The first type considered places two bases together with a single acid, while the reverse case of two acids sharing a single base constitutes the second type. These bifurcated systems are compared with the analogous H-bonds wherein FH serves as the acid. In most cases, a bifurcated system is energetically inferior to a single linear bond. There is a larger energetic cost to forcing the single σ-hole of an acid to interact with a pair of bases, than the other way around where two acids engage with the lone pair of a single base. In comparison to FBr and FI, the H-bonding FH acid is better able to participate in a bifurcated sharing with two bases. This behavior is traced to the properties of the monomers, in particular the specific shape of the molecular electrostatic potential, the anisotropy of the orbitals of the acid and base that interact directly with one another, and the angular extent of the total electron density of the two molecules.Bifurcated halogen bonds are constructed with FBr and FI as Lewis acids, paired with NH Linus Pauling’s early explanation  of what 1 and D2 as depicted in 1 and D2. Another sort of bifurcated HB shares a common electron donor between two different proton donors, as indicated in Another extension of the HB concept arises in connection with the number of groups participating. A classical HB places the bridging proton squarely between a proton-donor and acceptor atom, as might occur for example between the two O atoms of the water dimer. While such a geometry might in fact represent a preferred structure, there have been numerous observations of what have come to be termed bifurcated HBs. What is usually meant by this term is the placement of the bridging proton between two different acceptors, D2 group.Bifurcated H-bonds of either type are quite common as surveys of crystal structures have revealed . In gene4 system. An I atom of a PtI2Br2 moiety [Due to their similarity to H-bonds it is not surprising that bifurcated halogen bonds are a distinct possibility, and they have undergone some scrutiny as well ,53,54. T2 moiety  can invo2 moiety  provides2 moiety  under ceLike halogen atoms, chalcogen atoms are also capable of engaging in bifurcated interactions, as for example  wherein A great deal has been learned about the halogen bond (XB) in recent years, and its parallels to the HB. Both sorts of bonds involve a strong electrostatic attraction between the electron donor and acceptor groups. While the bridging proton takes on an overall positive partial charge within its A-H subunit, the opposite is true of the X atom. However the electrostatic attraction to the nucleophile is made possible by a small positive region lying along the extension of the A-X bond, commonly referred to as a σ-hole. Both sorts of bonds are stabilized further by charge transfer from the nucleophile into the σ *(A-H/X) antibonding orbital. Also in common, electron-withdrawing substituents on the Lewis acid strengthen either bond by enhancing the positive charge residing on the H or X atom.While much is now understood about the fundamental properties of standard halogen bonds, there is far less information about the corresponding bifurcated XBs. Under what conditions might they occur, and which or both of types A and B in 3 is a stronger base than NCH, in part due to its sp3 hybridization in comparison to sp for the latter. Each of the three Lewis acids is paired with both NH3 and NCH, and for each case both the acid-shared In order to answer these questions, a number of systems are devised here and evaluated via quantum chemical calculations. FBr and FI are both capable of forming halogen bonds by virtue of fairly deep σ-holes on the Br and I atoms, respectively. The larger size of the I atom imbues it with the ability to form somewhat stronger XBs of the two. FH is employed as the corresponding H-bonding acid for purposes of comparison. Two different bases are considered. NH3 and FH··NCH, respectively. There are two types of bifurcated bonds that might be imagined. In the first place, two separate bases can compete for the same σ-hole on the halogen atom (or the H atom in the case of H-bonds) as delineated in In the case of simple dimers, whether halogen or H-bonded, the geometric preference is for a linear arrangement, as delineated in 3 and NCH as bases, and each combined with a pair of FBr, FI, or FH molecules. None of the triads pictured there represent a true minimum. When two acid units were initially placed in positions similar to those in the figure, a full optimization led to large-scale motion of one acid, leaving a single linear dyad as in 3. In the case of the NCH base, the second FI molecule maintained a I··N XB with N of the base, but a highly distorted one. The geometries illustrated in The systems representing 3 is equal to 2.339 Å. The next row indicates this bond is stretched to 2.760 Å in the triad when a second FBr must share the N lone pair on NH3, as in A quantitative assessment of the bonding in these triads may be gleaned from The interaction energies in the next column were evaluated by comparing the energy of each complex with that of the sum of monomers which were left in their geometries within the confines of the dimer. These quantities verify the preference of the single XB within the dimer  to that of the triad  where the two FBr units must share a single N lone pair. The electron density at the Br··N bond critical points in the next column amplify this energetic preference, in that the single linear Br··N density of 0.056 au greatly exceeds the 0.021 au in each of the two bifurcated XBs within the trimer. The same pattern is evident in the NBO values of E(2) for the charge transfer from the N lone pair into the σ *(FBr) antibonding orbitals. This perturbation energy of 51.1 kcal/mol in the linear dimer is much higher than the 9.0 kcal/mol for each of the two XBs in the bifurcated trimer.It is anticipated that there are two factors contributing to the bond weakening in the triads. In the first place, each bond is distorted from its preferred linear arrangement which can only be achieved within the dimer. The second factor arises since there are two Lewis acids, both competing for the same N lone pair. The fact that the base must serve as electron donor to two acids simultaneously would lead to a negative cooperativity. The third row of each section of 2NH3 system as our example, the placement of a single FBr molecule in the position it occupies in the trimer reduces the interaction energy of the fully optimized dimer  down to 9.43 kcal/mol. So, the bifurcation configuration cuts the interaction energy nearly in half. The reduction in ρBCP is even greater, and E(2) is reduced to only about 1/5 its optimized value. Adding in the second FBr molecule does not double the interaction energy, as would be the case in the absence of any cooperative effects. Instead of 18.86 kcal/mol , the interaction energy of the trimer is only 15.60, so one might assess the negative cooperativity as their difference, i.e., 3.26 kcal/mol.Again taking the (FBr)Very similar trends can be observed when FBr is replaced by FI. The halogen bonds in the bifurcated triad are stretched relative to the fully optimized linear dimer. The interaction energy within the trimer is less than in the linear dimer, and there is a substantial negative cooperativity. The bifurcated H-bonds associated with a pair of HF molecules are slightly different. There is negligible weakening of the interaction energy on going from dimer to trimer, and the two HF molecules are placed a bit closer together, with θ(H··N··H) only 66°. However, the negative cooperativity remains.3 lone pair of NH3 is replaced by the sp-hybridization of NCH. In the first place, the latter is a weaker base, so interaction energies, and their AIM and NBO markers are similarly reduced, and there is a concomitant stretching of the corresponding XBs. However, there is also the change that the interaction energies of the triads are larger than those within the fully optimized linear dimers. The 7.2 kcal/mol interaction energy of the linear FBr··NCH dimer rises to 10.5 kcal/mol when a second FBr molecule is added, and the system is forced into a bifurcated arrangement. In other words, there is an energetic advantage to bifurcation about the NCH lone pair. Part of the reason for this change is that the energetic cost of moving each acid from its fully optimized position to that which it occupies in the bifurcated trimer is less stringent for NCH. For example, the interaction energy of the FBr··NCH dimer is 7.20 kcal/mol, which is reduced only slightly to 6.07 kcal/mol within the context of the trimer structure. The negative cooperativity observed for NH3 occurs as well for NCH, so that is a common feature of these bifurcated trimers.There is a qualitative change when the sp3 rather than NCH were not possible even with such a restriction, since one NH3 would rotate so as to engage in NH··N H-bonding with the other.An alternate form of bifurcation would have two separate bases sharing a single σ-hole on a Lewis acid molecule. Systems of this sort are depicted in The geometric and other properties of these bifurcated triads are reported in This opposite behavior is reflected to some extent in the geometries. The addition of a second Lewis acid lengthens the intermolecular distances by a fairly small amount, between 0.10 and 0.25 Å. These stretches are much longer when it is a second NCH that is added, in the range between 0.33 and 0.46 Å. The latter longer intermolecular distances are a major factor in the reduction of the distorted dimer geometries mentioned above. The pattern continues in the other measures of bonding as well. Both the bond critical point densities and the NBO E(2) quantities are reduced much more by the addition of a second NCH than by a second FX unit. The smaller θ angles in 3 as compared to NCH, consistent with the stronger basicity of the former. The elongations within the halogen-containing FX are more pronounced than within FH. The degree of stretching is reduced when the fully linear optimized dimer is distorted into the bent bifurcated structures. It might be noted as well that the reductions imposed in the trimers relative to the dyads are smaller when it is a second NCH that is added to FX··NCH, as compared to a second FX.It is understood that the formation of a HB or XB will affect the internal properties of each subunit. For example, the internal bond length of a FH acid will stretch when engaged in a H-bond, and its stretching frequency shift to the red. These changes will be accompanied by a downfield shift of the bridging proton’s NMR signal, caused in part by its deshielding. The effects of both dimerization and trimerization on the individual monomers are reported in 3 as compared to NCH. Also consistent with bond length changes, there is less of an effect arising from addition of a second NCH to the dimer than occurs from addition of FX.The next column of The last three columns of The data show that enforcing a symmetric sort of bifurcated pair of bonds is energetically inferior to a single linear such bond in most cases. The exceptions arise when a pair of H or X bonding acids interact with a single weak NCH base, wherein the total interaction energy of the triad is slightly larger than that of a single linear bond. A refined examination of the numerical data indicates that there is less of an energetic cost when two acids share a single lone pair, than the reverse situation wherein a single acid σ-hole must accommodate two bases. It is not only the energy itself, but the other indicators of noncovalent bond strength, AIM, and NBO markers, as well as geometric and spectroscopic data, that affirm the angular deformations necessary to align two bases with a single σ-hole is more damaging than when two acids share a single base lone pair.3 and NCH bases, whose negative MEP with its excess density might be termed a σ-lump. Their MEPs, displayed as the broken contours on the right sides of It is possible to understand the underlying reasons for this distinction by examination of the molecular electrostatic potential (MEP) that surrounds each molecule. 