in ferroelectrics is still far from complete. Here the authors report an enhanced conduction at domain walls in an ultra-thin (001) BiFeO The case of enhanced conductivity at the nominally uncharged ferroelectric domain walls was a particularly surprising finding8, as good ferroelectrics are generally conceived of as insulators, although in reality they are commonly semiconductors with wide bandgaps (>2.5eV9). Perhaps even more surprisingly, the ferroelectric domain wall conductivity  has now been found in a host of systems including BiFeO38, doped and mixed-phase BiFeO314, LiNbO315, LiTaO316, BaTiO317, and PbZrxTix1-O319. It is important, in reviewing the literature on the topic, to isolate two categories of experiments: those involving strongly charged domain walls, which are typically unfavored , and those of uncharged or nominally uncharged domain walls. Given the difficulty in producing charged domain walls in standard ferroelectrics, it is not surprising that their reports are fewer in the literature20. The classical explanation for their metallic conductance is the accumulation of carriers  at the charged domain walls to screen the polarization charge at head-to-head or tail-to-tail walls, resulting in the formation of a quasi 2D electron gas 21. Similar phenomena were also found to exist in nanodomains formed by an atomic force microscope tip18. In contrast, the explanations for domain wall conduction in nominally uncharged, or weakly charged domain walls appear more scattered. Various reports have suggested that the domain wall forms conducting paths through the film22 (similar to conduction channels in resistive switching memories), or that the reason for the conduction is the lowered bandgap23, with ionization of oxygen vacancies or vacancy clusters24, providing the free carriers for conduction localized to the domain wall. Hysteretic behavior has also been found at these domain walls25, and transient persistent conduction was reported by Stolichnov et al.26, where conductivity was isolated not to existing domain wall positions, but to their prior positions after their motion (induced by the applied field from the atomic force microscope tip). Clearly, a mechanism that posits intrinsically higher conduction at walls cannot be reconciled with a persistent conductivity that is observed and relaxes at previous wall positions. It has however been acknowledged that defect states play an important role in modulating the conductivity, typically oxygen vacancies that are ubiquitous in perovskites, and the conductivity of both domains27 and domain walls29 appears to be heavily influenced by their concentration.Localized metal–insulator transitions and other spatial modulations of electronic conductivity in complex oxides have been studied for more than two decades, with many celebrated examples including observation of a percolative transition in doped manganites7. An understanding of the inhomogeneity of the electric field generated by the biased tip is also needed, as the field will differ for the same applied potential based on the surrounding polarization profile. In parallel, there has been renewed interest in exotic domain topologies, spurred by the discovery of closure states32, quadrupole chains33, and continuous polarization rotations35 in ferroelectric thin films and, very recently, experimental demonstration of vortex-anti-vortex arrays in an oxide superlattice heterostructure36. Key to these novel states is the existence of non-zero curl of polarization. Yet the ability to twist existing domain walls  by the applied electric field and generate ∇ × P≠0 has not been heavily explored experimentally. Although such twisted structures are meta-stable, this is precisely the situation incurred during c-AFM studies on ferroelectrics38, and warrants investigation. Additionally, distinguishing between the conduction at the domain walls and the bulk of the film would benefit from ultra-thin samples (<~10 nm) (as current will invariably spread into the bulk7) while conductivity has only been reported in thicker films (>~30 nm).Given that the studies on wall conduction are often conducted using conductive atomic force microscope (c-AFM) technique, progress in reconciling experiments to possible mechanisms requires a strong appreciation of the role of the junction between the metal-coated AFM tip and the domain wallpc-oriented BiFeO3 thin films  grown on a (110) dysprosium scandanate substrate, and measure conductivity at pre-existing domain walls across the surface of the film. Analysis of high-resolution current–voltage (I–V) spectroscopy experiments shows that the Schottky barrier is modified near the domain walls at the surface, changing on average by ~20 meV, but the conduction mechanism between the domains and domain walls does not change. Importantly, phase-field modeling reveals the formation of a complex polarization nucleus in conjunction with domain wall twist. This structure enhances the electric field locally by between 1- and 50% at the tip apex, depending on the applied potential, with the nucleation of the reverse domain substantially enhanced near the domain wall. These results show an unexpected merging of domain wall conduction with field-induced topological defects, wherein the field introduces a domain topology where ∇ × P≠0 for the twisted domain