In Tas1r2 null mice the proportion of glucose nonresponsive tanycytes was greatly increased in these mice, but a subset of tanycytes retained an undiminished sensitivity to glucose. Our data demonstrate that the sweet taste receptor mediates glucosensing in about 60% of glucosensitive tanycytes while the remaining 40% of glucosensitive tanycytes use some other, as yet unknown mechanism.Hypothalamic tanycytes are glial‐like glucosensitive cells that contact the cerebrospinal fluid of the third ventricle, and send processes into the hypothalamic nuclei that control food intake and body weight. The mechanism of tanycyte glucosensing remains undetermined. While tanycytes express the components associated with the glucosensing of the pancreatic β cell, they respond to nonmetabolisable glucose analogues via an ATP receptor‐dependent mechanism. Here, we show that tanycytes in rodents respond to non‐nutritive sweeteners known to be ligands of the sweet taste (Tas1r2/Tas1r3) receptor. The initial sweet tastant‐evoked response, which requires the presence of extracellular Ca The sweet taste receptors are a family of GPCRs well known for their ability to respond to sweet tasting compounds, including glucose. The sweet taste receptor is a heterodimer consisting of Tas1r2 and Tas1r3 receptor subunits .All experiments and procedures in this study were performed in strict accordance with the UK Animals (1986) Scientific Procedures Act and the project approved by the Animal Welfare and Ethical Review Board of the University of Warwick and under the authority of PPL 80‐2592, or with German national and institutional guidelines approved by the animal welfare committee of the Ministry of Environment, Health and Consumer Protection of the federal state of Brandenburg Germany (Permit Number 23‐2347‐A‐1‐2‐2010) and the animal welfare committee of the federal state of Hamburg  with additional 10 mM MgCl2. The dissected brain was then placed under a microscope and the meninges covering the ventral surface of the brain were carefully removed using fine forceps. Coronal sections 300 µm thick were prepared using a vibrating microtome (Microm HM650). Each section was subsequently dissected along the midline separating the third ventricle and incubated in 35°C aCSF for 30–60 min to allow for recovery of adenine nucleotide levels  Act 1986. The brain was rapidly dissected and placed in ice‐cold artificial cerebrospinal fluid  at the stereotaxic coordinates: bregma 0 mm; midline – 0.72 mm; dorsal surface – 2.3 mm. After the procedure, a single injection of Metacam (Meloxicam) injectable  was given to the animal. The animals recovered for a week, and then acute slices were made, as described above.AdV‐pTSHR‐GCaMP3 was made by previously established methods . The fixed slices were washed 3 times for 15 min with a phosphate buffer saline (PBS) solution. A blocking solution  was applied for 1 hr and then directly followed by the primary antibodies as follows: GFP  1:500 (ab6556) and Vimentin 1:500 (ab24525); GFP (1:500 ‐ ab6556) and NeuN 1:500 ; GFP (1:500 ‐ ab6556) and Hexon protein 1:1000 (Pa1‐7201); GFP (1:500 ‐ ab6556) and glial fibrillary acidic protein (GFAP) 1:200 . These primary antibodies have been well characterized as being specific by others: vimentin  for 90–120 min in 1.0 mM glucose aCSF. Loaded slices were transferred to a flow chamber containing circulating 1.0 mM glucose aCSF and imaged with an Olympus BX51 microscope using a 60× water immersion objective (NA 0.95). An Andor Ixon EM‐CCD camera was used to collect the images. A ratiometric image of Fura‐2 loading was achieved by illuminating at 340 and 380 nm with a xenon arc lamp (Cairn Research) and a monochromator .To image CaFor live imaging of slices derived from mice expressing GCaMP3, the slices were mounted on a Scientifica Slicescope and observed via an Olympus 60x water immersion objective (NA 1.0). Illumination was provided via a 470 nm LED  and a Hamamatsu ImageEM EM‐CCD camera was used to collect the images. Metafluor imaging software was used to control the illumination and camera in all experiments.2.6Glucose or non‐nutritive sweeteners were applied via puffing from a glass patch pipette . For puffing experiments 290 mM glucose, 290 mM sucralose were dissolved into a solution of 10 mM HEPES pH 7.4 to give a solution with an osmolarity of 300 mOsm. 165 mM acesulfame K (AceK) was dissolved in 10 mM HEPES solution, pH 7.4, to give a solution with an osmolarity of 300 mOsm. A solution of 300 mM rebaudioside A (RebA) was diluted 1:1 with aCSF to give a final concentration of 150 mM in the patch pipette. The sweeteners were puffed directly onto the tanycytes in a series of 300–500 ms pulses spaced about 2 s apart. We previously established that this method gives a 30‐40 fold dilution of the pipette contents at the slice—as measured for glucose puffs by using a glucose biosensor to verify the applied concentration  was added to the slice through the bathing medium for at least 20 min to ensure emptying of internal Ca2+ stores. To remove extracellular Ca2+ from the bathing media, the Ca2+ was replaced by 2 mM Mg2+ and 1 mM EGTA added to the aCSF. This zero Ca2+ aCSF was applied to the slice for 10 min prior to testing.ATP was applied to the slice through the bathing medium at a concentration of 10 µM. The P2Y1 receptor antagonist, MRS2500, was applied at 100 nM for ∼10 min prior to tastant stimulation. For experiments investigating the contribution of intracellular Ca2.8F340/F380). For GCaMP3, an initial fluorescent baseline signal was computed (F0) and subsequent fluorescent values (F) were normalized to this (F/F0). Analysis of imaging data was performed offline using ImageJ. Regions of interest (ROIs) were drawn around individual tanycytes  derived from 129 mice strain as template, to amplify a 1.2 and 2.0 kb fragment, respectively. Homologous fragments were cloned into pKO‐V901‐diphtheria toxin A‐chain (DTA) plasmid (Lexicon Genetics), next to the coding sequence of the mouse opsin mws and a Cre‐recombinase‐ and neomycin resistance gene‐containing selection cassette (ACN) flanked by LoxP sites  was used, amplified using oligonucleotides 5′‐TTCCCCGCCTGTCTGCTTTTCT‐3′ and 5′‐TCCATGTGGACCCCAGGCAAAT‐3′. Correctly targeted stem cell clone was injected into C57BL/6 blastocysts, and chimeric mice were bred with C57BL/6 animals. Genomic DNA of the offspring was genotyped by hybridization with an additional internal probe (probe II) on Southern Blot, validating the self‐induced deletion of the ACN selection cassette by the Cre‐loxP system. Internal probe was generated by using oligonucleotides 5′‐GGGACGTCGCCACCATGGCCCAAAGGCTTACAGGTGAACA‐3′ and 5′‐GGGAGATCTTTATGCAGGTGACACTGAAGAGACAGATGAGAC‐3′. Further genotyping based on PCR analysis with oligonucleotides 5′‐GACACCAAATGAATGGATGAGGCTG‐3′ and 5′‐TGAGGTGAGAGACGCTCTTCACGTT‐3′ for wild type and 5′‐GACACCAAATGAATGGATGAGGCTG‐3′ and 5′‐CCCAGAACGAAGTAGCCATAGATTTGG‐3′ for mutated allele, to amplify a 526 and 713 bp fragment, respectively. Heterozygous animals were interbred to produce homozygous offspring B6;129SvJ‐Tas1r2tm‐Opsin mws. Genomic modification was further approved on RNA level by Reverse transcription polymerase chain reaction (RT‐PCR) in situ hybridization, performed as described previously for Tas1r1 , whereas probes for opsin mws contained the regions encoding either for exon 2 or exon 5, amplified by using oligonucleotides 5′‐GGGACGTCGCCACCATGGCCCAAAGGCTTACAGGTGAACA‐3′ and 5′‐TAAGGCCAGTACCTGCTCCAACCAAAGAT‐3′, next to 5′‐AGACATCCTGTGGCCCAGACGTGTT‐3′ and 5′‐GGGAGATCTTTATGCAGGTGACACTGAAGAGACAGATGAGAC‐3′. Tissue used for RT‐PCR and in situ hybridization based on animals on mixed 129/Sv and C57BL/6 genetic background, whereas mice used for analysis of tanycytes were backcrossed 10 generations on C57BL/6 (B6.129SvJ‐Tas1r2tm‐Opsin mws).The strategy for generation of 2.10Tas1r2 null mice or from wild type C57BL/6J mice, which is the closest wild type match to the genetic background of the knock out mice. A standardized protocol  was used to test the responsiveness of slices from the two strains of mice. For analysis ROIs were drawn around every tanycyte in the field of view. This enabled quantification of the responses to glucose across all tanycytes (responsive and nonresponsive), only those tanycytes that responded, and the number of responsive and nonresponsive tanycytes in a slice. A total of 22 slices from 7 wild type mice and 24 slices from 8 Tas1r2 null mice were used. The wild type and Tas1r2 null mice were matched for age and body weight 25.5 ± 2.8 and 25.3 ± 2.9 g, respectively.Slices were cut from 2.11In the case of characterization of tanycytes responses to sweet tasting compounds and analysis of the signalling pathway one slice was regarded as an independent repetition. Thus responses from individual tanycytes in one slice were used to calculate a mean response for that slice. All graphs illustrate the median response with the error bars representing the lower and upper quartiles. Nonparametric statistics were used because many samples had wide variation in the magnitude of the control responses prior to experimental manipulations. To show that tanycytes respond to sucralose, AceK and RebA were compared with pooled data from control puffs. The Kruskal‐Wallis test was performed on the data followed by the Mann‐Whitney U test to identify whether responses to an individual sweetener were different from control or other sweeteners. For the matched inhibition studies Friedman 2‐way ANOVA was performed to compare the control, drug, and wash responses. The Wilcoxon Matched Pairs Signed Ranks test was then used for pairwise comparison of drug treatments if required. The False Discovery Rate procedure was used to check significance when multiple comparisons were made ; and a natural sweetener derived from 3.12+ reporter Fura 2‐AM. Tanycytes were identified by their morphology and location at the boundary of the third ventricle as previously documented . Interestingly the responses to AceK in tanycytes of mouse slices were much greater than those for rat. Tanycyte responses to non‐nutritive sweeteners are seen in both rats and mice and, at least in mice, are retained throughout juvenile and adult life. In a few experiments we cut parasagittal slices to image the apical surfaces of the tanycyte somata at the ventricular surface. This showed that the Ca2+ waves triggered by glucose and RebA spread radially in all