3 is much steeper than that for NCH.An alternate manner of viewing the comparative anisotropies is via 3 σ-lump region as compared to NCH accounts for the observation that the angular deformations required to add two acids is more energetically costly for the former. Moreover, the high anisotropy of the FBr and FI σ-holes explains the large drop in interaction energy when two bases are placed around them that are forced to lie off of the F-X axis.These visualizations conform with the numerical data. The greater anisotropy of the NHOf course, the electrostatic interaction represents only one chapter of the story. As represented through the NBO formalism, the bonding is partly due also to charge transfer from the N lone pair orbital to the unoccupied σ *(FX) orbital of the acid. This component too ought to have an angular dependence that diminishes as the bond is forced from linearity. The angular characteristics of the relevant NBO orbitals are provided in 3 curve is a bit more anisotropic, reminiscent of the MEP in The near isotropy of the NCH lone pair is particularly obvious by the nearly flat nature of the green curve in 3. 3, so can tolerate a wider displacement, which in turn minimizes repulsions between the two acids. The same can be said for the structures exhibited in The anisotropies of the MEP and lone pair orbital of the bases fit squarely with the geometries of the bifurcated systems in 3. This maximum at θ = 0 comports with the idea of a σ-lump that aligns with the N lone pair. The absence of a higher electron density resulting from a displacement off the lone pair direction is another factor accounting for the greater ability of the bases to share a lone pair with two Lewis acids, as compared to the diminished capability of a Lewis acid to share a single σ-hole with two bases. This same feature also helps explain the greater separation of the two FX acids in Still another factor in the ability of a partner molecule to locate itself off of the primary axis is the anisotropy of the electron density. A high electron density would tend to increase the exchange repulsion with its partner. An example may be drawn from the purple FI plot of the electron density around FI, at a constant distance of 3 Å from the I atom in 3 behave oppositely from one another. While the latter has its least negative value along the lone pair direction, this quantity is most negative along the lone pair of NCH.With regard to the electrostatic potential surrounding each monomer, an alternate view of this quantity is known as the potential acting on one electron in a molecule (PAEM)  which coAccording to the calculations discussed above, a bifurcated arrangement which encompasses a pair of distorted noncovalent bonds is less favorable than is a single, fully optimized linear arrangement even though the former has two such bonds to the single bond in the latter. This situation applies to H-bonds as well as halogen bonds. In fact, a geometry encompassing a bifurcated pair of noncovalent bonds to either a common lone pair or a common σ-hole does not represent a true minimum on the potential energy surface of these triads. The exceptions occur when a pair of H or X bonding acids interact with a single weak NCH base, wherein the total interaction energy of the triad is larger than that of a single linear bond, even if only by a small amount.3 and the two FX acids is no longer less than that within a fully optimized linear FX··NH3 dimer. However, even removal of the repulsion between the two NCH bases in the FX(NCH)2 triads does not raise the remaining pair of FX··N attractions up to the level of a single linear FX··NCH dimer, underscoring the lesser ability of a σ-hole to accommodate two separate bases.This situation arises due to several principal factors. Angular deformations implicit to bifurcated bonds cause a significant weakening. Coupled to this issue is the negative cooperativity that arises when a single molecule acts as either double electron donor or acceptor. There are also repulsive interactions between the pair of molecules competing to bond with the central unit. These repulsions are less than 2 kcal/mol for a pair of FBr or FI units but are a bit larger for two FH or NCH molecules. Were these repulsions to be removed from the triad interaction energies, the remaining quantity representing only the attractions between the central NHAll energetics have been corrected for basis set superposition error by the counterpoise protocol. Failure to make this correction enlarges each of the interaction energies in 3 as an example, the interaction energy of a bent XB is 9.4 kcal/mol, which grows to 15.6 kcal/mol upon formation of a second FBr··N XB.Although there is an innate energetic preference in many cases for a single noncovalent XB or HB as opposed to a bifurcated arrangement, the latter obviously does arise rather commonly within the confines of larger macromolecular systems, or when crystal packing forces are present. Configurations found there reflect the fact that external forces preclude the optimal linear arrangement. In such a situation, it makes sense that a second Lewis acid can take advantage of the unoccupied side of the base’s lone pair, which is preferable to the former lying dormant. After all, a bent bond is preferable to no bond at all. The data in 3 base, as presented in The bifurcated bonds considered here are idealized in the sense that they are symmetric. That is, the two CN··X angles when a pair of FX acids interact with the NCH lone pair were held to be equal to one another, as was true for the NHThe work described here considers the case where the nucleophile contains only a single lone pair that must be shared between two Lewis acids. However, when this restriction is lifted, allotting more than one lone pair to the base, it becomes much easier for the latter to participate in multiple noncovalent bonds simultaneously. Recent examples can be drawn from the rapidly developing field of selective anion binding. The base in question is typically a halide anion containing not only several available lone pairs, but also a full negative charge. Bulfield and Huber  have revThere is precedent for the finding that is confirmed here that the halogen bond tends more toward linearity than does the H-bond . For purThe work presented here has focused on halogen bonds, and their potential to share their single σ-hole with a pair of nucleophiles. Specifically, the halogen atoms considered here are all univalent in the sense that they are involved in only one covalent bond, in this case to F. However, halogen atoms can participate in hypervalent situations where they may be involved in more than one covalent bond. In such a situation one can anticipate that there will be one σ-hole that corresponds to each such internal bond. Kirshenboim and Kozuch  exploredint, of each complex was computed as the difference between the energy of the complex and the sum of the energies of monomers in the geometries they adopt within the complex. Basis set superposition error was corrected via the standard counterpoise [All quantum calculations were carried out within the framework of the Gaussian-09 set of codes . DFT proterpoise ,111,112 terpoise . The topterpoise . The natterpoise ,116 was 3, which allows the former to better accommodate a bifurcated arrangement with two acid molecules. Likewise, the lesser anisotropy of FH as compared to FBr and FI allows H-bonds to better share a single proton than can XBs share their X atom. A shift of a base off of the bond axis of the Lewis acid would encounter a higher electron density, and associated exchange repulsion, another factor inhibiting the formation of a bifurcated bond with two bases, particularly for FI. In contrast, there is no such exchange repulsion factor inhibiting the sort of nonlinear geometry required to link two acids to a single base.With some exceptions, a bifurcated pair of either H-bonds or halogen bonds is energetically inferior to a single such bond within a dimer. There is a larger energetic cost to forcing the single σ-hole of an acid to interact with a pair of bases, than the other way around where two acids engage with the lone pair of a single base. This distinction can be traced to details of the MEP that surrounds the various molecules, as well as to the shape of the pertinent molecular orbitals, and the total electron density. The acid’s σ-hole is more anisotropic than is the negative MEP at the site of the base’s lone pair. The same is true of the σ *(FX) antibonding orbital which is more directional than is the N lone pair orbital. On both of these scores, the weaker NCH base is less anisotropic than is NH


There is extensive literature debating whether perceived size is used to guide grasping. A possible reason for not using judged size is that using judged positions might lead to more precise movements. As this argument does not hold for small objects and all studies showing an effect of the Ebbinghaus illusion on grasping used small objects, we hypothesized that size information is used for small objects but not for large ones. Using a modified diagonal illusion, we obtained an effect of about 10% on perceptual judgements, without an effect on grasping, irrespective of object size. We therefore reject our precision hypothesis. We discuss the results in the framework of grasping as moving digits to positions on an object. We conclude that the reported disagreement on the effect of illusions is because the Ebbinghaus illusion not only affects size, but—unlike most size illusions—also affects perceived positions. Inspired by the two visual systems hypothesis  participated in the experiment. Assuming that the standard deviation in the effect of an illusion on maximum grip aperture is about 1.8 mm  to record the movements of markers attached to the nails of finger and thumb at 200 Hz. For the perceptual judgements, we placed a laptop on the wooden board, above the start location of the hand. This laptop showed a black screen  with a 2 mm thick vertical white line presented at the centre of the screen; the length of the line could be adjusted by sliding one’s finger on the trackpad.To ensure that participants looked at the bar before starting to move their hand, we had participants always report the perceived size of the object directly before they grasped it. This previewing may have increased the influence that perceptual judgements of size have on the grasping movement Glover , so the Each trial started with the experimenter placing a stimulus: a combination of a bar and a piece of paper with a drawing of the corresponding context Fig. . A line Every stimulus was presented 10 times in a pseudorandom sequence, so in total there were 80 trials, each consisting of a judgement and a grasping movement. If the participant dropped the bar before reaching the end position, or if the bar was not grasped at the ends with a precision grip, we repeated the grasping part of the trial . After every 20 trials, the participants were asked whether they wanted a short break.We analysed the kinematic data of each grasping movement. We determined the grip aperture by taking the 3D distance between the markers of finger and thumb. We subsequently determined the maximum of this distance (MGA) in the part of the trajectory between movement onset and the first moment that the thumb came close to the target. The latter was defined as the first moment after the thumb had