wall, and which increases the local electric field, allowing for a measurement of the intrinsic conductivity of the underlying ferroelectric. Our findings support a mechanism whereby domain walls need not host intrinsic conductivity, but rather act as nucleation centers for a twisted structure, which serves as field-amplifying confined sites that gate the ferroelectric at nanoscale dimensions. This picture of induced conductivity by geometric field confinement is general and can be widely applicable not only to domain walls, but to any configuration of order parameter topological defects where twisted structures can favorably nucleate.In this study, we perform c-AFM measurements in ultra-high vacuum (UHV) conditions on ultra-thin (~10 nm) (001)3 (BFO) thin film is shown in Fig. 3 samples on (110)pc DyScO3  ~10 nm-thick BiFeOcO3 Fig. 39. It sh ~10 nm-tI–V spectroscopy in a 50 × 50 grid over a 500 nm × 500 nm area of the BFO film. The log of the measured current at Vsam = 1.24 V is shown in Fig. I–V curves on the domain face and the domain wall itself appear to be very similar. We further plotted the ratio of wall and domain currents, shown as a solid green line. The remarkable stability of this curve through the measured voltage range suggests that the mechanism driving the conduction at the domain walls and the domains themselves is the same, as suggested by some of the earlier works41. To identify the conduction mechanism, we began by attempting to fit the I–V curves to candidate conduction models. Of all the mechanisms investigated , Fowler–Nordheim tunneling, and variable range hopping), Schottky emission returned the most reasonable results, as reported in ref. 22. Specifically, we fit toJ is the current density, T is the temperature (298 K here), V is the applied voltage, εr is the high-frequency dielectric constant, ε0 is the permittivity of vacuum, d is the film thickness (=10 nm), and kB is Boltzmann’s constant. The Schottky emission mechanism dictates that the log(J) vs. I–V data, as shown in Fig. 22 suggesting a difference in the trap distributions between our work and that by Noheda and Farokhipoor), but the residuals from fitting were slightly larger than for the Schottky emission fit. We therefore utilized Schottky equation as opposed to SCLC . However, upon fitting it was evident that many I–V curves captured during the spectroscopic experiment do not conform to the single linear equation. Rather, they appear better suited to a two-linear segment model . An example of such data is shown in Fig. I–V curves was 0.68 < V < 1.25 V, and robust fitting was used to exclude the effects of outliers. To explore this more systematically, we employed the Bayesian information criterion approach  precludes further detailed investigations of the conduction mechanism. Nonetheless, the results in Fig. Shown in Fig. 48. A central aspect of c-AFM measurements reported here and in previous investigations of domain wall conductivity are the very large local electric fields generated by the biased AFM tip, which is expected to cause substantial polarization reconstruction and consequently modify the local electric field distribution. One of the key assumptions in such investigations is that given a fixed applied potential to the AFM tip (or sample), the field distribution is identical across the sample, so that the conductivity values obtained from c-AFM are directly comparable from site to site. This assumption does not hold generally in ferroelectrics, given that the applied field couples to the initial polarization configuration which is variable across the surface, especially across domain walls. We directly quantified the degree to which this effect has an impact on the measured conductivity by phase-field modeling  planes is shown in Fig. Vtip = 1.5 V), while the (x–z) cross-sections are shown in Fig. P to crystallographically allowed orientations)49. However, when the tip is near the domain wall, there appears a twisted wall structure, where in one case the wall is bowed towards the nucleus  and (1–11) domains separated by 71° domain walls as shown in Fig. Vtip = 1.5 V) in locations 1 and 3, in Fig. x–y plane. The curl of the polarization, i.e. z-component of polarization through the z-direction from the tip apex to the bottom of the film is plotted in Fig. To further identify the nature of the twisted polarization nucleus, we plot the polarization vector fields for the biased tip , which is small at 1.0 V, but which grows with increasing voltage to ~50% for an applied tip potential of 1.5 V. This result shows that application of an electric potential to the tip will cause local-field enhancement, due to the polarization nucleus structure formed. In fact, such enhancement will even exist for the purely linear dielectric case;50 however, the enhancement is much greater for the domain wall-twist structure, as a result of a much more labile domain wall and lowered nucleation barriers for the reverse domain.To explore the modulation of the local electric field by domain wall twisting, we plotted the  Vtip = 1.5 V. The fractional degree of field enhancement is plotted in Fig. I–V from the domain wall regions in Fig. 26), and potentially, the change in the dielectric permittivity around domain walls. Such a scenario can explain most of the conductivity ‘enhancement’ seen around