directions .The responses to non‐nutritive sweeteners were not species or age specific. In slices derived from B6 wild type male mice aged between 6 weeks to 6 months Figure —sucralos2+ .In our studies, we have identified Fura 2‐loaded tanycytes by their characteristic morphology and position at the border of the third ventricle. To additionally verify that tanycytes do indeed respond to the non‐nutritive sweeteners, we targeted the expression of GCaMP3  and total responses . These data suggest that the responses evoked by activation by AceK and RebA most likely involve mobilization of Ca2+ from intracellular stores and an influx of extracellular Ca2+, possibly through G protein‐mediated activation of a nonselective cation channel.We next investigated whether the sweet responses required extracellular Cat Figure a. The reA Figure b. Removas Figure c. This i3.4Tas1r2 and Tas1r3. With the aid of a microscope, we dissected tissue immediately adjacent to the boundary of the third ventricle to obtain samples highly enriched in tanycytes and compared this to the more lateral regions of the hypothalamus and the tongue. We found Tas1r2 and Tas1r3 expression in the tanycyte layer, the wider hypothalamus and, as expected, in the tongue . When the magnitude of only the cells that responded to glucose was examined the mean change in F340/F380 was 0.06 ± 0.015 (n = 7) in wild type and 0.064 ± 0.017 (n = 8) in the Tas1r2 null mice  in wild type and 0.0184 ± 0.0224 (n = 8) in the Tas1r2 null mice . Given that these compounds are unlikely to enter the brain in any appreciable quantities following ingestion into the gut  are present in tanycytes  or glucose insensitive (the majority). If both mechanisms were to coexist in the same tanycytes then a partial loss of sensitivity would be apparent in all glucosensitive tanycytes. It is interesting to note that this may be a further similarity between tanycytes and taste cells. Taste cells also possess two distinct sugar sensing mechanisms: the Tas1r‐dependent pathway; plus a second pathway that is independent of Tas1r but instead involves glycosidases, GLUTs, SGLT1, and an ATP‐gated K+ channel and is thus reminiscent of the glucose sensing mechanism in the pancreas (Sukumaran, Yee, Iwata, Kotha, Quezada‐Calvillo, Nichols, … Margolskee, Previous authors have suggested that tanycytes sense glucose in a manner analogous to the pancreatic β cell, because the components of that mechanism (the relevant glucose transporters, glucokinase, the K4.2Tas1r2 gene affects the regulation of insulin secretions from pancreatic β cells (Kyriazis, Soundarapandian, & Tyrberg, Tas1r2 gene alters how mice adapt to a high fat low carbohydrate diet (Smith, Hussain, Karimian Azari, Steiner, Ayala, Pratley, & Kyriazis, Several reports have documented the expression of Tas1r receptor family subunits in extraoral tissues such as the gut, pancreas (Kyriazis, Soundarapandian, & Tyrberg, 4.3+ channels (Dunn‐Meynell, Routh, Kang, Gaspers, & Levin, + channel and an additional mechanism involving the sodium‐linked glucose transporter (Gonzalez, Reimann, & Burdakov, A variety of cells sensitive to glucose have been described in the CNS. In the arcuate nucleus and ventromedial hypothalamus, neurons excited by glucose use a mechanism dependent on glucokinase and ATP‐sensitive KSupporting Information Movie 1Click here for additional data file.Supporting Information Movie 2Click here for additional data file.Supporting Information Movie 3Click here for additional data file.


Among these, the nitrogen and carbon interstitial compounds exhibit all of the required properties such as a saturation moment of 1.6 T, Curie temperature of 700–750 K, however easy magnetic axis lies in the planar direction making them less attractive for permanent magnet applications. The calculated magnetocrystalline anisotropy energy is found to be −2.7 MJ/m3 for Nd2Fe17C3 and −4.7 MJ/m3 for Nd2Fe17N3. We further explored the possibility of changing the easy axis direction through La/Ce alloying at Nd site. Although the MAE is found to be smaller in magnitude for all the La/Ce alloys it still maintains planar direction.The electronic and magnetic properties of Nd A large value of MAE is essential for wide range of applications, from permanent magnets to magnetic storage devices8. The SOC is strong in heavy elements such as rare-earth compounds which consequently exhibit large MAE. Together with large MAE a good permanent magnet requires large value of magnetization, large coercivity, high temperature stability and high Curie temperature7. For example, one of the best magnetic materials, Nd2Fe14B, has Tc 588 K, Ms 1.28 MA/m, K1 4.9 MJ/m3 and an easy magnetic axis along the c-axis9.The Magnetocrystalline anisotropy energy (MAE), the energy required to switch crystal magnetization from the favorable direction to other spatial directions is the most important property of a ferromagnet. As formulated by Van Vleck2Fe17 is another Nd based interesting magnetic material. Nd2Fe17 is known to crystallize in rhombohedral Th2Zn17 structure type with the space group R−3 m. This compound has gained back interest since it was shown that insertion of interstitial atoms could raise the usually low Curie temperature (Tc) of this ferromagnetic compound. In the case of Nd2Fe17, carbon and nitrogen insertion increases Tc from 325 K to 658 K for carbon interstitial (Nd2Fe17C2) and to 732 K for nitrogen interstitial (Nd2Fe17N2.3) compound10. Although both carbon and nitrogen interstitial compounds exhibit high magnetic moments and Curie point15, unlike Sm2Fe17C3(N3)19 they suffer from planar MAE. One possibility for changing the easy axis direction is through alloying. Previous theoretical calculations21 for these compounds treat Nd f electrons as core electrons  which do not take part in hybridization. However for the rare earth elements the most of the contribution to MAE is shown to be from f electrons, hence a proper treatment of f electrons is crucial for accurate description of MAE24. To this end, here we have applied a Hubbard U correction on the Nd-f orbitals which splits the f bands into lower and upper Hubbard bands. Our results are in better agreement with experiments than previous theoretical studies where f orbitals were treated under open core approximation rather than as valence electrons. We also investigate the effect of elemental substitutions of La and Ce on Nd site on MAE. Our calculations show these substitutions do not switch the easy axis direction.Nd2Fe17 and its carbides and nitrides. To estimate the ground state, calculations were performed by assuming, non magnetic, ferromagnetic , and anti-ferromagnetic  configurations. For all three systems Nd2Fe17, Nd2Fe17C3, and Nd2Fe17N3 we find the ground state to have the Nd spin moment opposite to that of the Fe and energy difference between ground state and non magnetic was found to be as 360, 460, and 510 meV on per Fe atom basis. The calculated magnetic moments are summarized in Table 2Fe17N3 and Nd2Fe17C3 is presented in Fig. L) is positive for rare earth atom, indicating that the direction of the orbital magnetic moment is opposite to the spin magnetic moment. This is consistent with the Hund’s rule for half filled rare earth ions. The calculated orbital moment of Nd atoms without U is 1.49 μB for both Nd2Fe17N3 and Nd2Fe17C3. On varying U in GGA + SOC + U calculations, although the orbital moment of Nd atoms increases, overall the orbital moment displays a weak dependence on U. The rate of increase of orbital moments with U is higher for carbon interstitial compound than in nitrogen interstitial compound. The variation of Nd spin moments with U parameter is also shown in the Fig. We begin our study by determining the ground state for Nd2Fe17 lie between 0.04 to 0.05 μB. On introducing interstitial nitrogen/carbon atom the Fe orbital moments are increased, and the enhancement is higher for Nd2Fe17C3. The total magnetic moments for the crystallographically nonequivalent Fe sites are plotted in Fig. f and Fe-18h sites, which are close to N/C atoms decreases, the moments on the distant Fe-9d site are enhanced as shown in Fig. 21. Similar variation in magnetic moments upon nitrogenation and carbonization has also been observed for Sm2Fe17. Regardless of this slight variation the average Fe-spin moment of these systems remains in the range of 2.3–2.5 μB, which is significantly higher than the value for BCC Fe 2.2 μB. The experimental values of Fe moments for Nd2Fe17N3 by neutron powder diffraction measurements show large variation depending on the stoichiometry of the nitrogen in the system. For example the total magnetic moments of Nd2Fe17N2.85 and Nd2Fe17N2.91 are 32.5 μB and 40.6 μB, respectively19, indicating the sensitivity of magnetic properties on nitrogen stoichiometry. This large variation is mainly due to the difference in Fe-Fe distance depending upon on nitrogen stoichiometry. As explained above due to the sensitivity of magnetic properties on nitrogen/carbon content, a good quantitative agreement between experiments and theory is unlikely. Nonetheless our calculated total magnetic moment of 36.3 μB for Nd2Fe17N3 lies well within the range proposed by experimental measurements as shown in Table f electrons, Nd atom has a large (~1.47 μB) orbital moment. For both carbon and nitrogen interstitial compounds the orbital moment of Nd is slightly increased from 1.47 μB to 1.50 μB. The magnetic moment for La/Ce substituted compounds are also listed in Table f electrons and Ce has only one outermost f electron.The calculated Fe orbital moments for Nd1, which is shown in Table f electrons is essential by applying a onsite Hubbard U parameter. Here a U value of 3 and 5 eV is used for Ce, and Nd, respectively. The convergence of MAE with respect to U parameter is shown in Fig. 2Fe17N3 where the experimental data of MAE is available, our calculated MAE with UNd as 5 eV is in reasonable agreement with the experimental data. We found the parent compound Nd2Fe17 to be planar with K1 value as −2.1 MJ/m3, which is in fair agreement with the experimentally measured value of −3.7 MJ/m312. Unlike Sm2Fe17 where carbon and nitrogen interstitial atoms switch the direction of MAE from planar to axial, Nd2Fe17N3 and Nd2Fe17C3 does not exhibit this change in MAE sign and still maintain planar anisotropy. This planar anisotropy is less desirable for permanent magnet applications. However the magnitude of K1 