reached its peak height at which the thumb had descended to less than 1 cm above its initial height at the starting position. We chose this moment to ensure that the digits had never made contact with the object by the moment of MGA  for each participant, stimulus and task. The advantage of using the median rather than the mean is that it is robust for outliers. Moreover, it is strange to assume that MGA is normally distributed, because MGA cannot be smaller than the object size on successful grasps and it is limited by the anatomy of the participant’s hand. Therefore, the mean would be a systematically biased estimate of the central tendency of the MGA distribution. The difference between the median values for the two illusion configurations of objects of the same size provides us with four raw illusion effects for each participant for each task. To estimate the true change in size that corresponds with these raw illusion effects  and task . The precision hypothesis primarily predicts an interaction, because it predicts that the illusion effect on matched length will be the same for both sizes  in separate 4 (size) × 2 (illusion) repeated-measures ANOVA.As a second additional analysis, we determined whether the illusion effects are correlated across participants. If the precision hypothesis is correct, we expect the illusion effects for MGA to be correlated with the effects for matching for the small objects, but not for the large objects. If grip aperture is always based on the perceived size, we expect the illusion effects for MGA to be correlated with the effects for matching for both object sizes. A similar correlation between the effect of the illusion on MGA for small and large objects is expected according to all hypotheses except the optimal precision hypothesis, according to which there should be no correlation. No correlation is expected if grip aperture is based on judged positions. Correlations are quite difficult to establish for this type of data because several sources of variability play a role  with neither an effect of size  nor an interaction . As there is no interaction between task and size (the non-significant slightly larger effect of the illusion when grasping larger objects is even in the opposite direction than predicted), we can reject our precision hypothesis . The variability in the matched size was three times as high for the largest object than for the smallest one. There was a small effect of the illusion , with no interaction with size (p = 0.81). Figure The matched size increased with object size with a gain of 1.10 ± 0.02 (mean ± SEM), and increased from 1.2 cm for the 1.5 cm object to 5.0 cm for the 5 cm object. We checked whether the pattern of variability corresponds to the pattern reported by Ganel et al. . For theF3,54 = 12.5, p < 0.001), without a significant effect of illusion (p = 0.13) or interaction (p = 0.68). We will come back to this finding in the discussion. The non-significant tendency for smaller appearing objects to be grasped with a more variable maximum grip aperture than larger appearing ones : it increased from 4.8 cm for the 1.5 cm object to 8 cm for the 5 cm object. The within-participant standard deviation in grip aperture was on average 4.2 mm Fig. c, so grip > 0.5; upper row in Fig. p < 0.05; lower row in Fig. We find no correlation between the illusion effects for matching and maximum grip aperture for either of the bar sizes  is larger than in our pencil-and-paper version of the experiment (5%; Smeets and Brenner A recent extensive experimental paper demonstrated convincingly that grasping is affected by the Ebbinghaus illusion and that this effect was not due to the flankers acting as obstacles (Kopiske et al. A remaining question is why the illusions have such different effects on perceived positions while influencing perceived size in a similar manner. An explanation might be that the two illusions originate at different levels of visual processing. If so, our results suggest that the Ebbinghaus illusion affects visual processing at a stage that is common for determining size and position, whereas the modified diagonal illusion affects visual processing at a later stage. A somewhat similar distinction has been proposed to account for effects of two orientation illusions (Dyde and Milner The present study provides a comprehensive explanation for the apparently conflicting results that have been found when examining the effects of visual size illusions on grip aperture in reach-to-grasp movements. Size illusions themselves have no effect. The position illusion that is present in the Ebbinghaus figure is responsible for the robust effect of that ‘size’ illusion.https://osf.io/9cmz6/.The data for grip aperture and perceptual settings for each trial and subject are available at


Brexit represents an unprecedented moment for the European integration  project. Despite the evident difficulties in assessing the impact of the still ongoing process on the cladenstine activity of illicit tradde, all in all, the potential effects of Brexit on illicit tobacco trade in the UK could be rather modest. One thing which emerges with certain clarity is that just as in many other policy areas, the control of the illicit trade of tobacco by the UK will depend ultemately on the Brexit scenario negotiated with EU. In the event of a leaving the single market, while losing economically on many ends, the UK would retain higher policy flexibilites. While still bound by the ITP, this may mean greater speed and flexibility in learning and improving upon existing global or regional norms in this field to its own benefit. Brexit represents an unprecedented moment for the European integration  project. Despite the evident difficulties in assessing the impact of the still ongoing process on the cladenstine activity of illicit tradde, all in all, the potential effects of Brexit on illicit tobacco trade in the UK could be rather modest. One thing which emerges with certain clarity is that just as in many other policy areas, the control of the illicit trade of tobacco by the UK will depend ultemately on the Brexit scenario negotiated with EU. In the event of a leaving the single market, while losing economically on many ends, the UK would retain higher policy flexibilites. While still bound by the ITP, this may mean greater speed and flexibility in learning and improving upon existing global or regional norms in this field to its own benefit.en route to other EU Member States. The focus is therefore mainly on how Brexit may impact illicit trade into the UK. The case of Gibraltar, where illicit products tend to move from the UK territory into (the EU) Spain, represents an exception. Gibraltar already currently enjoys a special status and maintains a rather ‘hard’ border with neighbouring Spain. The 2019 Withdrawal Agreement foresees a Protocol accompanied by a bilateral ‘Memorandum of Understanding on Tobacco and other Products’ with Spain, capping the price difference between adjacent territories and fostering cooperation and enforcement. This should limit the risks of an increase of illicit activities related to tobacco targeting the EU market from Gibraltar as a result of Brexit.The data presented in this study shows that the UK is a target country for illicit tobacco trade. The country has no significant domestic production (licit or illicit), and is not a transit country for illicit products Hard Brexit’. The acceptance of the 2019 Withdrawal Agreement by the UK Parliament after the December 2019 elections and the start of the transition period (now set to last until the end of 2020) should prevent this. Much will of course depend on the UK’s ability to implement the changes necessary for (licit) trade to be carried out in an orderly manner.More generally, opportunities for smuggling into the UK may arise from an unorderly implementation of its withdrawal from the EU, a scenario that could materialize in case of ‘Brexit is not expected to have dramatic effects on the relevant legislation analysed. Most of it is already domestic (UK) legislation and directly applicable EU law will be retained in UK law after exit day. Whether and how in the future the UK will remain aligned to relevant EU legislation depends on several factors. The December elections brought clarity as to the most likely approach to future UK–EU relations. Now that the UK has officially withdrawn on January 31st 2020, the two parties are expected to negotiate an ambitious trade agreement.Tackling illicit tobacco: From leaf to light’) suggests that the country will maintain its strict tobacco regulations.Little, however, has been said regarding the unavoidable trade-off between ease of access to the EU markets and autonomy to diverge from EU regulation. This is particularly important for the future application of select aspects of the EU 2014 Tobacco Products Directive (TPD-2), of relevance to the control of tobacco trade. In general however, the past record and the current HMRC and UK Border Force joint national strategy in place  may push the UK into raising its import duties or internal taxes on tobacco. Under the three other Brexit scenarios, including the case of conclusion of a trade agreement, the parties would exchange tobacco products without applying import duties. If the UK remains part of the single market, it would have to abide by the EU minimum excise requirements (in this scenario the UK is till free to raise its excise given the concern of the EU legislation with minimum levels). Under all scenarios analysed, the UK would have the latitude to adopt the tobacco personal allowances threshold it wishes, without consideration for EU imposed minimum levels.Two additional aspects that may have an indirect impact on illicit trade also depend on the final form of Brexit. These are import tariffs and thresholds for personal allowances for tobacco products. In an environment of elevated prices (currently among the highest in the world), the adoption of excessively  restrictive measures on tobacco may indirectly strengthen the demand for illicit products. The option of levying import tariffs on tobacco products originating from the EU would only be available in case of ‘2019 Withdrawal Agreement and the non-binding 2019 Political Declaration) only contain limited references to this important aspect. The institutions, agencies, and EU bodies considered, all provide for partnership mechanisms for non-EU members which, while not comparable to full membership, may offer a viable solution. Cooperation in the fight against illicit trade in tobacco products should also be fostered at the national level, with the aim of developing a comprehensive UK national anti-illicit trade strategy covering other areas than tobacco.The analysis above also suggests that cooperation with EU institutions, agencies, and bodies in the areas of investigation and enforcement, as well as with the EU Member States, is an aspect that deserves specific consideration. This is of outmost importance since the UK is a target country for illicit tobacco—and a particularly attractive one because of the high price for licit products. Brexit will impact the current format of active participation in the work of the different agencies that operate to tackle illicit tobacco trade in the EU. Today, the UK takes part in EU police and judicial cooperation in criminal matters, including in the context of information exchange databases and systems, such as OLAF, Europol, Frontex and Eurojust. Cooperation has led to tangible results. Preserving the highest possible degree of interaction after Brexit is in the interest of both sides. The texts negotiated between the parties . As a party in its own right to the ITP, the UK would stand to gain from the broadest possible participation in such international initiatives.


