Increased taro flour (10–20 g) per 100 g of wheat flour resulted in an increased water absorption capacity (57.38%–58.23%) and mixing tolerance index (67.33–70.21 FU). The sensory analysis had revealed that as taro flour blending ratio increased the acceptability of blended breads were reduced. With respect to physical and sensory properties, the control bread had better acceptability than that of 10, 15, and 20 g taro flour‐mixed bread. The study revealed that there is possibility of incorporating taro flour up to 15 g per 100 g of wheat flour with acceptable sensory attributes of the composite bread.The study was conducted to evaluate the effect of taro drying methods and blending ratios on the physical quality attributes and sensory quality of wheat–taro bread and rheological properties of the blend dough. Farinographic properties like water absorption capacity, dough development time, dough stability time, time to break down, mixing tolerance index, and farinographic quality number were significantly ( The major or mandatory ingredients in bread making are flour, water, salt, and yeast. However, due to the high cost, geographical scarcity, and high demand of wheat flour, efforts are being directed toward the provision of alternative source of flour. Because of this, cocoyam, cassava, taro, and other root and tubers crops have been found to be additional ingredients of major raw materials for bread making.Colocasia esculenta (L.) Schott) is a major tuber crop cultivated in the tropical and subtropical regions of the world. The world average production of taro is about 6.2 tons/ha while African average is 5.1 tons/ha  in the forms of soluble oxalic acid and insoluble oxalate salts  and taro varieties (Boloso I) were based on bread‐making potential Habtu, , respect2.3Wheat was milled to particle size of less than 750 μm using the procedure described in the cereal grain processing manual, using the local miller  according to AACC .Dough strength was measured by Farinograph  and the loaf volume was determined using rapeseed displacement method Chopin,  and refe100 = Volume calculated for 100 g of the bakery productWhere, Vsr = Reading of volume in cm3.VG = Weight of one piece of bakery product.The specific volume of each loaf was calculated as follows:2.6.2The bread sample was cut into slices of 1.5 cm thick using a sharp knife. The outer crust of samples was carefully scrapped with kitchen‐type bread knife. The 1 g cuts from each point were combined to make a final weight of about 5 g. The moisture content was determined using connective oven set at 130°C for 1 hr  to 5 (extremely like). Bread was served at room temperature using the more widely used practice of three digit code during sensory analysis  with three replications using Statistical Analysis System  software version 9.0. Means were compared using Duncan's multiple range test (DMRT) at 33.13.1.1p < .05) affected by the drying methods, blending ratio and their interaction. The highest was observed in the 20 g and 15 g taro per 100 g wheat flour and the lowest was between the solar‐dried taro flour of 10 g per 100 g wheat flour. In general, with increase in the taro flour, an increase in the loaf weight was observed. Blending ratio appears to be dominant factor compared to the drying methods. Loaf weight is basically determined by the quantity of dough baked gluten functionality and the amount of moisture and carbon dioxide diffused out of the loaf during baking  affected by blending ratio and the interaction of drying methods and blending ratio. Drying method had no significant (p > .05) influence. The highest was observed for control bread (240.98 cm3) and loaf volume decreased with increase in the substitution of taro flour. This is may be due to the gluten protein contents of wheat flour. Lack of the gluten protein contents of taro flour is responsible for the reduction in the loaf volume of leavened taro–wheat flour bread  affected the specific volume. As taro flour content increased, the specific loaf volume decreased. This is may be due to the high fiber contents of taro flour that affects the loaf volume of blended bread by diluting the gluten network, which in turn impairs gas retention rather than gas production  affected by the drying methods, blending ratio and their interaction  and farinographic quality number (FQN) were evaluated. The water absorption was significantly (p < .05) influenced by the drying method and blending ratio. Water absorption is the point chosen by the baking industry which represents a target water to flour ratio in bread dough. It is important to determine taste, texture, and dough performance during proofing and baking. The WAC plays a major role in the functionality of dough. In particular, WAC has been shown to be related to dough consistency. WAC plays a major role in the functionality of dough. An increase in the taro flour blending ratio resulted in an increased water absorption capacity of blended flour. Such an increasing trend in WAC with increase in taro flour proportion has been reported in earlier studies  influenced by blending ratio and the interaction between drying method and blending ratio. There was a general increase in DDT with increase in the taro flour content particularly in sun‐ and solar‐died taro flour. The highest was observed for 20 g/100 g sun‐dried taro flour blended flours. The lowest was observed for control flour (4.00 min.). This might be due to the low gluten protein contents of the blended flours and relatively high amount of bran particles in high extraction rate flours, which may interfere in the quick development of gluten and hydration of endosperm. Dough development time increases with the increase in the proteolytical degradation of protein and with a decrease in the size of starch granule and the increase in the content of damaged starch due to the increase in specific surface area which absorbs water  is the time from first addition of water to that of maximum consistency immediately before first indication of weakening. The DDT was significantly (3.2.3p < .01) affected by drying time and the interaction between drying time and blending ratio. There was a general decreasing trend of the stability time with increase in taro flour proportion. The stability time is the gluten quality parameter which describe the viscoelastic properties of formed gluten complex. The stability time of dough is an indicator of the strength, with higher values suggesting stronger dough . The stability time was significantly (3.2.4p < .01) affected by the drying method and the interaction of drying method and blending ratio. The highest was observed for 15 and 20 g taro flour under sun‐dried taro flour and the lowest was for control flour (7.20 min.). This may be due to the low gluten protein contents of taro flours which take long mixing times to make consistent and uniform blended dough. The difference in the time break down of blended flours due to blending ratio (Table p > .01) except control flour. This is may be due to the high carbohydrate contents of taro flour and high fiber contents of whole wheat flour. This result is in the range of 2.90–25.4 min reported by Maghirang, Lookhart, Bean, and Pierce (Time to break down is a time from start of mixing until there has been a decrease of 30 FU from peak point. The time to break was significantly (d Pierce  for hard3.2.5p < .01) influenced by drying method, blending ratio and their interaction. Mixing tolerance index (degree of softening) is measured as the distance between the center of the curve at the end of analysis time and the central line which passes through the maximum of the curve. Blending wheat flour with taro had somewhat reduced the mixing tolerance index showing dough stability increased with taro flour addition.Mixing tolerance index is used by bakers to determine the extent that dough will soften over a period of mixing. The mixing tolerance index was significantly  influenced the FQN. An increase in taro flour generally showed an improvement in the FQN. Even though there is such improvement, the bread quality may be not high because this may not necessarily indicate the leavened products produced. The highest the farinographic quality numbers the better dough handling features. Such positive contribution to the blend may be contributed by the high small starch granules in the taro flour.The FQN indicates the quality of flour for bread making. If the flour has poor quality, it gets weakened early and quickly. The drying method, blending ratio and their interaction significantly (3.33.3.1p < .05). There was no significant (>0.05) difference in color of bread due to blending ratio and the interaction between drying method and blending ratio. However, there was a general decreasing trend in the score with increase in proportion of taro flour dried under solar and the sun. The highest score  was observed for the control sample. Among the experiments, the highest score of 4.6 (like very much) was observed for oven‐ and solar‐dried taro flour blended bread and 4.2 (like moderately) was for sun‐dried taro flour blended bread. The color of bread tells about the appearance of the bread, how it looks like, if it is appealing to the eyes, inviting, and bright. This result was similar to that reported by Sanful (The sensory scores for color are presented in Table y Sanful  where 103.3.2p < .05) in the taste of bread due to blending ratio and the interaction between blending ratio and the drying methods. Except taro sun dried, there was a general decrease in the taste score with increase in taro flour proportion. The highest score was 4.6 (close to extremely like) for taro flour dried in oven with taro proportion of 10 g/100 g flour. The least scores were for samples dried under solar and sun with taro proportion of 20 g/100 g. Similar studies reported a decrease in the taste scores of wheat–taro flour composite bread with increased proportion of taro flour  affected by the drying method, blending ratio and their interaction. Composite bread from taro flour dried under solar dryer with taro flour proportion of 10 g/100 g resulted in the highest score . The flavor scores decrease with increase in taro flour proportion which could be attributed to the high starch contents of taro flour with bland flavor. Flavor is a combination of aroma odor and taste. A decrease in odor and taste score of wheat–taro flour composite bread with increase in taro flour proportion has been reported in earlier studies  affected by drying method, blending ratio and their interaction. The appearance score for most of the treatment groups was around moderately like. However, composite bread from solar dried taro had higher appearance score whereas the control exhibited the highest appearance score. This might be due to the low gluten protein contents of taro flour which contributes to make less leavened characteristics of blended breads.Appearance is the surface characteristics of food materials which attracts the consumer perception. The appearance of taro–wheat bread was significantly (3.3.5p < .05) influenced the overall acceptability. The score ranged from 3.95 to 4.55 which could be associated with like moderately and like very much. However, there was a general decreasing trend in the acceptability score with an increase in the proportion of taro flour. The study revealed that there is possibility of incorporating wheat flour up to 15 g per 100 g of wheat with acceptable sensory attributes of the composite bread. In general, the solar‐dried taro flower resulted in better score in the overall acceptability and other sensory attributes. Similar trend has been reported by Ammar et al. (The overall acceptability scores of wheat–taro composite bread are presented in Table r et al. .4This study showed that physical characteristics, sensory properties of taro–wheat bread, and rheological properties of taro–wheat flour blend dough were significantly affected by drying methods and blending ratio. The acceptability for taro–wheat bread had decreased with increasing taro flour blending ratio due to the presence of salty taste and unusual flavor in the blended bread.None declared.