is quite significant and a value of −2.7 MJ/m3 and −4.7 MJ/m3 was obtained for Nd2Fe17C3 and Nd2Fe17N3, respectively. The measured anisotropy values for Nd2Fe17N3 are also listed in Table 1 one might obtain a coercivity higher than 1.5 Tesla in these compounds if the MAE was uniaxial.Next, we calculate the magnetocrystalline anisotropy energy (MAE) constant K17X3 and NdCeFe17X3 (X = C or N) with uniaxial anisotropy. We thus began with substituting one out of two Nd atoms in the primitive cell by La/Ce. Results of these calculations are summarized the in Table To explore the possibility of switching the sign of MAE and reducing the rare earth content in these magnets next we study the effect of La/Ce substitution on MAE. As the MAE is sensitive to the nature of electronic structure around the Fermi energy, it can be controlled by tuning the band structure around the Fermi energy. In practice this can be done, for example, by doping/alloying. Theoretical calculations were done in order to find a possible alloy based on NdLaFe2Fe17N3. The total DOS illustrates behavior of a ferromagnetic system. In the Fig. f and Fe-3d states are shown. On comparing the total DOS with partial DOS in the lower panels, we can see that the states in the vicinity of the Fermi level are mainly composed of Fe-3d states. The RE-4f states are not occupied in the spin-up channel and are partially occupied in the spin-down channel, confirming that the Nd spin-moments align in the opposite direction compared to the Fe moments. Due to Hubbard U parameter the Nd spin down band split into lower and upper Hubbard bands separated by 6 eV  in Fig. 2Fe17N3 and Nd2Fe17N3 respectively. The force theorem calculations produce a MAE of 12.1 MJ/m3 for Sm2Fe17N3, and −4.1 MJ/m3 for Nd2Fe17N3, which is in nice agreement with the values obtained from total energy calculations. Since the total MAE is positive for Sm2Fe17N3, as can be seen in Fig. 2Fe17N3 although the overall profile of MAE with respect to k-points remains the similar as Sm2Fe17N3, MAE takes negative values in the majority of brillouin zone.The important insights into the effect of SOC on magnetic properties can be obtained from perturbation theory2Fe17N3, and Sm2Fe17N3. As shown in the Fig. EF), the majority spin DOS is similar for the two compounds. However, as Sm is exchanged for Nd more electrons are added into the system and the minority spin states become occupied, whereby these 4f levels are shifted more towards the left in the middle panel of Fig. f level in the minority spin DOS of Sm2Fe17N3 shifts towards Fermi level. The Fe-DOS for both majority and minority spin in both Nd2Fe17N3, and Sm2Fe17N3 is quite similar. The important observation is the presence of minority 4f spin channels at the the Fermi level of large uniaxial anisotropy in Sm2Fe17N3, although other differences in the electronic structure are also important.Figure et al.26 by using perturbation theory that if deformations of the Fermi surface can be neglected and the MAE is dominated by spin-diagonal coupling, the MAE and orbital magnetic-moment anisotropy are proportional. The relation between MAE and orbital magnetic anisotropy is presented in Fig. 2Fe17C3, and Sm2Fe17N3 where a uniaxial anisotropy is obtained the orbital magnetic moments is also maximum along easy axis. On the other for Nd2Fe17C3, and Nd2Fe17N3 where the anisotropy is in hexagonal plane, orbital magnetic moment is maximum along the hard magnetization axis.The MAE of a permanent magnet is dependent on SOC, and can be further quantified by considering its dependence on orbital moments. As shown by Bruno 2Fe17, Nd2Fe17X3 and related compounds. For all compounds the rare earth and Fe atoms spin moments are anti-aligned in the ground state. The calculated magnetic properties of the base compounds agree well with available experimental data. The treatment of the Nd-f electrons in our calculations is different from the previously reported first principles calculations. For example the calculation of Lai et al.15 which were performed within orthogonalized linear combination of atomic orbitals (OLCAO) method, reported a decrease in total magnetic moments from 37.3 μB/unit-cell (for to Nd2Fe17) to 36.3 μB/unit-cell (for Nd2Fe17N3) which is contradictory to the experimental observation19, where on nitrogenation an increase in total magnetic moment has been reported. As shown in Table 2Fe17 upon nitrogenation (for Nd2Fe17N3) is nicely captured by our GGA + SOC + U calculations. Similarity Drebov et al.20 used open-core method for treatment of f-electrons by assuming RE3+ configuration. Such treatment of f electrons cannot reproduce some of the observed magnetic properties. For instance within open core method a moment of 2.5 μB has been reported on Nd site, which is overestimated on comparing with the available experimental data19 (1.6 μB). On the other hand, within our calculations the f-electrons are treated as valence electrons and, as a result we could correctly reproduce the reported moment for Nd site. Overall our all-electron calculations can reproduce a number of experimentally observed properties. For example, experimentally19 for Nd2Fe17 Fe-9d site has the lowest magnetic moment of 1.6 μB. Upon introducing the interstitial nitrogen the moment of Fe-9d increases significantly and for Nd2Fe17N3 the Fe-9d site has the maximum moment. This is nicely produced by our calculations as shown in Fig. 2Fe17 and Nd2Fe17N3 is in fair agreement with the available experimental data as shown in Table We present a computational study of intrinsic magnetic properties such as saturation magnetization and MAE for Nd2Fe17X3 compounds has many of the desirable properties for a permanent magnet such as high Curie point, large saturation magnetization, the easy axis of magnetization lies in the hexagonal plane hindering it practical application. To investigate the possibility of switching the MAE direction from hexagonal plane to c-axis we also investigated the effect of La/Ce substitution at Nd site. These calculations indicate that although the MAE on La/Ce substitution is reduced by 30–40%, its still lies in the hexagonal plane, which is not very promising for technological applications. The MAE has been also analyzed in terms of the electronic structure and by using the magnetic force theorem to compute k-point resolved contributions to the MAE. For Nd2Fe17N3 the density of states at the Fermi energy is dominated by 3d states, 4f states only contribute notably to the MAE in small regions of the Brillouin zone, however for Sm2Fe17N3 the DOS around the Fermi level is dominated by both Fe-3d, Sm-4f states. As the Fe-d states exhibit similar features for Nd2Fe17N3 and Sm2Fe17N3, the origin of uniaxial MAE could lie in the 4f-3d hybridization.Although Nd31 (GGA) were performed with the full-potential linearized augmented plane waves (FP-LAPW) method as implemented in WIEN2k34. The sphere radii were set to 2.50, 1.88, 1.61, and 1.61 Bohr for Nd, Fe, N and C. All calculations were performed with the experimental lattice parameters36. The structure relaxations were performed within spin-polarized calculations without spin orbit coupling and all internal coordinates were relaxed until internal forces on atoms were less than 1 mRyd/Bohr. For structural relaxation 500 k-points were in the full Brillouin zone. SOC was included in the MAE calculations within a second variational approach37. For all the calculations a RKmax = 7 was used. RKmax is typically defined as the product of the smallest muffin-tin sphere and the largest reciprocal lattice vector, and describes the basis set size for a calculation. For La/Ce substitution at Nd site, one out of two Nd atoms in the primitive cell was replace by La/Ce. These substituted structure were subsequently relaxed to their ground state by minimizing the forces on all the atoms.Density functional theory (DFT) calculations in the generalized gradient approximationa − Ec, where Ea and Ec are the total energies for the magnetization oriented along the a and c directions, respectively. Positive (negative) K corresponds to uniaxial (planar) anisotropy. For MAE calculations the convergence with respect to K-points was carefully checked all the MAE results reported in this paper correspond to 2000 reducible K-points in full Brillouin zone. To correctly treat the strong interactions between the Nd/Ce-f electrons, the Hubbard “U” correction was applied with UNd = 5.0 eV, and UCe = 3.0 eV with the Hund’s coupling parameter J as zero. For DFT + U calculations, the standard self interaction correction (SIC) method39 was used where onsite Coulomb interaction for localized orbitals is parametrized by Ueffective = U − J. The values of U parameter were obtained by optimized various magnetic properties with respect to available experimental data, and lies in the typical range that has been used successfully to describe various properties of Nd/Ce compounds before43.All the calculations are performed in the collinear spin alignment. The magnetic anisotropy energy (MAE) is obtained by calculating the total energies of the system with spin obit coupling (SOC) as K = E