the domain walls. Moreover, the field enhancement appears to occur at a threshold voltage. Practically, this is likely to vary substantially in the real experiment due to local geometry of the domain walls (which are more complex than the simple structures modeled), distribution of pinning sites, presence of chemical or structural inhomogeneities, etc. Nonetheless, we do observe that at many points, there appears to be a cross-over in the slope, as evidenced in Fig. We further quantified this behavior for different tip positions, plotting the differences in the electric field at the tip–film interface when compared with the tip placed within the domain in Fig. 26 showed persistent conductivity after domain walls had moved from initial positions, and other related time dynamics of wall conductivity25 have been reported previously. Our report here is consistent with those observations, namely, the mechanism suggested in that study was the change in barrier height due to the unscreened polarization in the freshly switched area. However, in our case we do not need to invoke defects to explain the apparent domain wall conductance. Further, the key differences in our study are that we posit that the domain wall becomes conducting through essentially field-induced excitation of the polarization order parameter, and therefore its conductivity is a transient effect. This is in contrast to models with strong carrier accumulation and metallicity that imply the domain walls to remain conducting at zero field.We note that repeated scans by the AFM tip, by disabling the slow-scan axis, appears to increase the domain wall conductivity , but generally for uncharged walls the mechanism of domain conduction remains the same as for the domain walls41, and the enhancement is not in general very large. A similar charged domain twist structure was invoked previously, for the conductivity observed at ferroelectric vortex cores51. Here we build on the concept, and suggest it can be generalized to explain the observed conductivity of both domain walls and domain junctions. The question also arises of whether such wall twists can be observed experimentally. We note much experimental evidence exists to show that such wall complex structures are indeed possible in both rhombohedral53 and tetragonal56 systems at high fields, some of which have also been modeled by phase-field simulations58. Bowing of the domain wall has also been directly imaged59, and modeled analytically60. In addition, there is a wealth of evidence in the last decade, primarily by scanning transmission electron microscopy, for polarization topologies with non-zero curl61. Based on this data, we suggest that extrapolation of the phase-field simulations to lower fields is justified.This mechanism to explain domain wall conductivity relies on no electronic property changes at domain walls, and does not invoke oxygen vacancies. Naturally, the concentration of oxygen vacancies and cation vacancies62. It can be said that there should be effects on the emitted electrons due to interaction with trap states, and that bulk mobility is also a parameter in the modified form of the Schottky equation . Recent work has suggested that the intrinsic AC conductance of domain walls is indeed different64, as explored through scanning microwave impedance microscopy, where it was posited that due to domain wall roughness induced by disorder, local perturbations of the wall can accumulate charge even in nominally uncharged walls63.We finally note that given that the conduction is interface limited, this limits our understanding of the bulk conduction mechanismIn summary, we propose a mechanism for apparent conductance of ferroelectric domain walls, relying solely on the field-induced changes to ferroelectric topology. Based on extensive phase-field simulations of polarization changes induced by the AFM tip, we infer local electric field enhancement at the tip apex from a complicated polarization-twist structure with non-zero curl that facilitates interfacial electronic conduction. The mechanism presented here can reconcile some discrepancies in the existing literature, and strongly suggests that the field-induced perturbations to the domain structure should be taken into account in conductive AFM measurements. This mechanism can be generalized to explain observed conduction at domain vertices and other topological defects in ferroelectrics.3 thin films were deposited on etched (110) DyScO3 substrates via pulsed laser deposition with a KrF excimer laser (248 nm) striking a stoichiometric target at 700 °C in an oxygen pressure of 100 mTorr and cooled in 1 atm of oxygen. The BiFeO3 thin films have high crystal quality  and are coherently strained to the substrate . Before fitting of the All data used in this manuscript is available from the authors on request.Supplementary InformationPeer Review File