Basic techniques and recent progress of a variety of important approaches for domain structure simulation, including first-principles calculation, molecular dynamics, Monte Carlo simulation, effective Hamiltonian approach and phase field modeling, as well as multiscale simulation are then elaborated. For each approach, its important features and relative merits over other approaches for modeling domain structures in ultrathin ferroelectric films are discussed. Finally, we review recent theoretical studies on some important issues of domain structures in ultrathin ferroelectric films, with an emphasis on the effects of interfacial electrostatics, boundary conditions and external loads. This review covers methods and recent developments of the theoretical study of domain structures in ultrathin ferroelectric films. The review begins with an introduction to some basic concepts and theories (e.g., polarization and its modern theory, ferroelectric phase transition, domain formation, and finite size effects,  In the literature, considerable attention has been devoted to issues of ferroelectric domain structures, for the very practical reason that most of the important applications of ferroelectric materials  are determined to a great extent by the stability and evolution of the domain structures. During the past decades, fundamental physics and device applications of domain structures in ferroelectric crystals have been extensively studied and well understood.Ferroelectrics are a certain family of materials possessing spontaneous polarization in a definite range of temperature, which can be reversed, in particular, by the application of an external electric field ,2. When etc. Recently, the main focus of ferroelectric thin films has been turned to ultrathin ferroelectric films (UFFs) with the film thickness ranging from a few to dozens of nanometers. Being nanoscale in one dimension, the finite size effect is considerable to diversify the domain patterns and properties of the UFFs from those of their bulk counterparts. For example, the dielectric constants being very high in bulk material (like in SrTiO3 and Pb(Mg1/3Nb2/3)O3 (PMN)) are strongly reduced in the films, while the values of the coercive fields are elevated O3 (PZT) films)  state and daac[R(ac)] state as shown in . Moreover, the domain morphologies in (001)-oriented PbZr0.8Ti0.2O3 and PbZr0.2Ti0.8O3 thin film have also been simulated by Li et al. [Phase diagrams of PbTiO3 ,274, SrT3 [3 , BST [303 [3 ,308, and3 [3 ,312,313 3 [3 . It is ny et al.  based oni et al.  using phet al. [3 and BaTiO3 thin films, resulting in fruitful misfit strain-misfit strain domain stability diagrams at certain temperatures. It is noteworthy that previous discussions are focused on the isotropic misfit strain, while there are conditions anisotropic misfit strain can occur practically. For example, the misfit strain can be anisotropic when UFFs are grown on tetragonal substrates instead of cubic substrates. There have been articles concentrating on this issue based on thermodynamic analysis . Using aet al. ,315 alsoet al. [3 UFFs subject to a substrate constraint using a phase-field method. With taking into account different oriented dislocation, they predicted that the presence of dislocation modifies locally the ferroelectric transition temperature and leads to the preferential formation of ferroelectric domains around misfit dislocations. Meanwhile, Alpay et al. [et al. [etc. Li et al. [Misfit dislocations, generated to release the strain energy arising from the lattice mismatch and or thermal expansion coefficient differences between the film and the underlying substrate, are commonly observed in UFFs when their thickness exceeds a critical value . They plet al.  investigy et al.  develope [et al. ,322,323 i et al.  further i et al. , generali.e., treating mechanical loads in a similar role of electric field) is surprisingly few. Using thermodynamic analysis, Emelyanov et al. [et al. [a/b-domains can be induced by large tensile strain, leading to the evolution of initial 180° cylindrical domain pattern into a complex domain pattern. Interestingly, results show that mechanical erasing can still be achieved in some cases even though a/b-domains were induced.Considering the possible problems of electrical means nowadays , and potential applications of ferroelectrics in non-electrical environments, effective strategies for processing ferroelectric domains by mechanical means would be meaningful. However, compared with the numerous articles being focused on utilizing film-substrate misfit strains to obtain novel ferroelectric domain structures in UFFs, attentions on manipulation of domain structure in UFFs by mechanical loads -oriented BaTiO3 ultrathin films with stripe domain structures alternating along [3 ultrathin films. For the latter ones, before switching, Before switching directions, the dipoles antiparallel to the electric field decrease in magnitude at the very beginning which is not found in PZT. Others include the appearance of the zigzagged domain walls and the direction where the bubbles contract along. Furthermore, Sichuga and Bellaiche [i.e., the dipolar wave state and transitional state, are predicted to occur. Therefore, the domain structure of the PZT film under such a field application exhibits a transformation in a sequence of stripe state, dipolar wave state, nanobubble state, transitional state and finally monodomain state. The 20 Å-thick compressively strained film adopts stripe domain structures alternating along  directioed for 00-orientedng along ,7,8,9,10ng along . Neverthellaiche  investigellaiche  directioet al. [3/SrTiO3 superlattices, first-principles based calculations have shown that the domain pattern of the BaTiO3 layer and the SrTiO3 is similar at zero applied field. Interestingly, a difference in the domain pattern between the two layers is observed when the applied filed increases [3 and SrTiO3 layers induced by electrostatic field, as shown in Artemev et al.  studied ncreases . This imet al. [3 thin films under a biased SFM tip was demonstrated by Vasudevan et al. [et al. [There are many cases where we apply an inhomogeneous electric field rather than homogenous electric field to manipulate domain structures in UFFs. Compared with homogenous field, which usually requires sufficiently large parallel electrodes and thus occupies a large space, inhomogeneous field can be generated by a nanoscale conductive tip in combine with a bottom electrode and thus can be rather localized and strong, making it advantages in performing local probing and control on the domain structure. Driven by the application of scanning probe microscope (SFM), in literature, there are quite an amount theoretical works focusing on the stability of nanodomains and domain walls under SPM electric field. In particular, researchers paid attention to dependence of 180° nanodomains stability on SPM electric field  ,336. Theet al.  suggesten et al. . Recentl [et al.  have preetc. In general, the domain walls affect the properties of a ferroelectric thin film in two ways, i.e., the microscopically and macroscopically. In the first case, the existence of domain walls locally modified the properties of thin film, such as local enhancement of piezoelectric responses and increase of conductivity near the domain wall [Domain walls in UFFs are of fundamental importance and technological relevance, since they are at the core of the polarization switching and make important contributions to most of material responses, thus playing central roles in the practical applications of UFFs such as ultrahigh-density ferroelectric memory, sensors, and actuators, ain wall . Even thain wall . In the ain wall ,345,346.i.e., first-principles calculations and first-principles derived atomic level methods. Phenomenological models available nowadays are focus on domain walls with reasonably large thickness according to available experimental and theoretical estimates, and UFFs with well-defined free energy [To address the issue of domain walls, phenomenological level of description were previously adopted. There were several articles aiming to estimate the thickness of domain wall using a Landau-Ginzburg type of continuum theory ,350,351.e energy ,354,355.et al. [3 and confirmed their Ising-like nature along the tetragonal z axis employing the effective Hamiltonian method combined with MC simulations at finite temperatures. With first-principles calculations, Pöykkö and Chadi [3, followed by the investigation of 180° and 90° domain walls in PbTiO3 carried out by Meyer and Vanderbilt [3-type ferroelectrics. Based on this framework, various domain walls of ferroelectric BaTiO3 were further studied. Interestingly, the internal structure of the lowest energy domain wall separating antiparallel rhombohedral ferroelectric domains was shown to be analogous to the so-called Bloch wall known in ferromagnets [et al. [et al. [3 and LiNbO3  in multiferroic BiFeO3 are attracting increasing attention [Recently, the structural, electronic, and magnetic properties of the ferroelectric domain walls  by mechanical bending strain based on the flexoelectric effect. The difficulty in theoretical calculation resides in lacking of accurate flexoelectric coefficients. Note that the flexoelectric coefficients adopted in most available theoretical investigations nowadays are roughly estimated. Although several attempts based on first-principles calculations to determine the coefficients have been carried out [Flexoelectric effect due to the coupling between polarization and strain gradient are attracting considerable attention quite recently. Such a universal phenomenon which was predicted to be ignorable compared with other effects in solids is of keen interests in UFFs for practical reasons ,360: as et al.  exploredried out ,367,368,RMn2O5 , bismuth compounds BiFeO3 and BiMnO3, and others such as BaNiF4, etc. Among these multiferroics, perovskite-structured BiFeO3, exhibiting ferroelectrictiy, antiferromagnetism, and a weak magnetization resulting from a spin canting at room temperature, is an intriguing example. There have been several theoretical investigations concentrating on the domain structures in BiFeO3 thin films. Importantly, using a first-principles-derived effective Hamiltonian method, Prosandeev, Lisenkov and Bellaiche [3 ultrathin films. Based on first-principles calculations, Ren et al. [3 films subject to compressive strain. Despite these efforts, theoretically probing the behaviors of the domains requires a fully understanding of both the ferroelectric and magnetic characteristics. Most simulation methods nowadays are severely limited to the lacking of materials parameters. In particular, in phase field simulation, even more of a problem is the lack of a well-defined free energy describing the magnetic characteristics. Phase field modeling of BiFeO3 available are limited to the ferroelectric behaviors.Recently, multiferroics possessing magnetoelectric coupling are quite hot researching objects as they are promising for appearing abundant phenomena and indicate prospective applications. Typical multiferroic materials include ellaiche  obtainedn et al.  investigThis paper has