The concentrations of TNF α were significantly elevated in patients with malignant tumors of the adrenal cortex and in patients with Conn’s syndrome compared to control. In patients with non-functioning adenomas and pheochromocytomas, TNF α levels were similar to those detected in the control. In subjects with myelolipomas, the serum concentration of TNF α was lower compared to the control. After adrenalectomy, the levels of TNF α were decreased in patients with malignant tumors and in patients with Conn’s syndrome, nonfunctioniong adenomas and pheochromocytomas compared to the concentration before surgery. The serum concentrations of soluble receptors of TNF α did not differ among different patient groups and compared to the control. After adrenalectomy, the blood concentrations of TNF α R1 and TNF α R2 were decreased in patients with Conn’s syndrome. However, to confirm practicality of the evaluation of TNF α and its soluble receptors in differential diagnosis in patients with adrenal tumors, a larger study group is needed.The peripheral blood levels of TNF  Most of them are incidental findings. Although the majority of adrenocortical and adrenomedullary tumors are benign, there are no clinical and laboratory markers to distinguish most of them from malignant tumors ,2. The mα is a pleiotropic cytokine that plays a central role in inflammation and apoptosis. It modifies the inflammatory reactions and immune reactions in response to injury and infection . Seven patients with malignant cortical tumors, seven patients with Conn’s syndrome, 10 patients with non-functioning adrenal adenomas, 12 patients with pheochromocytomas and three patients with myelolipomas (according to pathology data) were studied. The clinical diagnosis of the malignant tumors was suspected in patients with weight loss, high serum concentration of dehydroepiandrosterone sulfate and irregular, large  mass found by computer tomography imaging. The diagnosis of the Conn’s syndrome was based on elevated level of plasma aldosterone, low level of plasma renin and an aldosterone/renin ratio of greater than 5.4. The diagnosis of pheochromocytoma was suspected in patients with poorly controlled hypertension associated with bouts of sweating, headache, palpitations, a high serum concentration of chromogranin A and a high level of metanephrins in urine. In patients with nonfunctioning adenomas and myelolipomas, the tumors were incidentally found during computer tomography imaging.The study group was composed of 39 subjects [28 females aged 25–82  aged 36–60 (49.5 ± 8.38). The presence of adrenal tumor was excluded on the basis of physical examination, laboratory tests and ultrasound examination of the abdomen. All patients and control group were included in the study only after giving informed consent. The study was approved by the Local Ethical Committee.α and its soluble receptors were measured by enzyme immunoassay (ELISA) . The intra-assay precision for TNF α was 4.67%, for TNF α R1 – 4.43%, and for TNF α R2 was 3.53%. The inter - assay precision for TNF α was 5.8%, for TNF α R1 – 6.1%, and for TNF α R2 was 4.07%. The statistical analysis was performed using the non - parametric Kruskal-Wallis test and Wilxocon test. A p-value of less than 0.05 was considered statistically significant.The concentration of TNF 3.α, TNF α R1 and TNF α R2 before surgery and after surgery are presented in x ± SD) concentration of TNF α in the control group was 12.09 ± 4.31 pg/mL, the mean concentration of TNF α R1 was 1,479.2 ± 388.23 pg/mL, and the mean concentration of TNF α R2 was 2,456.7 ± 521.12 pg/mL. The concentration of TNF α was significantly elevated in patients with malignant tumors of the adrenal cortex, compared to the control , and in patients with Conn’s syndrome, compared to the control . The highest levels of TNF α in patients with malignant tumors of the cortex were noted. In patients with non-functioning adenomas and pheochromocytomas, TNF α levels were not significantly different from those of the control, (p > 0.05) and were respectively 11.0 ± 2.03 pg/mL; 9.14 ± 4.58 pg/mL. In patients with myelolipomas, the serum concentration of TNF α was lower, compared to control  (The concentrations of TNF  < 0.05) .α were lower in patients with malignant tumors , and in patients with Conn’s syndrome , non-functioning adenomas  and pheochromocytomas  than before surgery    and 6.α and its receptors were found.No significant correlations between the size of the tumors and the serum level of TNF 4.α is a transmembrane protein described originally as a protein which causes the haemorrhagic necrosis of experimental tumors [α possesses proinflammatory activity and plays a key role in apoptosis [α and its receptors may increase susceptibility to infections [α is also probably involved in development of lymphoid tissue [TNF l tumors . Furtherpoptosis –12. A lofections . TNF α id tissue .α in pathogenesis of malignant tumors is not clearly understood. Mocellin and Nitti [α may represent one of the molecular links between chronic inflammation and further development of malignant tumors. Leibovich et al. [α may promote angiogenesis but others [α. Schweigerer et al. [α on the proliferation of endothelial cells derived from blood vessels of the brain and adrenal cortex. In both types of these cells, TNF α decreased the proliferative activity of endothelial cells.The role of TNF nd Nitti  suggest h et al.  revealedt others  did not r et al.  assessedα acts also as a paracrinal and autocrinal regulator of the growth and development of the adrenocortical cells. Call et al. [α and IL-6. They also proved, using immunoassay techniques, that the glomerular zone of the adrenal cortex is the site of the greatest synthesis of TNF α. The increased level of TNF α in our study in patients with Conn’s syndrome may confirm that the glomerular zone of the adrenal cortex mainly secretes TNF α. The secretion of TNF α by human adrenal glands confirmed the studies of González-Hernández et al. [et al. [α has an influence on synthesis and secretion of steroid hormones in adrenal cortex [α occur in the blood of patients with malignant tumors. Cimino et al. [α  in the blood of patients with acute leukemia. Elevated levels of TNF α were also confirmed in the peripheral blood of patients with melanoma [TNF l et al.  revealedz et al. ,20 and P [et al. . It is al cortex . Moreoveo et al.  observedmelanoma , renal cmelanoma  and pancmelanoma .α in patients with malignant tumors . It may be due to activation of the immunological system and cytokine net in patients with malignant tumors. The elevated levels of TNF α in patients with malignant tumors may be also due to activation of angiogenesis and overexpression of TNF α in the transformed cells. Interferon γ is known as the most important factor to increase the expression of TNF α. The regulation of the expression is at the transcriptional level [α may lead to overexpression of integrins and adhesive molecules [et al. [α on the expression of ZC3H10 (zinc finger protein) and GRHL-3 (grainyhead-like 3) in breast cancer cell lines (MCF-7). Expression of these proteins was increased after addition of TNF α to the incubation medium of the cells in vitro. However, ZC3H10 acts as an antagonist of cell growth in vitro, but GRHL-3 stimulates the migration of the endothelial cells.The results of our study showed the highest level of TNF al level . It has olecules . Guardio [et al.  assessedα was decreased after adrenalectomy. These results may be due to elimination of the tumor, which was the main local source of TNF α.In our patients with malignant tumors of the adrenal cortex, in patients with Conn’s syndrome and in patients with non-functioning adenomas and pheochromocytomas the concentration of TNF α in the pathogenesis of adrenal tumors is unknown. Elevated levels of these receptors were found in the amniotic fluid of pregnant women [α receptors may bind TNF α and inactivate this molecule. On the other hand, these forms of TNF α receptors can stabilize the TNF α molecule at low concentrations of TNF α [The role of the soluble receptors of TNF nt women ,31, in tnt women  and patint women . Some auof TNF α .α receptors did not differ, either among study groups or compared to the control. After adrenalectomy, the levels of the soluble forms of TNF α decreased in patients with Conn’s syndrome. It could be argued that the soluble forms of TNF α receptors are also involved in the process of tumorigenesis. The low level of TNF α and high level of soluble forms of TNF α receptors in patients with myelolipoma may confirm the hypothesis that soluble forms of these receptors inactivate this molecule. However, such a small group (3 patients) is not sufficient to draw any significant conclusions.The results of our study revealed that the concentration of soluble forms of TNF α and its soluble receptors in differential diagnosis in patients with adrenal tumors, a large study group is needed. The evaluation of the concentration of numerous cytokines in the blood has been used only in the clinical studies so far. To take advantage of these molecules in clinical practice, further studies are needed and clinical examinations, laboratory tests and imaging techniques will still be the most important means of diagnosing patients with adrenal tumors.To confirm practicality of the evaluation of TNF 5.α and its soluble receptors appear to be involved in adrenal gland oncogenesis. The peripheral blood concentrations of this cytokines before surgery may be useful for the discrimination between malignant and benign adrenal tumors.TNF


Employing recombinant (r) CK18 and caspase cleaved CK18 (CK18 Asp396 neo-epitope) as external standards, kit to kit reproducibly was <6% (n=19). rCK18 was stable in plasma for 4 months at −20°C and −80°C, for 4 weeks at 4°C and had a half-life of 2.3 days at 37°C. Cytokeratin 18 Asp396 NE, the M30 Apoptosense Elisa assay antigen, was stable in plasma for 6 months at −20°C and −80°C, for 3 months at 4°C, while its half-life at 37°C was 3.8 days. Within-day variations in endogenous plasma concentrations of the M30 and M65 antigens were assessed in two predose blood samples collected from a cohort of 15 ovarian cancer patients receiving carboplatin chemotherapy and were shown to be no greater than the variability associated with methods themselves. Between-day fluctuations in circulating levels of the M30 and M65 antigens and in XIAP mRNA levels measured in peripheral blood mononuclear cells by quantitative (q) RT–PCR were evaluated in two predose blood samples collected with a 5- to 7-day gap from 23 patients with advanced cancer enrolled in a phase I trial. The mean variation between the two pretreatment values ranged from 13 to 14 to 25%, respectively, for M65, M30 and qRT–PCR. These data suggest that the M30 and M65 Elisa's and qRT–PCR as PD biomarker assays have favourable performance characteristics for further investigation in clinical trials of anticancer agents which induce tumour apoptosis/necrosis or knockdown of the anti-apoptotic protein XIAP.Data are presented on pharmacodynamic (PD) method validation and preliminary clinical qualification of three PD biomarker assays. M65 Elisa, which quantitates different forms of circulating cytokeratin 18 (CK18) as putative surrogate markers of both apoptotic and nonapoptotic tumour cell death, was shown to be highly reproducible: calibration curve linearity  For PD A vital component in PD biomarker validation is the establishment of its pretreatment range and inherent biovariability, in order to assess the likelihood that the technology of choice and trial design have the statistical and technological resolving power to discriminate between normal background biologic