Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase  from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.The basidiomycete  CcPDH named as CcSDH in our previous paper) that represents a new type of pyrroloquinoline quinone (PQQ) quinohemoprotein from the basidiomycete Coprinopsis cinerea  strain 5338. This CcPDH is the first such protein found among all eukaryotes [CcPDH indicates that the extracellular enzyme contains three domains: an N-terminal cytochrome domain, a C-terminal carbohydrate binding module (CBM), and a central PQQ domain.Cellulolytic fungi are known to produce many extracellular oxidoreductases, such as peroxidase and laccases, as well as cellulolytic enzymes –4. RecenCcPDH has 32 to 42% identity with the cytochrome b domains of basidiomycete cellobiose dehydrogenases (CDHs). CDH is a major oxidoreductase secreted by many cellulolytic fungi during growth on cellulose, and the genes that code for CDHs have been identified in numerous cellulolytic fungi [b containing a cytochrome domain, and a linker region connecting the two domains. CDH catalyzes the dehydrogenation of cellobiose and cello-oligosaccharides to their corresponding δ-lactones. The oxidation of cellobiose takes place in the FAD domain, subsequently followed by inter-domain electron transfer to the cytochrome domain [b-type heme with unusual Met-His ligands [www.cazy.org), the FAD and cytochrome domains of CDH are classified in subfamily 1 of Auxiliary Activity family 3 (AA3_1) and AA8, respectively [CcPDH has an AA8 cytochrome domain but a cellulose-binding domain attached to a PQQ domain instead of an FAD domain.The N-terminal cytochrome domain of ic fungi . CDH is e domain . The cyt ligands . Because ligands , 10. Accectively . CcPDH hc-type heme cofactors [Acinetobacter calcoaceticus [Escherichia coli [Pyrobaculum aerophilum [A. calcoaceticus sGDH. These Asds have oxidation activity towards a broad range of aldose sugars, with a weak affinity for substrates. The central PQQ domain of CcPDH has low identity with sGDH from A. calcoaceticus (16%). The putative homologues of the PQQ domain of CcPDH are widely distributed in bacteria, archaea, amoebozoa, and fungi. These quinoproteins belong to a different phylogenetic category from the known PQQ-quinoprotein family [CcPDH is currently identified as a new Auxiliary Activities family 12 (AA12) in the CAZy database.Several known prokaryotic dehydrogenases contain PQQ as their prosthetic group and mainly catalyze the oxidation of a number of alcohols or aldose sugars in the periplasm of Gram-negative bacteria –14. In tofactors , 18. Theofactors , 20 and aceticus , 22. In hia coli  and Pyrorophilum  are PQQ n family . TherefoCcPDH indicates the presence of a family 1 CBM (CBM1). The majority of cellulose-binding domains attached to fungal cellulolytic enzymes belong to CBM1. The main role of CBM1 in cellulolytic enzymes is to enhance the hydrolysis of crystalline cellulose by increasing the effective enzyme concentration on the cellulose surface [Many carbohydrate active enzymes including hydrolytic and non-hydrolytic proteins contain one or more non-catalytic CBMs that have functions in recognizing and adhering to carbohydrates –27. Base surface , 30. CBM surface –35. The  surface .CcPDH is an attractive multi-domain oxidoreductase that has three unique domains with different backgrounds. Until now, CcPDH had been overexpressed in the methylotrophic yeast Pichia pastoris, and initial characterization of the recombinant protein has demonstrated the PQQ-dependence of the enzyme reactivity for the first time in a eukaryotic enzyme. Despite its low amino acid sequence homology with known PQQ-binding enzymes, this enzyme binds PQQ strongly and shows oxidation activity towards some sugars. In this study, we thoroughly investigated the enzymatic and electrochemical properties of recombinant CcPDH.As noted above, c were purchased from Sigma-Aldrich Japan G. K. . 2-Keto-D-glucose was purchased from Santa Cruz Biotechnology, Inc. . l-fucose, l-lyxose, and l-xylose were purchased from Wako Pure Chemical Industries . d-arabinose and l-mannose were purchased from Sigma-Aldrich Japan G. K. . l-galactose, d-talose, and l-rhamnose were purchased from Funakoshi Co., Ltd. . l-gulose and l-glucose were purchased from Tokyo Chemical Industry Co., Ltd. .Pyrroloquinoline quinone and equine heart cytochrome CcPDH and PQQ containing dehydrogenase domain (DHPDH) of CcPDH (DHPDH: residues 240–649) were heterologously expressed in the methylotrophic yeast Pichia pastoris and purified as described previously [CcPDH” in this paper means the heterologously expressed wild type. The purity of CcPDH was confirmed by SDS/PAGE and by absorption spectroscopy. The CcPDH concentration in the solution was estimated from the absorbance at 420 nm (ε420 = 130 mM-1 cm-1) [Recombinant intact eviously . The enz-1 cm-1) .CcPDH and DHPDH were recorded at room temperature with a JASCO V-660 spectrophotometer . CcPDH was diluted in 50 mM HEPES buffer, pH 7.0, containing 1 μM PQQ and 1 mM CaCl2. Ca2+ is ligated with PQQ and several amino acid side chain atoms and is essential for the formation of active holo-enzyme of quinoproteins. The reduced form of CcPDH was prepared by addition of 1 mM ascorbic acid or l-fucose. Apo-DHPDH was stored in 50 mM HEPES buffer, pH 7.0, containing 1 mM CaCl2. Holo-DHPDH was obtained by addition of PQQ to apo-DHPDH according to our previous report [PDH was prepared by addition of 1 mM l-fucose.Electronic absorption spectra of s report . The red2-cooled CCD detector . Raman scattering was excited by the 413.1 nm emission from a Kr ion laser , and the incident powers were 1 mW. The spectra were obtained with data accumulated over 30 sec and a spectral resolution of 1.47 cm-1. The spectra were calibrated against indene and CCl4 as external standards. A two-point baseline was performed with GRAMS/386 software. All measurements were carried out in 50 mM HEPES buffer (pH 7.0) containing 1 μM PQQ and 1 mM CaCl2, at room temperature. The reduced form of CcPDH was prepared by the addition of 1 mM ascorbic acid or l-fucose.Resonance Raman (RR) spectra were measured on a JASCO NRS-1000 spectrometer using a Kaiser Optical holographic notch-plus filter and a liquid NCcPDH were analyzed by cyclic voltammetry . The potential was determined by averaging the anodic and cathodic peak potentials. The electrochemical responses of CcPDH were obtained by using a carbon nanoparticle-modified plastic-formed carbon as a working electrode, as described elsewhere [2 using a three-electrode cell under a nitrogen atmosphere at room temperature.Redox responses of heme in lsewhere . An Ag/ACcPDH were assayed using cytochrome c as an electron acceptor [c at 550 nm (Δε550 = 17.5 mM-1 cm-1) at 30°C. To obtain the kinetic parameters of oxidation for l-galactose, d-talose, l-xylose, and l-glucose, the assay was performed with various concentrations of monosaccharides  in 50 mM Tris buffer, pH 8.5 containing 1 μM PQQ and 1 mM CaCl2. To determine the Michaelis constant (Km) and turnover number (kcat), experimental data were fitted with the Michaelis-Menten equation with using Origin 8.1  The activity of DHPDH was determined using a dye-linked assay with the artificial electron acceptors phenazine methosulfate (PMS) as a primary electron acceptor and 2,6-dichlorophenolindophenol (DCPIP), according to our previous report [PDH toward l-fucose was measured in 50 mM MES buffer, pH 6.5, containing 0.1 mM DCPIP, 1 mM PMS, 1 mM CaCl2, 100 mM l-fucose, 50 nM DHPDH, and varying concentrations of PQQ (2.5–100 nM). The reaction rate was measured by following the decrease in the absorbance of DCPIP at 520 nm (Δε520 = 6.9 mM-1 cm-1) [The enzyme activities of acceptor , 39, 40.s report . The eff-1 cm-1)  at 30°C CcPDH sequence were subjected to multiple-alignment analysis using MAFFT (ver. 6.85) [The amino acid sequences of CBM1s within carbohydrate-active enzymes were collected from the CAZy database. The CBM1 sequences and the r. 6.85) , 43, andr. 6.85) .Cladophora sp. and phosphoric-acid-swollen cellulose (PASC) prepared from Avicel according to the method described previously [CcPDH (0.5–32 μM) were incubated with 0.1% w/v celluloses for 1 h at 30°C and then were centrifuged for 20 min at 16,100 g. The absorbance at 420 nm, which corresponded to the Soret band of a b-type heme (ε420 = 130 mM-1 cm-1), was measured with UV-vis spectroscopy to determine the concentration of free enzyme in the supernatants. The equilibrium dissociation constant (Kd) and the maximum amount of adsorbed enzyme (Amax) were estimated as described previously [Adsorption experiments were carried out in 50 mM HEPES buffer, pH 7.0, using crystalline cellulose from eviously  as an ameviously , 47.CcPDH are shown in CcPDH by ascorbic acid, sodium dithionite, or l-fucose, which is a substrate, induced the same behavior, and no difference was observed in the resulting spectra. The absorbances of the γ-, β-, and α-bands were increased and the maxima of the bands shifted slightly to 429 nm, 532 nm, and 562 nm, respectively. The spectra shifts due to the reaction with l-fucose suggests that the electron generated by l-fucose can be transferred from the PQQ domain to the cytochrome domain, like that from the FAD domain to the cytochrome domain in the cellobiose oxidation of CDH.The absorption spectra of the oxidized CcPDH, RR spectra were measured for both the oxidized and reduced forms of CcPDH with excitation at 413 nm as shown in 4 of CcPDH in its oxidized form appears at 1370 cm-1, indicating a ferric heme. The core size marker bonds ν2 and ν3 of the oxidized form, occurring at 1574 and 1502 cm-1, respectively, identifies the ferric heme as having a hexacoordinate low-spin (6cls) heme state. The reduced form of CcPDH prepared with either ascorbic acid or l-fucose showed the same RR spectral bands with the ν2, ν3, and ν4 to 1577, 1492, and 1360 cm-1, respectively. These values indicate that the reduced CcPDHs have a ferrous heme with a 6cls state. The RR spectral data of the oxidized and the reduced forms of CcPDH are summarized in P. chrysosporium. The RR frequencies of oxidized and reduced CcPDH were near agreement with those of CDH, suggesting that these cytochrome proteins have a similar microenvironment around the heme cofactor.Both the oxidized and reduced heme cofactors are RR active chromophores, and the vibrational modes can be enhanced with laser excitation in the 400–500 nm range. To further investigate the properties of the heme in CcPDH was estimated by cyclic voltammetry. As shown in CcPDH and the electrode. The redox potential determined from the midpoint of the oxidation-reduction peak potentials is 130 mV vs. NHE at pH 7. This is in accordance with previous reports describing the cytochrome domain of CDH from P. chrysosporium in solution  [3+/Fe2+ redox couple of the heme b center in CcPDH.The reduction-oxidation (redox) potential of V, pH 7) . Thus, tCcPDH, we used DHPDH that was separated from a cytochrome domain and a CBM1 by a linker region. The UV-visible spectrum of the purified DHPDH was typical of an enzyme without a cofactor. It had an absorbance maximum at 279 nm with aromatic amino acid residues  of 0.99. This value increases in linear manner to the equivalence point of the PQQ, indicating that the affinity of apo-DHPDH for PQQ is high, similar to bacterial