The mechanism of assembly of the extracellular matrix protein fibronectin (FN) into elastic, insoluble fibrils is still poorly understood. FN fibrillogenesis requires cell-generated forces, which expose cryptic FN-FN binding sites buried in FN Type III domains. The number and location of cryptic binding sites have been debated, but experimental evidence suggests multiple domains may contain FN-FN binding sites. The requirement of cell-dependent forces to generate FN fibrils restricts investigation of the mechanism of assembly. To address this, we use a recently developed biophysical model of fibrillogenesis to test competing hypotheses for the location and number of cryptic FN-FN binding sites and quantify the effect of these molecular alterations on assembled FN fibril properties. Simulations predict that a single FN-FN binding site facilitates either negligible fibrillogenesis or produces FN fibrils that are neither robust nor physiological. However, inclusion of multiple FN-FN binding sites predicts robust fibrillogenesis, which minimally depends on individual domain properties. Multiple FN-FN binding site models predict a heterogeneous fibril population that contains two distinct phenotypes with unique viscoelastic properties, which we speculate may play a key role in generating heterogeneous mechanical signaling in the extracellular matrix of developing and regenerating tissues. FN fibril assembly serves as an early and crucial step in embryogenesis7 and wound healing9, while misregulation of FN assembly is associated with diseases, including cancer10, liver disease11, and lung disease12. Assembly of FN fibrils requires the application of cell-generated traction forces via integrins13: cells bind to FN via transmembrane integrins, which are coupled to the actin cytoskeleton via focal adhesions. Myosin-driven contractility applies force to the focal adhesion complex, which leads to deformation of the attached FN dimer, identified as essential for FN fibrillogenesis14.Soluble fibronectin (FN) is present in blood plasma at high concentration and is assembled by cells into insoluble, elastic fibrils, which play a major role in cell migration, cell adhesion, and formation of a provisional extracellular matrix (reviewed in the literature)14. Once stretched, a cell-attached FN dimer can bind a soluble FN dimer. An FN dimer is comprised of two disulfide bonded FN monomers, which each consist of a series of individually folded domains that comprise one of three structures, which are referred to as Type I, II, or III  simulations have indicated that stretched FN Type III domains have a stable intermediate conformation in which β-strands along the edges of the domain are extended and exposed33. This conformation would be capable of binding other proteins by β-strand addition. Since β-strand addition consists exclusively of backbone hydrogen bonding, it is reasonable to envision a mechanism by which all 15 Type III domains are capable of binding the 70 kDa N-terminus.The 70 kDa N-terminus of FN is capable of binding to various proteins via 36. Given the similar structure of all Type III domains, it is feasible to hypothesize that all 15 domains may be capable of FN-FN binding, and that the degree of binding would be determined by the relative magnitudes of the domain stiffness. Work from Harold Erickson’s group has demonstrated that several FN Type III domains are opened during FN fibrillogenesis37, while work from the Ingber group work has indicated a specific role of the ‘B’ β-strand in facilitating FN fibrillogenesis39. Taken together, there is a strong rationale that FN fibrillogenesis may be facilitated by stretching of FN Type III domains, exposure of β-strands, and subsequent β-strand addition of the 70-kDa N-terminus of a soluble FN molecule.While all 15 Type III domains in FN have a homologous structure, biophysical studies have demonstrated that the domains vary widely in their mechanical and chemical stability40. Briefly, the model simulates FN fibril growth by modeling each FN dimer as a series of Hookean springs that represent each elastic Type III domain  and the stretched length  increase in time, approaching equilibrium values after several hours  the mechanical stiffness of the domain, and 2) the domain stretch length required to exposes the cryptic FN-FN binding site? To address this question, we vary the values of resting domain stiffness εt, specifically considering the III2 domain. Note that we expect similar results for any of the individual Type III domains N-terminal of III10. In Fig. 2 domain . These results suggest that, while a single binding site C-terminal of III10 can result in fibrillogenesis, the FN fibril assembly process is fragile, failing nearly half of the time. Physiologically, FN assembly is a robust and reproducible process, suggesting alternative hypotheses for the location of FN-FN binding sites: multiple cryptic FN-FN binding sites are located within the 15 Type III domains.We next investigate in more detail the properties of FN assembly for a single binding site C-terminal of IIIain Fig. . The FN 15–16 = 32752 combinations), we consider several combinations of previously identified sites, along with our hypothesis of a binding site located in all 15 Type III domains. Specifically, we consider the following four (4) hypotheses for FN-FN binding: (1) binding sites located in each of the first three Type III domains (III1–3); (2) binding sites located in all of the Type III domains N-terminal of III10 (III1–9); (3) binding sites located in the first three Type III domains and one domain C-terminal of III10 , and (4) binding sites located in all 15 Type III domains (III1–15). For each hypothesis, we characterize the fibril size and morphometry; extensibility, given by the