The conformation of the German shepherd dog (GSD) varies considerably within the breed. These differences may result in large variation in the movement and limb loading and undesirable consequences to their musculoskeletal health. This study aimed to investigate the relationship between conformation and biomechanical measures in 60 GSDs. Full body kinematic and kinetic measures were computed from 3D motion capture and pressure data. The dogs were divided into groups based on their back slope and curvature. Correlation analysis and statistical differences between groups showed that GSDs with a greater back slope have a greater contact area in their forelimbs and place them closer together when standing (n = 60). During trot, the dogs with sloped back showed a greater vertical force in the forelimbs and a greater mid-thoracic flexion (n = 60). Unilateral differences were found in the stifle flexion, hock flexion and hock adduction, suggesting greater movement asymmetry with an increase in the back slope (n = 30). In conclusion, several biomechanical parameters are affected by the GSD’s slope of the back and not by its curvature. Further studies are required to determine whether the variation in movement, posture and conformation within the breed relates to an increased susceptibility to musculoskeletal disorders. The different conformations are described in specific breed standards3, however, they tend to evolve with time. Currently, there are notable differences in the conformation of the purebred German shepherd dog (GSD), which is commonly observed as variation in the incline and curvature of the back and in the angulation of the hindlimbs. The modern purebred GSD is a breed that first started as a herding dog, and later as a working dog used by the police and the armed forces. Nowadays, there are two distinct types based on the dog’s activity; a working GSD and a showing GSD.Conformation varies enormously between dog breeds, even between large breeds of similar weight2. The current breed standard of the United Kingdom for the GSD3 states that “the topline (back) runs without any visible break from the set on of the neck … falling away slightly in a straight line to the gently sloping croup”. It is further stated that the croup should be “slightly sloping and without any break in the topline (and) merges imperceptibly with the set on of the tail”; where a flat or excessively inclined croup is undesirable3. The British breed standard of the GSD hindlimbs states that “Angulations (should be) corresponding approximately with front angulation, without over-angulation”, the standards further emphasise that over-angulation of the hindlimbs are undesirable3. However, despite this standard, a GSD is considered to have a favourable topline by many judges with an incline that progressively increases towards the croup, as this seems to be based on people’s perception of aesthetics of the breed rather than function and health. This perception has led to a profound change in the conformation of the breed over the last century, with the back profile evolving from relatively straight, rectangular conformation to a curved (Germanic type) and sloped conformation (British type)4. The various interpretations of the breed standard have led to a wide variety of conformations of GSD, with some conformations being perceived as having health related side effects and leading to unhealthy hindlimb mechanics in gait. For example, the Crufts 2016 show was hit with controversy and a media outcry when a GSD with a ‘sloping back’ won best in breed but appeared to be struggling to walk5. However, there is limited understanding of whether the evolution of conformation in the breed does indeed have significant effects on movement and if this relates to any clinical presentations.Despite little variation in the breed standard across the world, there are considerable differences in its interpretation by judges, breeders and dog’s owners; resulting in large variation in the breed, in particular their conformation, which can be categorised by the slope and curvature of the topline8, Doberman9 and Greyhound6, but not the GSD. When comparing the kinematics and kinetics of different breeds and between dogs of the same breed, it is important to consider if differences are due to variations in the way the dogs move or due to variation in body mass and size. After normalising kinetic parameters for size and body mass, Bertram et al.6 concluded that the movement and limb loading of the Labrador and Greyhound were dynamically similar during trot.Some studies have used kinematic and kinetic analysis of walk, trot and square standing to describe the gait and posture of specific canine breeds, such as the Labrador12, force plates13 or weighing scales14 to record parameters such as limb loading, peak vertical forces, paw pressures and temporospatial (time and distance) parameters. This has shown that in normal stance, dogs support 60% of their weight on the forelimbs when standing square, although this value will change depending on the head and neck position15. The vertical forces and weight distribution during walk are also similar between breeds when normalised to the weight of the dog10.Other studies which measure the kinetics in dogs have used a pressure walkway16 and a lower peak vertical force compared to normal dogs13. These findings clearly indicate that the degree of hip dysplasia is related to the weight bearing and lameness in the GSD. Fischer and Lilje2 analysed the stride parameters and the angles of the joints in the sagittal plane in 10 working and 14 showing GSDs. They showed that the limb’s forward and backward range of motion during the stride was bigger in the show lines compared to the working lines, and that the show lines had a shorter femur and lower leg. This is probably the only attempt in the current literature to analyse the two breed lines with gait quantification methods, however the statistical significance of their findings was not reported. The kinetics and 3D kinematics of healthy GSDs across a range of conformations and types have not yet been fully investigated, hence it is not known if differences in conformation within the breed affects movement and standing posture and if this relates in any way to the risk of musculoskeletal disorders in this breed.Very few biomechanical studies have investigated the GSD. In a study with 10 healthy GSDs and 10 GSDs with hip dysplasia, GSDs with dysplastic hips were shown to have a greater angle of flexion at the hip and a greater rotational velocity of the hip when trottingThe present study aimed to quantitatively describe standing posture and trot of 60 healthy GSDs by analysing the kinematics and kinetics. The relationship between the kinematic and kinetic parameters and conformational measures of the back curvature and slope was investigated to determine if the conformation is likely to affect the standing posture and movement in the healthy GSD.Sixty healthy, lameness-free, purebred GSDs of both sexes, any age from 6 months old, and any conformation type were recruited. All dogs had been previously hip scored and were eligible to participate if their hips were structurally sound [denoted by a hip score (HS) which was less than the mean for the breed (HS ≤ 16 from BVA/KC hip score statistics)]  . Owners All the methods have been approved by the institutional Ethical Committee from the University of Surrey’s (NASPA ethics committee). All protocols carried out in these animals were in accordance with relevant guidelines and regulations of this committee and the institution, and all owners gave written consent to participate in the study.17.Dogs attended a single session in the Biomechanics Laboratory at the School of Veterinary Medicine in the University of Surrey. Dogs that showed any signs of anxiety or aggression , were considered unfit to participate or were lame  were excluded from the study. Dogs were allowed some time to familiarise themselves with the biomechanics lab prior to data collection. This was followed by measurements of conformation and fitness. The conformation measures were as follows: the slope of the back calculated as the slope of the line connecting the withers and mid-sacrum to the horizontal and the length of the back (withers to mid-sacrum). To obtain these measures, a height measurement stick was used to measure the positions of the wither and mid-sacrum when the dogs were standing square and a tape measure was used to measure the length of the back. Indicators of the dog’s fitness were also recorded, these were: the body weight, muscle tone of the thighs (i.e. quadriceps muscles) and upper forelimb (triceps muscle) using a semi-objective scale based on palpation: (1) hypotonic, (2) normal tonicity and (3) hypertonic, body condition score , mid-thigh circumference using a measuring tape calibrated for the handler’s pulling strength (GULICK II TAPE MEASURE MODEL 67020) and fat thickness using a fat calliper at the mid-lumbar region . All meaReflective markers were attached to the dog’s skin using hypoallergenic double-sided tape; markers were placed at anatomical landmarks and clusters of reflective markers were attached to the middle of each segment, as shown in Fig. Kinematic and kinetic data were recorded while dogs stood square on the pressure walkway for 10 s; the paws were positioned so they were directly below the hip joint or humeral head Fig. , right. Dogs trotted over the pressure walkway at a comfortable, self-selected speed for twelve valid trot stride cycles; recordings were rejected if dogs were not looking ahead, tripped or changed speed. The tracked reflective markers were assigned for each dog to create a 3D model of the dogs and these were used to compute kinematic parameters in standing and trot.T) and lumbar (RL) regions18 as shown in Fig. The centre of each cluster on the back was used to calculate the radius of curvature of the back in the thoracic  and limb positioning. The flexion/extension and abduction/adduction angles of each joint were calculated from the anatomical marker positions in the sagittal and transverse planes and axial rotation was quantified in VISUAL3D  using the clusters placed on the limbs and Euler sequence ZXY.The protraction/retraction angles of the forelimb and hindlimb were also calculated for each limb, using the position of the metacarpal/metatarsal anatomical markers and the acromion/greater trochanter markers respectively. The data were filtered using a second order low pass Butterworth filter (with 6 Hz cut-off frequency) to minimise the errors associated with marker movement.The markers placed at anatomical landmarks were also used to quantify the height