PQQ quinoproteins [PDH binds PQQ with a 1:1 stoichiometry and has strong affinity with a dissociation constant (Kd) of 1.11 nM [CcPDH is a PQQ-dependent dehydrogenase.proteins , 49, 50.CcPDH catalyzes the dehydrogenation of monosaccharides having a 1C4 chair conformation, such as l-fucose and d-arabinose [l-galactose, d-talose, l-xylose, l-glucose, l-lyxose, l-mannose, and l-rhamnose (= 6-deoxy- L-mannose), noting the configuration of the substrate as shown in kcat value towards l-galactose was comparable to l-fucose, which is a 6-deoxy sugar of l-galactose, whereas the Km values of l-galactose were twice that of l-fucose. The affinity for d-talose was about the same as for l-fucose, but the kcat value was decreased by 30%. The Km values for l-xylose and l-glucose were approximately 236 mM and 363 mM, respectively, which are markedly greater than the Km obtained for the former monosaccharides. Slight oxidation activities of CcPDH were observed for l-lyxose and l-mannose, but little was observed for l-rhamnose. Thus, the catalytic efficiency was highest for d-glucosone, followed by l-fucose > d-arabinose ≈ l-galactose > d-talose ≈ l-gulose > d-lyxose > l-xylose ≈ l-glucose.Our previous studies suggest that rabinose . We examCcPDH indicated the presence of a CBM Family 1 C-terminal domain of CcPDH. The CBM1 homologous sequence of CcPDH was aligned with other CBM1s from fungal cellobiohydrolases (CBHs), ß-glucosidase (BGL), carbohydrate-binding cytochrome b562 (CBCyt. b562) and the basidiomycete CDH using MAFFT multiple alignment, as shown in CcPDH contains three conserved aromatic residues  that are proposed to contribute to CcPDH binding on the surface of carbohydrates. It also shows conserved cysteines  involved in disulfide bond formation. The amount of CcPDH adsorbed on insoluble cellulose was measured at various protein concentrations after incubation for 2 hours. A highly crystalline cellulose Ia from Cladophora and a PASC, which is an amorphous cellulose obtained by phosphoric acid treatment, were used as carbohydrate materials. Amax and Kd from the corresponding plots are shown in Amax on PASC showed higher adsorption by CcPDH than on cellulose Ia from Cladophora and the binding efficiency (Amax/Kd) for PASC was approximately 11 times higher than that for cellulose Ia. This is likely due to the difference in the surface area where the enzyme can adsorb. Therefore, the results suggest CcPDH is a redox enzyme that has the ability to adsorb on cellulose surfaces as well as CDHs.The amino acid sequence of P. chrysosporium, CcPDH was discovered in the C. cinerea 5338 strain as a new quinohemoprotein. CcPDH has a three-domain organization, with a catalytic domain containing PQQ as a cofactor (DHPDH), a b-type cytochrome domain as a redox site, and a CBM1. As far as we know, this is the first example of an extracellular quinohemoprotein connected to a cytochrome domain of CDH and CBM1. In this study, the spectroscopic and electrochemical properties, substrate specificity, and carbohydrate-binding properties of this novel PQQ-dependent enzyme from C. cinerea were determined.In a previous homology search with the cytochrome domain of CDH from CcPDH shows significant homology to that of CDH, with well-conserved Met/His ligands for heme binding and a disulfide bond. The structural homology modeling of the cytochrome domain of CcPDH by the Phyre program provided structural folds into an immunoglobulin-like ß-sandwich consisting of a five-stranded and a six-stranded ß-sheet with a short α-helical structure at the C-terminus, which is an unusual module among cytochromes [CcPDH in the oxidized and reduced forms are in good agreement with those previously reported for the cytochrome domain of CDH from P. chrysosporium. Those studies confirm a 6cls heme b in both the ferric and ferrous states with Met/His ligands, like CDH. In addition to spectroscopic results, electrochemical studies indicate that the redox potential of the heme is +130 mV, which is comparable to CDH from P. chrysosporium. These observations suggest that the cytochrome domain of CcPDH may have a similar electron transfer function to the cytochrome domain of CDH, i.e., intra- and inter-molecular electron transfer between the cytochrome domain and the catalytic domain and also Fe (III)-reducing ability. Indeed, the electron transfer between CcPDH and cytochrome c can be observed, as it is commonly used as an electron acceptor in the CDH reaction. CDHs are well known as redox enzymes capable of DET between the enzyme and the electrode and the cytochrome domain can act as a built-in electron-transfer mediator [CcPDH was also capable of DET. Thus, CcPDH is an excellent candidate for construction of DET-system bioelectronic devices such as biosensors and biofuel cell anodes, as well as CDHs.The N-terminal cytochrome domain of ochromes . The elemediator , 10, 51.CcPDH exhibited dehydrogenase activity towards monosaccharides in a 1C4 chair conformation, in contrast to the substrate specificity of A. calcoaceticus sGDH, which has an absolute preference for l-arabinose and d-xylose in the 4C1 conformation over the d-arabinose and l-xylose enantiomers [E. coli also lacks activity for l-hexoses or pentoses that have a 1C4 chair conformation, such as l-glucose, l-mannose, l-rhamnose, and l-xylose and d-pentoses [E. coli exhibited no preference for different enantiomers with d- and l-arabinose [Interestingly, ntiomers . The mempentoses . Additiorabinose .CcPDH for these substrates and the side group orientation of each monosaccharide when in a 1C4 conformation are summarized in d-glucosone and l-fucose in d-glucosone (= 2-keto-D-glucose) has four tautomer forms reported by Freimund et al [1C4 conformation of 2-keto-D-glucopyranose is the one of the tautomer, at 21% abundance in aqueous solution, as shown by d-glucosone are shown with the corresponding carbon number of the other monosaccharides in a 1C4 conformation . The Km value for l-gulose, which is a C-3 epimer of l-galactose, was approximately 1.7-fold higher than that of l-galactose. This value indicates that the C-3 hydroxyl group in the equatorial conformation is preferred over the axial for CcPDH. The affinity for pentose sugars, which lack a hydroxymethyl group at the C-5 position, or a 6-deoxy-hexose, which has methyl group at C-5 (in L-fucose), increased over hexose sugars that have a C-5 hydroxymethyl group, suggesting that the hydroxymethyl group at this position causes some steric hindrance. However, the result suggests that C-5 methyl group is not critical for binding to CcPDH, as indicated by the oxidation of l-fucose. In light of the stereospecificity of other PQQ-dependent dehydrogenases [CcPDH may catalyze the dehydrogenation reaction of regioselective substrates at the C-1 hydroxyl group, although we can not rule out side reactions at other positions. Further studies are necessary to identify a product of CcPDH oxidation and they are currently under consideration.The compared catalytic activity of nd et al . The 1C4ormation . The reslucosone . The equogenases , 52, 55,CcPDH shows high affinity toward microcrystalline and amorphous cellulose. This result suggests the possibility of localizing CcPDH on the surface of cellulose, as is the case for CDHs. Further cytochemical analysis will be needed to identify the localization of CcPDH in nature.Many extracellular fungal carbohydrate hydrolytic enzymes carry a CBM1, which is thought to facilitate binding to the surface of cellulose chains and is related to the catalytic function of these enzymes. Although these CBM1 are not found in basidiomycetous CDHs, the flavocytochrome enzymes are also capable of binding to cellulose by its flavin domain and localizes on cellulose surfaces –58. In tCcPDH could allow direct electron transfer for LPMO as well as that of CDH. However the physiological functions remain obscure in vivo. It is not only some redox proteins instead of CDH, but some reductive compounds are the possibly electron donor for LPMO in those fungi. Indeed, l-ascorbic acid is typically used as an electron donor for LPMO in vitro [d-glucosone by CcPDH could be an intermediate in the l-ascorbic acid biosynthesis [l-ascorbic acid [Penicillium cyaneo-fulvum has an extracellular d-erythorbic acid (isoascorbic acid) synthesis pathway by using glucose oxidase and gluconolactone oxidase [CcPDH, therefore, may play a role in the l-ascorbic acid biosynthesis pathway and then LPMOs could be provided with electrons from these reductive compounds. Anyhow, CcPDH must be related to the enzymatic degradation of the plant cell wall because of its capability of binding to cellulose.Under aerobic conditions, cellulose degradation by cell-free culture filtrates of various fungi has shown much higher rates compared to anaerobic conditions, suggesting that an oxidative reaction may play a crucial role in the fungal degradation of cellulose. CDH is the first example of an oxidoreductase related to the extracellular oxidative process for cellulose degradation. Furthermore, it has been recently reported that the cytochrome domain of CDH can transfer electrons to fungal copper-dependent lytic polysaccharide monooxygenases (LPMOs) that were classified in AA9. The combination of CDHs and LPMOs synergistically enhances cellulase activity –62. The in vitro . The oxiynthesis . PQQ quibic acid , 65, 66. oxidase . CcPDH, CcPDH, which was classified as a member of the new AA12 family in CAZy using spectroscopic and electrochemical methods. These enzymatic properties provide new insight into other known PQQ quinoproteins as well as the basidiomycetous oxidoreductase network. The N-terminal cytochrome domain of CcPDH contains a 6-coordinated low-spin heme b in both the ferric and ferrous states with Met/His ligands as well as CDH and it enables DET between CcPDH and the electrode. The catalytic domain shows oxidation activity toward monosaccharides in a 1C4 chair conformation in the presence of PQQ as a cofactor, and the most efficient catalysis was observed for d-glucosone. CcPDH exhibited binding affinity for insoluble cellulose. These results suggest that CcPDH may be involved in the extracellular oxidative degradation as well as CDH. Therefore, these proteins are an attractive component of cellulolytic enzymes for use in biodegradation and biomass conversion or as an anode catalyst for bioelectrochemical applications.We have demonstrated the full characterization of