reviewed methods and recent developments of theoretical investigations on domain structures in UFFs, aiming to provide readers with the latest knowledge of this field and insights into how factors, e.g., surface, interface, thickness, electrical and mechanical loads, determine the stability and evolution of ferroelectric domain structure and related properties of ultrathin films. It begins with the basic concepts and theories that are relevant to study of domain structures in ultrathin ferroelectric films. Then, it turns to a survey on approaches powerful on domain structure simulation, including first-principles calculation, MD, MC, effective Hamiltonian approach and phase field method. Finally, recent theoretical studies on some important issues of domain structures in ultrathin ferroelectric films are discussed.To have an outlook of this field, we would like to point out some aspects that are worthy of future attention. Firstly, the development of an efficient multiscale investigation scheme is in demand. Among the various theoretical approaches, atomistic level simulations such as MD, MC and effective Hamiltonian, and phase field method are powerful to directly simulate the domain structure with details. Nevertheless, to fully understand the different aspects of domain structure of UFFs, other approaches should be also relied on. In particular, we need first-principles calculations to reveal electronic structure, the accurate energy profile as a function of different degrees of freedom, and detailed physics at the surfaces and interfaces. Such information is necessary for better understanding the behaviors and related properties of domain structures at larger scale. By appropriately choosing thermodynamic variables and construction of free energy, thermodynamic analytic modeling can be an important method of providing a clear description of the thermodynamic behaviors of domain structure. Although in the literature there are some efforts on combining different approaches , a well-defined and efficient multiscale investigation scheme is lacking. 3, PbTiO3, and PZT, etc.). The investigated boundary conditions and external electrical and mechanical loads are also simple, with a lacking of consideration on complicated situations as well as on the detailed physics of surface and interface. As a consequence, the approaches often only take into account a few degrees of freedom, particularly polarization, strain/stress and electric field. In reality, other degrees of freedom may not be neglected, such as octahedral rotation that is common in perovskite ferroelectric thin films, and magnetic ordering in multiferroic thin films. The incorporation of more degrees of freedom into the theoretical model is definitely important to predict coupling effects and multi-functionalities of domain structure in UFFs.Secondly, improving the computational methods to be capable of modeling a wide range of materials, more realistic systems is quite essential in future. Presently, simulations on domain structures in UFFs are mainly performed on conventional perovskite ferroelectrics (e.g., UFFs of perovskite type like BaTiOMoreover, note that currently the true dynamics of domain structure in ferroelectrics is a topic relatively lacking in research, despite its importance in both fundamental research and technological applications. This is largely due to the fact that many approaches, such as MC simulation and phase field method, is not capable of give the true dynamics of domain structures, making them deficient in tackling related problems. On the other hand, MD simulations, as they have the advantage of tracing the intrinsic dynamics of polarization reversal and domain evolution, may be exploited to determine the true kinetic coefficients of the TDGL equation employed in phase field simulations. As the phase field method is capable of dealing large systems and a longer time scale, it would be significant if it can tackle the real dynamics of domain structure. Nevertheless, so far there is no such kind of work being reported. Anyway, the field of domain structures in UFFs is now rapidly expanding, with increasing materials and phenomena being under investigation. We are awaiting breakthroughs on theoretical investigations on domain structures in UFFs, particularly on the above mentioned aspects. Although there are many challenges, it is certain that many research efforts will continue to focus on this field. It is quite reasonable to be optimistic that powerful theoretical methods will finally give rise to a systematical understanding of the domain structure in UFFs and provide us instructive information on designing revolutionary devices based on domain structure in UFFs.


The two isoforms of the Bcr-Abl tyrosine kinase, p210 and p190, are associated with different leukemias and have a dramatically different signaling network, despite similar kinase activity. To provide a molecular rationale for these observations, we study the Dbl-homology (DH) and Pleckstrin-homology (PH) domains of Bcr-Abl p210, which constitute the only structural differences to p190. Here we report high-resolution structures of the DH and PH domains and characterize conformations of the DH–PH unit in solution. Our structural and functional analyses show no evidence that the DH domain acts as a guanine nucleotide exchange factor, whereas the PH domain binds to various phosphatidylinositol-phosphates. PH-domain mutants alter subcellular localization and result in decreased interactions with p210-selective interaction partners. Hence, the PH domain, but not the DH domain, plays an important role in the formation of the differential p210 and p190 Bcr-Abl signaling networks. The Bcr-Abl tyrosine kinases p210 and p190 are linked to different leukemias and differ by the Dbl homology (DH) and Pleckstrin-homology (PH) domains. Here the authors characterize structures of the Bcr-Abl p210 DH and PH domains and find that the PH domain is important for the cellular localization and signaling network of p210. Bcr-Abl is a constitutively active tyrosine kinase and is selectively inhibited by imatinib (Gleevec), which became a paradigm for targeted cancer therapy2. Different Bcr-Abl protein isoforms are expressed, all of which contain exons 2–11 of the ABL1 gene, but, depending on the location of the translocation breakpoint in BCR, include different portions of the BCR gene3. The most common Bcr-Abl isoforms are p210 and p190. p190 lacks residues 427–927 of Bcr, and thus is 501 amino acids  shorter than p210, but otherwise contains the same domains with identical sequences 3. In addition, 20–30% of adult B-cell acute lymphoblastic leukemias (B-ALL) are Bcr-Abl-positive, of which approximately 1/4 of cases express p210, whereas 3/4 express Bcr-Abl p190. The treatment of CML patients with the Bcr-Abl tyrosine kinase inhibitor (TKI) imatinib results in deep remissions and long-term survival5. Although TKIs prolong survival in Bcr-Abl-positive B-ALL, acquired TKI resistance is frequent and overall survival is still dramatically low7. Therefore, a deeper understanding of Bcr-Abl p210 and p190 signaling may guide the identification of additional drug targets for combinatorial therapy in B-ALL.The Bcr-Abl oncoprotein is expressed from the Philadelphia (Ph) chromosome, which is formed upon the inase ABL1. Bcr-Ab9. Unexpectedly large differences in the interactome and tyrosine phosphoproteome were found, with more than a dozen proteins that preferentially interact with either p210 or p190 and ~100 differential phosphotyrosine (pY) sites indicating activation of distinct signaling pathways by the two Bcr-Abl isoforms9. In contrast, no difference in degree and sites of p210 and p190 autophosphorylation or in vitro kinase activity of Bcr-Abl was detected that could have rationalized the observed signaling differences. These observations suggested that the functional disparities of p210 and p190 arise from regions outside the kinase domain. Hence we decided to study the differences in structure of the two isoforms. Due to the different translocation breakpoints, p210 contains a predicted Dbl-homology (DH) and Pleckstrin-homology (PH) tandem domain that p190 lacks  that was proposed to lack GEF activity was able to induce leukemia with shorter latency as compared to wild-type p210 in a mouse model13. Thus far, the activity and selectivity of the Bcr-Abl DH domain remains unclear and no convincing direct link between Rho GTPase activation and the Bcr-Abl DH domain could be made.DH–PH tandem domains are the canonical structural motifs of the Dbl-family guanine nucleotide exchange factors for Rho GTPases (RhoGEF), with the DH domain mediating GEF activity14. As part of the DH–PH unit, the PH domain has been implicated in multiple functions, including membrane localization, allosteric modulation of GEF activity, and in certain cases direct contributions to DH GEF activity15. Deletion of the entire PH domain from Bcr-Abl p210 slightly increased transformation and mildly decreased survival in mouse bone marrow transplantation models, showing a disease phenotype that resembles p190-driven B-ALL16.PH domains are well-characterized lipid-binding domains found in a variety of proteins, in which they mediate interactions with phosphoinositide lipids in biological membranes19 . Crystallographic analysis of the N-terminus of Bcr-Abl showed that a coiled-coil domain forms antiparallel homo-tetramers26 and the very C-terminus of Bcr-Abl contains an F-actin binding domain that has been studied in solution by NMR27.In contrast to the DH and PH domains, the other domains of Bcr-Abl have been structurally well characterized, including the central SH3-SH2-kinase domain unit in both the autoinhibited formIn summary, the absence of a structural model and poorly defined biochemical specificity and regulation of the Bcr-Abl DH and PH domains, in combination with conflicting data on the role of both domains in Bcr-Abl-dependent leukemogenesis and signaling necessitates an in-depth structural and functional characterization. Here we provide high-resolution structures of the DH and PH domains that unravel unique structural features. Detailed functional characterization give no evidence for RhoGEF activity of the DH domain. In contrast, we characterize in detail PH-mediated phosphoinositide binding that affects cellular localization and the signaling network of Bcr-Abl p210.15N,1H]-TROSY spectrum of the DH domain was clearly resolved and was indicative of a well-folded protein. We therefore embarked on the structure determination of this domain by NMR. Given the considerable size of the DH–PH tandem domain  and poor behavior of the isolated PH domain, we first concentrated on the DH domain .The DH and PH domains constitute the only predicted folded domains of Bcr-Abl whose structure remains undetermined. Therefore, we set out to structurally characterize them by crystallography. Among several constructs, the Bcr-Abl DH- (residues 487–702) and DH–PH-domain (residues 487–893) constructs expressed in high yields and showed good biophysical behavior ) that bound to the DH domain with low nanomolar dissociation constants , Supplementary Fig. The Bcr-Abl PH domain exhibited limited stability at