of each segment above the ground  and the segment length. Parameters were normalised to the dog’s height measured at the withers.Trot stride cycles were split using the time of initial contact of the left forelimb recorded from the pressure mat. The maximum and minimum joint rotations and the range of motion for each joint were measured in four valid stride cycles for each dog and joint angles were plotted against cycle time.Limb loadings, peak pressures, contact areas and weight distribution were quantified from kinetic data during standing and trotting; the loading of the digital pads and metacarpal/ metatarsal pads were also quantified for each limb to describe intra-paw variation. Data were normalised to body mass recorded from the pressure mat to ensure parameters were not affected by the weight of the dog. The stride length was normalised to the height at the withers to ensure that the size of the dog did not affect the findings.p = 0.371), the back slope and the curvature of the back were used to investigate relationship between the back profile and kinematic and kinetic parameters using linear and non-linear regression analysis, where appropriate.Means of the kinematic and kinetic parameters were computed for each dog and used to calculate the intra and inter-subject standard deviations. All data recorded was tested for normality using the Shapiro–Wilk test and the slope and radius of curvature of the back were tested for independence using the Chi-squared test see . As bothT) were also used to assign each dog to a theoretical conformation group  and curvature group   were used to check for differences between the groups. To avoid type 1 errors, biomechanical measures were only considered to be affected by conformational measures if (both) significant Pearson’s correlations with the conformational measure (back slope or curvature) and differences between the conformational groups were found. Finally, Freidman tests were used to test for differences between standing positions (square and stacked).The back slope and thoracic radius of curvature (R m) Fig. . Chi-squp = 0.006). The height of the sacrum was significantly higher in the levelled group than in the intermediate (p = 0.012) and sloped (p < 0.001) groups. Dogs with levelled backs had a greater thoracic radius of curvature compared to dogs in the sloped back group (p = 0.017).A total of 60  GSDs were recruited in the study with a mean age of 4 years and 2 months (SD: 2 year and 3 months), mean body mass of 39.8 kg (SD: 7.5 kg) and a mean combined hip score of 8.1 (SD: 3.8). Back inclinations ranged from 0° to 10.34°  and the mean radius of curvature of the thoracic region of the back was 2.26 m (SD: 4.00 m). When categorised by slope, 18 dogs were in the levelled back group, 21 in the intermediate and 21 in the sloped back group. When categorised by curvature of the back, 29 dogs were in the arched back profile group and 31 in the straight back profile group. In total, 38% of the GSDs were trained in agility and 42% of dogs had participated in breed shows; in particular, most GSDs in the sloped back group (68%) participated in dog shows, whereas 17% of GSDs in the levelled back group participated in dog shows  and differences between conformational groups were also found. The results showed that a number of kinetic and kinematic parameters were related to the back slope (detailed below) but none were shown to be affected by the curvature. The full results of the statistical tests are shown in supplemental material –S7.It was not always possible to acquire full 3D kinematics of the 60 dogs because of obstructions to the marker trajectory by the owner on one side of the dog; this was particularly the case for forelimb joints kinematics in trot. In addition, the quality of data were compromised in some occasions due to the dogs’ fur. As a result, all the figures depicting kinematic results have been limited to high-quality data obtained from 30 dogs.p < 0.01; stacked: r = left 0.44, right 0.45, p < 0.001); GSDs in the sloped back group had a greater contact area than dogs in the levelled back group . Additionally, in the stacked position, the percentage of weight bearing on the forelimbs was correlated with the slope of the back and inversely correlated in the hindlimbs .In standing, the main kinetic parameter affected by the slope of the back was the contact area of the forelimbs which was correlated with the slope of the back during both standing positions (square: correlation coefficient (r) = left 0.37, right 0.44, p < 0.001). Dogs with a levelled back had a mean retraction angle of 6.10° (SD: 2.19°) in the left and 4.68° (SD: 2.96°) in the right hindlimb and dogs with a sloped back had a greater retraction angle of 10.71° (SD: 5.26°) in the left (p = 0.003) and 10.45° (SD: 6.12°) in the right hindlimb (p = 0.002). The abduction/adduction of the left and right hindlimb joints were not found to be (consistently) affected by the slope of the back.In the hindlimb, the angle of retraction was correlated with the slope of the back during square standing . When comparing groups in square standing, GSDs in the levelled back group positioned their forelimbs with a distance of 40.2% (SD: 3.8%) of the wither’s height, whereas GSDs in the sloped back group positioned their forelimbs closer, at a distance of 34.0% (SD: 4.1%) of the wither’s height (p < 0.001). The flexion of the thoraco-lumbar joint was also found to be affected by the back slope in the two standing positions, where a greater thoraco-lumbar flexion was found is dogs with a greater back slope .The forelimbs were positioned closer together during both standing positions in dogs with a greater slope of the back (expressed as a percentage of wither’s height)    compared to the loading in the digital pads when standing square  (05) Fig. .Figure 5p = 0.001) and increased the flexion of the left stifle (p = 0.035) while retracting the left hindlimb. The flexion of the hock in the retracted limb did not change compared to standing square, but in the opposite hindlimb the hock joint became more flexed (p = 0.005)  and protracted their right hindlimb , whereas during square standing the left hindlimb was retracted by 8.55° (SD: 4.25°) and the right hindlimb by 8.30° (SD: 5.07°). To stand in the stacked position, GSDs extended their left hip , where dogs in the sloped back group had longer swing times and shorter stance times (58:42) than dogs in the levelled back group (56:44); interestingly, this difference did not lead to a change in speed.In trot, the swing phase duration of the hindlimbs were correlated with the slope of the back . GSDs in the sloped back group had a mean vertical force in the forelimbs of 128.6% of the body weight (SD: 17.6%), which was greater than in the levelled back group with a mean of 106.9% of the body weight (SD: 11.6%), (p < 0.001). Loading of the digital pads was greater in both forelimbs  and both hindlimbs (right and left p < 0.001) in GSDs with a more sloped back, but the loading of the metacarpal and metatarsal pads were not consistently affected by the slope of the back . GSDs in the sloped back group had a mean flexion angle of 7.9° (SD: 3.4°), which was greater than the levelled back group with a mean of 3.6° (SD: 2.4°). The flexion angles of the other regions of the back were similar for all GSDs . GSDs in the sloped back group had a mean flexion angle of 94.1° (SD: 7.9°) which was greater than the levelled back group  (p = 0.012). The left hock remained more flexed throughout the trot stride cycle in dogs with a greater back slope , where dogs in the sloped back group had a mean minimum flexion angle of 38.6° (SD: 7.0°) which was greater than the levelled back group  (p = 0.028). The flexion/ extension angles of the other joints were not affected by the slope of the back . The right hock of GSDs in the levelled back group had a greater abduction angle  compared to the sloped back group , (p = 0.001). The abduction/adduction angles of the other joints were not affected by the slope of the back  for the left hip and 67.1° (SD: 16.7°) for the right hip. During the swing phase, the hips were externally rotated, as shown in Fig. ° for theThe slope of the back had no significant effect on the internal or external rotation of the back during trot. The thoraco-lumbar and lumbo-sacral regions of the back were axially rotated to the right side of the dog (positive) during the late swing phase of the right front limb and left hindlimb, as these limbs are lowered to the ground; whereas, towards the end of stance phase, these axially rotated to the left (negative) Fig. . ConversThis study gives detailed quantification of the three-dimensional movement during trot, standing posture and limb loadings of GSDs of a range of conformations; and provides evidence that these are affected by the slope of the back. Each parameter was tested for correlations with measures of conformation including the slope of the back and its curvature. In addition, dogs were categorised into groups according to their back slope and back curvature. The results showed correlations between kinetic and kinematic measures with back slope. Conversely, the curvature did not have an effect on any of the biomechanical measures.19. Therefore, care must be taken when making conclusions related to mediolateral and transverse plane rotations. Findings showed inconsistencies in the effect of back slope on some kinematic measures between the right and the left, for example, back slope was shown to be correlated with an increase in the right stifle flexion, the left hock flexion and right hock adduction. These results did not reach statistical significance on the contralateral side. To ensure that these results are real, we have visually assessed the synchronised video recordings for confirmation and indeed more GSDs with sloped backs presented an irregular motion pattern of one or both sides when trotting compared with the more levelled back dogs which is likely to be a cause of the inconsistencies found between the two sides.Due to differences in fur, the quality of the kinematic data in the trot trials in some dogs were compromised. Therefore, to ensure the trot kinematic data were reliable and repeatable, a representative sample of the best 30 GSDs data were presented during trot. This did not affect the reliability of the standing kinematic data or in the kinetic data. Besides of problems with the fur, skin displacement alone is reported to affect non-sagittal motion analysis in dogs20 indicating that GSDs naturally stand with their forelimbs well under the forechest. Other breeds carry approximately 60% of their weight in their forelimbs and 40% in the