variation and a drug-induced effect  and certified low and high quality controls , and was operated according to the manufacturer's instructions. In brief, 25 μl of sample  was added to each well, which was coated with a mouse monoclonal ‘catcher’ antibody that binds to an epitope on CK18  solution was added to act as the detection antibody  for XIAP mRNA analysis were collected from a cohort of cancer patients entered into the AEG35156 Phase I trial. Two predose samples were obtained from the patients with normally a 5- to 7-day gap between collections as part of the full PD protocol. Venous blood samples were collected into 2.5 ml PAXgene Blood RNA extraction tubes . The PAXgene tubes were inverted 8–10 times and cells lysed at room temperature for 2–24 h before being frozen at −20°C after which they were transferred to −80°C storage.g for 20 min in a swing out rotor and the supernatants discarded (IEC Centra GP8R bench top centrifuge). Pellets were resuspended in RNase-free water (5 ml), further centrifuged as above and finally resuspended in 360 μl buffer BR1  by vortexing. Tubes were then incubated for 10 min at 55°C in a 280 g shaker-incubator with 40 μl Proteinase K and 300 μl buffer BR2. At the end of the incubation 350 μl of 100% ethanol was added and the total sample was then applied on to the PAXgene extraction column together with 350 μl of buffer BR3 and centrifuged at 8000 g for 1 min in a minifuge discarding the flow through waste. Next the extraction columns were incubated with DNase I (10 μl DNase I stock solution to 70 μl Buffer RDD) for 15 min at room temperature. Extraction columns were then washed with 350 μl buffer BR3 as above, followed by two washes and spins with 500 μl of buffer BR4, the later performed at maximum speed (16 000 g) for 3 min to dry the PAXgene column membrane. The columns were finally eluted with 40 μl of buffer BR5 and the eluent collected in a 0.5 ml tube after a spin at 8000 g for 1 min. The final eluent was incubated for 5 min at 65°C in a heating block, then chilled on ice and the harvested RNA was stored at −80°C. qRT–PCR was then performed essentially as described in detail  value of 0.996±0.003 (n=27). The linear response is believed to be due to the high affinity constants (10−13 M) of the two mouse monoclonal antibodies utilised, M6 and M5  . This bias was introduced exclusively by kits from the earliest production batch, and appeared to be eliminated in the subsequent two production batches. Utilising the external standards, mean kit to kit variations in the concentration of antigen determined was 5.3% with rCK18 and 4.8% with CK18 Asp396 NE, normally well within the manufacturer's acceptance criteria of 10%, (http://web.peviva.se/index.asp).The M65 Elisa also proved to be highly reproducible with precision and accuracy (P and A) based on values obtained using the in kit QCs (200 and 1000 U l−1) . Furtherasma see . These s396 NE), their readouts are directly comparable and the ratio of the signal measured by each enables differentiation between cell death modes  (data not shown). At 4°C clear fluctuations in concentration were recorded, whereby after 4 months a small but significant reduction in value was observed followed by a larger increase after 6 months.The M30 antigen (CK18 AspP<0.01) reduction in concentration of full-length rCK18 occurred after 6 months at both −20°C and −80°C    indicati−1, 396 NE  for its epitope. Possible explanations for the increase in concentration are currently under investigation.An unexpected finding of this work was that statistically significant increases in concentration of both intact CK18 and its caspase cleaved fragment occurred during the stability studies. A similar effect has also been observed in long--term stability of CK18 AspWithin-day variations in the baseline levels of circulating M30 and M65 antigens detected in a cohort of 15 ovarian cancer patients are presented in In the case of between-day , a much Traditionally, detection of a keratin antigen in serum or plasma, such as tissue polypeptide antigen , TPS (CK18 fragments and complexes) and CYFRA 21-1 (caspase cleaved CK19), has been viewed as a marker of bulk tumour burden  is to found in The measurement of XIAP mRNA expression in PBMCs represents the primary PD assay being utilised in the AEG35156 trial . TherefoIt should be noted that the number of patients employed in both trials to evaluate variability in baseline values of M30 and M65 antigens and XIAP mRNA are based on typical phase I trial numbers (15–23). The present studies are intended as a first stage, and have yielded encouraging results. We are in the process of conducting further and larger clinical studies utilising the M30 and M65 assays as biomarkers of tumour cell death, not just from the point of view of method validation and baseline studies, but eventually to provide definitive proof or otherwise as to the clinical validity and utility of M30 and M65 as surrogate PD biomarkers of tumour cell death – biomarker qualification (396 NE in human plasma. The stability studies have revealed that caspase cleaved CK18 is more stable than the intact protein and that long-term storage of samples is not recommended for more than 4 months even at −20°C and −80°C. Finally, we show in patient studies that the typical baseline variation in the analytes of the M30 and M65 Elisa's and a qRT–PCR method for XIAP mRNA is comparatively small at 14, 13 and 25%, respectively.In summary, data are presented demonstrating that the M65 Elisa is a highly reproducible assay for the determination of CK18 and CK18 Asp


All patients were postmenopausal women presenting with large ERα-positive breast cancers. Clinical response to treatment was assessed by tumour volume changes as determined from sequential ultrasounds and pathological response by comparison of the tumour morphology before and after treatment. Of 33 cases, 23 (70%) were classified as having a clinical response and 16 (48%) as having a response pathologically. All tumours stained positively for ERα and ERβ and 15 out of 33 (45%) for ERβ2/βcx. There were no significant differences in quantitative expression of any receptor between tumours that subsequently responded and that did not, whether response was assessed clinically or pathologically. Tamoxifen treatment was associated with a decrease in ERα, but an increase was the most frequent change (17 out of 33) in ERβ, and no consistent change was evident in staining of the ERβ2/βcx variant. In summary, ERβ1 and ERβ2/βcx variant protein are detected in ERα-positive breast tumours but their expression is not associated with a response to tamoxifen. Differential changes in ERα and ERβ were seen with treatment.In order to elucidate the relative importance of oestrogen receptor (ER) Howevery β gene . Studiesβ, hereafter referred to as ERβ1, has been used previously to delineate the pattern of expression of this isoform of the receptor in breast cancer biopsies  and had no evidence of distant metastatic disease. None had received prior treatment with hormonal agents for breast cancer or were taking hormone preparations at the time of study. Tumour size was monitored clinically  and by breast ultrasound before and at monthly intervals during treatment. Therapy comprised daily administration of tamoxifen (25 mg) for 3 months; four patients electively continued on therapy for a further 3 months. A total of 72 consecutive patients were entered into the study; however, data are presented in 33 for ERα and ERβ1 and the ERβ2/βcx variant because in the remainder either pre- and/or post-treatment tumour blocks were exhausted by use for other studies. The demographics of the investigated cases were not different from the recruited population. The studies were performed with the patients’ informed consent and ethical permission (LREC nos. 2001/8/80 and 2001/8/81).All patients were referred between 1992 and 1995 to the Edinburgh Breast Unit and had histologically confirmed diagnosis of breast cancer  ethanol before processing into paraffin wax at the Department of Pathology using standard procedures.Samples of each breast cancer was obtained by biopsy (before treatment) and by definite surgery  after treatment. Tumours were fixed in 10% neutral buffered formaldehyde for 16–24 h, then stored in 70%  was obtained from Dako . Monoclonal antibodies specific for C-terminal peptides within wild-type human ERβ  were mounted on Superfrost coated slides , dewaxed and rehydrated in gradient alcohols and distilled water before staining with specific antibodies as outlined below.Sections , and applied to the sections for 60 min at room temperature. The secondary antibody, biotinylated anti-mouse Ig  was diluted 1 : 2000 in ‘background reducing diluent’ (Dako) and applied to the sections for 30 min at room temperature. The tertiary system  was applied as per the manufacturer's instructions for 30 min at room temperature. The tissue was visualised by immersing sections in 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 5 min. Sections were counterstained using Mayers haematoxylin , dehydrated through gradient alcohols and mounted.All staining for ER2O. Antigen retrieval was carried out by pressure cooking in 0.05 M glycine 0.01% EDTA pH 3.5 for 3 min setting 2  and sections left to stand undisturbed for 20 min. Sections were blocked for 30 min in normal rabbit serum  diluted 1 : 4 in TBS containing 5% BSA (NRS/TBS/BSA), rinsed briefly in TBS and an avidin–biotin block performed using reagents from Vector . Anti-ERβ antibodies were diluted in NRS/TBS  and incubated on sections overnight at 4°C. Sections were washed twice for 5 min each in TBS and incubated with biotinylated rabbit anti-mouse immunoglobulin (Dako) diluted 1 : 500 in NRS/TBS/BSA. Bound antibodies were visualised by incubation with DAB ; the DAB was added to sections at 8 s intervals and the colour allowed to develop for exactly 3 min (ERβ1) or 5 min (ERβ2/βcx). Control sections previously used to determine the dilutions of antibodies were included in all experimental runs. Sections were counterstained with haematoxylin.Tissue sections were dewaxed in Histoclear  and rehydrated in descending grades of alcohol to dHImages were captured using an Olympus Provis microscope  equipped with a Kodak DCS330 camera .t-test for small numbers of samples.Quantitation was based on a scoring system reported in detail previously  were classified as having a clinical response and 16 (48%) as having a response in tumour pathology. Although the majority of clinical responders/non-responders had corresponding changes in tumour pathology, one tumour that did not change in tumour volume with treatment showed significant changes in tumour morphology; conversely, eight tumours shrinking clinically with treatment did not change their morphological appearance.