A systematic study of a related set of crystalline sponge adsorbate compounds has enabled the investigation of the interplay between guest molecular structure and intermolecular interactions, which furthers our fundamental understanding of the applicability of this characterization method. 2)3(tpt)2·x(solvent)]n} method has enabled characterization of a novel family of synthetic organic oils. The systematic structural differences and diversity of functional groups offered by 13 related molecular adsorbates provide a detailed quantitative understanding of the relationship between the guest structure, its conformation, and the type of intermolecular interactions adopted with neighbouring guests and the host framework. This analysis is extended to assess the connection of these factors to the resulting quality indicators for a particular molecular structure elucidation.Utilization of the crystalline sponge {[(ZnI There are two key features of the host which allow it to effectively encapsulate and order guest molecules. First, the solvent-filled pores enable thermodynamic exchange of target molecules with weakly interacting host solvent. The controlled diffusion of molecules through MOF cavities assists the action of the second feature: molecular recognition points. These points are regions of the framework that are favourable for interaction and hence are commonly adopted by guest molecules. In particular, the tpt linkers provide highly aromatic, electron-deficient, planar surfaces for interaction. These hydrophobic regions can adopt a variety of contacts with guests via the pyridine and triazine rings, as well as hydrogen bonding with nitrogen atoms in tpt ligands and negatively charged iodine from ZnI2 units. The lack of specificity for interactions with the framework results in compatibility with a diverse range of compounds and chemistries. It also offers a variety of sites within cavities which multiple molecules can concurrently occupy.The MOF most commonly associated with CS analysis is {[, nitrile functionalities (Group 2), sterically and electronically demanding methoxy substituents (Group 3), multiple halogen types with varying position and number (Group 4), and sterically demanding and aromatic substituents (Group 5).2.2.1.supporting information. An illustrative numbering scheme for atoms in the host framework is available in Fig. S1 of the supporting information.The five groups of molecules can be further characterized according to molecular similarity as measured by the Tanimoto coefficient . The guest occupancies were then fixed before anisotropic refinement to prevent expansion of thermal ellipsoids and erroneous assignment of residual electron density within the porous framework. In some cases, low-occupancy guests and disordered exchange sites prevented stable anisotropic refinement. Rather than applying severe thermal restraints, these molecules were modelled isotropically with restrained thermal parameters (Uiso = 0.08). Further details on the crystallographic refinement strategy can be found in Section S1 of the supporting information.For consistent determination of guest occupancies and therefore reliable comparison between structures, in all cases non-hydrogen atoms were refined isotropically with molecule free-variable (FVAR) and restrained thermal parameters , hydrogen–aromatic (H⋯Ar) and oxygen–hydrogen (O⋯H). The criteria and process employed for identification of interactions are detailed in Section S2 of the 3.1.2.The first trend observed is a steady increase in Ar⋯Ar across the series. Further investigation reveals that these are predominantly host–guest interactions with the framework. However, it is the guest–guest interactions and the motifs adopted which provide insight into the Ar⋯Ar interaction trend and the steric influence of methyl substitution. BBA-9-Me can only form Ar⋯Ar contacts with its C1–C6 ring and relies on other interactions to accommodate the C7–C12 ring in the structure. In contrast, BBA and BBA-3-Me form complementary Ar⋯Ar interactions with both rings, as shown in Fig. 3This behaviour occurs because BBA and BBA-3-Me can adopt ‘tail-to-tail’ arrangements which allow Ar⋯Ar contact without steric clashing of methyl groups. In contrast, the additional methyl on the C7–C12 ring for BBA-9-Me would result in all possible arrangements possessing clashing, non-complementary methyl groups. Consequently, BBA-9-Me adopts the fewest Ar⋯Ar interactions in the series.3.1.3.To rationalize the increase in Ar⋯Ar interaction between BBA and BBA-3-Me, consideration of torsion angles between the aromatic rings is required. Both compounds have three guest molecules modelled in the asymmetric unit, but BBA-3-Me possesses a smaller torsion angle range (0.85° versus 3.52°). This small physical change is likely to be a consequence of the increased steric hindrance introduced by the additional methyl group at the C3 position, near the central aryl–aryl C–C bond. The restricted torsion angle results in the adoption of only two exchange sites for BBA-3-Me, compared with three exchange sites for BBA, visualized in Fig. 4et al., 2016supporting information) and acts as an important reminder that the CS method characterizes ‘isolated’ molecules in cavities throughout the framework.This arrangement is notable because of the disorder observed at exchange site G2 for BBA-3-Me, where the aromatic ring positions are consistent but there is variation of the benzyl alcohol arm. It is suggested that the more restricted torsion angle of BBA-3-Me enables adoption of almost identical locations from multiple orientations, thereby maximizing Ar⋯Ar interaction. In contrast, the greater variation of the torsion angle for BBA results in adoption of three individual sites which cannot all utilize aromatic contacts to the same extent. This highlights the influential role that molecular shape and Ar⋯Ar contacts play in determining the guest location and orientation within the pore. Consideration of this behaviour, alongside the matching primary exchange sites (B1 and G1) for BBA and BBA-3-Me, provides further evidence for the reliability of guest positions described in previous CS studies , which is significantly lower than BBA and BBA-3-Me . The main disparity is likely to be caused by the differences in Ar⋯Ar contacts between analytes, which highlight the consequences of altering aromatic motifs. The almost identical average occupancies of BBA and BBA-3-Me are particularly interesting because of the variation of contributions to their interactions. The influence of this is visible in the average number of interactions, where BBA requires 25% more interactions to achieve the same occupancy as BBA-3-Me. This suggests BBA-3-Me adopts stronger contacts within the pore and emphasizes the greater number of H⋯Ar and O⋯H contacts required, compared with a slight increase in Ar⋯Ar. It also shows that with fewer Ar⋯Ar contacts there is a much greater reliance on alternative interactions, as observed for BBA-9-Me.3.1.6.In a wider context, this group of molecules illustrates the direct influence that small sterically hindering substituents can impose on the interactions adopted. For BBA-3-Me, the proximity of the methyl group to the central aryl–aryl bond restricted rotational flexibility and led to more consistent adoption of favourable exchange sites within the pore. By contrast, the more flexible structures of BBA-9-Me and BBA occupy a greater variety of locations. It is also shown that substitution of these groups may not directly influence molecular conformation but can cause significant disruption to otherwise favourable interactions. For BBA-9-Me, the loss of Ar⋯Ar resulted in greater reliance on H⋯Ar and O⋯H, which was accompanied by increased movement of the benzyl alcohol arm. This shows that adsorbing molecules without alternative functionalities or conformational flexibility could be severely impaired by this loss of interaction and this is likely to impede the long-range ordering required for CS analysis.3.2.et al., 2017Group 2 contains BBA and BBA-10-CN, providing a direct comparison to understand the electronic influence of nitrile functionalities. Although a common functional group in organic chemistry, at the time of writing only 9 of the 400+ CS structures deposited in the Cambridge Structural Database (CSD) contain nitrile groups.supporting information.To explore the effect of the nitrile group, the three main interactions investigated are Ar⋯Ar, O⋯H and CH⋯N. An overview of these contacts is illustrated in Fig. 6The most apparent difference is a significant increase in the number of CH⋯N interactions. The prevalence of this interaction for BBA-10-CN compared with any other molecule in the study would have been expected because it is the only compound that can act as both a donor and an acceptor. Consequently, BBA-10-CN participates in both host–guest and guest–guest CH⋯N interactions.The increase in CH⋯N contacts is also accompanied by an increase in Ar⋯Ar interactions. This indicates that the nitrile group does not appear to limit adoption of Ar⋯Ar and therefore the electron-withdrawing resonance effects that reduce aromatic character do not initially appear to play a significant role. However, like in Group 1, the changes in the molecular structure result in considerably different aromatic motifs adopted by guest molecules, as shown in Fig. 7The major difference between guest–guest interactions observed for Group 2 compounds is the location of the benzyl alcohol arm relative to the Ar⋯Ar interaction of the C7–C12 rings. In BBA the substituent is directed downwards on the same side as the central interaction, whereas in BBA-10-CN the groups are oriented away. Two main factors drive this change in motif: steric repulsion between the benzyl alcohol arm/nitrile group and satisfying the electronic properties of the nitrile group. The impact of this difference in motifs is that BBA-10-CN can still adopt