stretched-to-relaxed length ratio (Ls/Lr); and geometry, characterized by the relaxed length-to-thickness (Lr/T) ratio. Average values for fibrils assembled with these subsets of multiple FN-FN binding sites are shown in Table While testing all possible combinatorial combinations of multiple binding sites is not feasible .Finally, we consider the hypothesis of cryptic binding sites located in all 15 Type III domains  domain stiffness for all 15 Type III domains are set to the mean value from the baseline case; (4) domain stiffness for all 15 Type III domains are set to the maximum value from the baseline case; (5) baseline domain stiffness values are maintained, but the FN-FN binding site exposure threshold εt is half of the nominal threshold of 15 Å; or (6) baseline domain stiffness values are maintained, but the FN-FN binding site exposure threshold εt is twice the nominal threshold of 15 Å. Average values for fibril size, morphometry, and geometry for these variations on the Type III domain properties are shown in Table We consider six (6) variations on fibrils with binding sites in all 15 Type III domains: (1) domain stiffnesses are scrambled: the same 15 domain stiffness values are used, but for each simulation, the specific order has been randomly rearranged; 2) domain stiffnesses for all 15 Type III domains are set to a constant, equal to the  domain sεt , the fibril stretch  gradually increases as a function of time, before approaching a constant or stable value, while the substrate traction force  also gradually increases, with a few deflections, before reaching the stall force  that prevents further actomyosin-mediated fibril stretch , both fibril stretch and substrate force initially gradually increase, followed by persistent fluctuation around an “average” stretch and substrate force value , compared with SSFs (mean of 16.8 ± 0.25 pN/μm)  Fig. . Furtherr = 0.924 and 0.980, respectively). In contrast, there is a much weaker correlation between the number of FN molecules in SSFs and the stable fibril stretch  and no correlation with the stable substrate force . The substrate force and fibril stretch for the FSFs are also positively correlated (r = 0.922), with a slope of 52.5 ± 0.63 pN/μm . The Type III domain spring constants and forces are related by Hooke’s law and are time-dependent .Simulations were performed using our recently developed model integrating FN assembly and cell-generated traction forces. Full details of model equations, parameters, implementation, and numerical methods can be found in Weinberg  FN Fig. . Each Ty FN Fig. . FN mole10 domain. Thus, each FN molecule contains two integrin binding sites, located at both III10 domains in the homodimer. Integrin binding is represented as a stochastic first-order reversible reaction, with constant on-rate and force-dependent off-rate  soluble FN binding to the exposed binding site, represented by a stochastic irreversible reaction. In a subset of simulations in this study, we increase or decrease the domain threshold εt in one or all Type III domains.Simulations are initialized with a single FN molecule, at rest, bound to an elastic substrate with stiffness ner Fig. , enablin34. Previous studies have demonstrated that Type III domains have unique mechanical unfolding properties; however these unique properties only exist until secondary structures are disrupted, after which point, domains behave as entropic springs that can be represented using the wormlike chain (WLC) model51. We account for both the domain-specific mechanical properties when domains are folded, and the domain-independent, WLC behavior at larger stretches, by specifying a stretch-dependent stiffness relationship in which the stiffness is a unique specific value at rest , and approaches an identical WLC-predicted stiffness as stretch increases . In our prior work, we estimated the unique Type III domain resting stiffness values from several experimental studies40. In this study, we also investigate several variations for the Type III domain stiffness values, specifically 1) Type III domain stiffness value Prior experimental and computational studies suggest that the Type III domain stiffness changes as the domain unfoldsLr, which is determined from the Hookean spring network architecture assuming all springs are in their respective equilibrium position; iii) the stretched fibril length Ls, the total fibril length under tension from actomyosin-induced forces; iv) the number of bound integrins; v) fibril thickness T, calculated from the fibril cross section diameter; and vi) substrate force fsub, given by the product of ksub and substrate deflection xsub. We also calculate measures of extensibility Ls/Lr, given by the ratio of the stretched-to-relaxed length, and fibril geometry Lr/T, given by the relaxed length-to-thickness ratio. Fibril measurements are averaged over the final 10 minutes preceding assembly termination, to account for stochasticity due to integrin bond formation and rupture. Mean values are presented plus/minus the standard error of the mean.For each simulation, we quantify several important measures of fibril morphometry and mechanical properties, including the following: i) the number of FN molecules in the assembling fibril; ii) the relaxed or resting fibril length The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.