Merops apiaster has only rarely – and if so mostly anecdotically – been documented in the literature record, although such behavior would substantially save time and energy. In this study, we quantify burrow re-occupation in a German colony over a period of eleven years and identify ecological variables determining reuse probability. Of 179 recorded broods, 54% took place in a reused burrow and the overall probability that one of 75 individually recognized burrows would be reused in a given subsequent year was estimated as 26.4%. This indicates that between-year burrow reuse is a common behavior in the study colony which contrasts with findings from studies in other colonies. Furthermore, burrow re-occupation probability declined highly significantly with increasing age of the breeding wall. Statistical separation of within- and between-burrow effects of the age of the breeding wall revealed that a decline in re-occupation probability with individual burrow age was responsible for this and not a selective disappearance of burrows with high re-occupation probability over time. Limited duty cycles of individual burrows may be caused by accumulating detritus or decreasing stability with increasing burrow age. Alternatively, burrow fidelity may presuppose pair fidelity which may also explain the observed restricted burrow reuse duty cycles. A consequent next step would be to extend our within-colony approach to other colonies and compare the ecological circumstances under which bee-eaters reuse breeding burrows.Re-occupation of existing nesting burrows in the European bee-eater  Merops apiaster)  Fig. is a widter) Fig.. Bee-eatter) Fig.. Eggs arter) Fig. composedBee-eaters regularly use the same breeding walls over several years  indeed found no evidence for any burrow reuse ; or alternatively (2) a decline in re-occupation probability is due to the fact that during colony establishment, unconstrained construction of high-quality burrows is possible, while in later years, burrows are built increasingly in suboptimal parts of the breeding wall with regard to, for example, soil properties or predation risk .In this study, we investigate between-year burrow reuse and its dynamics in a European bee-eater colony in southern Germany across a period of eleven years. We document that, in contrast to what is reported in the literature, between-year burrow reuse is common in this study colony. Furthermore, we test competing hypotheses that could explain mechanistically an observed general decline in re-occupation probability with increasing age of the breeding wall by statistically separating within- from between-burrow effects. More precisely, we test the hypothesis that (1) a decline in re-occupation probability is due to a limited duty cycle of individual burrows, for example caused by increasing contamination with detritus or parasites or a decrease in burrow stability over time  with binomial error structure and logit link function. We transformed the original covariate age of the breeding wall (ranging from 1 to 10) by subtracting 5.5 such that  estimates for the intercepts in our models are given for the median age of the breeding wall . We included study year and breeding burrow identity as random intercept effects as well as random slope effects for age of the breeding wall on breeding burrow identity in our models to control for pseudoreplication due to the lack of independence resulting from multiple observations of the same burrow or from the same year. We refrained from including in addition a random slope effect for age of the breeding wall on study year due to the observed lack of convergence when fitting such models. We estimated the overall re-occupation probability using a binomial GLMM with the intercept as only fixed effect and burrow identity and study year as random intercept effects to obtain an unbiased estimate for overall mean re-occupation probability (including appropriate standard errors) which is controlled for pseudoreplication.We tested for the fixed effect of the within-burrow age of the breeding wall effect precisely models the age of individual burrows and thus tests our burrow age hypothesis, while the between-burrow age of the breeding wall effect tests our burrow quality hypothesis.Not all the burrows could be monitored across the entire study period due to first appearance of burrows after establishment of the breeding wall in 2003) and/or disappearance before the end of the observation period  took place in a nesting burrow which had already been used in a previous year, indicating that reusing burrows is a common breeding site selection strategy in the bee-eater breeding wall under study. Altogether, 424 burrow-year observations have been recorded for 82 different, individually recognized burrows over the whole observation period  or due to a selective disappearance of burrows with relatively high re-occupation probabilities with increasing age of the breeding wall . Using within-burrow centering of the covariate age of the breeding wall to disentangle within- from between-burrow effects, we found that a decline in re-occupation probability of individual burrows with age , but not selective (dis-)appearance  was responsible for the observed pattern across time  indicated that individual burrows differed in their re-occupation trajectories with increasing age. To control for the fact that the data are right-hand censored, that is, more data points are available for older than for younger burrows, we restricted the analysis to 316  observations of 59  burrows which were first occupied between 2003 and 2010. By excluding those burrows that could be monitored for only 2 or 3 years, we test for the robustness of our analysis. The analyses of the restricted data set yielded very similar results .Re-occupation probability showed a highly significant decline with increasing age of the breeding wall  (Heneberg Merops philippinus) . This might simply be due to an elevated risk of collapsing for older burrows which might not always be observable from outside. Additionally, bee-eaters do not normally remove indigestible regurgitates of their offspring or other detritus from their burrows . Thus, it seems not to be the case that high-quality burrows with an associated higher reuse probability were built early during colony establishment. Still, the first burrows in newly established breeding walls of burrowing birds are usually built along the upper margin, whereas lower burrows follow only later (Ursprung Martes foina) and European badgers (Meles meles) is indeed higher when burrows are located in closer proximity to the ground (Sieber Vulpes vulpes) excavate burrows from the top (Heneberg Declining burrow reuse probability over time was not attributable to the selective disappearance of high-quality burrows (Ursprung . While td Sieber , red foxHeneberg . BesidesHeneberg  or rainwHeneberg  should pNaturally, the present study is limited to a within-colony approach when trying to understand the ecological conditions that promote or hinder nesting burrow reuse in European bee-eaters. It would therefore be worthwhile to extend the approach taken here over the breeding range of the species as, for example, the availability and quality of breeding walls and the demographic and genetic composition of populations may well differ, especially in the margin regions of the distribution. Based on quantitative data from many different populations, such an extension will promote a better understanding of the ecology and ultimately conservation of a European flagship species for conservation and for studying population responses to environmental change.


The purpose of this study was to investigate the epidemiological situation of the caprine herpesvirus 1 (CpHV-1) infection in nine districts in mainland France, mostly in the south, near Italy or Spain, where high seroprevalence has been observed. Two more central areas were also included in the study. The serosurvey was carried out in 9564 goats (275 herds) using bovine herpesvirus 1 (BoHV-1) glycoprotein B and E ELISAs. To confirm the presence of specific CpHV-1 antibodies, some of the samples were tested in neutralization assay. Results demonstrate, for the first time, CpHV-1 infection in goat herds on the French mainland. The analysis found cases of alphaherpesviruses infection in each district studied, with different levels of seroprevalence observed within each district . Moreover, in the Alpes-Maritimes district, the seroprevalence seemed to be higher in older goats  than in younger animals (40.99% of one-year-olds). This result suggests frequent virus re-excretion and circulation in herds. Results analysis also shows that the seroprevalence was higher when the herd size increased. In addition, the first French CpHV-1 strain was isolated from nasal swabs taken on an infected goat. The data reported herein demonstrate that CpHV-1 circulates in mainland France, which should henceforth be taken into consideration in cases of unexplained abortion in goats. Like etc.) . Althougetc.) , vulvovaetc.) ,9 and baetc.) , and neoetc.) ,12,13,14etc.) ,16,17. ACpHV-1 infection was observed worldwide, e.g., in the USA ,14,18, CTo obtain a broader epidemiological view of the CpHV-1 status of goats in France, a serological survey was undertaken in nine districts in mainland France. The survey focused on districts located in southern regions, close to Italy or Spain and on two main goat-farming areas. The serological results show that CpHV-1 infection was detected in all districts studied. The influence of age and herd size on seroprevalence was examined. In addition, a CpHV-1 strain was isolated from seropositive goats.CpHV-1 infection was studied in several French districts in the south east, close to Italy , in the south west, close to Spain , and in two main goat farming areas (Deux-Sèvres in the Poitou-Charentes Region and Aveyron) . In all,The serological status of the goats against CpHV-1 was obtained by using a combination of commercial bovine ELISA tests designed to detect gB and gE antibodies, and cross seroneutralisation assay against CpHV-1 and BoHV-1 . As showAll gB positive sera were tested with the BoHV-1 gE ELISA commercial kit. Most sera were gE negative (86%), suggesting that goat herds were infected by an alphaherpesvirus related to BoHV-1 .These results were confirmed by comparative virus neutralisation tests (VNT) against CpHV-1 or BoHV-1 . The dati.e., Var and Alpes-Maritimes, 30% and 31.5% respectively).Positive herds were found in all the districts studied, thus demonstrating goat CpHV-1 infection on the French mainland . The serIt is worthy of note that despite the high level of seroprevalence at herd level (up to 46.1%) in the Pyrenean districts (Ariège and Hautes-Pyrénées) and Aveyron, individual apparent seroprevalence remains low (about 3%). In contrast, the individual seroprevalence in Deux-Sèvres, which is the main goat farming area, is only 0.2%. Overall, among 275 analyzed herds located in mainland France, 30% present a CpHV-1 infection for an individual apparent seroprevalence of 14%.The analysis of the distribution of within-herd prevalence of CpHV-1 in 83 infected herds shows that 42.2% of infected herds (n = 35) had a within-herd prevalence lower than 10% . In 27.7In addition, the relationship between herd size and individual seroprevalence or infection status of herds was investigated. The 115 herds analyzed in Alpes-Maritimes were sorted into 3 groups depending on the herd size. In this district, most of the herds are small and many goat breeders own fewer than ten goats. There were 22 herds of 50 goats or more , 43 herds of from 10 to 50 goats  and 50 herds of fewer than 10 goats . gB ELISA results corresponding to the 3161 goats tested were analyzed. The larger herds have significantly higher individual apparent seroprevalence than the other groups .These large herds are also more frequently infected . Thus heThe effect of the age of animals on individual seroprevalence was also studied. The analysis was done one 14 large herds located in Alpes-Maritimes . These animals were stratified according to their age into 6 different categories from one year to six years or more. The number of animals in each group ranged from 124 to 322 animals. Results show