hindlimbs2 during normal standing, where this percentage can increase by 10–15% if the dog extends or lowers the head and neck15. Furthermore, in data analysed by the researchers on 12 sound Labrador retrievers, it was found that they carry 69% (SD: 5%) of their weight in the forelimbs, which is higher than the sloped back GSDs of the present study in spite of being both similar sized breeds. This means that although GSDs with a sloped back have an increased loading of the forelimbs, the loading distribution is comparable to other breeds and is still less than in a similar large breed. It can be speculated that this can either lead to, or, be due to more well-muscled forelimbs compared to the musculature in the hind quarters of sloped back GSD. Although this study recorded the muscle tone on the forelimbs and hindlimbs and the circumference of the thighs as indirect measures of muscle development and strength of the GSDs, there was no difference between the groups. Although it should be noted that these are semi-quantitative analyses that might not be very precise, hence further conclusions on differences in musculature and its relation to back slope in GSDs cannot be made from this study alone.The increased loading of the forelimbs in dogs with a greater back slope suggests that sloped dogs were leaning forward, thus increasing their forelimb weight bearing. This agrees with the observations by HollenbeckIn both standing positions, square and stacked, the greater contact area of the forelimbs in dogs with a sloped back was independent of the increased weight bearing. When dogs stood in the stacked position, there was no difference in the loading of the forelimbs and hindlimbs compared to standing square, which means that dogs were able to maintain a symmetrical weight bearing in both standing postures. In order to achieve this during stacking, the dogs extended the hip and flexed the stifle of the retracted limb and loaded the paw mainly through the digital pads; whereas the limb which was not retracted had a more flexed hock to transfer more of the weight onto the metatarsal pad. Although the stacked position seemed more difficult to achieve for a few dogs, particularly those not trained for shows, their limbs were not loaded differently, suggesting no disadvantage or discomfort when standing in either position. Dogs with a more inclined back also positioned their forelimbs significantly closer together during standing, and this was not due to a difference in the widths of the thorax, defined in this study by the distance between the scapulae.21, the vertebral columns of 9 GSDs was found to have less mobility at L7–S1 than in dogs of other breeds, it was suggested that these findings were related to the detailed anatomy of vertebral facets in this breed. Further in vivo studies would be necessary to compare spinal motion with other breeds, in particular of the caudal spine, as the morphology and morphometry of the lumbosacral region has been assumed to promote lumbosacral disease in the GSD22.Flexion of the mid-thoracic region of the back was the only spinal region significantly correlated to back slope at trot. In most of the GSDs with straight back profile, the greatest flexion initiated at the thoraco-lumbar and lumbo-sacral regions, whilst dogs with an arched back profile had a curve originating from the thoraco-lumbar region. In an in vitro study by Benninger et al.23 found that Belgian shepherd dogs (BSDs) with borderline hip dysplasia had a more flexed stifle joint compared to healthy BSDs and Miqueleto et al.16 reported greater flexion angles in GSDs with hip dysplasia. However, the current study showed no correlation between the back slope and the maximum hip flexion angles. Although it should be noted that dogs in this study were only included if they had hip scores less than the mean of the breed and therefore all dogs in this study were cleared from hip dysplasia. The inclusion criteria served to ensure that dogs included in this study were not subclinically lame. However, it may be argued that such a criterion biases against certain conformations. Anecdotal evidence from dogs recruitment suggests that dogs with more sloped backs were more difficult to recruit. Nevertheless, despite having this criterion dogs included in the study had a large range of back slopes and back curvatures which were the two conformational measures of interest.The slope of the back was significantly correlated with increased values of several angle rotations of joints of one or the other hindlimb suggesting asymmetry of the hindlimb motion. Bockstahler et al.20. These findings might be linked as compensatory mechanisms to maintain a better balance, however, non-sagittal rotations cannot be directly attributed to biological reasons considering the great latero-medial skin displacement reported in the literature for the hindlimb in dogs19. It is still unclear why the GSDs with a more sloped back show more variable right-left sagittal plane movements of the stifles and hocks. The relevance of this findings might be important for the GSD’s musculoskeletal health, or at least, it might explain why some sloped back dogs seem to move more irregularly in the show ring without presenting explicit lameness.During trot, there was a greater adduction angle and greater range of motion (ROM) of one of the hocks in dogs with a sloped back (i.e. ‘cow hock’), which is undesirable in the GSD breed, as it can be considered as broken columns of limb support, however it appears to provide lateral stability13, suggesting the reduced vertical force in the hindlimbs during standing and trot may be related to the development of dysplasia of the hip.The higher loading of the forelimbs and lower of the hindlimbs of sloped GSDs during trot and stacked standing suggests that this could lend the forelimbs a greater chance to exercise and develop musculature, while the reduced use of the hindlimbs in comparison could be a contributing factor to GSDs being perceived as having weaker hindquarters. Previous studies have found that GSDs with hip dysplasia also have a lower peak vertical force in their hindlimbs compared to normal dogs6, however, all dogs moved at a comparable self-selected trot speed. Therefore, most likely, the differences in loading are likely to be related to differences in body conformation6. More levelled backed dogs distributed the loading more evenly between digital pads and metacarpal and metatarsal pads, however, neither of these ways of loading the limbs seem to have a clear advantage or disadvantage.During trot, the increased loading of the digital pads in all limbs in sloped GSDs would normally be attributed to differences in speed2 in 10 working and 14 showing GSDs where they found that limb’s forward and backward range of motion during the stride was bigger in the show lines compared to the working lines, and that the show lines have a shorter femur and lower leg. The authors did not report the back slope of their dogs, which could have been different than in our study. Other anatomical differences may have been present in their dogs since they were recruited in Germany and it is known that morphological differences between the Germanic type and British type exist4.In our study, the slope of the back did not affect the segments’ lengths or the protraction/ retraction angles significantly, which disagrees with the study performed by Fischer and Lilje24, the GSD had a more extended hip joint by approximately 20°, more flexed stifle by approximately 20° and a greater maximum flexion angle of the hock of approximately 38° during trot. Conversely, the GSD had comparable flexion and extension angles of the forelimb to other larger breeds25. These differences emphasise how the kinematics can be directly affected by the conformation of each breed.When comparing with other large breeds reported by Fu et al.24, with the greatest amplitude of external rotation occurring at the end of the swing phase. Furthermore, the hocks were more externally rotated in the GSDs of this study compared to other larger breeds with a mean range of motion of axial rotation from 13° external rotation to 5° external rotation24; the GSDs also had considerably greater inter-subject variation in the hock axial rotation angle. The externally rotated hocks may be a compensating mechanism for the internally rotated hips. It is widely known that GSDs tend to present externally rotated hocks, in which case the metatarsus are externally rotated in relation to the tibia, resulting in the lower limbs rotating outwards, a feature of some dogs presenting the so called ‘cow hocks’. Hollenbeck20 explains that lack of muscle balance in the hindlimbs in dogs with a spread stance can result in cow hocks. This suggests that maintaining a healthy muscle mass in the hindlimbs can be essential in this breed with tendency to rotate hindlimbs externally.All dogs internally rotated their hip joints for the majority of the stride cycle, only showing external rotation at the end of the swing phase. However, the hips of other larger breeds were externally rotated throughout the stride cycle, with a mean range of motion of axial rotation from 24° external rotation to 7° external rotationAn extensive range of kinetic and kinematic parameters were analysed in this study. However, parameters were only reported in the results if they were correlated with the slope of the back in addition to showing differences between the back slope groups, as this meant the parameters had a statistically significant and relevant relationship with the slope of the back. This statistical approach ensures that the reported results are truly significant and any possibility of randomness is discarded.In conclusion, this comprehensive description of limb loading and joint angles of the GSD shows that this breed moves and stands differently compared to other breeds; and when comparing different lines within the breed as a result of variation in conformation, particularly the inclination of the back and not by its curvature or other conformation features. GSDs with sloped back carry more load in their forelimbs during standing and trot, and load their digital pads in all limbs more than the metacarpal and metatarsal pads; they also showed greater right-left differences in their hindlimb movement, particularly in the stifles and hocks. Although there are clear differences in standing posture and movement that are a result of changes in conformation, further research is needed to investigate whether the observed biomechanical changes are also related to the prevalence of musculoskeletal disorders in the GSD.Supplementary Information 1.Supplementary Information 2.