Ar⋯Ar interactions with both C1–C6 and C7–C12 rings rather than losing potential contacts, as observed for BBA-9-Me in Group 1.Another difference between the two is the direction of ring slippage. For BBA this results in elongation of the interaction and is likely to be adopted to minimize steric repulsion. Conversely, BBA-10-CN has a more laterally shifted overlap but still results in similar centroid–centroid distances to BBA. This benefits the formation of guest–guest CH⋯N contacts for BBA-10-CN, while achieving similar ring separation to maximize aromatic interaction.The final difference between Group 2 compounds is the decrease in the number of O⋯H interactions for BBA-10-CN. Investigation of the oxygen position away from the ring planes in BBA-10-CN shows a deviation of 84 (5)° to adopt the only O⋯H contact observed. This demonstrates that the molecule can utilize its conformational flexibility to gain more interactions, even when adopting the different Ar⋯Ar motif. Interestingly, unlike H⋯Ar and O⋯H in Group 1, the O⋯H and CH⋯N interactions observed in Group 2 do not appear to operate in a synergistic manner and instead compete. This further highlights the indirect effect imposed by the functionalization of aromatic rings on the interactions adopted.Adopting this range of interactions results in a significant difference in average guest occupancy: BBA averages 44.3%, whereas BBA-10-CN averages 27.2%. The low occupancy of BBA-10-CN also necessitates the application of considerable restraints in modelling and is accompanied by a relatively high amount of residual electron density. The average number of interactions also provides insight into the overall strength of the contacts present, with both compounds relying on a similar average per guest, and BBA-10-CN is assumed to be much more weakly interacting.Group 2 has provided another example of nitrile-containing compounds resulting in a poorly defined CS structure. It is thought that the electronic environment desired by the nitrile group, as well as its size and rigidity, play significant roles and affect the exchange locations and motifs adopted by the guests. In BBA-10-CN it is suggested that, although the local ordering with guest–guest interaction is satisfied, this can have detrimental effects on the regular long-range ordering.3.3.Group 3, and subsequently Group 4 and Group 5, provide an opportunity to compare PBA and BBA systems. The presence of a phenol group will introduce electron-withdrawing inductive (−I) and electron-donating mesomeric (+M) effects which influence the aromatic character of the C1–C6 ring. The consequence of this is not clear; the overall result is a more electron-rich π-system which will favour interaction with the electron-deficient framework, but it also increases the negative quadrupole associated with the aromatic ring which will disfavours Ar⋯Ar interaction , whereas BBA-8,10,12-OMe has the second highest (41.8%) and BBA-8,12-OMe has the highest (47.5%). This suggests that the more diverse host–guest and guest–guest interactions of PBA-12-OMe lead to weaker contact. The average number of interactions follows an inverse trend to occupancy, which further suggests that PBA-12-OMe is more weakly interacting. For BBA-8,12-OMe the greatest average occupancy and fewest average interactions may be aided by the significant preference for host–guest interaction, which can offer more reliable support than neighbouring guests.Group 3 highlights the interplay between steric and electronic properties that must be considered for bulky groups with a propensity for hydrogen bonding. This also emphasizes the ‘induced fit’ which guest molecules adopt when encapsulated within the host framework and the influence this has on the subsequent interactions adopted. The best example of this is shown by comparison of A and B exchange sites in BBA-8,10,12-OMe, which rely on O⋯H and Ar⋯Ar, respectively, but produce similar models. This showcases the versatile modes of interaction available for the same molecules and the equal level of stabilization that can be achieved.3.4.et al., 2020Group 4 contains PBA, PBA-3-F, PBA-2-F and PBA-2,6-I. This set of molecules examines the influence of halogen atoms on the aromatic and oxygen-based interactions that were dominant in previous groups. A comparable size to hydrogen, together with its strongly electron-withdrawing nature, makes fluorine an attractive and commonly used functionalization in organic chemistry. Therefore, for the widest possible adoption of the CS method, understanding the electronic effect of fluorine is crucial. Iodine is significantly less electron-withdrawing but is commonly found in aromatic intermediates to facilitate the addition of organic and heteroatom substituents. Further understanding its influence would assist in the characterization of such intermediates in a manner akin to the approach used for drug metabolites . The average interaction summary for the series is illustrated in Fig. 10Investigation of PBA enables comparison with the BBA compound discussed in Group 1 and Group 2. Both molecules represent the simplest, or least functionalized, structures of their respective groups. They possess similar averages for the number of Ar⋯Ar contacts, 1.50 and 1.67 per guest, respectively, but this similarity is not reflected in the extent to which a particular ring is involved in the interaction. PBA has a significant preference for the C7–C12 ring with it being involved in 77.8% of its Ar⋯Ar interactions, compared with 40.0% for BBA. Exemplar interactions are shown in Fig. 11In Group 4, Ar⋯Ar interactions do not exhibit a clear trend across the series. The −I nature of fluorine appears to have a varied effect, with no change in Ar⋯Ar contact between PBA and PBA-3-F but a significant decrease for PBA-2-F. PBA-2,6-I also experiences fewer aromatic interactions than PBA-3-F, even though the substituents are much less electronegative. The lack of distinguishable trends suggests a more complex influence of the halogens than simply altering the properties of the aromatic rings. These observations cannot currently be rationalized and require more attention in subsequent research.ortho-position, the hydroxyl group is expected to dominate over fluorine in the competition for interactions because it has a stronger hydrogen-bonding ability .Finally, there are twice as many X⋯H interactions for PBA-2,6-I compared with PBA-2-F, suggesting that the interaction may scale linearly with an increasing number of halogen substituents. This is also dissimilar to Group 3 which showcased a more complex trend when increasing the number of methoxy groups and is proposed to be caused by the introduction of steric bulk. The major contribution from X⋯H is also accompanied by a significant number of O⋯H interactions. It is proposed that the location of substitution and longer C—I bonds enable a more co-operative action between the two contacts, which then benefits the guest stability. These interactions are shown in Fig. 12The overall influence of the varying interactions on the molecular structure determination of PBA, PBA-3-F and PBA-2-F is minimal, as the three molecules achieve similar average guest occupancies of 26.8, 21.0 and 23.5% respectively. There is a slight decrease in the average number of interactions across this series, which suggests that PBA-2-F may adopt the strongest contacts. One of the clearest differences between these compounds is the number of exchange sites . This hints that the halogen atoms cause intermolecular interactions to become more directional and specific. Therefore, although there are numerous locations which suit the size and shape of the molecules, a limited number have the requisite interactions available for regular ordering throughout the crystal. This hypothesis is supported by PBA-2,6-I which has two exchange sites and a significant increase in average occupancy to 74.7%. This large increase only requires a few more interactions for PBA-2,6-I than PBA which again suggests that the introduction of halogens can improve the strength of host–guest and guest–guest contacts.This series of molecules demonstrates that halogen atoms can adopt interactions within the pores and therefore have a more direct influence than just altering the electronic properties of the corresponding aromatic rings. This highlights the relative freedom which guest molecules possess in the framework, as they can change orientation to satisfy these different functionalities. The influence of halogen interactions is greatly affected by three factors. First, the element type, where poorly hydrogen bonding fluorine is not as effective as larger halogens which have better orbital overlap for interaction. Second, substituent position, where substitution away from the crowded central aryl–aryl bond allows for better intermolecular contact. Finally, the number of atoms, where more halogen atoms improve the number of directions which can be utilized for interaction within the enclosed environment.3.5.Group 5 contains PBA, PBA-2-Me and PBA-2-Ph. This series explores functionalization with increasingly bulky substituents away from the central aryl–aryl bond. The additional aromatic ring in PBA-2-Ph also provides insight into whether molecules with more aromatic groups have greater stability in the CS pore.supporting information.To investigate the influence of this functionalization, Ar⋯Ar, H⋯Ar and O⋯H interactions are examined. The average interaction per guest across the series is illustrated in Fig. 13There is minimal variance in Ar⋯Ar interactions across the series, with PBA and PBA-2-Me having very similar structures and no distinct electronic differences. However, the slight decrease observed for PBA-2-Ph is unexpected because of the significant role of the Ar⋯Ar contacts, as indicated throughout this study, and the apparent molecular shape complementarity with the tpt linker, as illustrated in Fig. 