that seroprevalence increases with the animal’s age . SimilarViral isolation was performed to further confirm the CpHV-1 infection. To this end, 2 goat herds located in Var in which CpHV-1 antibodies were found in sera samples taken in 2008 were investigated again in 2011. Serum samples, nasal and genital swabs were taken from adult seropositive goats and kids. 18 goats from Herd A and 13 goats from Herd B were sampled. Interestingly, although apparent seroprevalences of Herds A and B were 70% in 2008, no significant change of seroprevalence was observed for Herd B in 2011 while seroprevalence of Herd A became null.. Viral multiplication was assessed by specific PCR for glycoprotein B and glycoprotein E.Viral strain culture was performed on MDBK cells from nasal or genital swabsWeak band were obtained for genital swabs from Herd A, but most appeared negative by PCR after cell culture. However PCR products with the expected size (443 bp and 624 bp respectively) were obtained from nasal swabs taken from this herd after two rounds of amplification on MDBK cells. In addition, no amplification of viral CpHV-1 DNA from Herd B was obtained. PCR products obtained from gB and gE PCR were sequenced. The phylogenetic analysis obtained with compiled gB and gE gene regions is presented in Sequences were compared with those from different ruminants alphaherpesviruses found in the literature. They display a strong homology with the CpHV-1 Ba1 strain, close to 100% . Similar results were obtained when the phylogenetic analysis was performed separately with the 414 nt of the gB region or the 519 nt of the gE region. We were therefore able to confirm that we had successfully isolated a CpHV-1 strain from France, named 11F83.Our results demonstrate, for the first time, the presence of CpHV-1 infected goat herds on the French mainland. The current study presents the evidence of CpHV-1 infection occurring in mainland France, with an apparent seroprevalence of 13.73%. The highest rates of individual seroprevalence were found in the two Mediterranean districts studied, Alpes-Maritimes and Var. This is in agreement with previous reports describing CpHV-1 infection preferentially located in Mediterranean countries ,24,25,26In south-western districts, CpHV-1 individual seroprevalence is lower. Nevertheless, many herds contain one or several seropositive animals. In this region, about 27% of the herds tested showed CpHV-1 seropositive goats. Herd seroprevalence is especially high in the two districts located close to Spain. The two previous studies performed in Spain were done in Andalusia ,27. IndiResults obtained in Deux-Sèvres and Aveyron district showed that CpHV-1 infection also occurs in the main French goat farming areas, even if the prevalence of the infection is low. However, in Deux-Sèvres district, only 20 herds out of about 1200 herds listed were tested . It would be interesting to perform a larger study in the entire Poitou-Charentes Region (which includes three other districts) where about 2500 herds are listed .Marked regional differences were observed. Differences in agricultural structure and geography can partially explain the regional variation of seroprevalence among goats. In the Alpine and Pyrenean regions, pasturing of goats is a traditional farming practice. Animals from many farms generally graze during summer on Alpine pastures where wild and different domestic ruminants frequently share grazing areas and water sources, which increases the risk of transmitting infectious disease agents, and then return in the autumn to their respective home farms. This proximity between ruminants during pasturing may partially explain the higher herd and individual seroprevalence in the South-East and also in the South-West region close to Spain. From this point, it will be interesting to continue the serological survey in France in the major goat farming areas and in neighbouring countries.An analysis of within-herd seroprevalence shows that for more than 42% of infected herds, this was less than 10%. This result is consistent with the low individual seroprevalence observed in several districts. On the other hand, total within-herd prevalence was more than 51% in some herds located in Alpes-Maritimes, Var and also in Savoie and Aveyron districts. These results are consistent with the high individual seroprevalence in these districts.Results analysis shows that both seroprevalence and herd infection status are correlated with herd size. Larger herds are more frequently infected and present significantly higher within-herd seroprevalence. The venereal route is considered the main route of transmission , in factTo isolate a CpHV-1 strain, samples from two herds (A and B) located in Var district were taken at two different times of year, first in 2008 and then over a second phase in 2011. Interestingly nasal swabs were positive whereas vaginal swabs were found negative by PCR. This surprising result may be related to the period of sampling. The results obtained for Herd A shows that serological status of herds and/or animals could change over time. This result is supported by other results obtained in Alpes-Maritimes district (data not shown). Specific antibody titres may not persist in goats for many years as was previously suggested . As a coThe Madin-Darby bovine kidney (MDBK) cell line ATCC CCL-22) was maintained in Modified Eagle’s Medium (MEM) supplemented with 10% of foetal bovine serum (FBS) and glutamine (complete medium). The viral stocks of BoHV-1 Los Angeles strain (ATCC VR188) and CpHV-1 E/CH strain [2 was maiGoat serum samples were collected in several districts in south east France , south west France  and the main goat-farming areas (Deux-Sèvres and Aveyron district) . For allThe combination of two enzyme-linked immunosorbent assays (ELISA) based on the detection of BoHV-1 glycoproteins B (gB) and E (gE) was previously shown to be effective for distinguishing between BoHV-1 and related alphaherpesvirus infections in goats . We theri.e., to discriminate between CpHV-1 and BoHV-1 antibodies, a panel of sera showing different response profiles with respect to the two ELISAs was analysed in VNT. Several animals per profile were tested. After filtration, serum samples were heated to 59 °C for 30 min. Serial twofold dilutions of each serum sample tested were incubated in 96-well microplates at 37 °C for 24 h either in the presence of 100 DECP50 of BoHV-1 Los-Angeles strain or in the presence of 100 DECP50 of CpHV-1 E/CH strain. MDBK cells (2.0 × 105 cells/mL) were then added to each well. Readings were made after incubation for 3 days at 37 °C. Viruses used in the running test were titrated (3 twofold dilutions) to verify the infectivity of the virus. The CpHV-1 titer was evaluated to 101,15 TCID per well in the same time of the neutralizing test. The affinity of a serum with a herpesvirus strain was expressed as the reciproqual of the highest dilution of serum sample inhibiting cytopathic effect. Positive reference sera were obtained by experimental infection of calves infected intranasally with CpHV-1 E/CH strain or BoHV-1 Los Angeles strain.To further check the specificity of the antibody response, Viral isolation was performed on nasal and vaginal swabs collected from animals from positive herds. Briefly, swabs were resuspended in 1 ml of sterile phosphate buffered saline (PBS) and frozen at −80 °C until use. The PBS suspension was inoculated into 24-well microplates containing an MDBK cell monolayer, grown in complete medium supplemented with antibiotics . Cell monolayers were checked daily for cytopathic effects due to herpesvirus infection. When the first cytopathic effects appeared, supernatants were collected, centrifuged at 1500 rpm for 20 min and inoculated again into MDBK cells. When the cytopathic effect reached 50%, culture medium was removed, clarified by centrifugation, and analysed by conventional PCR.Total DNA was extracted from each cell culture supernatant using Dneasy Blood & Tissue kit  according to the manufacturer’s instructions. Two different sets of consensus primers were used to amplify parts of glycoprotein B and glycoprotein E genes from different ruminant alphaherpesviruses ,30. The 2 test was used to compare the apparent prevalence between the different sizes of herds. The non-parametric Mann-Whitney test was used to compare the individual seroprevalence between the two different groups of herds. The limit of statistical significance of the conducted test was defined as p < 0.05.The 95% confidence interval (95% CI) of the apparent prevalence was estimated using a binomial exact distribution. . StatistThis CpHV-1 infection in France could have a possible role in cases of unexplained abortions in several goat herds. This virus should also be taken in consideration since it may provoke severe genital lesions, respiratory disorders and neonatal mortality. The correlation between serological response and clinical features in herds (abortion) needs further study.Finally, this study suggests that it would be interesting to investigate more widely the serological status of goat herds in France and neighbouring countries. In France particularly, investigations should focus on areas of intensive goat farming and the south of France.


Ferroelectric domain walls are of great interest as elementary building blocks for future electronic devices due to their intrinsic few-nanometre width, multifunctional properties and field-controlled topology. To realize the electronic functions, domain walls are required to be electrically conducting and addressable non-destructively. However, these properties have been elusive because conducting walls have to be electrically charged, which makes them unstable and uncommon in ferroelectric materials. Here we reveal that spontaneous and recorded domain walls in thin films of lead zirconate and bismuth ferrite exhibit large conductance at microwave frequencies despite being insulating at d.c. We explain this effect by morphological roughening of the walls and local charges induced by disorder with the overall charge neutrality. a.c. conduction is immune to large contact resistance enabling completely non-destructive walls read-out. This demonstrates a technological potential for harnessing a.c. conduction for oxide electronics and other materials with poor d.c. conduction, particularly at the nanoscale. Conducting charged ferroelectric domain walls, as potential building blocks for future electronic devices, are unstable and uncommon in ferroelectric materials. Here, Tselev et al. show that neutral insulating domain walls in PbZrO3 and BiFeO3 thin films are conductive under microwave excitation, allowing for non-destructive read-out. In ferroelectrics, domains of uniform polarizations are separated by domain walls. Domain walls can be created and reconfigured by electric fields13579101112131491011121311110121414The problem of contact resistance is particularly important in domain wall electronics. The contact interface Schottky barrier conceals the intrinsic mechanisms of charge transport along the in-depth region of a domain wall. Overcoming contact resistance necessitates electric fields that can become comparable or exceed the threshold for domain wall motion101010Here we reveal an alternative regime of ferroelectric domain wall conduction at a.c. frequencies in the gigahertz