α-positive tumour in order to be eligible for the study. There was no quantitative difference in the initial staining score of tumours that either responded or did not, clinically or morphologically (data not shown). Median (range) was 7 (6–8) for responding tumours and 7 (4–8) for non-responders . Although only one score fell to 0, the difference between pre- and treated tumour was highly significant (P<0.0001 by paired Wilcoxon test). No significant differences in change of score were apparent between tumours that responded and that did not, whether response was assessed clinically or by morphology (Comparison of biopsies taken before and during treatment with tamoxifen showed that ERrphology .P=0.03 by paired Wilcoxon test). The levels of ERβ1 were not significantly different in tumours that morphologically changed with treatment as compared with those that did not. There was no correlation between ERβ1 and ERα scores and expressing results as the ratio of ERα to ERβ1 failed to increase discrimination between responding and non-responding tumours  because of the difference in the distribution of values, levels were significantly higher in non-responding tumours . However, the pattern of change in the ERβ1 score with treatment did not differ according to response, whether this was assessed clinically or by tumour pathology.In contrast to ERβ2/βcx variant was detected by immunohistochemistry in 15 of 33 tumours (45%). Before therapy, the level of expression of ERβ2/βcx did not relate to expression of ERα or ERβ1, and examples of divergent results are shown in β1 immunoexpression was found in all tumours, 55% were ERβ1 positive/ERβ2/βcx negative. There was no difference in the status/level of expression of ERβ2/βcx variant between tumours responding to and those that did not to tamoxifen (α or ERβ1 failed to increase discrimination between responding and non-responding tumours (results not shown).Expression of the ERamoxifen . Expressβ2/βcx in 11 cases, increased expression in six tumours and no changes in 16. Changes in expression were not related to response or to changes in ERα and ERβ1  and the ERβ2/βcx variant  in reporter assays , the literature relating to ERβ and response to tamoxifen is confusing and conflicting. In part, this is because studies have been performed in two different settings, giving tamoxifen either as an adjuvant to surgery and measuring recurrence rates/times  but the group was unusual in that all three ERα-negative tumours also responded to treatment. The other study (β2/βcx expression to be associated with less chance of response to tamoxifen. Given that the present study failed to find either a positive or a negative association between ERβ2/βcx and response to tamoxifen, it seems unlikely that ERβ2/βcx will be predictive of response in individual cases.These cell-based studies provide a rationale for considering whether the expression of ERβ1 protein was detected in all tumours, there was no correlation between the levels in those that responded to treatment and those that did not. Furthermore, there was no difference in the status/level of expression of ERβ2/βcx between tumours responding to and those that did not to tamoxifen. In conclusion, to date, there is as yet no consensus among the different studies in which response to tamoxifen has been correlated with expression of ERβ and/or the ERβ2/βcx variant as to whether measuring ERβ expression in ERα-positive breast cancers is likely to be informative as regards response to anti-oestrogen therapy.In the current study, although ER


Learning of stimulus sequences is considered as a characteristic feature of episodic memory since it contains not only a particular item but also the experience of preceding and following events. In sensorimotor tasks resembling navigational performance, the serial order of objects is intimately connected with spatial order. Mammals and birds develop episodic(-like) memory in serial spatio-temporal tasks, and the honeybee learns spatio-temporal order when navigating between the nest and a food source. Here I examine the structure of the bees’ memory for a combined spatio-temporal task. I ask whether discrimination and generalization are based solely on simple forms of stimulus-reward learning or whether they require sequential configurations. Animals were trained to fly either left or right in a continuous T-maze. The correct choice was signaled by the sequence of colors  at four positions in the access arm. If only one of the possible 4 signals is shown (either blue or yellow), the rank order of position salience is 1, 2 and 3 (numbered from T-junction). No learning is found if the signal appears at position 4. If two signals are shown, differences at positions 1 and 2 are learned best, those at position 3 at a low level, and those at position 4 not at all. If three or more signals are shown these results are corroborated. This salience rank order again appeared in transfer tests, but additional configural phenomena emerged. Most of the results can be explained with a simple model based on the assumption that the four positions are equipped with different salience scores and that these add up independently. However, deviations from the model are interpreted by assuming stimulus configuration of sequential patterns. It is concluded that, under the conditions chosen, bees rely most strongly on memories developed during simple forms of associative reward learning, but memories of configural serial patterns contribute, too. Learning of stimulus sequences requires memory of the temporal order of occurrences. Under natural conditions temporal sequence is often combined with spatial sequence, e.g. in navigational tasks. Position in space of objects is defined both by the temporal sequence of experience for the navigating animal and by its relation to surrounding cues, giving the position of each item a unique spatial character. Memories developed for sequential spatial positions of items may, therefore, be embedded in a large-scale relational spatial memory . The memory formed under these conditions has been recognized as episodic or episodic-like resembling key features of memories that allow humans to mentally experience a previous occasion in space and time The role of order in a purely temporal sequences has been studied intensively after Ebbinghaus’ground braking discovery of the primacy and recency effect Here we ask whether an insect, the honeybee, learns sequences of two colors in a context that attempts to simulate a navigational task. The bees fly in a T-maze which due to its narrow channels stimulates their neural distance measuring device (odometer) so strongly that they appear to experience multiples of the actual flight length I find that bees learn to discriminate a series of two colors in four positions. Stimulus salience follows a rank order according to the distance of stimulus position from the choice point with highest salience to the closest position indicative of a recency effect. Discrimination is predicted to a large extent by positional salience, but divergence from this rule and data from the transfer tests also indicate configural phenomena.Apis mellifera carnica) were trained from the hive to a T-maze (at a distance of 35 meters) during two summer periods. The T-maze was located between trees which allowed the bees a view of the canopy and the sky. The T-maze consisted of a 2.50 m long entrance tunnel (30×30 cm), two 1.10 m long tunnels to the right and left of the T intersection, and two 2.60 m long connecting tunnels that led the bees back to the entrance area after they had been feeding . Thus any one bee made only one decision during a within-training test. During the between-training tests all doors were open, no feeder was available and the two feeder areas were covered with new paper of the same black-and-white random pattern. Bees were allowed to fly through the tunnel in any direction, but they usually flew into the tunnel via the entrance tunnel and exited via the respective connecting tunnel. Decisions were counted when a bee approached the T intersection via the entrance tunnel and flew at least half the length into one of the two arms of the T-maze. Each experimental bee made 3–5 choice flights during the between-training tests which lasted for 10 minutes. No difference was found between the results of the within-training and the between-training tests. Therefore, the data were pooled. Tests for the same signal patterns were repeated during the 3–5 hours of the test phase, and the different test patterns (including the transfer tests) followed each other in a pseudorandom fashion. Training for both patterns was continued during the test phase, and test patterns were always different from the last training pattern.The serial position tasks involved two colors  at four positions . Training patterns differed with respect to numbers and positions. The bee learned two patterns in sequential approach flights, one that was associated with a right turn in the T maze, and one associated with a left turn in the T maze. The notation of the training pattern will show the respective color  at the respective position and the training side. Ø represents a position without a color signal. For example, [Ø B B Ø]r vs. [Ø Y Y Ø]l means bees learned to fly into the right arm when a blue signal appeared at positions 2 and 3 and no signal appeared at positions 1 and 4, and during the same training session the same bees learned to fly into the left arm when yellow signals appeared at positions 2 and 3 and none at positions 1 and 4. Experimental design and statistics: Each test for a given pattern resulted in about 3 to 6 decisions made by each experimental bee, leading to up to 20 decisions per test. Tests for the same pattern were repeated several times for the same group of experimental bees on the same day, and many experiments were repeated with different experimental bees , numbers (1–4), and positions (1–4). The role of these variables in guiding the bee in their choices at the T-intersection