room temperature despite extensive optimization efforts. Homology modeling indicated the presence of a unique insertion of 59 amino acids (residues 770–829) between predicted strands β5 and β6 constituting almost a third of the entire Bcr-Abl PH domain. Since the presence of such a large, putatively flexible element can dramatically interfere with any structure determination approaches, we deleted this part of the PH domain. Still, we were not able to obtain crystals. Encouraged by our success with the DH domain, we generated additional monobodies that bound to the PH domain. Here we made use of the phage-display pool of monobodies originally enriched with the Bcr-Abl DH–PH tandem domain, but performed the secondary yeast-display screening using the PH domain as the target . In other DH–PH units, the PH domain may regulate GEF activity either via intramolecular interactions with the DH domain, which could block GTPase binding or by forming an additional interface with the GTPases to facilitate nucleotide exchange34. The resulting model indicated that residues 770–829 of the Bcr-Abl PH domain protrude away from the core of the globular PH domain and add a significant volume to the overall shape of the domain 36. In particular a glutamate residue in CR1 and a lysine residue in CR3 are important for catalytic activity38. Furthermore, residues in the ‘seat-back-region’ of the DH domains, comprising α-helix 4, α4-α5 loop and α-helix 5, determine GTPase specificity15. Notably, these residue that are important for GTPase interaction, catalysis, and selectivity are not conserved in the Bcr-Abl DH domain. Furthermore, the extended α4–α5 loop points directly into the GTPase binding interface, where it would sterically clash with binding of a Rho GTPase  mass spectrometry experiments in the Bcr-Abl p210-expressing cell line K562 using the isolated DH–PH tandem domain to assess direct interactors of this fragment, but we found no Rho GTPase captured with the Bcr DH–PH tandem domain -binding site, which is formed by a positively charged patch of amino acids (arginine and lysine) in the β2–β3 hairpin of PH domains40. In this context, we observed that the Bcr-Abl PH domain bound preferentially to bis- and tris-phosphorylated PIPs over mono-phosphorylated PIPs, as well as preferentially to PS-containing liposomes over other phospho- and sphingo-lipids , as well as p190. Additionally, we combined the PH domain mutations with a mutation disrupting the binding of the F-actin binding domain PBcr-Abl p210 showed diffuse, predominantly cytosolic, staining, and quantitative image analysis additionally revealed a strong co-localization with F-actin Fig. . Both mu8. Bcr-Abl immunoprecipitation experiments showed a strongly reduced amount of Sts1 with both PH mutants, as compared to wild-type p210, almost reaching a similar level of Sts1 interacting with p190 that lacks this domain , as well as on the Bcr-Abl interactome from others and us, did not indicate that Rho GTPase directly interact with Bcr-Abl43. We also carefully checked the structural location and possible functional role of the S509A mutation that was previously proposed to modulate leukemogenesis in a mouse model13, but found no supporting evidence for these findings, as the DH domain structure or binding remains unaltered  that builds part of the GTPase-interaction interface. The biological impact of the phosphorylation of these tyrosines may merit deeper analysis. However, in cases where the regulation upon DH domain phosphorylation has been studied46, GEF activity was only mildly modulated upon phosphorylation. Collectively, based on our detailed study, there is no evidence that the Bcr-Abl DH domain acts as a RhoGEF. Hence, it may therefore be dubbed ‘pseudo-GEF’, along with six other Dbl family members that lack key catalytic residues47. Still, the DH domain may play a possible important role for the overall structural organization and integrity of Bcr-Abl p210. We postulate that the activation of RhoA that had been observed for Bcr-Abl p210, but not for the Bcr-Abl p190 isoform in two previous studies12, may be the consequence of the drastically altered signaling networks between the two isoforms rather than the presence of the DH–PH domain in p210.Diverse mechanisms for the regulation of DH-mediated GEF activity have been described, including direct intramolecular interactions with adjacent linkers of the PH domain, allosteric regulation via phospholipid binding to the PH domain, as well as post-translational modifications of the DH domain, such as phosphorylation27. In line with this observation, the localization of the Bcr-Abl p210 and p190 isoforms does also not differ drastically, as both forms of Bcr-Abl contain the FABD. Consequently, the membrane targeting of the PH domain is not as evident as might be expected and compared to other PH domain examples, such as Btk. Still, given the complex spatial organization and dynamics of signaling complexes in mammalian cells, even globally small differences in protein subcellular localization can have strong consequences in signaling. Moreover, as demonstrated here for the Bcr-Abl/Sts1 interaction, even small differences in the ‘microenvironment’ of the two Bcr-Abl isoforms lead to a different subset of interacting or substrate proteins modifying the entire signaling network.Our results indicate that the PH domain contributes to the signaling network of Bcr-Abl through PIP-binding. While Bcr-Abl has been studied for decades, and interaction domains with both unmodified  and post-translationally modified (SH2) protein partners were shown to be critical for localization and signaling, the PIP binding of the PH domain has not been implicated in Bcr-Abl signaling thus far. Here, we show that PIP binding of the Bcr-Abl PH domain has important consequences on the Bcr-Abl signaling network. Given that Bcr-Abl oligomerizes via its N-terminal coiled-coil domain, the lipid-binding affinity of the isolated PH domain determined here likely additionally profits from an avidity contribution of multiple PH domains in the Bcr-Abl tetramer Fig. . Regardi30.Finally, the overall architecture of Bcr-Abl is another important aspect to consider with regard to cellular localization and recruitment of interaction partners. The presence of the DH–PH tandem domain in the p210 isoform contributes a quarter of the size of the whole protein and it is not unlikely that these additional domains strongly affect the overall structural organization of this multi-domain oncoprotein. Currently it is not well understood whether and how the DH–PH module interacts with its neighboring domains or if, in the context of higher oligomers, the DH domain could dimerize or simply act as a long spacer separating the coiled-coil domain from the kinase domain. Furthermore, as monobodies tend to target functional surfaces, it is not unconceivable that the epitopes for the DH and PH monobodies might represent sites of yet undiscovered functional importanceOur study closes the last gap of high-resolution structural data on individual Bcr-Abl domains that have been studied over the past 20 years. The structures of the DH and PH domains now enable future structural on higher order structural organization of multi-domain constructs and full-length Bcr-Abl p210 by SAXS, cryo-EM and crystallography. The results of these studies will be essential to understand the structural differences between Bcr-Abl p210 and p190 and relate them to the Bcr-Abl signaling networks and its interactors.48. Similarly, the DH–PH and PH constructs for monobody generation were cloned in the pHBT1 vector with an N-terminal Avi-tag for biotinylation. Expression of the DH–PH, DH and PH constructs was conducted overnight in Rosetta(DE3)  in LB medium after induction with 0.5 mM IPTG at an optical density of ~1.0. The temperature during the expression was reduced to 18 °C. Biotinylation of the DH–PH and PH constructs with Avi-tag was achieved upon co-expression with the biotinylase BirA in BL21(DE3) in the presence of 50 μM biotin49. The monobodies were expressed in BL21(DE3) using auto-induction medium . During overnight expression the temperature was lowered to 20 °C. Finally, the GTPases were expressed overnight at 18 °C in BL21(DE3) using Terrific Broth (TB) medium and 0.5 mM IPTG for induction. The purification buffer consisted of 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% glycerol, and 1 mM DTT. For purification of the GTPases the buffers additionally included 10 mM MgCl2. Lysis of all samples was conducted on an EmulsiFlex-C5 homogenizer (Avestin) using 18,000 PSI output pressure and three lysis cycles. The cleared supernatants were in a first step purified by gravity flow Ni-NTA Agarose (Qiagen) prior TEV cleavage during overnight dialysis . For the formation of the monobody complexes (DH/Mb(Bcr-DH_4) and PH/Mb(Bcr-PH_4)), the proteins were mixed after affinity purification with a twofold excess of monobody prior the dialysis and TEV cleavage. TEV protease, the His-tag and un-cleaved protein were removed by a reverse Ni-affinity step. The final purification step for all constructs was a size-exclusion-chromatography step depending on the size of the constructs either on a Superdex75 or Superdex200 16/600 column . The samples were concentrated for the subsequent downstream application by Amicon-Ultra centrifugal filters (Millipore) and all proteins could be stored at −80 °C.The DH–PH residues 487–893) and DH (residues 487–702) constructs were cloned with an N-terminal His-tag and TEV cleavage site into pETM-11, the PH domain (residues 704–893) and all GTPases were cloned with an N-terminal GST-tag and TEV-cleavage site into the pETM-30 vector. Primer sequences are shown in Supplementary Data 87–893 an29. Two different monobody phage-display libraries were screened, the loop only library50 and the loop and side library29. A biotinylated DH–PH domain sample in the presence of 25 µg ml−1 poly lysine51 was used for sorting of phage-display libraries. Four rounds of phage-display library selection were performed at concentrations of 100, 100, 50, and 20 nM of biotinylated DH–PH domain for the first, second, third, and fourth rounds, respectively. After amplification of the cDNA encoding the resulting pool of enriched clones, the sub-library was transferred to the yeast display format, as described previously29 and selection performed at 20 nM DH–PH or 100 nM of PH domain. Clones were analyzed for target protein binding in the yeast-display format and sequenced. Binding assays in the yeast display format were performed as previously described29 with a concentrations range of the biotinylated targets from 10 nM to 1 μM in Tris-buffered saline (TBS) with 0.1% BSA. Incubation of target protein, yeast cells, and a mouse anti-V5 antibody were for 30 min at RT. Streptavidin-DyLight650 and a FITC-coupled antimouse IgG was added after two washing steps and incubated for 30 min at RT. Samples were analyzed on a Gallios (Beckman Coulter) or BD Accuri C6 (BD Bioscience) flow cytometer. Plots of the mean fluorescence intensity vs. target concentration and fitting a 1:1 binding model to the data using Prism (GraphPad) was used to determine the apparent Kd values. We have established that the apparent Kd values determined using this method generally agrees with those determined from biophysical measurements of purified proteins29.The general approach for monobody generation have been previously reportedIsothermal titration calorimetry (ITC) measurements were performed on a MicroCal iTC200 (GE) instrument. All proteins were degassed after extensive dialysis against 25 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM TCEP. Protein concentrations were determined by measuring UV absorbance at 280 nm. The titrations were performed in 16 steps with 0.49 μL injections for the first and 2.49 μL for the other injections. Concentrations were selected based on assumed affinities and the signal strength of the interaction. The MicroCal software was used for data analysis.