range. Measurements at high frequencies are insensitive to the contact barriers at the electrode–ferroelectric interface, enabling quantitative non-destructive measurement of the domain wall conductance. We found that nominally uncharged domain walls in conventional ferroelectric thin films of lead zirconate and bismuth ferrite can be strongly conducting, on par with charged walls. The a.c. conductivity is at least 100 times higher than at d.c. with the same probing voltage. From the behaviour of the a.c. domain wall conductance as a function of bias voltage, temperature and time, we conclude that the cloud of free carriers forming around the transient charged domain walls during polarization switching incompletely dissipates after the domain stabilization and partially remains at the wall. The domain wall assumes a rough morphology assisted by a disorder potential in the film. Such a configuration hinders d.c. conduction, but remains many fold more conducting at gigahertz frequencies than the surrounding domains, paving a way to practical utilization of domain wall circuits.0.2Ti0.8)O3 (100) (PZT) grown using pulsed laser deposition on a single-crystal TiO2-terminated SrTiO3 (001) substrate with 50 nm SrRuO3 as a bottom electrode  of the structure taken with a zero d.c. bias at the probe and an a.c. voltage amplitude ≲300 mV. Domain walls are clearly conducting unlike the bulk of the surrounding domains, while no contrast between the two is seen in the simultaneous permittivity image . This depletes a small amount of oxygen and creates oxygen vacancies. The vacancies act as electron donors increasing the number of charge carriers in the film. The sMIM-G response of the domain walls in the annealed film was ∼1.5–2 times larger than the value of the as-grown film .G response to complete the picture. Such vibrations would result in microwave energy dissipation, which can be indistinguishable from the mobile charge high-frequency conduction in the sMIM measurements. However, it should be taken into account that displacement of domain walls in response to the applied electric field contributes to material polarizability and permittivity3 as an example, while the on-field sMIM-C signal O3 (100) thin film was grown using pulsed laser deposition on a single-crystal TiO2-terminated SrTiO3 (001) substrate with 50 nm SrRuO3 as a bottom electrode. The growth temperatures were 700 and 630 °C for SrRuO3 and PZT layers, respectively. The growth oxygen pressure was the same for both materials—0.13 mbar. After growth, the sample was cooled down at 1 bar oxygen with a cooling rate of 5 °C min−1. The thickness of the PZT film was controlled by the growth rate and was confirmed with X-ray reflectometry.The ferroelectric Pb(Zr3/50 nm SrRuO3/DyScO3 (110) heterostructure was grown using pulsed laser deposition from a Bi1.1FeO3 target. The SrRuO3 bottom electrode was grown at 645 °C in a dynamic oxygen pressure of 100 mtorr at a laser fluence 1.8 J cm−2 and a frequency of 17 Hz. The BiFeO3 film was grown at 700 °C in a dynamic oxygen pressure of 100 mtorr at a laser fluence of 1.0 J cm−2 and a frequency of 20 Hz. Following growth, the heterostructure was cooled in a static oxygen pressure of 760 torr at a rate of 5 °C min−1.The 100 nm BiFeOpp a.c. voltage at a frequency close to the contact resonance frequency of the probe (∼250 kHz) was applied for imaging. The c-AFM and corresponding PFM imaging were performed in an Omicron VT AFM in ultrahigh vacuum at a background pressure of 2 × 10−10 torr using Budget Sensors ElectriMulti75-G cantilevers.PFM imaging in ambient was performed in the same set-up and with the same probes as the sMIM imaging  using the Asylum Research Dual AC Resonance Tracking mode. A 1.5-Va.c.. Fully shielded sMIM cantilever probes  sMIM add-on unit installed on an Asylum Research MFP-3D atomic force microscope. The sMIM microwave output power was set to 100 μW (−10 dBm), at which the upper estimate for the a.c. amplitude at the probe tip is ca. 300 mVC and -G channels, the ScanWave's detector phase was adjusted while imaging a set of 1-μm diameter capacitors of a SMM calibration kit from MC2 Technologies (France). The phase was adjusted so that the variation ΔsMIM-G of the reflectometer sMIM-G signal from the capacitors was zeroed. To correctly define the sMIM-C channel sensitivity to admittance changes was calibrated using an MC2 Technologies SMM calibration kit based on the approach of ref. G channel as well and can be used to measure the real part of the admittance of the tip–sample system on contact with a sample. The microscope sensitivity was determined as ΔVout/ΔRe,Im(Y), where ΔVout is the raw, uncalibrated, voltage outputs at sMIM channels (in mV), and ΔRe,Im(Y) are the corresponding changes of the tip–sample admittance real and imaginary parts (in nS).The sMIM-ɛPZT was measured using the sMIM-C microscope response in respect to air (probe is out of contact with a sample) as a reference signal and an MC2 calibrations kit. The measurements yield ɛPZT≈70±5, which is in a good agreement with the literature value 70–100 for the same compositionG signal change in respect to air. Since the sMIM outputs depend on the probe–sample contact area, the contact diameter was measured using the apparent domain wall thickness in PFM images. The real part of the admittance over domain walls was measured from domain wall images based on the sMIM-G signal change in respect to the domain bulk. The data for measurements were taken from images of the domain walls obtained after writing. While creating a suitable domains structure, a special attention was paid to stability of tip–sample contact through all writing sequence to preserve the validity of the probe calibration. Quantifying measurements for the film bulk were carried out with larger tip–sample contact areas to ensure a sufficiently large signal-to-noise ratio. The calibration measurements and measurements of the tip–sample contact diameter were performed before and after each image intended for quantification of permittivity and/or conductivity.The PZT relative permittivity PFM images of the domain walls were used to determine the tip–sample contact diameter. Since the domain wall thickness is 1–2 nm, that is, much smaller than the contact diameter, the apparent width of the domain walls in the PFM images can be taken as the tip–sample contact diameter with a high accuracy. This method was used for quantification of the film conductivity from both sMIM and c-AMF data. In sMIM measurements, the contact diameter was 100–350 nm, depending on the probe wear. In turn, in c-AFM measurements, the contact diameter was 25–30 nm.For imaging at elevated temperatures, the sample was glued to a heater with a silver paint. Sample temperature was measured with a miniature thermocouple attached to the sample. The sample temperature of 115 °C was maximal, when properties of the sMIM probe remained stable and imaging was possible. For better thermal stability, the experiments were started following a 10-min waiting period after the temperature of 115 °C was reached. Because heating of a sample warms up the probe as well, the phase setting of the sMIM electronics was adjusted at 115 °C after that. The probe was kept retracted from the sample surface by 3–5 μm during heating and waiting periods. To avoid tip-apex ware and contamination , we did not perform a search of the structure written at room temperature after a temperature point was reached, but prepared another similar structure. The domain structures were prepared and imaged at electrically identical conditions at room temperature and at 115 °C.f=3 GHz was performed using an a.c./d.c. module of the COMSOL v.4.2a Multiphysics finite elements analysis package (COMSOL AB). Two-dimensional axisymmetric models were used in the simulations. The conductivity of the SrRuO3 film was set to 105 S m−1 O3 domain structure was simulated using a phase-field model by numerically solving the time-dependent Landau–Ginzburg–Devonshire equationsThe spatial and temporal evolution of Pb, fgrad(∇Pi), felas and felec represent the Landau–Ginzburg–Devonshire free energy density of the uniform strain-free bulk, the gradient energy density, the elastic energy density and the electrostatic energy density, respectively. Details of the expressions for each of the energy density terms are summarized in refs Ebi, which can be associated with charged defects randomly distributed in the film volume. The x, y and z components of Ebi at a point in the film volume are:where α and β describe the random field orientation and are uniformly distributed random numbers between −2π and 2π. A is the amplitude of the random field A=M·f(μ), in which M is the disorder magnitude and f(μ) is the Gaussian function with μ being a uniformly distributed random number between −1 and 1. To determine the local electric potential and the spatial distribution of free charge carrier in the presence of the charged defects, coupled equations:where Nd, Na, n and p denote the local concentrations of donors, acceptors, electrons and holes, respectively. Nc and Nv are the effective density of states of electrons in the conduction and holes in the valance bands, respectively .Supplementary Figures 1-10


Dbx1 is a homeodomain transcription factor involved in neuronal fate specification belonging to a widely conserved family among bilaterians. In mammals, Dbx1 was proposed to act as a transcriptional repressor by interacting with the Groucho corepressors to allow the specification of neurons involved in essential biological functions such as locomotion or breathing.Sequence alignments of Dbx1 proteins from different species allowed us to identify two conserved domains related to the Groucho-dependent Engrailed repressor domain (RD), as well as a newly described domain composed of clusterized acidic residues at the C-terminus (Cter) which is present in tetrapods but also several invertebrates. Using a heterologous luciferase assay, we showed that the two putative repressor domains behave as such in a Groucho-dependent manner, whereas the Cter does not bear any intrinsic transcriptional activity. Consistently with in vitro data, we found that both RDs are involved in cell fate specification using in vivo electroporation experiments in the chick spinal cord. Surprisingly, we show that the Cter domain is required for Dbx1 function in vivo, acting as a modulator of its repressive activity and/or imparting specificity.Our results strongly suggest that the presence of a Cter domain among tetrapods is essential for Dbx1 to regulate neuronal diversity and, in turn, nervous system complexity.The online version of this article (doi:10.1186/s13227-016-0055-5) contains supplementary material, which is available to authorized users. In the eumetazoan group, development of the nervous system relies on the progressive diversification of neuronal cell types and the establishment of appropriate connections between them. The architecture of the nervous system is therefore shaped by evolutionary constraints. For instance, transition from aquatic to terrestrial life was accompanied by an increase in cell diversity and circuit complexity specifically allowing breathing and locomotion. Neuronal cell identity and connectivity are regulated by complex gene regulatory networks that typically involve homeodomain (HD) transcription