will be studied by systematically varying them independently and in combination. The measure of performance will be the probability of correct choices in discriminating the two patterns associated with the left and right turn  provide the same salience, and that the two directions of turns in the T-maze  are fully symmetrical.and positions of the signals, but not in their color. I first asked whether bees discriminate between two alternatives associated with right or left turns that differed in the numbers If a single signal differs in color for the two alternatives, bees learn the task particularly well when the signals are at position 1, equally well when at positions 2 and 3, but do not discriminate between the two alternatives when the signals are at position 4 . Choice In the case of two signals at different positions, one can reduce the multitude of permutations to patterns in which the same position in the two alternatives presents either the same (S) or different (D) colors. Thus for positions 1 and 2 patterns of [B B Ø Ø] vs. [Y B Ø Ø], or [Y B Ø Ø] vs. [B B Ø Ø], or [Y Y Ø Ø] vs. [B Y Ø Ø], or [B Y Ø Ø] vs. [Y Y Ø Ø] belong to the same group of [D S Ø Ø] patterns. Since it was found that the two colors B and Y have the same salience, and no preference existed for one of the two sides, there was no need to test all possible permutations. Thus our notation for the three types of patterns DS, SD, DD at 6 permutations of 2 positions out of four is: DS at positions 1 and 2, 2 and 3, 3 and 4, 1 and 3, 1 and 4, 2 and 4 ; SD at tBees discriminate two signal patterns whenever differences appear in either position 1 or 2 or in both of these positions . If the DSØØ vs SDØØ = 0.3; PØDSØ vs ØSDØ = 0.04; PØØDS vs ØØSD = 0.3; PDØSØ vs SØDØ<0.0001; PDØØS vs SØØD = 0.01; PØDØS vs ØSØD = 0.0003). One would expect that different signals at both positions may add up and make it easier for the animal to discriminate the two alternatives. This is not the case, as a comparison between DSØØ vs DDØØ = 0.8; PØDSØ vs ØDDØ = 0.5; PØØDS vs ØØDD = 0.6; PDØSØ vs DØDØ =  0.08; PDØØS vs DØØD = 0.1; PØDØS vs ØDØD = 0.4). This effect may indicate that the single signals at the 4 positions are not learned independently, but rather as a sequential pattern, which allows better discrimination if the two signal sequences differ when compared to similar sequential signals. This question will be further addressed with transfer tests (see below).The combined effect of differences and similarities in these two signal patterns on the rank order of positions 1–4 can be seen in a comparison of several other cases presented in D** vs S**<0.0001). Differences only in position 2 are discriminated, but those in position 3 are not discriminated. The latter case is particularly interesting because the number of choices in these tests is rather high (n = 181).Seven triple patterns are examined for the three positions 1, 2 and 3 . Position 4 is excluded from these experiments because it was found earlier that signals at position 4 may not contribute at all or only marginally to discrimination. In each of the seven triple patterns, two experiments are performed which, because of the earlier findings showing that both colors and direction in the T-maze are interchangeable, are pooled in DDD vs DSD = 0.0002). The same tendency is observed when position 2 provides the same signal and the other two positions the differences . These results show that position 1 have the higher salience, followed by position 2. Position 3 does not contribute to discrimination in these triple patterns indicating that similar signals at positions 1 and/or 2 can overshadow the small contribution of position 3 as seen in the dual patterns  do not lead to significant transfer . In another case of a non-significant training effect ([B B B B] vs [B Ø Ø Ø]) the transfer to [B Ø B Ø] (n = 53) is significantly different from those to [Ø B B Ø] (n = 31) and [Ø B B B] (n = 13)  indicating that a transfer test can uncover a weak learning effect with respect to differences in signals at positions 2–4.Taking all these transfer results together, we can derive the following rules: (1) Generalization to different colors is readily performed. In this case, decisions are made with reference to position and number of signals. (2) Signals at position 1 provide the highest salience, and a shift to position 2, 3 or 4 lead to a gradual reduction of salience. This gradient resembles the one found in the training experiments, which showed that position 1 has the highest salience, position 2 a somewhat lower one, position 3 a much lower one and position a very low salience. (3) Transfer is reduced or lost if position 1 provides the signal for discrimination during training, but is empty in the transfer patterns . (4) If The results from both the discrimination and the transfer experiments clearly show that serial position is the main parameter. The question I shall address next is how well the data can be understood by assuming a particular rank order of positional salience, and whether additional factors may guide the bee's decisions.Position 1 provides the most salient signal and position 4 the least, if any. To address the question of whether position is the only relevant parameter I modeled the effect of positional salience on discrimination by assuming two relations between position and a positional salience score (PSS). The linear model relates positions 1, 2, 3, and 4 to the PSS of 1.0, .7, .4, and .1, and the stepwise model assumes an equal PSS of 1.0 for positions 1 and 2, .5 for position 3, and zero PSS for position 4. Since the animals learn the difference between the two signal patterns the model creates a cumulative PSS that takes into account the differences at the respective positions weighted with their respective salience scores. The PSS are calculated for the discrimination tests, transfer tests are not considered.It is not obvious how the animals might have related the differences at each position. Therefore, two calculations of the cumulative differences in PSS are run. In calculation 1  it is assumed that animals learn each signal in both alternatives, weight them according to the PSS, and the cumulative differences in PSS result from the sum of the differences. No PSS is given to a position without a signal. For example (numbers in brackets are the respective PSS according to the linear model): [B (1.0) B (.7) B (.4) B (.1)] vs [B (1.0) Ø (0) Ø (0) Ø (0)] lead to differences in cumulative PSS: 0+.7+.4+.1 = 1.2. In calculation 2  it is assumed that only those signals are learned that are different. The respective numbers for the same examples are (again according to the linear model): [B (1.0) B (.7) B (.4) B (.1)] vs [B (1.0) Ø (0) Ø (0) Ø (0)] lead to differences in cumulative PSS: 0+.7+.4+.1 = 1.2. The same two calculations are performed for the stepwise model. Since choice performance is expressed in % for each of the two alternatives and the model requires the calculation of differences between the choice performances, the % values are first linearized by calculating the corresponding probit values and then subtracting these probits. This gives a probit value for each pair of trained patterns.2 = 0.49; linear model, calculation 2: P<0.0001, r2 = 0.48; step model, calculation 1: P<0.0001, r2 = 0.39; step model, calculation 2: P<0.0001, r2 = 0.41. The correlation coefficients for the four models are only marginally different. The best correlation is found for the linear model and calculation 1 . This result is shown in The four models give the following results (Pearson correlation): linear model, calculation 1: P<0.0001, rNine data points in The task honeybees had to solve in these experiments was to fly either to the right or to the left in a T-maze to obtain food reward depending on two different patterns of up to 4 sequential blue or yellow signals. The sequential patterns were presented in a flight tunnel with black and white patterns on the floor and walls. The aim was to simulate a navigational task during a foraging episode, with the parameters involved in sequential landmark experience during approach flights more precisely controlled than it would be possible in a natural foraging range. Bees are known to interpret the length of their flights through a narrow tunnel as up to five times longer than their flights in the open due to their distance estimation via visual flow field These results can be interpreted in several ways. I first consider the possibility that training in a narrow tunnel may not simulate a natural navigational situation. Several reasons may account for this effect. a) Similar context conditions: Although bees may experience longer flight distances in the tunnel the same location in the navigational space of the bee may not allow the bee to associate different serial positions of “landmarks” to different flight directions. This possibility is important because similar training conditions with bees flying over short distances in boxes or tunnels were used in the past to study questions related to navigation in bees review: . My resuIt thus appears that the training conditions in the T-maze favor associative learning of multiple sequential stimuli experienced within the time span of sensory/working memory as tested in trace conditioning paradigms. Two parameters may be instrumental for the rank order of positional salience, limited time span and limited capacity of sensory/working memory. A multitude of associative learning phenomena favor temporal recency to the evaluating (reinforcing) stimulus  is limited in time and capacity. Serial memory tests have been used since James Next I want to ask whether there is any evidence for configural phenomena in the T-maze serial signal learning task. Since the signals are experienced well within the time span of working memory one might expect that these signals might be grouped together to form a unique compound. Although the serial salience effect is strong there is evidence that bees indeed learn the sequential patterns also as stimulus sequence patterns. (1) A comparison of the results in Table S1Discrimination tests. The table gives the choice data of all experiments for discrimination tests. Rows (column a) are numbered as in (0.07 MB DOC)Click here for additional data file.Table S2Transfer tests. Rows and columns a, b, c and d are the same as in (0.05 MB DOC)Click here for additional data file.