−1 15NH4Cl and either 2 g L−1 13C-glucose (Cambridge Isotope Laboratories) or 4 g L−1 unlabeled glucose. Finally, the samples were dialyzed into an NMR compatible buffer containing 50 mM HEPES, pH 7, 50 mM NaCl, 1 mM DTT. For GTPase titration experiments, the buffer additionally contained 1 mM imidazole and 2 mM EDTA.For NMR structural studies, the DH domain was expressed in minimal M9 medium supplemented with 1 g L15N, 13C-labelled DH domain (487–702) was prepared at a concentration of 850 μM. Measurements for the assignment were carried out at 303 K on Bruker Avance spectrometers equipped with cryogenic 1H{13C,15N} probes operating at proton resonance frequencies of 600–950 MHz. Backbone assignment was achieved using TROSY-type53 BEST55 versions of HNCO, HN(CA)CO, HNCACB, HN(CO)CACB, and HN(CA)HA triple resonance experiments. Side chain resonances were assigned based on three-dimensional, TROSY-type versions of the H(CCCO)NH-TOCSY and (H)C(CCO)NH-TOCSY, a 3D 13C-start CCH-TOCSY, as well as 3D 13C-separated NOESY experiments. Aromatic side chains were assigned by the following 2D experiments: H(N)CD (Trp), H(NCDCG)CB (Trp), (HB)CB(CGCD)HD, (HB)CB(CGCC-TOCSY)Har. NOE distance restraints were derived from 15N and 13C-separated NOESY spectra. In addition to the manual assignment strategy, we employed the FLYA automated resonance assignment algorithm for backbone and side chain assignment56. The 1H/13C/15N resonance assignments were complete for all CH_n groups and the backbone NH groups. About 90% of these assignments (from FLYA) were verified manually. Automated NOE assignment and structure calculation was conducted in CYANA57. The structure calculation was based on 4298 NOE upper distance limits, of which 845 were long-range. Structure calculations with torsion angle dynamics were started from 200 random conformers58. A consensus structure bundle of 20 conformers was subjected to restrained energy minimization in explicit solvent against the AMBER force field with OPALp61. RMSD values were calculated for the well-defined regions of the protein, residues 496–619, 638–693, as determined with CYRANGE62.For the NMR structure determination, uniformly 15N-labeled DH domain at a concentration of 50 μM. GTPase stock solutions ranged between 600 and 900 μM. All proteins were dialyzed into the same buffer prior the experiment. Chemical shift changes were monitored using -TROSY experiments recorded on a Bruker Avance spectrometer with cryogenic 1H{13C,15N} probe operating at a proton resonance frequencies of 800 MHz. The composite 15N and 1H chemical shift perturbation was calculated with a scaling factor for the 15N shift changes of 0.1463. Binding constants were fitted in Prism (GraphPad) according to a 1:1 binding model.The interaction studies with the GTPases were done using a uniformly −1. Initial screenings were set-up in a 96-well plate with sitting drop crystallization using the Mosquito robot (TTP Labtech). Drops were composed of a mixture of protein and buffer in a 1:1 ratio (v/v) and crystals grown at 291 K. The DH/Mb(Bcr-DH_4) complex crystallized in condition F5 of the Morpheus HT screen  corresponding to 0.12 M monosaccharides , 0.1 M buffer system 2 pH 7.5 ) and 50% precipitant mix 1 . The PH/Mb(Bcr-PH_4) complex crystallized in condition 81 of the JCSG+ suite (Qiagen) that contained 0.1 M potassium thiocyanate and 30% (w/v) PEG MME 2000. PH/Mb(Bcr-PH_4) crystals could be improved in the presence of 20% glycerol.Samples for crystallization trials were concentrated to ~10 mg mLhttp://xds.mpimf-heidelberg.mpg.de/). Phasing was achieved by molecular replacement in Phaser using homology models created in SwissModel65. The homology model for the DH and the PH domain was based on the template with PDB ID 2Z0Q, the monobody was modeled based on template PDB ID 3TEU. Manual model building, solvent addition and refinement employed phenix.refine in the Phenix software suite66.X-ray diffraction data were collected at the synchrotron SLS , beamline ×06DA using a single wavelength at 100 K. The diffraction data were processed with the X-ray Detector Software . SAXS data were collected at EMBL P12 beamline, DESY, Hamburg, Germany with protein concentration ranges of 1.6–14.4 mg mL−1. The data were cropped from the first point of the Guinier region until the end of the useful range defined by SHANUM67. High- and low- concentration curves were merged to counter concentration effects, such as inter-particle interference using the program PRIMUS from the ATSAS package68. GNOM was used to obtain the p(r) and determine the corresponding Dmax and Rg values69. SREFLEX was used to generate and refine high-resolution hybrid models using high-resolution structures as the starting point34. EOM was used to generate an ensemble of conformations and to assess the inter-domain flexibility of the DH–PH domain35. The scattering curves from the high-resolution models were calculated using CRYSOL70. SAXS figures were prepared using PyMOL (www.pymol.org) and SASpy71.Proteins for SAXS analysis were concentrated to 10–15 mg mL74. Homology models were created using SwissModel (swissmodel.expasy.org)64. Structural analysis and figure preparation was conducted in PyMOL (www.pymol.org).Sequence and secondary structure analysis relied on webservers for BLAST (blast.ncbi.nlm.nih.gov), PSIPRED (bioinf.cs.ucl.ac.uk/psipred) and HHpred (toolkit.tuebingen.mpg.de/#/tools/hhpred)2-containing buffer  overnight at 4 °C. Half of the sample was then directly loaded onto an analytical Superdex75 10/300 size-exclusion column. For pull-down analysis, the samples were twofold diluted with binding buffer , mixed with Ni-NTA Sepharose  and incubated for 2 h at 4 °C. The slurry was transferred into a micro spin column and the unbound fraction was collected by centrifugation at 500×g for 1 min. The resin was washed with a total of 10 column-volumes buffer  prior to elution of the bound proteins in 3 column-volumes buffer . The samples were analyzed on Coomassie-stained SDS-PAGE. Equal amounts of input, flow-through, wash and elution fractions were analyzed on SDS-PAGE, scanned with the Odyssey imager (Li-Cor) and quantified using the Image Studio Software (Li-Cor). The percentage of untagged protein in the respective gel lane normalized to the input fraction was calculated.DH–PH and DH constructs were mixed with GTPases in a 1:2 molar ratio and dialyzed into either EDTA- or MgCl2 with an additional incubation time of 30 min on ice. Excess nucleotide was then removed with a PD-10 Desalting column  and the protein concentration determined by absorbance measurement.GTPases were dialyzed in loading buffer  overnight at 4 °C to remove bound nucleotide. The nucleotide-free GTPases were then mixed with a fivefold excess of mant-GDP (Thermo Fisher Scientific) and incubated for 90 min on ice. The mant-GDP-loaded GTPases were stabilized upon addition of 10 mM MgClNucleotide exchange reactions were set-up in a black 96-well plate with 1 μM GTPase/mant-GDP and 10 mM GTP in reaction buffer . The exchange reaction was started upon addition of 20 mM EDTA or 0.5 μM Dbs DH–PH as positive controls, and 5 μM or 50 μM Bcr-Abl DH–PH and DH domains and immediately measured at the SpectraMax M5 plate reader (Molecular Devices). The single-wavelength measurement was done at 25 °C with an excitation at 360 nm and emission at 445 nm. The decrease of the fluorescence upon GDP-to-GTP exchange was monitored up to 20 min, when the intrinsic exchange reached the baseline. The exchange rate constants were fitted in Prism (GraphPad) and the enhancement factor was calculated in comparison to the intrinsic exchange rate without addition of EDTA or GEF protein.PIP strips (Echelon Biosciences) were blocked in PBS-T + 3% BSA for 1 h at room temperature before adding 20 nM of the target proteins for another 1 h at room temperature. Membranes were then washed three times in PBS-T and incubated with an anti-His antibody (Qiagen) in PBS-T + 3% BSA overnight at 4 °C. After washing again three times in PBS-T, detection was achieved with a secondary antimouse antibody coupled to an IRDye800  on the Li-Cor Odyssey imaging system (Li-Cor Biosciences) and quantification was done in the Image Studio software (Li-Cor Biosciences).40. Briefly, liposomes were formed in the assay buffer  from lipid mixtures containing various combinations of 17 signaling lipids .The fabrication of liposome microarrays, the experimental procedure of protein-liposome interaction assay, the image analysis, and the calculation of normalized binding intensities (NBIs) values were described previously−1 in PBS). Finally, three times 5 min washes in PBS + 0.1% Triton X-100, coverslips were mounted on a microscopic slide using Mowiol. Images were obtained on a Zeiss LSM780 confocal microscope using a Plan-Apochromat 63x/1.40 Oil DIC M27.HeLa cells were transfected with vectors encoding p210, mutants thereof or p190 under the control of an SV40 promoter by using PEI transfection reagent . After 48 h of incubation, the medium of transfected cells was removed and the cells were quickly washed with phosphate buffer saline (PBS) equilibrated at 37 °C. Coverslips were then incubated in 3% paraformaldehyde (PFA). After 15 min incubation on ice, the cells were washed three times 5 min with PBS. The cells were permeabilized for 10 min at room temperature with PBS + 0.5% Triton X-100 and blocked with a commercial 10% normal goat serum blocking solution (Invitrogen 50-197Z) for 10 min. Incubation with the primary antibody solution  was performed for 1 h and followed by three washes with PBS + 0.1% Triton X-100. Then, the secondary antibody solution  was applied for 30 min. After application of the secondary antibody, all incubation steps were performed protected from light. After washing, the actin cytoskeleton was stained by incubation of the cells with phalloidin-rhodamin for 20 min according to supplier instructions . After three times 5 min washes in PBS + 0.1% Triton X-100, DNA was stained with Hoechst for 10 min . Cells with similar shape and actin morphology were selected for all conditions. For co-localization of Bcr-Abl with actin, we computed the Manders coefficient of co-localization75 using the ImageJ software, as follows: we background-corrected the Bcr-Abl channel  and applied a threshold on the actin channel; next we used the thresholded actin image to mask the background-corrected Bcr-Abl channel (Bcr-Abl actin); image stacks from ‘Bcr-Abl total’ and ‘Bcr-Abl actin’ were sum-projected along the z-axis. The fraction of Bcr-Abl co-localizing with actin was calculated by dividing the sum intensity of ‘Bcr-Abl actin’ by the sum intensity of ‘Bcr-Abl total’ in a manually drawn ROI around each cell in the projected images.All images were acquired with the same confocal microscope settings  and cell lysis and purification was performed in TAP buffer  NP-40 Alternative) containing protease and phosphatase inhibitors . Normal mouse IgG antibody  and the monoclonal Abl antibody (clone 24–21)76 were covalently coupled to NHS-activated sepharose . The lysates were pre-cleared for 1 h at 4 °C with the IgG-NHS resin and the supernatant then transferred to the Abl-NHS resin for another 3 h at 4 °C. Elution fractions containing Bcr-Abl were pooled.A detailed description of the Bcr-Abl immunoprecipitation was described previously77. Other data are available from the corresponding author upon reasonable request.Coordinates have been deposited in the Protein Data Bank with the accession codes: 5N6R (DH NMR structure), 5N7E (DH/Mb(Bcr-DH_4) and 5OC7 (PH/Mb(Bcr-PH_4)). NMR chemical shift assignments are deposited in BMRB entry 34101. The collected SAXS data and the generated high-resolution hybrid models have been deposited at SASBDB Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