Temporal interference (TI) stimulation was recently proposed that allows for the stimulation of deep brain structures with neocortical regions being minimally stimulated. For human brain modulation, TI current patterns are known to be considerably affected by the complex structures of the human head, and thus, it is hard to deliver TI current to a specific deep brain region. In this study, we optimized scalp electrode configurations and injection currents that can deliver maximum TI stimulation currents to a specific deep brain region, the head of the right hippocampus in this study, considering the real anatomical head structures of each individual. Three realistic finite element (FE) head models were employed for the optimization of TI stimulation. To generate TI current patterns, two pairs of scalp electrodes were selected, which carry two sinusoidally alternating currents with a small frequency difference. For every possible combination of electrode pairs, optimal injection currents delivering the maximal TI currents to the head of the right hippocampus were determined. The distribution of the optimized TI currents was then compared with that of the unoptimized TI currents and the conventional single frequency alternating current stimulation. Optimization of TI stimulation parameters allows for the delivery of the desired amount of TI current to the target region while effectively reducing the TI currents delivered to cortical regions compared to the other stimulation approaches. Inconsistency of the optimal stimulation conditions suggest that customized stimulation, considering the individual anatomical differences, is necessary for more effective transcranial TI stimulation. Customized transcranial TI stimulation based on the numerical field analysis is expected to enhance the overall effectiveness of noninvasive stimulation of the human deep brain structures. These techniques have been shown to be effective in modulating a variety of brain functions and facilitating neurorehabilitation in Parkinson’s disease, stroke, Alzheimer’s disease, and aphasia5. It has been known that tDCS facilitates excitability of cortical neurons whereas tACS entrains the endogenous brain oscillations7. The target brain areas stimulated by tDCS and tACS have mostly been confined to shallow cortical areas. This is because it is generally difficult to selectively stimulate deep brain structures with tDCS or tACS while avoiding unwanted modulations of the neocortical neurons. Although previous studies showed that a certain level of electric field can be delivered to subthalamic regions with tDCS8, a computational simulation study has demonstrated that the delivery of much larger electric fields to neocortical regions is inevitable9.Transcranial direct current stimulation (tDCS) and transcranial alternating current stimulation (tACS) are noninvasive brain stimulation techniques that can modulate cortical excitability by transmitting weak currents via electrodes attached to scalp surfaces10. Two temporally interfering sinusoidal electric fields with a small frequency difference (Δf) can form a TI pattern with a frequency equal to the average of the two electric fields and an envelope equal to Δf at specific brain areas. This low frequency component, often referred to as a beat frequency, is delivered only to neurons in a deeper site of the brain. As high frequency electric fields are known not to directly evoke neuronal activities11, this novel technique can selectively modulate neurons in the deep brain structures. An animal study with mice also verified that a TI stimulation with two modulation frequencies of 2 kHz and 2.01 kHz could directly activate neurons in the hippocampus with a frequency of 10 Hz, while neurons in the shallow cortical areas were not affected10. However, a recent simulation study on transcranial TI stimulation with a realistic human head model showed that it is generally difficult to focalize the TI pattern only around a specific target brain area because of the inhomogeneous conductivity distribution inside the human head12. A recent simulation study demonstrated that TI stimulation can deliver electric field large enough to entrain brain oscillations at the frequency of TI envelope in deep brain structures13; however, since this study focused only on validating that deep brain areas can be modulated more strongly by using the TI stimulation than the conventional tACS, it did not fully demonstrate that deep brain areas could be modulated selectively with the unwanted modulation of neocortical areas being minimized. Furthermore, this study utilized only a single human head model. Since it is well-known that individual anatomical differences can affect the electric field distributions in the brain during transcranial electrical stimulation15, feasibility of the optimized TI stimulation needs to be further validated with different head models.Recently, a new noninvasive electrical brain stimulation method called temporal interference (TI) stimulation was proposed to modulate deep brain regions while avoiding unwanted modulation of neocortical neurons17. The temporal changes of electric field distributions in the brain were then evaluated by successively conducting the finite element analysis (FEA), followed by Hilbert transform, resulting in the spatial distribution of the TI envelope amplitude at a beat frequency. In this study, the head of the right hippocampus was selected as the target brain structure and three realistic head models generated from individual magnetic resonance imaging (MRI) data were employed for the simulation and optimization. Four electrodes were selected from 61 electrode candidates, and then, optimal injection currents that form the maximum amplitude of TI envelope in hippocampus while reducing the unwanted modulation of the shallow cortical areas were determined. The overall efficacy of the proposed optimization process was compared with that of conventional approaches.To address the abovementioned issue, in this study, we optimized the parameters of TI stimulation, including electrode configurations and magnitudes of injection currents, with three individual head models, to deliver the maximum TI currents to a designated target region, with TI currents in cortical areas being minimized as much as possible. To evaluate the distribution of TI patterns in the brain, we first solved a quasi-static Laplace equation using the finite element method (FEM) with a realistic head model, enabling the calculation of the electric fields generated by a pair of electrodes, each of them carrying a certain amount of injection currentsub1, Hsub2, and Hsub3, respectively. T1-weighted MR images were acquired from a 3-T MRI scanner with a resolution of 1 × 1 × 1 mm. All subjects were required to provide a written informed consent after they had been informed of the purpose of the experiment. They also agreed the publication of their potentially identifiable head images in an online open-access publication by signing the written informed consent. The experimental protocol was approved by the Institutional Review Board (IRB) Committee of Hanyang University (HYI-17-180-5). All data acquisitions were performed in accordance with the guidelines and regulations set by the IRB of Hanyang University. We used SimNIBS v2.018 for automatic segmentation of tissues including scalp, skull, cerebral spinal fluid (CSF), gray matter, and white matter  and a certain point in the head of the hippocampus was set to the target in this study  head models were created from three young male subjects  with no clinical history of psychiatric disorders and with no abnormal findings in their magnetic resonance (MR) images. We named their head models H20. This process was repeated so that the electric field distributions for all 61 cases  were evaluated and stored in the computer21. We assumed that all the tissue compartments are homogeneous and their electrical conductivity values were set to be the same as those used in a previous study20. The variation of electrical conductivity values in the high-frequency condition was ignored12. The electrical conductivity of the hippocampus was set to be the same as that of gray matter, and the conductivity of the electrodes was assumed to be 1 S/m22.The three FE models were used for the determination of optimal electrode configurations and injection currents. Sixty-one electrode candidates were attached according to the international 10–10 EEG electrode system and a reference electrode was assumed to be attached at the left mastoid of each model. The shape of the electrodes was a thin cylinder with a diameter of 1 cm and thickness of 2.5 mm. The electric field intensity vector at each node of the given head model could be evaluated by solving the FEM formulated using the electrostatic Laplace equation given by 21. Then, no current flows in the reference electrode. We can apply the same process to the other electrode pair, thereby resulting in two independent electric field (vector) distributions denoted by t, denoted by Four electrodes were selected from 61 electrode candidates to form two electrode pairs. Assuming that a specific electrode pair combination was determined, electric field distributions generated by each of the two electrode pairs were calculated as follows: When injection currents of 1 mA and − 1 mA were applied to one electrode pair, the resultant electric field distribution could be readily evaluated by the superposition of two pre-calculated electric field distributions, which are the electric field distribution when currents of 1 mA and − 1 mA are applied to one active electrode and the reference electrode, respectively, and the electric field distribution when currents of 1 mA and − 1 mA are applied to the reference electrode and the other active electrode, respectively α and β are scalar constants that need to be determined. In this study, E(t), the maximal envelope amplitude A of E(t) was determined as12φ represents the angle spanned by θ denotes the angle between θ ranges from 0 to 2π. The maximal TI envelop amplitude A was calculated at every node in the head model. The FEM solver to evaluate electric fields inside the human head was based on that used in the Comets2 toolbox (https://www.cometstool.com) developed by our group, which has been extensively employed by more than 40 research groups all around the world25 and validated through the comparison with analytic solutions25. To deliver maximum TI current with an envelope frequency of Ahippo should be larger than 0.2 V/m according to a previous study reporting that the alternating electric fields larger than 0.2 V/m are sufficient to entrain endogenous brain oscillations in the target region27:Ahippo and Acortex represent the peak amplitudes of the TI envelope with a frequency of α and β, were determined so that the objective function in (3) could be maximized. We used an exhaustive search approach for the determination of α and β when the value of α was increased from 0.5 to 1.5 mA with a step size of 0.05 mA, while the sum of α and β was fixed at 2 mA. We applied the above process for all three combinations of electrode pairs that can be made using four selected electrodes, and then selected one electrode pair combination with injection currents of α and β that maximizes the objective function (3). The same process was repeated for all possible selections of four electrodes among 61 electrode candidates , and then the optimal electrode configuration and injection currents could be determined.10. Note that the same electrode pairs were selected for all three homogeneous head models by chance. For simulating the single frequency tACS, the same electrode pairs as the optimized ones were employed, while two sinusoidal currents both with a constant frequency of 10 Hz were applied to the two electrode pairs. The amplitudes of the two injection currents were also set to be the same as those of the optimized ones. To determine the optimal tACS condition in the 61-channel electrode configuration, an optimization process used in Guler, et al.28 was employed, when the total injection current and the maximum individual injection