The presence of the ApoE4 isoform is considered to be a contributing risk factor for AD. However, the detailed molecular properties of ApoE4 interacting with the Aβ peptide are unknown, although various mechanisms have been proposed to explain the physiological and pathological role of this relationship. Here, computer simulations have been used to investigate the process of Aβ interaction with the N-terminal domain of the human ApoE isoforms . Molecular docking combined with molecular dynamics simulations have been undertaken to determine the Aβ peptide binding sites and the relative stability of binding to each of the ApoE isoforms. Our results show that from the several ApoE isoforms investigated, only ApoE4 presents a misfolded intermediate when bound to Aβ. Moreover, the initial α-helix used as the Aβ peptide model structure also becomes unstructured due to the interaction with ApoE4. These structural changes appear to be related to a rearrangement of the salt bridge network in ApoE4, for which we propose a model. It seems plausible that ApoE4 in its partially unfolded state is incapable of performing the clearance of Aβ, thereby promoting amyloid forming processes. Hence, the proposed model can be used to identify potential drug binding sites in the ApoE4-Aβ complex, where the interaction between the two molecules can be inhibited.The relationship between Apolipoprotein E (ApoE) and the aggregation processes of the amyloid  Unraveling the molecular details of the interaction between apolipoprotein E and the amyloid β peptide will yield insights into the relationship between Alzheimer's disease and lipid transport and metabolism. The isoform E4 of apolipoprotein E has been shown to be closely related to Alzheimer's disease. We have therefore used a computational approach to depict a detailed interaction map for this peptide-lipoprotein interaction. The simulation shows that the specific formation of the lipoprotein isoform E4 and the peptide complex affects the structure of the lipoprotein and the peptide. We suggest that this is related to some of the pathogenic effects in Alzheimer's disease. Our results provide a molecular model to work with for the design of potential therapeutic agents capable of modulating this interaction. The disease is characterized by the formation of neurofibrillary tangles and plaques in the brain, leading to neuronal dysfunction, neuronal loss and finally death. The main component of the plaques is the amyloid-APOE ε2/ε2 homozygotic individuals to have a higher predisposition to diseases related to high amounts of cholesterol and triglycerides APOE ε4/ε4 homozygotic individuals have higher risk for coronary heart disease and a significantly greater risk for developing AD.The connection between Apolipoprotein E (ApoE) and AD is well established βThe only difference between the ApoE isoforms is found in residues 112 and 158, where Cys112/Cys158 corresponds to ApoE2, Cys112/Arg158 to ApoE3, and Arg112/Arg158 to ApoE4. The presence of cysteines at these positions confers oligomerization properties to ApoE. Indeed, ApoE2 and ApoE3 are able to form disulfide-linked homo- and hetero-oligomers due to the presence of “respectively” two and one Cys residue. ApoE4 lacks the possibility of strong disulfide linking; however, it is unclear whether weaker interactions could promote the oligomerization of ApoE4. The Cys/Arg substitution in ApoE4 also has molecular impact in terms of intra-protein polar contacts: the orientation of Arg61 is different in ApoE4 compared to ApoE3; the orientation of Arg61 towards the C-terminal domain See  facilitaβ peptide, the details of the interaction between ApoE and Aβ at a molecular level are unknown. Such detailed knowledge is however important for the understanding of the pathological mechanisms of AD, and may also help to identify potential therapeutic target sites where the interaction between ApoE4 and Aβ can be blocked.Although different mechanisms have been proposed to explain the physiological and pathological relationship between ApoE and the Aβ with the N-terminal domain of the different ApoE isoforms in order to determine potential Aβ peptide binding sites in ApoE. In the next step, molecular dynamics (MD) calculations are undertaken to explore the conformational dynamics of ApoE under Aβ interaction and evaluate the stability of each of the ApoE-Aβ complexes. From the analysis and the statistics of the electrostatic interactions of the three ApoE isoforms, we present a model explaining the role of the Aβ-ApoE interaction and its relevance for AD.In the present study we are using molecular docking simulations based on global minimum energy to investigate the interaction process of Aβ with the different isoforms of ApoE. In order to study the Aβ peptide binding site on the N-terminal domains of the three ApoE truncated isoforms we used the Aβ(1–40) peptide as ligand, employing an SDS-induced α-helix solution structure previously determined by NMR spectroscopy β monomeric state (non-aggregated) has been shown to be the most populated one in highly hydrophobic environments Molecular dockings followed by MD simulations were used to study the interaction of Aβ complexes were found to be quite different. Although the Aβ(1–40) peptide assembles between the first and fourth ApoE helix for all ApoE isoforms, the orientation of the peptide was found to depend on the ApoE variant , while the double change of Cys112 and Cys158 to Arg112 and Arg158 in ApoE4 distributes the ionic residues in an ApoE2-like way.All 3D models of the ApoE-Aβ peptide was carried out on each of the lowest energy ApoE-Aβ models obtained by docking calculations as well as on each isolated species. β or the ApoE3-Aβ complexes. However, the ApoE4-Aβ complex showed a large conformational transition indicated by a significant RMSD change of about 10 Å in the 10 ns timescale  peptide. At 10 ns, the hydrophobic groups inside the ApoE4 helices had become exposed to the solvent. The interruption of the stable salt bridge network by external electrostatic interactions (coming from the peptide) was thus transmitted from the dense helix region to the whole protein, causing a severe loss of α-helical structure.In β was carried out through the analysis of the distances between charged residues. For this analysis, direct salt bridges have been assumed to be around 4.3 Å, whereas indirect or water-mediated salt bridges have been assumed to have a distance between 4.3 and 7.0 Å as reported by Dzubiella et al.β complex, the peptide interacted with helices I and IV of ApoE4. The Aβ residues responsible for these interactions were the negatively charged Asp1 and Asp23, which interacted with positively charged arginines in ApoE4 (Arg38 in helix I and Arg142 in helix IV respectively). The direct salt bridge between AβAsp23 and ApoE4Arg38 was very strong  was affected and showed a maximal fluctuation from 2.5 to 7.5 Å and then back to 2.5 Å in the 10 ns time window  broke down , whereas for ApoE4 alone, the salt bridge was more indirect or water mediated. The MD results for the Arg112-Asp110 pair . In the ApoE4-Aairs see . Also Asairs see . Arg158 β. Although the docking was plausible for ApoE2 and ApoE3, the interactions did not generate any conformational transition in the 10 ns time window while for ApoE4, the interaction promoted unfolding of the ApoE4, as shown by the MD simulations. This result is compatible with earlier thermal and chemical denaturation studies using circular dichroism and scanning calorimetry, which have indicated stability differences (ApoE4<ApoE3<ApoE2) among the three isofoms  vs. seconds/minutes). But in the case of ApoE4, it is likely the Aβ peptide behaves as an unfolding catalyzer. Thus, effects on the stability of ApoE2 and ApoE3 exerted by Aβ peptide at longer time scale cannot be discarded.Our computational approach assumes a direct interaction between ApoE and Aβ complex forms between helices I and IV of ApoE4  and Glu66 (helix II). The MD simulations show that this initial chain of events induced by the presence of the Aβ peptide and occurring in helices I and II of ApoE4 (but not ApoE3) would soon be transmitted to helix III stabilizing the Arg61-Glu66 and breaking the Arg61-Glu109 salt bridges in this N-terminal domain, and probably affecting also the Arg61-Glu255 salt bridge in the full protein form. Disruption of this domain interaction by the ApoE4 R61T mutation has been shown to reduce Aβ production β production βAsp1 and ApoE4Arg142 appears less relevant for the destabilization of the salt bridge network; however, Arg142 is within the heparin and receptor binding region . This direct interaction may shield the ApoE4 binding region, affecting the cell membrane recognition of ApoE4 interacting with Aβ.The fluctuation of the salt bridges in helices I and II could be explained by the interruption of the Arg38-Asp25 salt bridge in ApoE4. This effect is most likely induced by Asp23 of Ain vitro studies, both ApoE3 and ApoE4 interact with Aβ and form SDS stable complexes. ApoE-Aβ complexes have been isolated from AD brain extracts and shown to be stable and as tightly packed as Aβ fibrils β: the interaction triggers the partial unfolding of ApoE4 into a misfolded intermediate which we suggest is incapable of performing the clearance of Aβ, and leading to pathogenic effects such as the promotion of amyloid forming processes. In our results, mostly the N-terminus of the peptide is involved in the ApoE4-Aβ complex formation (residues 1 and 23). Previous studies with Aβ peptide have shown that electrostatic interactions are the main cause for the formation of larger oligomers and that the C-terminus region is important for the formation of such oligomers β (1–42) pentamers β C-termini, thus provoking the aggregation of the ApoE4-Aβ complexes.As shown by β effect could probably be overcome by the usage of agents (such as GIND-25 and GIND-105) β and ApoE derived peptides have also been used as blocking therapeutic agents of both the protein and the peptide This Aβ with ApoE4 induces a partially unfolded intermediate by the frustration of the existent network of salt bridges. The four-helix bundle of ApoE4 opens up and the hydrophobic core becomes exposed due to the ApoE4-Aβ complex formation, presumably rendering the protein incapable of performing Aβ clearance. The interaction with Aβ affects the proposed binding site formed by Arg61/Glu109/Arg112 in ApoE4, a binding site that has been shown to be relevant for substances capable of reducing the Aβ production. The model here presented has implications for therapeutic drug design for AD, as it defines on a molecular level the ApoE-Aβ complex as a potential drug target.We propose that the interaction of Aβ peptide solution structure, determined by NMR in 10% SDS/Water (ID:1BA4) and used as the docking model. Crystallographic waters were removed and the structures were fully solvated before energy minimization. Energy minimization was performed for the macromolecules using the GROMACS3.3.2 software with GROMOS96 as the force field β peptide, due to the flexibility of the N-terminus, the RMSD was 4.7 Å (RMSD of 0.8 Å for the α-helix Aβ residues 13 to 40).Crystal structures of the three ApoE truncated isoforms  were downloaded from the PDB database , together with the Ahttp://bioinfo3d.cs.tau.ac.il/), where candidate solutions were generated by rigid-body docking methods http://bioinfo3d.cs.tau.ac.il/) β complex.The structures obtained after energy minimization were used in PatchDock  was used in an unrestrained simulation. Water molecules were equilibrated in the presence of the protein complex for 10 ps before running an unrestrained molecular dynamics simulation for 10 ns. For unrestrained molecular dynamics simulation, the temperature coupling and pressure coupling were conducted in the NpT ensemble by using a Berendsen thermostat of 300 K and 0.1 ps relaxation time. The pressure was 0.5 bar with 0.000045 compressibility and 1ps relaxation time, respectively. The simulations with 300 K were applied by 173529 seeds. Isotropic pressure coupling and Berendsen's temperature coupling were then used during a 10 ns molecular dynamics simulation. In addition, two MD simulations were run involving the three ApoE isoforms alone, following the above-mentioned process. All molecular representations in this study were generated using Chimera v1.4 (http://www.cgl.ucsf.edu/chimera/) Energy minimization, equilibration and molecular dynamics simulations were carried out at neutral pH using the GROMACS3.3.2 software with GROMOS96 as the force field Figure S1β is represented as a golden ribbon (ApoE is displayed as blue ribbons). The subsequent nine solutions for Aβ are plotted as grey cylinders.FireDock Clustering. Clustering of the ten lowest energy solutions ranked by FireDock for ApoE2 ; ApoE3 ; and ApoE4 . The lowest energy solution for A(0.14 MB PDF)Click here for additional data file.