The differentiation of human bone marrow derived skeletal stem cells  into osteoblasts involves the activation of a small number of well-described transcription factors. To identify additional osteoblastic transcription factors, we studied gene expression of hMSCs during ex vivo osteoblast differentiation.ZNF25, ZNF608 and ZBTB38. siRNA knockdown of ZNF25 resulted in significant suppression of alkaline phosphatase (ALP) activity. This effect was not present for ZNF608 and ZBTB38. To identify possible target genes of ZNF25, we analyzed gene expression following ZNF25 siRNA knockdown. This revealed a 23-fold upregulation of matrix metallopeptidase 1 and an 18-fold upregulation of leucine-rich repeat containing G protein-coupled receptor 5 and RAN-binding protein 3-like. We also observed enrichment in extracellular matrix organization, skeletal system development and regulation of ossification in the entire upregulated set of genes. Consistent with its function as a transcription factor during osteoblast differentiation of hMSC, we showed that the ZNF25 protein exhibits nuclear localization and is expressed in osteoblastic and osteocytic cells in vivo. ZNF25 is conserved in tetrapod vertebrates and contains a KRAB (Krueppel-associated box) transcriptional repressor domain.Clustering of gene expression, and literature investigation, revealed three transcription factors of interest – ZNF25, is associated with differentiation of hMSC to osteoblasts.This study shows that the uncharacterized transcription factor,  ClusTSC22D3) –44.i. The majority of these are zinc finger proteins  and two are zinc finger and BTB domain-containing proteins, ZBTB1 and ZBTB38. The final TF was in cluster ii, which was zinc finger protein ZNF608.We further examined the upregulated clusters for TFs that are currently not associated with differentiation and development. This revealed a total of 11 TFs of interest; ten of which were in cluster i-v in Fig. vi, vii and viii in Fig. i to v were examined to identify which TFs have previously been implicated in MSC differentiation, bone formation or related mesodermal differentiation processes such as adipogenesis. Fifty nine of the TFs were found to be differentiation-associated, whereas 16 TFs were identified as potentially novel osteoblast-differentiation associated TF candidates. These included ZNF181, ZNF697, ZNF295, ZNF22, ZNF532, ZNF302, ZNF217, ZNF721, ZNF25, ZNF608, ZNF419, ZNF558 and ZNF627, along with two zinc finger- and BTB-domain containing proteins ZBTB38 and ZBTB40 and zz-type zinc finger-containing protein ZZZ3.The Illumina TF dataset was also subjected to the same process of SOM clustering as described for the Affymetrix dataset. In this case, this produced 36 clusters of TF expression profiles. Each cluster contained between 3 and 44 genes. Five clusters showed up-regulation of TFs across the time-course (labeled ZNF25), zinc finger- and BTB-domain containing protein 38 (ZBTB38) and zinc finger protein 608 (ZNF608). The fold change increase in gene expression and p-values showed by these during differentiation for day 13 versus day 0 were ZNF25 , ZBTB38  and ZNF608  in the Illumina dataset .We employed a number of criteria to select TFs for further analysis. As shown in Fig. set Fig. . We furtnts Fig. . Similarnts Fig. . There wZNF25  in ALP activity. For the purposes of this study we further focused our analyses only on ZNF25.The temporal gene expression pattern of the candidate TFs suggested a role in osteoblast differentiation of hMSC. To test this hypothesis, we examined the effects of siRNA-mediated knockdown of each of the TFs on ALP activity, as a marker of the osteoblastic phenotype. A statistically significant decrease in ALP activity was seen on knockdown of ZNF25 gene expression, the production of the ZNF25 protein was found to increase during in vitro osteoblast differentiation, at days 6, 10 and 15  at days 0 and day 14, post-osteoblast differentiation . At day 14, by contrast, 520 genes were identified as differentially expressed between siZNF25 and control samples . Since ZNF25 contains a KRAB domain and is likely to be a transcriptional repressor, any genes that show marked upregulation could be of functional significance. At day 0, there were no genes that showed dramatic upregulation in siZNF25 cells. However at day 14 there were four genes that showed 18-fold to 26-fold upregulation. The most significantly upregulated gene in differentiated siZNF25 cells relative to control was matrix metallopeptidase 1 . The next most upregulated genes were leucine-rich repeat containing G protein-coupled receptor 5  and RAN binding protein 3-like . We also observed a very high upregulation in unannotated transcript cluster, 17118303 . On further investigation it was found that this transcript cluster maps to chromosomal location: chr7:94058513–94060553. This chromosomal location corresponds to exon 52 of the COL1A2 gene, however no other probes for this gene were upregulated to this level.ZNF25 contains a KRAB domain, and is therefore likely to act as a transcriptional repressor . To gainion Fig. . At day p value = 2.9 × 10−11, FDR = 4.6 × 10–8), ‘ossification’ , ‘bone development’ , ‘skeletal system development’  and ‘regulation of ossification’ . The genes involved in these enrichment categories are all upregulated  and Xenopus tropicalus zinc finger protein 180 . However these are unlikely to be true orthologs as they did not identify ZNF25 via reciprocal BLAST. Whilst a single KRAB domain was present in each protein, different numbers of C2H2 domains were present, with 12 in human but 11 in chicken and Xenopus. In zebrafish, there was partial homology to the ZNF25 C2H2 domain found in gastrula zinc finger protein XICGF57.1-like . In the fruit fly, there was also partial homology to the ZNF25 C2H2 domain in the crooked legs protein, isoform B . The KRAB domain was not present in these two proteins. These results indicate that the KRAB domain of ZNF25 protein is conserved in tetrapod vertebrates. Figure The evolutionary conservation of ZNF25, has a role in the differentiation of hMSCs to osteoblastic cells. We observed that expression of the transcription factor ZNF25 increased steadily during OB differentiation of hMSCs, and its deficiency significantly decreased alkaline phosphatase levels in differentiating osteoblastic cells. The ZNF25 protein is present in the nuclei of mature osteoblastic cells and osteocytes in human bone. These results suggest that ZNF25 plays a role in in osteoblast differentiation of hMSCs.This study has shown that a novel transcription factor, ZNF25 belongs to the Krueppel C2H2-type zinc-finger protein family and contains 12 C2H2-type zinc fingers and one KRAB domain [B domain . The KruB domain . The KRAB domain . The KRAB domain .ZNF91) are restricted to specific hematopoietic cell lineages and may play a role in lineage commitment, possibly silencing transcription from nonlineage-expressed genes [AJ18 (ZFP354C) was identified by Jhoen et al. as a repressor of osteoblast differentiation in rat embryonic tibia and calvariae [AJ18 suppressed RUNX2 activity and repressed the markers of osteoblast differentiation ALP and, BGLAP. Our BLAST analysis confirmed the KRAB domain is only found in tetrapod vertebrates. Previous studies have reported that KRAB-ZFPs have expanded to a large degree in mammals [KRAB-zinc finger proteins are known to play important roles during cell differentiation and development. Individual members of one subfamily of KRAB zinc finger genes   and otherecently  been reprecently  showed tn et al.  reportedspondins . LGR5 alnd colon . Its relZNF25, has a role in the differentiation of hMSCs to osteoblasts. ZNF25 appears to act as a transcriptional repressor via a KRAB domain and we identified three potential targets of ZNF25, matrix metallopeptidase 1, leucine-rich repeat containing G protein-coupled receptor 5 and RAN-binding protein 3-like. Future studies will determine the role of this transcription factor in the in vivo bone formation.In this study we have shown that the uncharacterized transcription factor,


Background: Mental healthcare needs person-tailored interventions. Experience Sampling Method (ESM) can provide daily life monitoring of personal experiences. This study aims to operationalize and test a measure of momentary reward-related Quality of Life (rQoL). Intuitively, quality of life improves by spending more time on rewarding experiences. ESM clinical interventions can use this information to coach patients to find a realistic, optimal balance of positive experiences (maximize reward) in daily life. rQoL combines the frequency of engaging in a relevant context (a ‘behavior setting’) with concurrent (positive) affect. High rQoL occurs when the most frequent behavior settings are combined with positive affect or infrequent behavior settings co-occur with low positive affect.Methods: Resampling procedures (Monte Carlo experiments) were applied to assess the reliability of rQoL using various behavior setting definitions under different sampling circumstances, for real or virtual subjects with low-, average- and high contextual variability. Furthermore, resampling was used to assess whether rQoL is a distinct concept from positive affect. Virtual ESM beep datasets were extracted from 1,058 valid ESM observations for virtual and real subjects.Results: Behavior settings defined by Who-What contextual information were most informative. Simulations of at least 100 ESM observations are needed for reliable assessment. Virtual ESM beep datasets of a real subject can be defined by Who-What-Where behavior setting combinations. Large sample sizes are necessary for reliable rQoL assessments, except for subjects with low contextual variability. rQoL is distinct from positive affect.Conclusion: rQoL is a feasible concept. Monte Carlo experiments