current were set to 2 mA and 1 mA, respectively.The effectiveness of our optimization was validated through the comparison with four conventional approaches: (i) unoptimized TI stimulation, (ii) single frequency tACS and (iii) optimized single frequency tACS. For simulating the unoptimized TI stimulation condition, two current-carrying electrode pairs, O1–Fp1 and T6–F8, with alternating frequencies of 2 kHz and 2.01 kHz, respectively, were assumed, with the amplitude of both sinusoids being set to be 1 mA. The two electrode pairs were determined using the exhaustive search so as to make TI amplitude of the target (head of right hippocampus) maximized in the head models with a single homogeneous conductivity value, which is the approach used in the Grossman et al.’s worksub1, the electrode pairs PO7–FC3 and T8–F8 were determined as the optimal electrode configuration  while reducing the stimulation intensity of neocortical regions. A numerical analysis based on FEM was employed to compute the three-dimensional distribution of TI currents inside realistic head models. The effectiveness of our optimization was validated through the comparison with the results of unoptimized TI stimulation and single frequency tACS. Our results demonstrated that the optimization of stimulation conditions taking individual head anatomies into account could enable the delivery of the desired amount of stimulation current to a target deep brain area with reduced delivery of stimulation currents to shallow cortical areas compared to the other stimulation conditions. In addition, the difference in the optimal conditions for three head models suggests that customized TI stimulation based on individual electric field analyses might be necessary to enhance the efficacy of TI stimulation.According to a previous study13. It seems difficult to prevent the modulation of other deep brain regions with just two electrode pairs. Therefore, it would be an interesting future topic to reduce the unwanted modulation of other deep brain regions by employing more electrode pairs or developing new objective functions.To further verify the feasibility of the optimization for different target locations, we changed the target location to the tail of the right hippocampus , when 61 electrode candidates were used. Please also note that approximately 8 h were needed to complete the segmentation of brain tissues using SimNIBS.In our individual optimization results, optimal stimulation conditions were not consistent between head models, which is believed to originate from the difference in individual anatomical structures. Previous studies on tDCS have demonstrated that the electric field distribution inside the head is significantly influenced by the difference in anatomical structures such as skull thickness, CSF thickness, and cortical folding32, researchers have tried to noninvasively stimulate the right hippocampus using transcranial electrical stimulation (tES)33. However, even though a certain amount of current can reach the head of the right hippocampus, modulation of the cortical neurons lying on the paths of stimulation currents is inevitable. This makes it difficult to discriminate whether the behavioral changes after the stimulation are solely due to the direct modulation of the hippocampal activity or partly due to the indirect modulation of the hippocampus via functional connections between hippocampal neurons and cortical neurons. Customized TI stimulation based on our optimization approach might address this issue because our approach could effectively reduce unwanted modulation of cortical neurons. Indeed, a previous study that attempted to modulate the temporal cortex34 observed modulation of the verbal declarative memory after tACS but could not clearly conclude whether this effect was produced by modulation of the hippocampus or the temporal cortex. Contrary to the single frequency tACS, our simulations showed that the right hippocampus can selectively be modulated with a frequency as low as 10 Hz without a considerable modulation of the temporal cortex.Among the various brain structures, the head of the right hippocampus was determined as the target region of interest (ROI). Because the hippocampus is closely associated with important brain functions such as the declarative memory and cognitive function35; however, a recent study reported that injection of 3 mA current through electrodes with a diameter of 0.8 cm did not cause any side effects listed above36. In addition, a recent tACS study reported that high-frequency stimulation with 5 kHz alternating current proved to be safe in human subjects37. Based on these reports, it is expected that TI stimulation with 1 mA – 2 kHz conditions can be safely applied to human subjects although further animal studies to confirm the safety of TI stimulation would be needed before human trials.In our study, high-frequency alternating currents up to 1 mA were injected though small-sized scalp electrodes, the diameter of which was 1.0 cm. Traditionally, it was believed that the use of small-sized electrodes might potentially cause skin burn, skin redness, and pain27. However, it should be noted that 0.2 V/m is not a definite threshold value. Indeed, the modulation threshold value is still controversial and may vary individually38. For instance, a previous human experimental study showed that the electric field strength ranging from 0.15 to 0.21 V/m can show stimulation effect39. In addition, bi-hemispheric phase synchronization of cortical rhythms could be induced even with the electric field strength as low as 0.1 V/m40. On the other hand, a previous simulation study with a human head model demonstrated that amplitude-modulated tACS, of which the waveform is similar to that of TI stimulation, needs stronger stimulation intensity than the conventional tACS to expect similar stimulation effect41. Despite these studies, the modulation threshold to elicit neural state changes in the human brain with TI stimulation has not been revealed yet. It is also possible that the modulation threshold may be dependent on the brain states and brain tissues, making it difficult to determine a hard threshold value. In our simulations, we assumed that 0.2 V/m is a threshold value that can induce neural state changes regardless of brain tissues just to validate the possibility of optimal stimulation at a target deep brain area; however, this assumption should be updated reflecting new experimental results that will be reported in the future.In this study, 0.2 V/m was used as a minimal electric field constraint in the optimization process based on a previous study with animal experiments42. We investigated the maximum feasible electric field that can be formed at the target deep brain region. We optimized the stimulation parameters again with a new objective function to maximize peak TI envelope amplitude at the target with a constraint of PR > 1. As shown in the Supplementary Fig. sub2 head model. Note that the transcranial TI stimulation does not aim to directly induce action potential of neurons but modulate states of the neurons in specific brain areas like many other transcranial current stimulation methods13.On the other hand, the resultant TI envelope amplitude was not high enough to induce action potentials in the neurons, for which electric field of approximately 1 V/m is necessary43.Compared to other stimulation approaches , the optimized TI stimulation could successfully increase PR values for all three head models, which can be seen more clearly in Supplementary Fig. 13, which is in line with our results. We reinforced the arguments of the previous study with more numbers of head models, and further suggested the need for the individual optimization for improving the effectiveness of TI stimulation. One of the differences between our study and the previous study is that we used the approach of Huang and Parra12 whereas the previous study employed the approach of Grossman et al.10 for the calculation of the maximal TI envelope amplitude in the brain; however, the resultant TI envelop amplitude distributions obtained by both approaches were globally similar to each other13. Interestingly, optimized electrode configurations obtained from our study and the previous study13 are quite different even though the same target area was assumed. This difference may originate from the different objective functions employed for the optimization. The previous study employed the objective function that minimizes the volume of the whole brain with TI current larger than the modulation threshold. On the other hand, the objective function used in the present study mainly focused on minimizing the stimulation of cortical neurons.A previous study reported that TI stimulation with the optimized electrode conditions allows for delivering TI envelop amplitude larger than the modulation threshold and the optimized TI stimulation enables more focal stimulation of deep brain regions compared to tACS29, suggesting that the difference in individual anatomical structures can lead to inter-subject variability in the electric field distribution. In addition, it was also reported that there can be a gender difference in the strengths of electric fields at targeted regions due to the difference in the anatomical structures44. Since the three head models used in our study may not be representative of the general population, employment of more numbers of head models would allow for a better understanding of the TI stimulation as well as statistical comparisons among different stimulation conditions.The small number of head models employed in our simulations is a clear limitation of our study. Owing to the limited number of head models, it was difficult to estimate how much PR values vary across the population or evaluate the gender differences. Indeed, a previous study reported that individual local hotspot and strength of electric field in neocortical region were slightly different among head models even though the same regions were targeted with tDCS47. Nevertheless, the anisotropic tissue conductivity will be considered in our future studies to enhance the accuracy of customized TI stimulation. Thirdly, we demonstrated the feasibility of our optimization approach with two targets (the head and the tail of the hippocampus). Testing various deep brain areas would be also an important future research topic. Lastly, the underlying mechanism of the TI effect has not yet been understood well. The fundamental assumption of our stimulation study as well as the previous relevant studies12 was that the modulatory effect of TI stimulation originates from the low frequency component included in the TI waveform; however, the experimental results presented in the Grossman, et al.10 suggest that the actual physical situation might be more complicated. Therefore, it would be a challenging but important research topic to build a more refined computational model better reflecting the actual mechanism of TI stimulation.We expect that the efficiency of TI stimulation might be further enhanced by employing new approaches in future studies. Firstly, we empirically set the objective function as the ratio of the peak electric fields in the hippocampus and on the cortex. However, as observed in Figs. In this study, we demonstrated that individually optimized TI stimulation enables stimulation of the deep brain structure (the head of the right hippocampus in this study) while reducing the amplitude of TI envelope in neocortical regions. Although the difference between TI envelope amplitudes of the target and the neocortical regions was not markedly large, we believe that there is still a room to increase the difference by employing new optimization approaches and introducing multiple pairs of electrodes. Inconsistency of the optimal stimulation conditions among different head models suggested that customized TI stimulation based on the numerical analysis with individual head models might be necessary for effective transcranial TI stimulation. In the future study, the efficacy of the customized TI stimulation needs to be further validated via human trials.Supplementary Information.