14The lack of increase in Ar⋯Ar in PBA-2-Ph can be rationalized by assessing the 3D conformation of the compound in relation to the space available within the framework. Steric repulsion between hydroxyl and methyl groups prevents a co-planar orientation of C1–C6 and C7–C12 rings. This induced twist would minimize intramolecular steric repulsion, but results in an unfavourable alignment for interaction with the planar tpt ligand. Instead, the guest molecule can adopt some overlap with the framework linker, while also utilizing the freedom of the cavity, as shown in Fig. 15The resulting sandwich-like cluster enables contact between the electron-rich C7–C12 guest ring and the electron-deficient framework. Simultaneously, the C1–C6 ring sits in the middle of the pore, which allows interaction with a symmetry-related molecule. Notably, Ar⋯Ar interactions are not adopted by the C13–C18 ring of the A exchange site, or any rings of the B exchange site for PBA-2-Ph. Instead, these rely on H⋯Ar interaction for stability. This reliance is reflected in a significant contribution to interactions for PBA-2-Ph in comparison with PBA and PBA-2-Me. This highlights the versatility of interaction within the pore and the varied action of aromatic rings, which makes them well suited to the CS method. In contrast, PBA-2-Me possesses the fewest H⋯Ar interactions, albeit a minimal decrease from PBA, but this is an initial indicator of the disruption that can be caused through addition of sterically dominating groups.This disruptive effect is revealed through investigation of O⋯H interactions and their reduction observed between PBA and PBA-2-Me. Investigation of H2 and H3 atoms of PBA and C15 of PBA-2-Me highlights the change in environments. There is an overall loss of three O⋯H interactions when comparing PBA with PBA-2-Me, which are replaced by a single H⋯Ar contact. This is likely related to a change in guest location because the additional methyl of PBA-2-Me would have considerable steric repulsion. Overlay of the two structures reveals a significant difference in host framework conformation, illustrated in Fig. 16Although this distortion prevents direct comparison of the exchange sites adopted, it clearly highlights the change of environment brought about by the additional methyl group of PBA-2-Me. Comparatively, PBA-2-Ph regains some O⋯H interactions but not to the same extent as PBA. This suggests that the location adopted by PBA-2-Ph achieves a better balance between the three principal interactions.Variation of intermolecular interactions has a negligible influence on the average guest occupancy for PBA, PBA-2-Me and PBA-2-Ph in Group 5 . Although differences in O⋯H interactions were identified between PBA and PBA-2-Me, they both achieve comparable average guest occupancies with a similar number of interactions. This may be aided by the more restricted torsion angle observed for PBA-2-Me, which allows it to adopt two exchange sites which maximize the interaction, as previously noted for BBA-3-Me. In comparison, PBA-2-Ph experiences a significant increase in the average number of interactions because of the greater reliance on weaker H⋯Ar contacts. A comparison between PBA-2-Ph and BBA-8,10,12-OMe (Group 3) shows that the two exchange sites in the asymmetric unit can rely on different interactions but result in similar guest occupancies and accompanying restraints. This also highlights the cumulative effect of many weaker interactions which can be utilized to match the stronger contacts.The series also demonstrates the impact that relatively small structural changes can impose on the location adopted by guests within a pore, although in this case the variation in guest exchange sites did not have a significant influence on the overall molecular structure elucidation. It has also been shown that additional aromatic rings do not have a direct correlation with the number of Ar⋯Ar contacts adopted. Instead, the substitution position, which is greatly influenced by the steric bulk of the molecule, and the electronic properties of the accompanying rings play an important role as to which interaction is the most favourable.4.Drawing from the summaries of each of the five groups analysed, it is shown that the location of the guest exchange site is determined by the steric requirements of the molecule. The subsequent orientation and molecular conformation of the guest are governed by the intermolecular interactions available at the exchange site. The interactions adopted will then define the ability of guests to achieve regular order and influence the quality of the resolved structure.Accordingly, structurally related compounds can exhibit considerably different behaviours. In particular, the addition or rearrangement of functionalities which have been shown to aid guest stability may actually have a destabilizing effect if not considered in conjunction with the 3D shape and conformation of guest molecules.It is also suggested that some functional groups favour localized interaction rather than regular long-range ordering, as introduced in the evaluation of nitrile functionalities in Group 2. This may manifest itself as guest–guest interactions which appear relatively strong, but the accompanying host–guest contacts are weak. The resulting CS structure may only be poorly resolved because of the lack of consistent stabilization throughout cavities in the framework.et al., 2023Follow-on investigations from this work will use crystallographic databases to identify functional group propensity to form strong intermolecular interactions with similar groups. This will provide insight into which functionalities are more likely to form strong guest–guest interactions and therefore may be more successfully analysed with other CS variants or different advanced crystallization methods  BBA-9-Methyl_CrystallineSponge, BBA_CrystallineSponge, BBA-3-Methyl_CrystallineSponge, BBA-10-Nitrile_CrystallineSponge, PBA-12-Methoxy_CrystallineSponge, BBA-8_12-Methoxy_CrystallineSponge, BBA-8_10_12-Methoxy_CrystallineSponge, PBA_CrystallineSponge, PBA-3-Fluorine_CrystallineSponge, PBA-2-Fluorine_CrystallineSponge, PBA-2_6-Iodine_CrystallineSponge, PBA-2-Methyl_CrystallineSponge, PBA-2-Phenyl_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059BBA_CrystallineSpongesup2.hklStructure factors: contains datablock(s) BBA_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059BBA-3-Methyl_CrystallineSpongesup3.hklStructure factors: contains datablock(s) BBA-3-Methyl_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059BBA-8_10_12-Methoxy_CrystallineSpongesup4.hklStructure factors: contains datablock(s) BBA-8_10_12-Methoxy_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059BBA-8_12-Methoxy_CrystallineSpongesup5.hklStructure factors: contains datablock(s) BBA-8_12-Methoxy_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059BBA-9-Methyl_CrystallineSpongesup6.hklStructure factors: contains datablock(s) BBA-9-Methyl_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059BBA-10-Nitrile_CrystallineSpongesup7.hklStructure factors: contains datablock(s) BBA-10-Nitrile_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059PBA_CrystallineSpongesup8.hklStructure factors: contains datablock(s) PBA_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059PBA-2_6-Iodine_CrystallineSpongesup9.hklStructure factors: contains datablock(s) PBA-2_6-Iodine_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059PBA-2-Fluorine_CrystallineSpongesup10.hklStructure factors: contains datablock(s) PBA-2-Fluorine_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059PBA-2-Methyl_CrystallineSpongesup11.hklStructure factors: contains datablock(s) PBA-2-Methyl_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059PBA-2-Phenyl_CrystallineSpongesup12.hklStructure factors: contains datablock(s) PBA-2-Phenyl_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059PBA-3-Fluorine_CrystallineSpongesup13.hklStructure factors: contains datablock(s) PBA-3-Fluorine_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059PBA-12-Methoxy_CrystallineSpongesup14.hklStructure factors: contains datablock(s) PBA-12-Methoxy_CrystallineSponge. DOI: 10.1107/S2052252523005146/lt5059sup15.pdfSupporting figures and tables. DOI: 2254202, 2254203, 2254204, 2254205, 2254206, 2254207, 2254208, 2254209, 2254210, 2254211, 2254212, 2254213, 2254214CCDC references:


Clinical proof-of-concept data have been obtained for the β3-AR agonists vibegron, mirabegron, solabegron, and ritobegron; however, the selectivities of these agents have not been compared directly under the same experimental conditions. Moreover, the bladders of some patients express lower β3-AR densities than those of healthy individuals, and the β3-AR density might be expected to affect agonist activity. This study assessed the β3-AR selectivities of four β3-AR agonists and examined the effects of β-AR density on their pharmacological profiles. Functional cellular assays were performed using Chinese hamster ovary-K1 cells expressing three human β-AR subtypes transfected with different amounts of plasmid DNA . The half-maximal effective concentration values, intrinsic activities (IAs), and β3-AR selectivities of vibegron, mirabegron, solabegron, and ritobegron were calculated to assess their pharmacological profiles. The β3-AR selectivities of vibegron, mirabegron, solabegron, and ritobegron were >7937-, 517-, 21.3-, and >124-fold higher than for β1-ARs, and >7937-, 496-, >362- and 28.1-fold higher than for β2-ARs, respectively, under the same experimental conditions. The IAs of mirabegron, solabegron, and ritobegron decreased in line with decreasing receptor density, while the IA of vibegron was maintained at the same level as that of the full agonist isoproterenol at various β3-AR densities. Vibegron has high β3-AR selectivity and exhibits full agonist activity, regardless of the β3-AR density. These results suggest that vibegron is a