should be used to assess the reliable implementation of an ESM statistic. Future research in ESM should asses the behavior of summary statistics under different sampling situations. This exploration is especially relevant in clinical implementation, where often only small datasets are available. Mental health care is becoming more patient-centered. Every person is unique and the current classification systems miss relevant nuances for customized therapeutic interventions  is a structured diary technique specially developed to appraise subjects in their daily interactions  is an important outcome indicator. Mental illness often decreases QoL, resulting in lowered subjective well-being and functioning  is rooted in ecological psychology where contextual embeddedness is widely recognized Heft, . Barker Imagine yourself on a market. Being there with friends will likely provide a different experience than being alone. In addition, activities influence the experience. You could be working, buying a last ingredient for dinner or simply enjoying yourself. Other factors play a role as well, such as the location of the market, its attributes, the weather and time of day. All ingredients form the behavior setting—the rich and meaningful context.Future research should emphasize the linkage between a person's affective experience and behavior setting characteristics, including the beliefs and know-how of this behavior setting Wicker, . With ESIn line with the literature above, subjects can improve QoL by engaging more often and for longer time in affectively rewarding situations. Some may argue that extremely rewarding, low frequency behaviors can boost QoL . However, exceptional situations do not rule moment-to-moment experiences, while frequent minor events that occur naturally in the flow of daily life have an impact on mental well-being  reflects the momentary fit between context and optimal affective experience. ESM can monitor this process in treatment  and the proof of concept of its applicability. A reward statistic is defined. It reflects individual daily life moment-to-moment variation in reward efficiency, by combining the actual (positive) affect with the occurrence frequency of the actual behavior setting. Data is collected using ESM. To our knowledge, this is the first study that uses mechanisms of reward to design a momentary quality of life measure.To assess the feasibility of the rQoL concept, ESM data from an existing dataset–the D-STIGMI study Experience Sampling Method (ESM) is a structured diary technique to assess moment-to-moment mental state changes in relation to situations in the daily life of individuals  is defined as the fit between frequency of situations (behavior setting) and actual mental state. rQoL can be computed for each moment and provides feedback to clinicians and patients to collaboratively select intervention strategies that optimize the mental state in daily life and lead to more overall well-being. The related rQoL statistic uses the subject's own data to assess if he/she optimizes the selection of contexts to maximize positive mental states, meaning that rewarding situations should occur often and less rewarding situations should be avoided.zPA(ij) = PA(ij) − PA(i)-with “i” for subjects and “j” for moments]. zPA(ij) yields positive scores for better than average mental states and negative scores for below average mental states. Better than average mental states are assumed to be rewarding.The ESM questionnaire contains mood items that assess PA: I feel “cheerful,” “satisfied,” “relaxed,” and “enthusiastic.” Momentary PA was normalized by subject  and weights it with the frequency of occurrence of the individuals' behavior setting [cp(BSij)i]. Reward efficiency occurs when high frequency situations yield positive moods. High rQoL occurs when high frequency situations are combined with positive mental states or low frequency is limited to negative mental states. Low rQoL situations combine poor mental states with high frequency situations or elevated mental states with infrequent behavior settings. These characteristics are reflected in the formula:The momentary rQoL statistic combines the frequency of the momentary behavior setting with the actual mental state. Specifically, the function uses the normalized positive affect score by individual . The range between minimum and maximum correlational scores in all three variations of behavior setting is large in smaller sample sizes (highest difference from min = −0.91 to max = 0.90) and decreases with larger sample sizes (lowest difference from min = −0.02 to max = 0.04).The Pearson's product-moment correlations of the Monte Carlo experiments on sampled sets of PA scores and rQoL are summarized in Figure The purpose of the current proof-of-concept study is two-fold. First, a momentary rQoL statistic was defined. Second, Monte Carlo experiments were performed with samples of beep-level data for virtual subjects to check the feasibility and initial validity of the statistic. Momentary rQoL integrates affective experience (positive affect) and situations (behavior setting). A cut-off score of 0.50 of the cumulative proportion was chosen to separate low and high frequency situations. This proved to be the best choice to detect relevant contexts for reward efficiency in most subjects. Results show that the rQoL statistic is feasible. The rQoL statistic is defined at the moment level, allowing assessment of change over time. At a specific moment in time, a positive rQoL score indicates good reward efficiency, whereas a negative score indicates bad reward efficiency.N = 8,000 for BS_WWW, N = 500 for BS_WW, and N = 100 for BS_W). Next, Monte Carlo experiments were performed to explore which sample size is needed to detect optimal rQoL variation. The range of rQoL scores increased up to generated samples of 500 beeps for BS_WWW and BS_WW, whereas the limit was reached at 60 beeps for BS_W. Therefore, a behavior setting defined by Who-What proved most useful: it provides sufficient behavior setting variation to generate a reliable frequency distribution at acceptable sample sizes. An optimal spread in rQoL variation is obtained after collecting 500 observations, meaning that all possible behavior-setting combinations are present in the sampling period , activity (What) and location (Where) were used to define different conceptualizations of a behavior setting, namely Who-What-Where (BS_WWW), Who-What (BS_WW), and What (BS_W). For Who-What-Where combinations, not all theoretical possibilities were available in the reference beep dataset. This dataset included 113 unique combinations out of the 216 theoretical options. Some situations had low frequencies (e.g. working with your partner or doing household/groceries at work) or simply did not occur in the group of patients with a severe mental illness. First, Monte Carlo experiments were performed to check which sample size is needed for reliable behavior setting calculations in individuals. Overall, large sample sizes were needed to generate realistic frequency distributions (time budgets) in virtual subjects (saturation was reached at N > 40), whereas larger sample sizes (N > 100) are needed for subjects with average and high behavior setting variations. It is difficult to assess which definition of behavior setting is sufficient for individual subjects. For now, behavior settings defined by Who-What-Where combinations seem the best option because the restricted living environments of individual subjects results in less overall situational variation.Further, Monte Carlo experiments were used to explore actual situational variations in real subjects with low, average and high behavior setting variation with combinations of persons present, activity and location as behavior setting. This behavior setting definition was chosen because it theoretically provides the largest chance of finding a decent spread in unique situations in this patient group. Results show that small sample sizes are possible in subjects with low behavior setting variations  = 0.33, p < 0.001], with mQoL explaining 11 percent of the variation in rQoL.Finally, Monte Carlo experiments were run to see whether momentary rQoL is distinct from PA. Only weak correlations were found and results confirmed that the concepts assess different aspects of daily life mental states. Additionally, the momentary rQoL scores were correlated with “In general, how is it going with you right now” (mQoL). For this, the overall beep sample of 1058 valid observations was used. Results show a moderate positive correlation [To our knowledge, this is the first study that combines affective experience with behavior settings in an operationalization of rQoL in daily life. A main strength is the use of ESM data collected in the flow of daily life, making the rQoL statistic highly specific to the situation of the subject. Monte Carlo experiments are especially suited for exploration of properties and sampling characteristics of specific combined statistics Mooney, . ResearcThe choices made to operationalize momentary rQoL can seem arbitrary. Other options could be explored. For example, the rQoL statistic can be computed with negative affect. Additional cut-off scores could be explored to differentiate low and high frequency situations. The behavior settings are defined on persons present, activity and location, although more aspects of the environment could be relevant. Behavior settings are complex entities which include a number of contextual variables Barker, . Here weFurthermore, the generated samples using real subject data  were oversaturated and the same records were used repeatedly (due to replacement). The used sample included insufficient subjects with large beep datasets (>1000 observations). It would be interesting to replicate these analyses when longer series become available.Another limitation is the use of a specific sample of patients with severe mental illness as seeds for the Monte Carlo experiments. These subjects often lead restricted lives with limited variation in daily life activities  measures of QoL. Furthermore, the statistic should be explored in other populations that engage in more diversified behavior settings.It is interesting to explore whether the rQoL statistic improves targeted communication with patients. The statistic could be used to identify situations that result in low rQoL, so that changes can be made in daily life and progress can be monitored see the  improvedSV and PD worked on the conceptualization of rQoL and subsequent analyses. SV wrote the initial article. All authors  provided substantial feedback to the rationale of the article and contributed to later versions of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We demonstrate that out-of-focus conditions of imaging produce contrast inversion, due to image distortion induced by charged surfaces, and also carry information on the polarization direction in the domains. Finally, we show that the intensity profile at domain walls provides experimental evidence for a local stray, lateral electric field.The understanding of domain structures, specifically domain walls, currently attracts a significant attention in the field of (multi)-ferroic materials. In this article, we analyze contrast formation in full field electron microscopy applied to domains and domain walls in the uniaxial ferroelectric lithium niobate, which presents a large 3.8 eV band gap and for which conductive domain walls have been reported. We show that the transition from Mirror Electron Microscopy (MEM – electrons reflected) to Low Energy Electron Microscopy (LEEM – electrons backscattered) gives rise to a robust contrast between domains with upwards (P Optical techniques, such as Raman micro-spectroscopy1112151819et al.3. The spatial resolution was 50–100 nm, much larger than the wall thickness, but nevertheless allowed imaging of the wall. This is possible when the signature of the domain wall is quite distinct from that of the surrounding domains.Photoelectron emission microscopy (PEEM) also enables imaging of domain walls, as Schaab A more recently introduced technique for studying ferroelectric materials is Low Energy Electron Microscopy (LEEM). It provides full field, non-contact imaging of surface potentials. The technique exploits monochromatic, low energy electrons to map the surface potential with a spatial resolution typically better than 20 nm5242728When probing surface charge with electrons, it is also important to bear in mind that, given the nanometric width of domain walls, local in-plane fields may be high and can easily dominate the image of the wall. This can make it impossible to resolve the physical wall since it will be masked by field effects extending over several tens of nanometers. However, these field effects can be very well characterized in LEEM and reveal signatures of the domain walls.up (polarization pointing upward to the surface) and Pdown (polarization pointing downward into the bulk), providing a simple reference material for LEEM experiments. The aim is to clarify the contrast mechanisms arising in the presence of domain surface charges and domain walls. We show (i) that a shift in the MEM-LEEM transition is a reliable way to identify the polarization in domains, (ii) that out-of-focus imaging conditions produces intensity contrast inversions, and we experimentally evidence for the first time (iii) the presence of stray, lateral electric fields at domain walls.Here, we investigate domains and domain walls in the prototypical uniaxial ferroelectric, magnesium-doped lithium niobate (Mg:LNO (0001)). Magnesium doping was chosen since it induces inclined domain walls with non-zero fixed charge providing possible conduction paths when illuminated by UV light2 5% Mg-doped single crystal of lithium niobate (0001) supplied by HC Photonics Corp. It was poled by the manufacturer with a regular pattern of ferroelectric domains: they cross the entire sample in the y-direction and are 9.3 μm wide. The sample was then chemically polished with HF. Immediately prior to insertion in the ultra-high vacuum LEEM system (base pressure 1 × 10−9 mbar), the surface was cleaned via 5 minutes ozone exposure to remove organic surface contamination.The sample was a 10 × 10 mm6 electron gun at an accelerating voltage of U0 = −20 kV. The sample was at U0 + SV, where the Start Voltage (SV) defines the incident electron energy with respect to the sample surface. At low start voltages (SV → 0 V), the incident electrons are reflected by the potential above the surface, i.e. Mirror Electron Microscopy (MEM). At higher voltages  they penetrate the sample and backscattering occurs, i.e. Low Energy Electron Microscopy (LEEM). The position of the MEM-LEEM transition measures the surface potential. The reflected or backscattered electrons are reaccelerated into the objective lens and finally pass through the imaging column. A double multi-channel plate, screen and camera recorded the electron intensity as a function of SV in a typical field of view of a few tens of microns. An angle-limiting aperture in the back focal plane of the objective lens cut off highly deviated electrons and improves spatial resolution. A schematic of the setup is shown in The experiments were performed using a LEEM III microscope (Elmitec). The incident electron beam was emitted by a thermionic LaBSince the band gap of lithium niobate is large 3.8 eV, electroPiezo-response Force Microscopy (PFM) images were obtained using an Innova (Bruker) microscope, after the LEEM experiments. PFM image acquisition occurs via contact mode using a 4 V AC bias voltage applied to a Cr/Pt-coated cantilever with a spring constant of 40 N/m (Tap300E from BudgetSensors). High voltages were applied with a home-made setup using a Keithley 2410. Silver paste on the rear of the sample provides a back electrode.up surfaces. The profile extracted from the topography image, as indicated by the red line in The domain structure of the sample was measured by PFM. down and the darker areas to Pup. down – Pup – Pdown, showing the characteristic PFM-phase of each domain. up and Pdown domains.In order to confirm the assignment of up/down polarization, we applied −200 V to the PFM tip to locally reverse the polarization of the surface. Here we present the main results of the LEEM imaging of domains and domain walls. First, the variation of the MEM-LEEM transition allows mapping of surface potential variations. Then the use of MEM contrast to determine the sign of surface charge is discussed. Out-of-focus imaging conditions do not change the surface potential contrast between domains but do influence the apparent domain size. The intensity profile at domain walls is then related to the domain surface charge. Contrast inversion in the surface potential is observed in out-of-focus conditions contrary to the surface potential far from domain walls, providing an example of caustic effects in imaging.up and Pdown domains appear as bright and dark stripes, respectively. up and Pdown domains, as can be seen from up and Pdown domains as a function of SV. At low SV, the reflectivity is maximized; hence all electrons are reflected before reaching the sample surface. For SV above 1.20 V the intensity starts to decrease as incident electrons have enough energy to overcome the surface potential barrier and penetrate the sample surface. The domain for which the intensity decreases at lower start voltage has a lower surface potential, corresponding to the presence of positive surface charge, i.e. an upwards pointing polarization (Pup)5up and Pdown domains cross giving rise to contrast inversion in the electron image. The MEM-LEEM transition for Pup (Pdown) is at 1.8 V (1.9 V). The shift in the MEM-LEEM transition between Pup and Pdown domains, and therefore the difference in surface potential is ~100 meV.At 1.75 V, the contrast between domains reverses with respect to that at 1.15 V. Similarly, in LEEM at 2.70 V, the contrast is inverted with respect to that at 2.10 V. These contrast inversions are the signature of a shift in the MEM-LEEM transition between P3 thin film where a work function difference of 300 mV was found in PEEM and a surface potential difference of 450 mV in LEEMThe difference in surface potential can be compared with work functions and surface potential values obtained on lithium niobate by PEEM and Electrostatic Force Microscopy (EFM). The contrasts in work function and surface potential should be of the same order but with opposite sign. This statement has been confirmed experimentally on a BiFeOdown domains while that of Pup domains was greater than ~6.2 V, i.e. a work function difference above 1.6 V, one order of magnitude larger than our value. EFM measurements performed on stoichiometric lithium niobate in high vacuum were used to study the influence of external screening charges on the evolution of the surface potentialet al., at 300 °C, we expect a higher surface potential value in Pup domains with respect to Pdown domains. We find the opposite.PEEM measurements on congruent lithium niobate3333The difficulty in comparing experimental values of surface potentials underlines the key role of screening, even in ultra-high vacuum experiments3 require UV illumination to show higher conductivityet al., the purely in-plane polarization gave rise to highly charged head-to-head and tail-to-tail walls and showed a band bending of the order of 1 eV3 is only 2.5 eVSuch a shift is negligible compared to the 3.8 eV band gap and is unlikely to significantly change the domain wall conductivity. We recall that inclined domain walls in LiNbOAt very low SV, well below the MEM-LEEM transition, reflectivity should be uniform across the field of view. Closer to the MEM-LEEM transition electrons are sensitive to positive or negative surface charge and hence to the ferroelectric domains. However, the surface charge creates stray fields which can deviate the electrons laterallyIn a LEEM, the presence of  convergent lenses in the imaging column modifies this. As can be deduced from down domains are bright while Pup domains are dark  charged area and a substrate increases, the image size of the charged area increases (decreases) and its intensity decreases (increases)up and Pdown domains because the same number of electrons is imaged in a smaller or larger area, changing the intensity. Note that this does not represent a shift in the MEM-LEEM transition. Simulations show that over-focusing, for an odd number of converging lensesdown) is dark on the screen38Nepijko up and increases for Pdown. As a result, the intensity of Pdown decreases with increasing domain size because the same number of electrons is now spread over a larger area in the image.Since the variation of domain size depends on the surface charge, it can be used to identify the domain polarization. This method can therefore be useful to characterize samples with a large band gap where it is impossible to reach the MEM-LEEM transition because of charging even at low start voltage. It could also be used to identify the polarization of, for example, kinetic structures appearing across a phase transition using just two focus values.down (Pup) domain of up and Pdown sizes are under-estimated and over-estimated, respectively.However, a precise measurement of the domain size is not easy since it depends on the effective surface charge, which in turn is determined by the degree of screening, a priori not known. Thus the most reliable direct measurement of the domain size is by PFM whereas the surface charge is better revealed by LEEM. We measure a 10.8 μm (6.7 μm) width of the Pup domains with respect to Pdown by ~100 mV, irrespective of the focus value, although the absolute surface potential may shift. This makes the measurement of the surface potential difference between Pup and Pdown robust against slight (here about 1%) defocusing.Although the focus value changes the intensity contrast between domains, it does not influence the MEM-LEEM transition. This can be checked by recording image series as a function of SV in both under-focusing and over-focusing conditions. The value of the surface potential for each pixel is then determined by a fit of a complementary error function to the MEM-LEEM curve. The maps of the surface potential over the full field of view are shown in down side of the domain wall while the bright lines are on the Pup side. In LEEM .