factors (TFs). The developmental expression and interaction of HD TFs with one another are evolutionarily conserved and tightly regulated in order to ensure the spatial and temporal coordination of target gene expression.Dbx genes are expressed in the developing nervous system [Drosophila [Xenopus [Members of the Dbx family of HD TFs have been identified in a range of bilaterian species. s system –7 and weosophila , zebrafiosophila , 9, Xeno[Xenopus , 5 and m[Xenopus . In the [Xenopus , 12. Mou[Xenopus , 14. Mor[Xenopus . Dbx1 thMost HD TFs controlling cell fate in the vertebrate spinal cord (including Dbx1 and Dbx2) are thought to act as transcriptional repressors via an Engrailed homology-1 (eh1) domain which recruits the co-repressor Groucho . This haProtein domains are defined regions of a polypeptide structure that often carry specific functions. Hence, the “domain architecture” of a protein represents a primary level to understand its function(s) . The vasin ovo electroporation. These experiments allowed us to demonstrate that the newly identified Cter domain is critical to regulate fate specification properties of Dbx1. We propose that the strong conservation of the Cter domain of Dbx1 among tetrapods reveals its contribution to the regulation of neuronal diversity and nervous system complexification during evolution.Here, we analyzed a multiple alignment of Dbx protein family members found in a representative range of bilaterians. In addition to previously suggested putative repressor domains (RDs), we identified a novel domain enriched in acidic residues at the C-terminus (Cter domain) highly conserved among tetrapods, but also found in several lineages among bilaterians, suggesting it yields an evolutionary conserved crucial function. We implemented in vitro luciferase reporter assays to assess the intrinsic transcriptional activity of Dbx1 domains and further tested their contribution to the in vivo function of the protein using chick Homo sapiens Dbx1: NP_001025036.2; Dbx2: NP_001004329.2), macaque , mouse , opossum , chicken , python , turtle , African frog , western frog , coelacanth , zebrafish , sea squirt , amphioxus (Branchiostoma floridae XP_002608529), acorn worm , sea urchin (Strongylocentrotus purpuratus XP_001198056.2), fruit fly (Drosophila melanogaster NP_647677.2), jewel wasp (Nasonia vitripennis XP_001599133.1), lingula (Lingula anatina XP_013413513.1), marine annelid (Platynereis dumerilii SAP35630.1).The following sequences of Dbx proteins family were retrieved from NCBI , AniseedLeucoraja erinacea), lamprey (Petromyzon marinus), acorn worm (Ptychodera flava), sea bat (Patiria miniata), sea snail (Lottia giganta), octopus (Octopus bimaculoides), annelid worm (Capitella teleta), water flea (Daphnia pulex) and centipede (Strigamia maritima) were recovered and manually reconstructed from NCBI [Ciona intestinalis was further confirmed by screening a cDNA library kindly provided by Dr. M. Branno . Despite reiterated analyses , we were unable to find Dbx-related sequences in all nematode genomes available in the UCSC database [Dbx protein sequences from little skate , T4 DNA ligase (Invitrogen), Shrimp alkaline phosphatase (Invitrogen) and Phusion polymerase (New England Biolabs) and produced using an Endo-Free Maxi prep kit (Qiagen).For luciferase assays, the following vectors  were usein ovo electroporation experiments, constructs were made in a pCAGG-IRES-EGFP plasmid. All subcloned sequences were preceded by a Kozak consensus sequence and an HA-epitope tag (YPYDVPDYA). The Ciona intestinalis and Danio rerio cDNAs were obtained by RT-PCR from cDNA libraries kindly provided by Dr. M. Branno  and Dr. S. Schneider-Maunoury , respectively. The Saccoglossus kowalevskii cDNA was kindly provided by Prof. C. Lowe . Deletion mutants of mDbx1 were obtained by removing sequences encoding residues 36–50 (∆RD1), 105–127 (∆RD2) and 311–335 (∆Cter). The sDbx∆Cter construct was generated by removing the last 21 aa of the Saccoglossus sequence, while the ciDbx + Cter was generated by adding the last 25 aa of the mDbx1 to the Ciona sequence.For COS-7 cells were cultured in DMEM containing Glutamax and supplemented with 10 % fetal calf serum, 100 IU/mL penicillin and 100 µg/mL streptomycin . Cells were seeded at approximately 20 % confluence in a 96-well culture plate. They were transfected 24 h later (at ~50 % confluence) and harvested the following day (~80 % confluence). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions using 25 ng of pMH100-hsp-TK-luc2, 10 ng of pRL-Renilla, 75 ng of pKW2T-mGrg4 (or empty vector) and 50 ng of the Gal4-Dbx1 domains constructs.The “Dual Luciferase Assay kit” (Promega) was used to measure firefly and Renilla luciferase activities sequentially using a TriStar LB 941 luminometer (Berthold Technologies). However, we found the Renilla luciferase activity to be significantly modulated by the different Gal4 fusion proteins. This was also observed using another reporter plasmid encoding β-galactosidase. We therefore decided to normalize the firefly luminescence obtained for each Gal4 construct by that of Gal4 alone. For each condition, measurements were taken on 10–15 wells obtained from at least three independent experiments. Experimental and control conditions were compared using Kolmogorov–Smirnov and Mann–Whitney nonparametric tests.+ or En1+ cells on each side of the spinal cord using paired Student’s t test.Fertilized chick eggs were obtained from Les Bruyères . DNA solutions  were injected into the spinal cord of HH10–12 embryos  using a Xenopus, the eh1-like domain of Dbx2 aligns with a sequence of Dbx1 that is distinct from the one identified by Muhr and colleagues [To identify conserved protein domains in Dbx proteins, we first aligned the mouse Dbx1 and Dbx2 proteins. This showed that the two sequences share an identity of ~40 %, mainly due to the HD  and RD2lleagues ).Fig. 1SIn the carboxy-terminal region (Cter) of Dbx1, we observed an acidic residues-rich domain: DEDEEGEEDEE  indicated that the average RSA values of the Dbx1 and Dbx2 RD1 domains were not significantly different  and crustaceans. The RD2 domain was well conserved in all species but, as already observed in mouse  progenitor domain  and estain ovo electroporation. Only one side of the neural tube was targeted, allowing comparison with the non-electroporated (control) side. Electroporated cells and transfection efficiency were visualized through the bicistronic expression of GFP. We monitored the ability of wild-type and RD-deleted Dbx1 proteins to modulate cell fate specification by immunostaining using Evx1/2 and En1 antibodies. As previously reported [+ interneurons fate at the expense of En1+ interneurons  and expressed them in the chick embryo neural tube by reported , full-leons Fig. . When eiord Fig. a, b, indbx1 Fig. c, d, ind+ or En1+ interneurons  was unable to promote or inhibit the generation of either Evx1/2ons Fig. . These din ovo electroporation. Opposite to the full-length mDbx1, we found the mDbx1∆Cter protein to prevent the generation of Evx1/2+ interneurons  and tested its activity by ons Fig. a, b. Incion Fig. c, d, indin ovo electroporation in the chick embryo neural tube  as a mDbx1 ortholog displaying a quite different composition in terms of domains (lacking both RD1 and Cter domains); (2) zebrafish (Danio rerio) Dbx1b (zDbx1b) as a closer ortholog but devoid of Cter domain (we favored zDbx1b over zDbx1a due to a slightly better conservation of the RD1 and RD2 domains with respect to the mouse protein); and (3) Saccoglossus kowalevskii Dbx (sDbx) as an ortholog from a distant specie but yet retaining a structure close to that of mDbx1 .In order to better understand how the evolutionary acquisition or loss of functional domains may have conferred specific activities to Dbx proteins, we tested the consequences of an overexpression of Dbx from different species on the production of v0 and v1 interneurons by Ciona protein could promote v0 generation. However, a ciDbx + Cter mutant protein had no measurable effect on the activity of ciDbx  bears a fairly conserved similarity in amino acids sequence in both the RD1 and Cter domain compared to the mouse protein. Indeed, when electroporated in the chick neural tube, sDbx promoted the differentiation of Evx1/2+ interneurons in the same extent as mDbx1  the Cter domain was already present in the common ancestor to all bilaterians and secondary losses occurred in multiple lineages or (2) convergent evolution drove a progressive enrichment and clusterization of acidic residues at the C-terminus independently in several species. A pattern of specific losses of genes and domains has already been documented by comparing the genomes of early divergent deuterostomes and vertebrates , 48, argSaccoglossus proteins harbor a long clustered Cter domain (~10 acidic residues) and reproducibly induce Evx1/2 expression in the chick spinal cord. At the other end of the spectrum, proteins showing no specific acidic enrichment at the C-terminus, whether naturally (ciDbx) or artificially (∆Cter mutants), robustly inhibit Evx1/2+ fate. Zebrafish Dbx1b displays a short cluster of acidic residues, as other teleost fishes, and was found able to neither promote nor repress Evx1/2+ interneuron fate. Since the mouse and zebrafish Dbx1 proteins have very well-conserved RD1, RD2 and HD, our results strongly suggest that differences in their respective activities lie in the Cter domain, although we cannot formally exclude that other less conserved regions are also involved. Our work therefore points to the idea that the evolutionary enrichment and clustering of acidic residues in the Cter domain could have been an important step in the expansion of gene regulatory networks controlling neuronal diversity, and ultimately nervous system complexity.We found a strong correlation between the enrichment and clusterization of acidic residues in the Cter domain and the ability of Dbx proteins to modulate Evx1/2 expression. Mouse and Drosophila Ubx has the ability to prevent limb development in thoracic segments through the transcriptional repression of dll [Drosophila embryos, it is unable to repress both dll and limb development. The ability of Drosophila Ubx to repress dll is due to a short C-terminal alanine rich domain that is absent in velvet worms. Thus, the acquisition of a new domain within Ubx has allowed the repression of appendage development on abdominal segments in insects.Such a hypothesis is reminiscent of the HD TF Ubx, whose domain composition was previously linked with the evolution of patterning in arthropods , 50. Dron of dll . In velvThe implications of the presence of an acidic-rich Cter domain in invertebrates remain an open question that will require comparative functional studies in these organisms. In mouse, the critical function of Dbx1 in controlling the fate of neurons essential for both locomotion –12 and bWe have identified a novel acidic-rich C-terminal domain within the Dbx1 transcription factor that is conserved among tetrapods as well as several invertebrates. We have shown that this domain is required for Dbx1-induced neuronal fate specification in the developing spinal cord. Our data are consistent with the idea that the acquisition of this domain during evolution is linked to increased neuronal diversity and nervous system complexity.


Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH) domain and a Phosphotyrosine Binding (PTB) domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs) via interactions of the β1/β2, β3/β4, and β5/β6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK. canonical binding mode and an alternative binding mode. Our simulations also demonstrate the presence of both a canonical and an atypical binding site for PIPs in agreement with recent crystallographic studies of the ASAP1 PH domain. Analysis of density landscapes for the interaction of the Dok7 PH domain with PIP-containing lipid bilayers is also able to identify both canonical and alternative binding modes. Our improved understanding of how Dok7 interacts with a membrane is key to examining Dok7/MuSK signaling.Neuromuscular junction formation and maintenance is an essential biological process, the disruption of which leads to congenital myasthenic syndromes and premature death. Dok7 is a key member in formation, maintenance and signaling in neuromuscular junctions. Dok7 is a peripheral membrane protein that is necessary for full activation of the receptor tyrosine kinase MuSK, a receptor tyrosine kinase residing in the postsynaptic membrane. The structure of Dok7 consists of both a PH domain and a PTB domain. The interaction of Dok7 with cell membranes is not well understood. Here, we use molecular simulations to show that the Dok7 PH domain preferentially binds to PIP lipid molecules when associating with a membrane. Dok7 interacts with the bilayer in both a  Mutatio domains ,17. The  domains ,19. It h domains . Thus, PUnderstanding the mechanism of association of the Dok7 PH domain with a model cell membrane will provide insights into the interactions stabilizing the Dok7/MuSK/membrane complex, since the PH orientation relative to the membrane is likely to play a key role in the Dok7/MuSK interaction. It has been shown that molecular dynamics (MD) simulations provide a tool for understanding the mechanism of association of peripheral proteins with models of cell membranes –23, havicanonical and alternative binding modes of the PH domain. Our results allow us to suggest a mechanism for PIP-mediated association of the PH domain of Dok7 with cell membranes.In the current study, we employ a multi-scale MD simulation approach  to explo2. An ensemble of 20 simulations each of duration 1 μs  for (derivatized) PIP2 and PIP3 of 4 and 2 μM respectively [During each simulation, the protein diffused in the aqueous solution before interacting with the bilayer . In the mulation  without ectively . In contectively ,31. The ectively ,33. HoweRzz component of the rotational matrix of the PH domain . In particular, Rzz is the zz component of the rotational matrix required for least squares fitting of a conformation onto a reference conformation, and dz represents the perpendicular distance between the centres of mass of the PH domain and of the lipid bilayer. Rzz may adopt values from -1 to +1, and is equal to +1 when we use the preferred orientation as a reference conformation for the calculation of the rotational matrix. We note that related methodologies have been used to calculate the energy landscapes e.g. of cholesterol flip-flop [To further analyse the orientation and interactions of the Dok7 PH domain relative to different lipid bilayers, we have constructed two-dimensional density landscapes of the lip-flop  and of tlip-flop .2-containing membrane reveals that there is a clearly preferred binding mode of the PH domain to the bilayer ; and the β3/β4 loop (residues 42–54), which contains a short α-helix region at the C-terminus of which is R54 . In this orientation, the long α-helix (residues 94 to 107) of the PH domain is at an angle of ~60° relative to the plane of the bilayer, with β1/β2 and β3/β4 loops still maintaining contact with the bilayer. The M1 binding mode seen is very similar to a recently solved crystal structure of Arf GAP ASAP1 [two anionic lipid binding sites: a canonical C-site between the β1/β2 and β3/β4 loops, and a second atypical A-site on the opposite face of the β1/β2 loops, towards β5/β6.Analysis of the orientation of the Dok7 PH domain relative to a PIPer M1 in . In the h is R54 . A simil were compared to 0.5 μs simulations of the ASAP1 PH domain (the latter are described in detail in ). Lipid 2 and PIP3, and a second peak at 1.0 nm, indicating that both PIP species preferentially cluster around the PH domain. Such clustering was not observed for PC or PS. Clustering of PIP lipids was observed only in the leaflet to which the protein was bound. Visual inspection of the simulation trajectories reveals that there are multiple PIP molecules surrounding the PH domain once it is bound to the bilayer, e.g. Evaluation of radial distribution functions (RDFs) of the different lipid species around the bound Dok7 PH domain  showed a2 or PIP3 as identified from the CG simulations were converted to atomistic representations and multiple (3 x 0.3 μs) atomistic simulations  orientations of the PH domain when bound to either PIPulations  were perulations , and thetions 2; . For botdid PIP3 . Examina PIP3 ; lipids used (PIPs vs. fluorescent derivatives of PIPs); and assay conditions (PIPs in a bilayer vs. in the presence of detergent). The fact that in CG simulations, Dok7 binds to both PIP2 and PIP3 with little apparent difference between binding probabilities, is encouraging, especially when compared to the fluorescence polarization assays. This suggests that the Dok7 PH domain may bind to both PIP2 and PIP3. However, in vivo PIP2 is likely to be the major ligand, given its higher content in plasma membranes.Calculation of density landscapes allowed us to quantify the Dok7 PH domain binding to the bilayer. Our analysis suggests that in the presence of PIP domains ,17,39. W to Dok7 . The sim2 lipids [2 and PIP3 observed in our simulations. This clustering of PIP molecules may aid the association of Dok7 with the bilayer and thereby facilitate formation of a complex with MuSK.Recently, a crystal structure of the PH domain of the Arf GAP ASAP1 was solved in complex with dibutyryl-PIP2 lipids . This prIn summary, the PH domain of Dok7 associates with the bilayer via a canonical binding mode observed for other PH domains, as evidenced by the two-dimensional density landscapes we have calculated. By virtue of possessing two binding sites for PIPs, Dok7 causes clustering of PIP lipids around it, potentially facilitating its interaction with MuSK enabling downstream signaling transduction. The simulation methodology employed in this work may be readily applied to other peripheral membrane proteins. Alongside methods such as pMD-Membrane  which enThe published structure of Dok7 (PDB id: 3ML4) was used as a starting point, using Modeller 9v8  to modelCG-MD simulations were performed using the MARTINI2.1 forcefield ,43 with www.gromacs.org) using the Berendsen thermostat and barostat for temperature and pressure coupling [All CG-MD simulations were run with GROMACS 4.5.5  multiplied by the total number of frames in each repeat simulation and by the bin area. After the calculation of the landscapes, the density in each bin was divided by the density in the global minimum to obtain normalized densities.To construct these density landscapes, we have merged all 20 simulations for each system. A two-dimensional histogram of Rzz value of M1 from the coarse-grain simulations. Note that “distance” refers to the distance between the COM of the protein and the COM of the bilayer. These were then used as starting points for atomistic simulations. The AT model of the protein was generated from the CG model as described in Stansfeld et al. [α atoms restrained (force constant 1000 kJ/mol/nm2), followed by 300 ns of unrestrained simulations. Analysis was performed using GROMACS [Representative CG snapshots were chosen which corresponded to the distance and d et al. . All simd et al. . The Pard et al.  and Bered et al.  was usedd et al.  used to d et al.  was used GROMACS , VMD [50S1 Fig2(A) and PIP3(B) lipids used in the simulations. The headgroups of the lipids are in van der Waals representation, whilst the tails are in licorice representation. PIP2 is in purple, and PIP3 is in orange.Images of the coarse-grained models of the PIP(TIF)Click here for additional data file.S2 FigDensity landscapes for the CG-PH-pip2 system when it is separated into 4 x 5 μs, 2 x 10 μs and 1 x 20 μs densities.(TIF)Click here for additional data file.S3 Fig(A) Dok7; (B) ASAP1 (see [(C) a structural alignment of the two PH domains for comparison. Dok7 is in yellow cartoon, ASAP1 is in ochre cartoon, the PIP2 that binds to Dok7 is in purple CPK and licorice, and the PIP2 that binds to ASAP1 is in magenta CPK and licorice. This is further analysed by calculating averaged normalised lipid contacts for the simulations of (D) ASAP1 and (E) Dok7. The β1/ β2 and β3/ β4 loops are highlighted on the graphs with red lines, and the canonical and atypical sites are labelled with a “C” and “A” respectively.PIP binding sites observed in atomistic simulations of AP1 (see  for deta(TIF)Click here for additional data file.S4 Fig(A), phosphates (B), and tails (C) of PIP2, and the headgroups (D), phosphates (E), and tails (F) of PIP3. PC and PS lipids are represented by either a red or green line, respectively; PIP2 lipids are represented by a purple line, and PIP3 lipids are represented by an orange line.Number of contacts of Dok7’s PH domain with the headgroups (TIF)Click here for additional data file.S5 Fig(A) Secondary structure analysis of all Dok7 PH atomistic simulations. Simulations containing PIP2 are in the left column, whilst the simulations containing PIP3 are in the right column. Each individual simulation is shown as a separate graph, with the different secondary structures highlighted in blue (α-helix), red (β-sheet), black (coil), green (bend), yellow (loop), orange (β bridge), purple (5-helix), and pink (3-helix). (B) Root mean square fluctuation (RMSF) of each residue on Dok7’s PH domain for simulations with PIP2 (left) and PIP3 (right). Each replicate is shown in a different colour.(TIF)Click here for additional data file.S6 Fig(A) PIP2 (above) and (B) PIP3 (below) from the three repeat atomistic simulations (rep1 to 3).Hydrogen bonds between the Dok7 PH domain with (TIF)Click here for additional data file.S7 Fig2(A) and PIP3(B). The colours correspond to the three repeat atomistic simulations.Hydrogen bonds of selected residues  on the Dok7 PH domain with PIP(TIF)Click here for additional data file.S8 Fig(A), phosphates (B), and tails (C) of PIP2, and the headgroups (D), phosphates (E), and tails (F) of PIP3. PC is shown in red, PS in green, PIP2 in purple, and PIP3 in orange.Atomistic lipid contacts broken down into headgroup (TIF)Click here for additional data file.S1 File2 bilayer that is aligned with the Arf GAP ASAP1 structure.PDB file of the Dok7 PH domain with a PC/PS/PIP(PDB)Click here for additional data file.