highly effective and safe drug for treating OAB. Overactive bladder (OAB) is a symptom complex characterized by a sensation of urgency, with or without urge incontinence, usually accompanied by frequency and nocturia . The medβ-ARs are subclassified into three subtypes: β1-, β2-, and β3-ARs. In 1999, three research groups independently reported that β3-AR mRNA was expressed in human detrusor muscle –10, and Expression levels of β3-ARs have been reported to vary according to the pathology. For example, β3-AR mRNA expression was significantly decreased in the bladder mucosa in patients with severe bladder outlet obstruction (BOO) compared with mild BOO and controls . In addiVibegron is a novel, potent, and selective β3-AR agonist that was recently licensed for the treatment of OAB in Japan and the United States ,21. In tVibegron was obtained from Kyorin Pharmaceutical Co., Ltd. , the active form of ritobegron was synthesized in our laboratory  . The fol2 at 37°C. For cAMP assay, 2×105 cells/mL were inoculated onto a 96-well plate at 100 μL/well. For membrane preparation experiments, the cells were inoculated onto 150 mm dishes with the same number of cells per culture area as for the 96-well plate. The cells were cultured for about 24 h and used for β-AR transfection (see below). The DNA sequences encoding β1-AR (P08588.2), β2-AR (NP_000015.1), or β3-AR (NP_000016.1) were cloned into a pCI-Neo expression vector . CHO-K1 cells expressing β1-AR, β2-AR, or β3-AR were constructed by transfection with the appropriate plasmid DNA  using Lipofectamine 2000 , according to the manufacturer’s instructions. Human β-AR plasmid DNA was diluted with Opti-MEM (Life Technologies Japan Ltd.) (0.1 μg plasmid DNA/25 μL Opti-MEM) for each subtype. Lipofectamine was diluted with Opti-MEM (0.5 μL Lipofectamine/25 μL Opti-MEM) and incubated for 5 min at room temperature. Solutions containing plasmid DNA and Lipofectamine, respectively, were mixed in equal amounts and incubated for 20 min at room temperature. The mixture (50 μL/well or 23.735 mL/dish) was added to the above CHO-K1 cells inoculated onto a 96-well plate or 150 mm dish. The transfected CHO-K1 cells were then cultured for about 24 h and used for the following experiments. Similar experiments were performed after reducing the amounts of transfected plasmid DNA to 0.05 and 0.025 μg.Chinese hamster ovary (CHO)-K1 cells  were cultured in Ham’s F-12 medium containing 10% fetal bovine serum under 5% COCells that had been cultured for about 24 h after transfection were subjected to cAMP assay. The cells were incubated with vibegron, mirabegron, solabegron, ritobegron, or isoproterenol for 30 min at 37°C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine, and intracellular cAMP accumulation was evaluated using a cAMP Gs Dynamic kit . Fluorescence emissions at 665 nm and 620 nm were detected using a PHERAstar FSX plate reader .g for 10 min, the cells were resuspended in membrane preparation buffer, and centrifuged at 1880 ×g for 10 min. After removing the buffer, the cells were resuspended in homogenate buffer  and frozen at −80°C for 1 h. The cells were thawed and homogenized using an ultrasonic homogenizer. Nuclei were removed by centrifugation at 1310 ×g for 10 min, and the membrane fraction was centrifuged at 92,691 ×g for 60 min at 4°C. The pellet was resuspended in 10 mM HEPES buffer (pH 7.4) containing 0.1 mM EDTA-2Na and stored at −80°C until use. Protein concentration was determined using a Pierce BCA Protein Assay kit .At 24 h post-transfection, cells in 150 mm dishes were washed with Dulbecco’s phosphate-buffered saline and detached with a cell scraper into 10 mM HEPES buffer (pH 7.4) containing 154 mM NaCl, 0.7 mM EDTA-2Na, and protease inhibitor cocktail  (membrane preparation buffer). The buffer was then removed by centrifugation at 1880 ×2, pH 7.4) were incubated with [125I]-iodocyanopindolol (ICYP) (PerkinElmer) for 90 min in a final volume of 200 μL at 27°C for each β-AR. β1-, β2-, and β3-AR saturation binding assays were carried out using [125I]-ICYP ligands, ranging from 16–1000, 16–1000, and 31–2000 pM, respectively. Nonspecific binding was determined in the presence of 10 μM alprenolol for β1- and β2-ARs and 100 μM SR59230A for β3-ARs. After incubation, the membranes were filtered onto a 96-well white microplate with bonded GF/B filter preincubated in 0.3% polyethylenimine for 30 min and washed four times with ice-cold wash buffer . Radioactivity on the filter was counted using a MicroBeta2 microplate counter (PerkinElmer) with quenching correction, after the addition of 100 μL of Microscint 20 (PerkinElmer) and shaking for 5 min. Saturation experiments were analyzed by GraphPad Prism 6.0  using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action: B = (Bmax × [F]) / (KD + [F]), where B is the amount of ligand bound at equilibrium, Bmax is the maximum number of binding sites, [F] is the concentration of free ligand, and KD is the ligand dissociation constant.Aliquots of membrane preparations resuspended in binding buffer  value was calculated for each agonist based on its concentration–response curve using GraphPad Prism 6.0 nonlinear regression analysis. The intrinsic activity (IA) of each drug was calculated as the proportion of the maximum response to 1×10−5 M when the maximum response to isoproterenol was taken as 1.00. When the IA of the drug was >0.5, an EC50 value was calculated. β3-AR selectivity was calculated by comparing the EC50 values. The concentration of each drug required to produce a 50% maximal response induced by isoproterenol (Iso50) was calculated to assess the potency of the drugs.In the cAMP assay, the half-maximal effective concentration  induced a concentration-dependent increase in the accumulation of cAMP in cells expressing β3-ARs , with ECectively . In cellectively . The β3-ectively . Vibegromax values in cells transfected with 0.1, 0.05, and 0.025 μg/well were 781, 245, and 153 fmol/mg for β1-ARs, 713, 231, and 108 fmol/mg for β2-ARs, and 347, 222, and 116 fmol/mg for β3-ARs, respectively  < 26.9 nM (mirabegron) < 252 nM (solabegron) < 943 nM (ritobegron) in β3-AR-expressing cells transfected with 0.025 μg/well, with similar trends for the other transfection conditions (The ECectively . Howeveroterenol . We thernditions .50 = 594 and 570 nM), with β3-AR selectivities of 517-fold for β1-ARs and 496-fold for β2-ARs. To date, no studies have clearly demonstrated the direct agonist activity of mirabegron in β1- and β2-ARs, although it has shown affinities for β1-and β2-ARs in binding assays, with inhibition constants (Ki) values of 383 and 977 nM, respectively [In the present study, we evaluated the agonist activity of the β3-AR agonists vibegron, mirabegron, solabegron, and ritobegron, developed for the treatment of OAB, against each β-AR subtype , using functional assays indexed to cAMP accumulation, and compared their β3-AR selectivities. Differences in β3-AR selectivity were observed among the four drugs, in the order vibegron > mirabegron > solabegron = ritobegron. Vibegron showed the highest β3-AR selectivity, which was >7937-fold higher than for β1- and β2-ARs. Mirabegron showed similar β3-AR agonist activity to vibegron, but it also demonstrated agonist activity for β1- and β2-ARs  differed between 99.2% and 80.4%, respectively [The β3-AR density in the urinary bladder has been reported to differ according to the disease pathology , and theectively . The curmax 116 fmol/mg) approximated the density in the urinary bladder in incontinent patients. Under these conditions, vibegron showed full agonist activity (IA = 1.07) and ritobegron showed strong agonist activity (IA = 0.85), but mirabegron and solabegron showed only partial agonist activity . Notably, there were clear differences in the IAs of vibegron and mirabegron, although both drugs are used in clinical practice.Significant reductions in β3-AR mRNA expression have been reported in the bladders of subjects with severe BOO compared with mild BOO and healthy individuals . In addimax) and minimum observed plasma concentrations (Cmin) of once-daily 50 mg vibegron and mirabegron [max,u and Cmin,u for the unbound forms, taking into account the plasma-protein-binding rate of each drug, were calculated as 56 nM and 14 nM for vibegron and 20 nM and 3 nM for mirabegron, respectively. Applying these values to the dose-response curve of β3-AR-expressing cells transfected with 0.025 μg/well, the cAMP activity of vibegron was calculated to be about 82% in Cmax,u and 59% in Cmin,u, while the cAMP activity of mirabegron was about 53% in Cmax,u and decreased to about 27% in Cmin,u. These findings indicate that vibegron maintained strong β3-AR agonist activity compared with mirabegron, which could affect the relative efficacies of the two drugs in clinical practice, with vibegron acting more strongly and continuously in bladder tissues expressed β3-ARs.Based on the maximum (Crabegron ,27, as tThe desensitization of receptors is an important factor that negatively affects drug efficacy. Compared with β2-AR, the β3-AR has been reported to be less susceptible to desensitization because it lacks the C-terminal phosphorylation site involved in desensitization . In addiβ1- and β2-AR densities vary in the regions of the human heart, with β1- and β2-AR densities in pacemaker cells in the sinoatrial node, which control the heartbeat, being 4.2- and 2.6-fold higher, respectively, compared with the atrium . The β1-In this study, we evaluated the activities of the β3-AR agonists vibegron, mirabegron, solabegron, and ritobegron on β-AR subtypes under the same experimental conditions, and determined the effects of receptor density on the pharmacological profiles of the drugs. Vibegron showed the highest β3-AR selectivity and demonstrated full agonist activity, regardless of the β3-AR density. These results suggest that vibegron is a highly effective and safe drug for the treatment of OAB.


One landmark application of evolutionary game theory is the study of social dilemmas. This literature explores why people cooperate even when there are strong incentives to defect. Much of this literature, however, assumes that interactions are symmetric. Individuals are assumed to have the same strategic options and the same potential pay-offs. Yet many interesting questions arise once individuals are allowed to differ. Here, we study asymmetry in simple coordination games. In our set-up, human participants need to decide how much of their endowment to contribute to a public good. If a group’s collective contribution reaches a pre-defined threshold, all group members receive a reward. To account for possible asymmetries, individuals either differ in their endowments or their productivities. According to a theoretical equilibrium analysis, such games tend to have many possible solutions. In equilibrium, group members may contribute the same amount, different amounts or nothing at all. According to our behavioural experiment, however, humans favour the equilibrium in which everyone contributes the same proportion of their endowment. We use these experimental results to highlight the non-trivial effects of inequality on cooperation, and we discuss to which extent models of evolutionary game theory can account for these effects.This article is part of the theme issue ‘Half a century of evolutionary games: a synthesis of theory, application and future directions'. That is, all individuals have the same sets of strategies and identical incentives to choose each strategy. Yet in most human and animal populations, inequality is ubiquitous. People frequently differ in their endowments, productivities, their shares of rewards, and the positions they occupy in social hierarchies ,25. SuchIn this paper, we discuss some of these questions in the context of a simple coordination game, the threshold public goods game. This game has many possible equilibria, but group members disagree on which equilibrium they prefer. To explore the effect of exogenous inequality, we consider two independent sources of asymmetry. Individuals can either differ in their endowments  or in their productivities (i.e. the efficiency of their contributions to the public good). We use this set-up to explore how inequality impedes overall coordination , and how it affects which equilibria are chosen. We address these questions with both a theoretical analysis and a behavioural experiment.As a part of this analysis, we also ask to which extent classical evolutionary dynamics can accurately predict the behaviours that emerge in the experiment. Perhaps somewhat surprisingly, we find that a naive application of evolutionary models fails to reproduce many of the empirical patterns. For example, models based on replicator dynamics with random initial conditions tend to overestimate how often people defect, and they underestimate how often less productive individuals cooperate. However, we also show that predictions become more accurate when initial conditions match the empirical first-round behaviours. These results suggest that especially in games with natural focal points, a naive application of evolutionary game dynamics may mis-predict which equilibrium is most likely to occur. These predictions improve dramatically, however, once the participants’ true initial play is taken into account.Overall, the contribution of our study is twofold. First, we provide novel empirical evidence on how people coordinate in the presence of exogenous asymmetries. Second, our data also allow us to test the predictive power of different types of evolutionary dynamics. Both contributions may help to further improve existing models on the evolution of cooperation in asymmetric social dilemmas.. 2Traditionally, a classical model to study the effect of inequality on cooperation is the linear public goods game ,27. In tIn general, however, the interaction of inequality and cooperation is non-trivial. A vast amount of experimental studies observe that endowment inequality tends to reduce cooperation even in the one-shot public goods game –32. FurtWhile in the linear public goods game, the dominant action in a single game is to defect, social dilemmas can also arise in games in which cooperation is an equilibrium. One example is the threshold public goods game –46. HereRecently, a particular variant of the threshold public goods game has attracted much attention, the climate game or collective-risk dilemma –53. For Most of the previous studies on the impact of asymmetry on coordination are based on a multi-period climate game. Here, players are required to reach the target at the end of a series of repeated contributions ,52–54,56. 3i  receives a fixed positive integer endowment of ei. Then the players individually decide which amount ci from their endowment they wish to contribute to a common pool, with ci ∈ {0, 1, …, ei}. Each individual’s contribution is multiplied by a productivity factor pi. We refer to p1c1 + p2c2 as the players’ collective contribution. If the players’ collective contribution reaches a predefined threshold θ, each individual i receives a reward ri in addition to their remaining endowment. Otherwise, both players only receive their remaining endowment. Therefore, pay-offs depend on the fixed parameters of the game  and on the contribution tuple . The pay-off of player i is:a; electronic supplementary material, table S1). As a control, we consider a treatment with ‘full equality’. Here, individuals coincide in all dimensions, and the game is symmetric. Next, we consider two treatments in which individuals only differ in their endowments. Here, player 1 either obtains twice the endowment of player 2  or three times the endowment of player 2 . Finally, we also consider two treatments in which individuals have different productivities. Again, player 1 is either twice as productive , or three times as productive as player 2 . To facilitate comparisons across treatments, the reward is kept constant, and the threshold θ is determined such that groups reach the threshold if both players contribute half of their endowment.To explore the effect of exogenous inequality on coordination, we use a comparably simple set-up. We consider an asymmetric threshold public goods game among two players, player 1 and player 2. The game proceeds as follows. First, each player To obtain some intuitive understanding of the strategic considerations that apply to this game, we first describe the respective Nash equilibria of the one-shot (non-repeated) game .b, we illustrate the set of Nash equilibria for the five experimental treatments. Two interesting observations follow immediately. First, for the parameters of the experiment, full defection is an equilibrium under full equality and under endowment inequality, but it ceases to be an equilibrium under productivity inequality. In the last two treatments, the more productive player has a sufficiently strong incentive to contribute even if the other player defects. Second, in all treatments there are many different cooperative equilibria. In all these equilibria the group reaches the threshold, yet they differ in how the respective costs are allocated among the two players.In general, the one-shot threshold public goods game can have two types of equilibria. First, there is a set of cooperative Nash equilibria in which the group’s collective contribution exactly matches the threshold. In addition, there can be a defective Nash equilibrium in which all players keep their endowment. In figure 1c1 = c2. Similarly, if they both strive to achieve equal relative contributions (ERC), they choose the equilibrium that satisfies c1/e1 = c2/e2. In addition, participants might also strive for equal collective contributions, equal pay-offs, and a maximal collective pay-off. In the electronic supplementary material, table S2, we summarize which predictions these criteria make for each of the five treatments. As one aim of the experiment, we explore which of the above rules is best able to explain the subjects’ behaviours.To further explore how participants might choose among the different cooperative equilibria, in the electronic supplementary material, S1.3 we describe a set of heuristics that participants might apply. For example, if participants strive for equal absolute contributions (EAC), they settle at the cooperative equilibrium for which . 4To explore how humans act in such threshold public goods games, we invited a total of 558 participants to interact in one of the five treatments illustrated in successful if their collective contribution matches or exceeds the threshold. Similarly, we call the group effective if their collective contribution exactly matches the threshold. In figure 2a, we compare the proportion of successful groups across the five treatments. Overall, we observe that under full equality and under productivity inequality, these proportions are largely similar, with approximately b, we show that this proportion increases in all treatments, suggesting that individuals learn to better coordinate their contributions over time. However, especially in the groups with endowment inequality, it may take a substantial number of rounds until groups have learned to coordinate effectively.To assess the effects of inequality, we consider two measures of a group’s performance. We call a group c,d). Under full equality and productivity inequality, the contributions of the two players are indistinguishable. When there is endowment inequality, however, high-endowment players contribute more in absolute terms but less in relative terms . To further explore these results, we look at the groups’ contribution patterns  over time, separately for each treatment . When there is either full equality or productivity inequality, most participants quickly coordinate on giving half of their respective endowment. This contribution pattern is consistent with the heuristics of EAC and ERC in these treatments . By contrast, in the treatments with endowment inequality, the picture is more mixed. Here, in about a quarter of groups, players continue to give half of their endowment (consistent with the ERC criterion). In addition, there is a substantial number of groups in which players either make the same absolute contribution (EAC), they contribute nothing at all, or their contribution pattern is somewhat between the patterns of ERC and EAC . Interestingly, the picture is much more clear when participants need to specify the contribution pattern they consider to be most fair. According to the post-experiment questionnaire, ERC is clearly the most common response .In the next step, we compare the contributions of players 1 and 2 across the five treatments c,d. UndeIn a last step, we also analyse why some groups fail to reach the threshold  and how players respond to failure . To this end, we take the outcome with ERCs as the baseline, and distinguish three categories of failing groups: (i) groups in which only player 1 contributes