Chemical synapses are asymmetric intercellular junctions through which neurons send nerve impulses to communicate with other neurons or excitable cells. The appropriate formation of synapses, both spatially and temporally, is essential for brain function and depends on the intercellular protein-protein interactions of cell adhesion molecules (CAMs) at synaptic clefts. The CAM proteins link pre- and post-synaptic sites, and play essential roles in promoting synapse formation and maturation, maintaining synapse number and type, accumulating neurotransmitter receptors and ion channels, controlling neuronal differentiation, and even regulating synaptic plasticity directly. Alteration of the interactions of CAMs leads to structural and functional impairments, which results in many neurological disorders, such as autism, Alzheimer’s disease and schizophrenia. Therefore, it is crucial to understand the functions of CAMs during development and in the mature neural system, as well as in the pathogenesis of some neurological disorders. Here, we review the function of the major classes of CAMs, and how dysfunction of CAMs relates to several neurological disorders. The brain is characterized by an enormous degree of complexity and diversity of neural networks, making it one of the most complicated organs. This complexity and diversity come from the vast numbers of neurons, but also from the variety of synapses where neurons pass electrical or chemical signals to other cells. Most of the synapses are small in size (1 μm in diameter), but biochemical studies reveal a high level of the molecular complexity. The post-synaptic proteome of the excitatory synapse of a mammalian brain contains more than 1000 proteins, indicating complicated protein-protein interactions occurring both within and between synapses  layer of extracellular space called a synaptic cleft  localize at the center of the synapse  and neuroligins (NLs) form one of the best studied molecule pairs in the CAM family. Nrxs, discovered as receptors for α-latrotoxin Südhof, , are typ2+-dependent  in the pre-synaptic terminal, while CASK binds Velis/MALs proteins and Mints/X11 proteins  mice showed that NLs and Nrxs are essential for synaptic maturation and function  and are mainly located at excitatory synapses. The LRRTM family is expressed in both developing and adult brains and is especially enriched in the post-synaptic density (PSD)  of two LRRTMs and NL-3 in cultured NL-1 KO neurons leads to a ~40 % reduction in excitatory synapses , are grouped into subfamilies of classic cadherins and protocadherins. These are also transmembrane proteins containing an extracellular domain with a repeated “cadherin motif” or “cadherin repeat” sequence   represent the largest family of receptor tyrosine kinases. Eph receptors contain an extracellular domain that comprises a globular ephrin ligand-binding domain, a cysteine-rich region, and two fibronectin type III domains; a cytoplasmic domain composed of a juxtamembrane region with two conserved tyrosine residues; a tyrosine kinase domain; a sterile alpha motif (SAM); and a PDZ-binding motif  is a glycoprotein of the immunoglobulin (Ig) superfamily, expressed in both the pre- and post-synaptic membranes. The extracellular part of NCAM has five Ig domains that bind to NCAM, and two fibronectin type III (FNIII) domains related to neurite outgrowth. At least 27 alternatively spliced NCAM mRNAs are present in rat brain, suggesting wide and diverse functions of NCAM  to synapses and is important for NMDAR-dependent LTP and LTD  leads to smaller NMJs and impaired accumulation of pre-synaptic proteins. The number of docked vesicles is reduced and the paired-pulse facilitation (PPF) is lacking at NCAM null junctions . At least four members are recognized in vertebrates: L1CAM, Close Homolog of L1 (CHL1), NgCAM-related cell adhesion molecule (NrCAM), and Neurofascin. L1CAM contains an ectodomain with six Ig-like domains and five fibronectin type III repeats, followed by a transmembrane region and a highly conserved cytoplasmic tail , and a reduction in the mean amplitude of putative unitary IPSCs, whereas the basal excitatory synaptic transmission is normal  has been reported to co-localize with nectins via the PDZ domain-binding domain of the latter  are a group of GPI-linked Ig-CAMs containing six N-terminal Ig-like domains and four fibronectin III-like domains. Six members are recognized in the CNTN family: CNTN-1, CNTN-2/TAG-1, CNTN-3/BIG-1, CNTN-4/BIG-2, CNTN-5/NB2, and CNTN-6/NB3. CNTNs play an important role in the formation of axon connections in the developing nervous system. Both CNTN-1 and CNTN-2 are involved in axon growth and guidance  have been identified as a family of proteins that contain three extracellular Ig-like domains, a single transmembrane domain and a cytoplasmic tail  are a newly discovered family of adhesion molecules: at least five members have been identified in the central nervous system. SALMs 1–3 contain an extracellular region consisting of a leucine-rich repeat (LRR), a fibronectin type III domain, Ig-like domains, a transmembrane domain, and a C-terminal PDZ-binding motif that interacts with PSD-95. SALMs 4 and 5 lack the PDZ-binding domain  proteins are a family of LRR-containing CAMs consisting of three members: NGL1–3. NGLs are mainly located post-synaptically at excitatory synapses. NGL-1 and -2 bind to netrin-G1 and netrin-G2 through their cytosolic tails in an isoform-specific manner , OBCAM (opioid-binding cell adhesion molecule), Ntm (neurotrimin), and Kilon. The LAMP, Ntm, and OBCAM molecules interact homophilically with themselves and heterophilically with each other  have recently been proposed to have a role in pre-synaptic development. The LAR-RPTP proteins have a single transmembrane domain, two intracellular PTP domains, and an extracellular domain .The incidence of neurological disorders is increasing in the human population. For example, in 2006, there were 26.6 million AD patients in the world  and cadherin 9 (CDH9) in the pathogenesis of autism  levels are higher in patients with AD  undergoes a tremendous reduction in BP disorder (−140%) in hippocampal tissue Vawter . QuantitAlthough only some superficial evidence that try to reveal the underlying mechanisms how CAMs are involved in neurological diseases pathogenesis can be observed, two points are seemed quite certain: on one hand, many CAMs could contribute in one neurological disease; on the other hand, one altered CAM could result in several neurological disorders, both in either separate or in cooperative way. Thus no ‘leading’ protein represents a common pathway for each of the neurological diseases, it is more likely that ‘many hands make light work’ in nature. Therefore more investments are required to deepen our understanding of the mechanisms of the molecular regulation of synapses.Neurons communicate via synaptic connection mostly mediated by precisely-controlled intercellular interactions. CAMs are involved in all stages of synapse formation and stabilization, providing ‘bridges’ between pre- and post-synaptic sites. At present, mounting evidence clearly indicates that no single pair of CAMs is necessary or sufficient for the organization of synapse developments from initiation to maturation, indicating overlapping or redundant functions of CAMs. The diversity of the isoforms and functions of CAMs may contribute to the complexity of neuronal network. Abnormalities in CAMs often cause neurological diseases.in vitro and in vivo (Christopherson et al., The involvement of CAMs in synaptogenesis and synaptic connection is now well accepted. However, it remains unclear that actually how many and which proteins are involved in the process of synaptogenesis, how these distinct CAMs contribute to the specific synapse sybtypes and functions and how the CAMs cooperate together. At present, in view of the large variety of synapses in the brain, the number of known CAMs is surprisingly low. It is natural to speculate that more proteins are involved in this process, perhaps even some ‘old’ molecules with recognized activities in other aspects of functions. For instance, SNAP25, one component of the SNARE complex required for synaptic exocytosis, regulates dendritic spine maturation and function through its expression level (Tomasoni et al.,


Spatial energy landscape that provides new insights into domain dynamics is also mapped based on the breathing motion of domain walls. The evolution of complex domain structure can be understood by the process of occupying the lowest available energy states of polarization in the energy landscape which is determined by defect-induced internal fields. Our result highlights a pathway for the novel design of ferroelectric domain-wall devices through the engineering of energy landscape using defect-induced internal fields such as flexoelectric fields.Fundamental understanding of domain dynamics in ferroic materials has been a longstanding issue because of its relevance to many systems and to the design of nanoscale domain-wall devices. Despite many theoretical and experimental studies, a full understanding of domain dynamics still remains incomplete, partly due to complex interactions between domain-walls and disorder. We report domain-shape-preserving deterministic domain-wall motion, which directly confirms microscopic return point memory, by observing domain-wall breathing motion in ferroelectric BiFeO Domain-wall (DW) motion in ferroic materials has been the subject of abundant researches1234567891011458910121345645618192223For the realization of the conceptual DW-based nanodevices, whose performance relies on the precise manipulation of DWs, a deeper understanding of DW dynamics under the influence of disorder is required. A conceptual one-dimensional energy landscape has been often used to describe DW motion in disordered system122125112829301920In this paper, we report an intuitive approach to understanding complex domain dynamics in ferroelectric thin film, including domain nucleation and the propagation of DWs, by visualizing spatial energy landscape that correlates the energy landscape with defect-induced local internal fields. In the scheme of energy landscape, domain switching kinetics corresponds to the process of sequentially occupying the lowest available energy states of polarization under an external field. Our work provides new insights into domain dynamics and highlights a new pathway for controlling DW motion in emerging technologies that exploit DWs by using defect-induced internal fields such as flexoelectric fields.3 (BFO) is known to have a very strong coupling between ferroelastic strain and ferroelectric polarization3 heterostructure grown on a vicinal SrTiO3 (STO) (001) substrate with a 2° miscut as a model system  images in BiFeOImportantly, the behavior of DW breathing in the BFO film reveals that DW motion is deteriminstic in large parts. The DW breathing motion shows an asymmetric growth behavior, where DWs at the right side of the switched domain move toward the [100] or [−100] directions, while the left side is strongly pinned, in agreement with the previous reportMicroscopic return point memory, which measures the reproducibility of microscopic domain structure after hysteresis loop cycling in ferroic systems, is strongly influenced by disorder and has been shown to increase with increasing random-field disorderEeff.u[P] is the free-energy density of polarization P(r) and the term Eext and Eint are external and local internal fields, respectively. The origin of the internal field includes depolarization field, ferroelastic strain, and other fields coming from defects etc. As for the effect of defects, Nattermann et al. introduced random-field and random-bond type interactions between DWs and random disorders to treat DW pinning in disordered systemsIn order to explore experimentally the features of energy landscape for domain dynamics in ferroelectrics, we first consider the energetics of polarization switching under local effective field, P states in equation If we assume that a ferroelectric system is initially poled to have a uniform polarization by applying a strong external field, the local energy of polarization determined by equation U[P(r)]. When the interaction is strong enough to overcome the effect of defect-induced internal fields, a stochastic (or non-deterministic) DW motion should occur. Thus, nanoscale study of domain dynamics can provide information on defect-induced internal fields as well as the dominant defect-type governing the domain dynamics.It should be mentioned that domain growth may influence local internal fields through the interaction between the polarization of adjecent sites37To gain further insights into the details of the deterministic DW motion, we investigate DW breathing for two different polarization states in the out-of-plane direction, as shown in 32311132Rcosθ) images acquired during the breathing motion, and then plotting the total intensity of each pixel in spatial coordinatesThe energy landscape of the BFO film can be mapped by superimposing the total piezoresponse  is relatively flat for both up- and down-polarization domain switching, which allows fast domain growth to the direction. Thus, the morphology of energy landscape well explains the asymmetric DW propagation and anisotropic domain growth in the film.The nucleation and subsequent growth behavior of domains are determined by the morphology of the energy landscape. DWs in the energy landscape are interfaces between the occupied and unoccupied energy states of up-/down-polarization. The nontrivial morphology of the energy landscape in 89va, which is equivalent to the slopes of the time dependence of the switched areas, is quite different during the expansion and shringkage of domains. The difference should result from the built-in internal field which prefer to a particular direction of polarization. The values of va are also quite dependent on the polarization direction of switched domains; 13.3 and 3.21 mm2 s−1 for the up- and down-polarization domains in the initial down- and up-poling states, respectively. For the sideways DW motion, we consider the energy cost V and ΔA are the changes in the volume of switched domains and the area of DWs, respectively, and d the thickness of film, and We argue that the roughness of energy landscape is also quite relevant to domain growth behaviors.  domains , multiplInterestingly, the overall switching time varies significantly depending on the preexisting domains. In the second stage of domain expansion  was grown on a vicinal STO (001) substrate with a 2° miscut via off-axis radio frequency magnetron sputtering2, were patterned through photolithography and sputtering. The epitaxial nature of the BFO film was confirmed by using high-resolution x-ray diffraction . The preferential monoclinic distortion of BFO lattices toward the downhill miscut direction  was also confirmed by reciprocal space mapping /BFO (200 nm)/SrRuO mapping . Ferroel mapping . Built-iVext and a width t were applied to the Pt top electrode of BFO capacitors (i and ξi) for each domain-switching state is the sum of the widths of a series of negative or positiveswitching pulses. We monitored the evolution of ferroelectric domains step by step after the application of each switching pulse by using stroboscopic PFM technique34R), phase (θ), and total piezoresponse (Rcosθ) signals under an ac voltage of 0.3 Vrms at a frequency of 19.1 kHz. The Vext values used for polarization reversal were −4.5 and 3.0 V for negative and positive switching pulses, respectively, which were close to the coercive voltages in the ferroelectric hysteresis loop. Additional details of the PFM experiment, including the R images that clearly show DWs, can be found in To visualize the breathing DW motion in a BFO (001) capacitor under the application of an external electric field, a modified stroboscopic PFM configuration was usedpacitors . The cumθ  signals of the PFM images acquired during breathing DW motion pixel by pixel, we were able to map the spatial energy landscape .