less than half of the endowment, (ii) groups in which only player 2 contributes less than half of the endowment, and (iii) groups in which both players fail to contribute at least half of their endowment. In the two treatments with endowment inequality (where we observe most failures), most groups either fail because player 1 (high-endowment player) falls short of contributing at least half of the endowment, or because both players contribute too little. Only in a clear minority of groups it is the low-endowment player who under-contributes. Overall, these results suggest that endowment inequality represents the biggest obstacle to coordination since no contribution pattern is a clear focal point. Finally, conditional behaviours are observed in failing groups with categories (i) and (ii), where players who contribute less than half of their endowment tend to increase their contribution in the next round . This can help to understand how players gradually coordinate on the pattern of ERCs.. 5When games have multiple equilibria (as in our treatments), evolutionary game theory is often interpreted as a means to single out one of these equilibria as the most salient outcome . In the a. The equilibria predicted by this method, however, fail to reproduce the experimental patterns. For three treatments , this method selects the mutual defection equilibrium as most salient. In the remaining two treatments , replicator dynamics predicts the more productive player to make all the contributions. We find a similar mismatch between evolutionary trajectories and experimental results when we use a random sample of initial populations, or when we use different evolutionary dynamics . In all cases, evolutionary models underestimate the contributions of both players in the treatments with full equality and endowment inequality. In addition, they overestimate the contributions of the more productive player in the treatments with productivity inequality.As a first approach, we assume that initially, all strategies are played with equal frequency and trace the trajectory of replicator dynamics ,64, see figure 3b). Similarly, also the fit of all other evolutionary models is now greatly improved, as illustrated in the electronic supplementary material, figure S6. Overall, these results suggest that a naive application of classical evolutionary dynamics and learning processes may fail to anticipate the regularities of empirical play (irrespective of whether the used dynamics is deterministic or stochastic). However, evolutionary models can recover these empirical regularities when taking into account the true empirical initial distribution.As one potential reason for this mismatch, we note that models of evolutionary game theory neglect information that is not directly represented by the pay-off matrix. This information may entail, for example, the presence of natural focal points . In addifigure 3To complement this one-shot analysis, in the electronic supplementary material, S4, we provide additional analyses to capture the repeated-game character of the experiment. Among other results, we explore how individuals learn to make decisions when they can choose among all reactive strategies. When individuals use reactive strategies, they condition their behaviour on their co-player’s behaviour in the previous round. Our simulations suggest that once individuals interact repeatedly with the same partner, they are more likely to reach the threshold over time . At the same time, however, the repeated game analysis does not recover the empirical observation that individuals have a clear tendency to favour outcomes in which both players contribute equally (either in absolute or relative terms). These findings may be taken as another indication that focal points and the subjects’ expectations prior to the experiment may play a decisive role.. 6In this paper, we study the effect of inequality in asymmetric threshold public goods games. Players decide how much of their endowment to contribute to a public good. Only if the group’s collective contribution reaches a given threshold, players receive a reward. To study the effect of exogenous inequality, individuals may either differ in their endowments, or in how productive their contributions are. In general, such threshold public goods games allow for many different equilibria. These equilibria differ in whether or not the threshold is reached, and in the relative contributions of each player.With a behavioural experiment, we show that endowment inequality is more of an obstacle to cooperation than productivity inequality. Interestingly, the strength of asymmetry (e.g. whether one player has two times or three times the endowment of the other player) seems to have a negligible effect. Our data suggest that endowment inequality makes it particularly difficult to coordinate because different individuals may consider different contribution patterns as the most salient. While some individuals may strive to make equal absolute contributions, others may strive to equalize the individuals’ relative contributions (relative to the players’ endowments) ,46. SuchInterestingly, although groups in the treatment with productivity inequality are extremely successful in reaching the threshold, they typically do not coordinate on the most efficient equilibrium. In these treatments, efficiency would require the more productive player to make all the contributions. While this equilibrium maximizes the group’s collective pay-off, it leads to a rather uneven distribution of these pay-offs, with the low-productivity player benefitting disproportionately. Instead, our experiment suggests that groups unanimously prefer the two players to make equal contributions. This observation suggests that there is an interesting efficiency dilemma on top of the usual social dilemma. Individuals may refuse to choose the most efficient equilibrium if doing so would lead to unequal pay-offs ,67.Using different models of evolutionary game theory, we have also explored to which extent classical evolutionary dynamics can account for these empirical regularities. To this end, we assume in the main text that individuals interpret their interactions as a series of one-shot games. This analysis does not directly mimic the set-up of the experiment, in which participants interact with the same co-player for 20 rounds. Nevertheless, in coordination games such as ours, a one-shot analysis can already give basic insights into the strategic logic of the game: in threshold public good games, individuals prefer to meet the threshold, yet they prefer their co-player to make the respective contributions. Repeated games may allow for additional equilibria. Yet the most efficient repeated-game equilibria correspond to the set of cooperative one-shot equilibria shown in When applied naively , we find that all considered evolutionary dynamics based on the one-shot game provide a poor match to the empirical data. They overestimate how often people would defect, and how often the more productive player would cooperate in the treatments with productivity inequality. One reason for this mismatch might be related to the participants’ beliefs and expectations prior to the experiment. In their daily lives, individuals rarely play any single coordination game in isolation. Rather they play a mixture of different games with different incentives to cooperate. As a result they develop simple heuristics based on inequality aversion ,69, refeOverall, our study demonstrates that evolutionary game theory remains a powerful tool for modelling behavioural dynamics in asymmetric interaction. However, when predicting human behaviour in coordination games with many equilibria, they may mis-predict which equilibrium arises if some of the equilibria are more salient to participants than others. In addition, the experiments also identify another source of complexity. For instance, some players withdraw from the cooperative equilibrium by decreasing their contribution, most likely because they expect the co-player to make up for their missing contributions in the future ,73. In t


Stem cells from the apical papilla (SCAPs) are used to regulate the microenvironment of nerve defects. KDM6B, which functions as an H3K27me3 demethylase, is known to play a crucial role in neurogenesis. However, the mechanism by which KDM6B influences the neurogenesis potential of SCAPs remains unclear. We evaluated the expression of neural markers in SCAPs by using real-time RT-PCR and immunofluorescence staining. To assess the effectiveness of SCAP transplantation in the SCI model, we used the BBB scale to evaluate motor function. Additionally, toluidine blue staining and Immunofluorescence staining of NCAM, NEFM, β-III-tubulin, and Nestin were used to assess nerve tissue remodeling. Further analysis was conducted through Microarray analysis and ChIP assay to study the molecular mechanisms. Our results show that KDM6B inhibits the expression of NeuroD, TH, β-III tubulin, and Nestin. In vivo studies indicate that the SCAP-KDM6Bsh group is highly effective in restoring spinal cord structure and motor function in rats suffering from SCI. Our findings suggest that KDM6B directly binds to the HES1 promoter via regulating H3K27me3 and HES1 expression. In conclusion, our study can help understand the regulatory role of KDM6B in neurogenesis and provide more effective treatments for nerve injury. Nerve defects caused by tumors and trauma can significantly impact the motor ability and quality of life of patients. The new incidence rate is approximately 40 cases per million population , with anEpigenetic modifications are known to play a crucial role in neurogenesis, learning, and memory. During the process of neural repair, histone lysine methylation modifications have been shown to influence the function of repaired neural tissue by interfering with the expression of specific genes ,11,12,13SCAPs are a type of stem cell derived from neural crest cells and have the potential to differentiate into various types of tissue. Additionally, they highly express various neural markers such as brain-derived neurotrophic factor (BDNF), Glial cell line-derived Neurotrophic Factor (GDNF), Angiopoietin-1 (ANGPT1), and vascular endothelial growth factor A (VEGFA) . SCAPs hIn our study, we investigated the role of KDM6B in neurogenesis in SCAPs. Our results suggest that the knockdown of KDM6B in SCAPs has a restorative effect on motor function in rats with SCI by enhancing the neurogenesis potential of SCAPs. These findings provide valuable insight into the potential applications of SCAPs in nerve repair and epigenetic modification.In this study, the effect of KDM6B on neural marker expression was investigated using SCAPs. To achieve KDM6B knockdown in SCAPs, lentivirus transfection was utilized, followed by detection of knockdown efficiency through Western Blot analysis after 3 days of treatment with 2 μg/mL of puromycin. The expression level of KDM6B exhibited a significant decrease in the KDM6Bsh group when compared to the control group A. MorphoTo investigate the effect of KDM6B on neural marker expression in SCAPs, we overexpressed KDM6B in SCAPs and observed morphological changes during neural induction. The efficiency of KDM6B overexpression was confirmed by Western blot analysis. The expression level of KDM6B presented a marked increase in the HA-KDM6B group when compared to the control group A. DynamiWe established a spinal cord hemisection rat model and transplanted SCAPs to evaluate their repair effect after 5 weeks A. The BBTo further assess the repair of spinal cord tissue, we stained the repair tissue using hematoxylin–eosin (HE) and toluidine blue (TB). HE staining was employed to visualize tissue cell structure, while TB staining was used to assess the degree of neuronal damage repair. HE staining showed that there were large tissue vacancies in the SCI group, while the SCI + Scramsh group displayed only a few tissue cavities resembling residual scar tissue. However, the tissue cavities in the SCI + KDM6Bsh group were almost invisible, closely resembling the spinal cord tissue of the sham group A. ResultTo examine neuronal communication and intracellular filament distribution, we stained the NCAM  and NEFM (Neurofilament Medium), respectively A. The exTo identify differentially expressed genes between KDM6B-overexpressed SCAPs and the control group, we performed microarray analysis. The results indicated 118 differential genes in the KDM6B overexpressed SCAPs compared to the control group. Among these genes, 40 were upregulated, while 78 were downregulated . Specifip-value and enrichment factor. Specifically, upregulated genes were associated with the estrogen signaling pathway, DNA replication, and cell cycle, while downregulated genes were associated with the Jak-STAT signaling pathway, MAPK signaling pathway, TGF-beta signaling pathway, TNF signaling pathway, signaling pathways regulating the pluripotency of stem cells, and FoxO signaling pathway (We carried out gene ontology (GO) analysis and pathway analysis to identify the statistical enrichment of various biological functions and pathways associated with these differentially expressed genes. GO analysis showed that upregulated functions included catalytic activity, binding, cellular processes, and biological regulation, while downregulated functions included negative regulation of the metabolic process, cellular processes, signaling, and biological regulation A,B. Path pathway A,B.Three downregulated genes, HES1, EGR1, and NR4A2, were selected from the microarray analysis and used real-time RT-PCR to detect their expression after KDM6B knockdown and overexpression. The results were consistent with the microarray analysis data, and in SCAPs overexpressing KDM6B, the expressions of HES1, EGR1, and NR4A2 were significantly downregulated A–C, and Nerve injury caused by trauma and tumors seriously affects patients’ quality of life. Current treatments mainly rely on surgical decompression, neurotrophic drugs, and physical therapy. However, their efficacy cannot meet clinical needs ,25. In rThe present study investigated the role of KDM6B in the development of neuron-like characteristics in SCAPs. The knockdown of KDM6B led to the formation of larger neuron-like cells, which displayed a typical morphological manifestation of neurogenesis induction . ImmunofAnimal experiments were carried out to confirm the effectiveness of KDM6B knockdown on nerve tissue regeneration. The transplantation of KDM6B-knockdown SCAPs led to faster and better motor function recovery after 5 weeks, as indicated by BBB scores, reaching about 70% of the sham group. Morphologically, the spinal tissue at the defect of the KDM6B depleted group exhibited satisfactory results in terms of texture, morphology, and fusion with the surrounding area. HE staining and toluidine blue staining revealed that the distribution of cells and stroma in the defective area of the spinal cord treated with KDM6B-knockdown SCAPs was similar to that in the normal spinal cord, indicating a better repair effect.Moreover, the observation of h-mitochondria revealed that transplanted human SCAPs remained localized within the operative area of spinal cord defects. It can be concluded that SCAP transplantation promotes the activation of neural precursor cells, the remodeling of neuronal axons, and the information exchange between nerve cells in the spinal cord injury model of rats, forming favorable conditions for nerve tissue regeneration and signal transduction, with KDM6B knockdown further enhancing this effect. NCAM is involved in axon growth and guidance, synaptic function, and neuronal survival ,34. Its To investigate the effects of KDM6B on SCAP regulation in the neural repair microenvironment, we conducted a microarray analysis to identify differentially expressed genes and signaling pathways. The expression levels of hairy and enhancer of split 1 (HES1), EGR1 (early growth response 1), and nuclear receptor subfamily 4 (NR4A2) were significantly downregulated in KDM6B overexpressed SCAPs. HES1 is a direct transcriptional target of the Notch pathway and plays a critical role in determining the fate of neural precursor cells . NOTCH sThe findings of this research have significant implications for the treatment of spinal cord injury. Currently, there are limited effective therapies available to promote functional recovery after spinal cord injury. The identification of KDM6B as a potential therapeutic target for improving motor dysfunction post-SCI is a meaningful discovery in the field. Additionally, our study demonstrates that combining KDM6B treatment with SCAP transplantation can provide a more effective strategy for promoting recovery after spinal cord injury. This combination approach has the potential to significantly improve the quality of life for individuals suffering from spinal cord injury.SCAPs used in this study were procured from Shanghai Anwei Biotechnology Co., Ltd. . SCAPs were extracted from the immature root tip tissue of wisdom teeth with appropriate patient consent. SCAP cultures at the third to fifth passages were utilized for conducting the subsequent experiments. Further details regarding the culture of SCAPs have been elucidated in our previous publication .In this study, plasmids were utilized as per standardized protocols. The pQCXIN retroviral vector was used for the insertion of the hemagglutinin (HA)-tag, along with the complete sequence of KDM6B, while the pLKO.1 lentiviral vector was employed for the insertion of short hairpin RNA (shRNA) specific to KDM6B. The virus packaging procedure was performed meticulously in accordance with the guidelines provided by Clontech Laboratories or Addgene, depending on the specific protocol adopted. The shRNA target sequence for KDM6B was reported as KDM6B shRNA (KDM6Bsh) 5′-AAGTGGAATGTTTAACAGTGC-3′, and the scramble shRNA (Scramsh) was procured from Addgene.6 cells into ultra-low adsorption dishes  and cultured them for 9 days. The neural induction medium employed was previously described [For the induction of neurogenic differentiation in SCAPs, we seeded 1×10escribed . Half chescribed . At 6 weescribed ,47.The primer antibodies were as follows: Nestin , β-III-Tubulin , NEFM , NCAM , and h-mitochondria .Protein extraction and gel electrophoresis methods employed in this study were consistent with the protocols outlined in our previous publication . The priTotal RNA was extracted from SCAPs using an extraction kit . The extracted RNA (1 μg) was reverse transcribed into cDNA according to the standard procedure of the Invitrogen kit. Real-time PCR reactions were carried out in a manner consistent with our previous publication . Glycera6 cells in 5 μL volume was added to a 30 μL ECM gel  and transplanted at the site of spinal cord injury. The Sprague Dawley rats were randomly assigned to four groups: Sham, SCI, SCI + Scramsh, and SCI + KDM6Bsh, (n = 6).Female Sprague Dawley rats aged 6–8 weeks were procured from Vital River Laboratory Animal Technology Co., Ltd.  for use in the spinal cord injury (SCI) model. Following a one-week period of adaptive feeding, surgical procedures were performed on all rats. Anesthesia was induced in the rats using sodium pentobarbital , following which the spine between T9 and T10 was exposed, and hemisection of the spinal cord at T10 was performed as per previously established methods . A suspeTo assess the exercise ability of the rats, we employed the Basso, Beattie, and Bresnahan locomotor rating scale  . Two expAt 5 weeks post-SCAPs transplantation, the rats were sacrificed, and spinal cords containing the injured site were collected. The collected spinal cord tissue was fixed for 24 h using 4% paraformaldehyde and 30% sucrose solution at 4 °C. Tissue sections of 5 μm thicknesses were prepared and stained with hematoxylin–eosin (HE). To evaluate the number and degranulation of mast cells, the spinal cord sections were subjected to toluidine blue staining following standard protocols. Initially, the sections were deparaffinized in xylene and then dehydrated through a series of graded ethanol solutions for 5 min each . Subsequently, they were placed in water for 5 min and transferred to toluidine blue for 4 min before being carefully blotted. Following this, the sections were treated with absolute alcohol for 1 min, cleared in xylene, and mounted on a glass slide using Eukitt . The staining protocol was consistent with established procedures and resulted in blue-stained sections and purple-stained mast cells .p-value < 0.05 as the threshold criteria for identifying significant changes in mRNA expression.For gene expression profiling, the PrimeView™ Human Gene Expression Array was employed. The differential gene expression analysis was carried out using Affymetrix GeneChip Operating Software, as per previously established methods . DiffereThe ChIP assay was conducted utilizing a ChIP assay detection kit , following established protocols . The DNAt-test, one-way ANOVA, or Kruskal–Wallis test employed accordingly. A p-value ≤ 0.05 was considered statistically significant for all analyses.Each experiment was conducted a minimum of 3 times to ensure the reliability of the results. Statistical calculations were carried out using GraphPad5  or SPSS 17 . The choice of statistical test was determined based on the nature of the dataset and its distribution, with either Student This study aimed to investigate the role of KDM6B in promoting SCAP-mediated nerve repair. We observed that SCAPs could enhance adhesion and spreading between neurons, while knockdown of KDM6B led to increased expression of neural markers and secretion of the neural matrix. Furthermore, we identified the KDM6B-HES1 signaling axis as a key mechanism involved in this process. Overall, these findings shed new light on the potential for epigenetic modification to assist SCAPs in promoting nerve repair, and provide a basis for future studies for dental tissue-derived stem cells in treating nerve defects.


FOXO family of proteins are transcription factors involved in many physiological and pathological processes including cellular homeostasis, stem cell maintenance, cancer, metabolic, and cardiovascular diseases. Genetic evidence has been accumulating to suggest a prominent role of FOXOs in lifespan regulation in animal systems from hydra, C elegans, Drosophila, and mice. Together with the observation that FOXO3 is the second most replicated gene associated with extreme human longevity suggests that pharmacological targeting of FOXO proteins can be a promising approach to treat cancer and other age-related diseases and extend life and health span. However, due to the broad range of cellular functions of the FOXO family members FOXO1, 3, 4, and 6, isoform-specific targeting of FOXOs might lead to greater benefits and cause fewer side effects. Therefore, a deeper understanding of the common and specific features of these proteins as well as their redundant and specific functions in our cells represents the basis of specific targeting strategies. In this review, we provide an overview of the evolution, structure, function, and disease-relevance of each of the FOXO family members. The four isoforms of FOXO transcription factors in mammals have both overlapping and non-redundant functions.Isoform-specific, non-redundant functions are regulated by intrinsic and context-dependent mechanisms including differences in the paralog genes, transcripts, and proteins, their regulation by miRNAs, post-translational modification (PTMs), and binding partners as well as their cell-specific expression and regulation.FOXO isoforms exert a differential role in human aging and in several age-related diseases.Pharmacological inhibition or activation of FOXO proteins for therapeutic or preventive purposes has become feasible.What is the contribution of tissue-specific expression and co-expression patterns versus isoform-specific upstream regulation and downstream signaling to non-overlapping functionalities of the FOXO isoforms?When and why do FOXO isoforms exert redundant functions under physiological and pathological conditions?Is isoform-specific manipulation of FOXO activity feasible and beneficial?Which are the clinical indications best suited for isoform-specific FOXO targeting?FOXO proteins represent a sub-family of the forkhead box family (FOX) superfamily of proteins. Originally identified in the fruit fly, the FOX proteins can be found in virtually all eukaryotes, from yeast to vertebrates, but not in plants –4. The FC elegans and in Drosophila, daf-16 and dFOXO, respectively give rise to various isoforms, while the FOXO paralogs FOXO1, 3, 4, and 6 in mammals produce similar proteins. FOXO2 is a reported synonym for FOXO3, and FOXO5 is the FOXO3 ortholog in fish. FOXO isoforms in humans are generated through both mechanisms. In this review article, the term isoforms refer to the paralog-derived isoforms while the term variants will be used for the isoforms derived from single FOXO genes. The paralogous FOXO genes encode for the FOXO isoforms FOXO1, 3, 4, and 6, here referred to as paralog-derived isoforms, and each of these single genes can generate variants of these proteins. Human FOXO proteins are encoded by four different genes and one pseudogene located on different human chromosomes. These FOXO paralogous genes account for the diversity of functions of FOXO family proteins. While single copy genes suffer from strong negative selective pressure to evolve conservatively, gene duplications can generate redundant copies of genes relieving the selection pressure on one of them and opening up the possibility to adopt new functions. Wang et al. suggested that two major duplications of FOXO genes had occurred early in the vertebrate lineages. The first duplication leading to the emergence of two lineages which evolved into FOXO3/6 and FOXO1/4, and the second duplication finally resulted in FOXO6 and FOXO3, and FOXO1 and FOXO4 [The term isoform has been ambiguously used in the literature as it can refer to different concepts. Isoforms can describe different proteins produced by a single gene or similar proteins derived from different genes. A single gene can give rise to several distinct proteins by using alternative promoters or through alternative splicing. Conversely, paralogous genes generated by duplication of a single original gene can produce very similar proteins. Indeed, the unique FOXO genes in nd FOXO4 . Whereasnd FOXO4 , 22. Indnd FOXO4 . It has nd FOXO4 . Historind FOXO4 . The difC. elegans, two identified isoforms, daf-16a and daf-16 d/f display differential tissue enrichment, preferential modulation by upstream kinases, and regulate distinct and overlapping target genes, but functionally cooperate to fine-tune insulin/IGF-1 signaling and lifespan of the worm [While several paralog-derived variants have been reported for each FOXO isoform Table , none ofthe worm . Anotherthe worm . The lonthe worm , 29 havethe worm . Two shoHydra, C elegant, and drosophila [As discussed above, during vertebrate evolution, four paralog-derived FOXO isoforms emerged from a single gene in invertebrates. A large body of evidence has indicated that FOXO1, 3, 4, and 6 have redundant and isoform-specific functions. Most remarkable, full body knockout of Foxo1 in mice leads to defects in vasculature and is embryonic lethal . Whereasosophila –36. In mosophila .Fig. 2ThAlthough it has been shown that all FOXO isoforms bind to the same consensus sequences, subtle differences in the environment of the enhancer and promoter elements might still influence the preferential binding of specific FOXO isoforms to target genes. In agreement with this hypothesis, it has been reported that non-canonical FOXO1 recognition sites, incomplete FOXO1 occupancies at the available insulin response elements, and FOXO1 dimeric interactions may play a role in stabilizing DNA looping . While F10 -helix  conformation, which may play a role in DNA recognition via nonspecific interactions (helix H4) [FOXO proteins have a structured winged-helix type of DNA binding domain (DBD), followed by a largely unstructured, flexible “tail” containing nuclear export sequences (NESs) and a transactivation domain (TAD). The forkhead, or “winged helix” DBD (WHD), is the trademark of the FOX protein family , 40 and elix H4) . AnotherThis insertion creates a longer connecting sequence between helices 3 and 4, which appears to be highly flexible, as revealed by both structural studies  and moleIn contrast to the high level of sequence conservation observed in the DBD, most of the remaining sequence of FOXOs is largely divergent with sequence identity as low as 20%, except for three short-conserved regions CR1, CR2, and CR3 Fig. . The divThe main negative and positive regulatory inputs that control the activity of FOXO proteins come from the growth factor-dependent PI3K/AKT pathway and cellular stress signaling, respectively. FOXOs are primarily regulated through a complex combination of PTMs, such as phosphorylation, acetylation, mono and polyubiquitination, methylation, glycosylation, and nitrosylation. Table In the presence of insulin, and many other growth factors, the canonical regulator AKT phosphorylates FOXO proteins in the nucleus, at three conserved RxRxxS/T residues. AKT phosphorylates FOXO1 at Thr24, Ser256, and Ser319, FOXO3 at Thr32, Ser253 and Ser315 and FOXO4 at Thr28, Ser193 and Ser258. These covalent modifications create a docking site for the chaperon protein 14–3–3 enabling the CRM1-mediated nuclear export of FOXO factors  FOXO nucOn the other hand, elevated levels of reactive oxygen species (ROS), nutrient deprivation, or DNA damage, can activate FOXOs to restore cellular homeostasis , 49, 51.Upon low energy levels, AMPK can phosphorylate FOXO3 in six different residues  to induce FOXO activity. Only FOXO3 and FOXO4 share the motif flanking Ser413 while the Ser588 motif is solely conserved between FOXO3 and FOXO1 . AlthougC. elegans, respectively. As the LxxLL motif in the human FOXO isoforms has been suggested to mediate their interaction with nuclear hormone receptors (NHRs), the isoform-specific differences of the LxxLL motif might play a role in the selective interaction between FOXOs and several NHRs [FOXOs activity significantly depends on the interaction with binding partners to regulate transcription. This co-regulation can even occur independently of direct binding of FOXO to DNA either through transcriptional synergy, recruitment of conventional cofactors, proteolytic degradation, transcription factor sequestration, or displacement of regulatory cofactors . These iral NHRs .FOXOs activity can also be regulated at the post-transcriptional level. Table Interestingly, FOXO proteins are involved in several feedback loops. FOXO factors can control their own expression adding a new level of complexity. FOXO1 can induce its expression, while FOXO3 can elevate FOXO1 and FOXO4 mRNA levels. The FOXO1 promoter exhibits a conserved FOXO-binding site involved in this positive feedback loop, whereas no conserved binding site is present in FOXO4 . Furtherwww.proteinatlas.org), human FOXO transcripts except FOXO6 mRNAs, are ubiquitously expressed, but at different levels depending on the tissue site   did not differ under conditions of caloric restriction compared to wild type controls [Caloric restriction (CR) is the most reliable intervention to regulate aging and increase the healthy lifespan in diverse species, including rodents and non-human primates  and FOXOcontrols . Althougcontrols . DisruptAs aging is not a positively selected process, evolutionary theories of aging have raised the question on natural selection of longevity genes. Indeed, FOXO factors serve as paradigmatic examples of different mechanisms underlying aging theories  includinFOXO factors have been established as context-dependent tumor suppressors that are frequently inactivated in human cancers , 113. HoAs mentioned above, DAF-16/FOXO proteins are important mediators of insulin signaling, regulating a variety of metabolic functions in a broad range of organisms , 123.Diabetes is a metabolic disease characterized by elevated levels of blood glucose caused by impaired capability to produce or respond to insulin. Since FOXO proteins are key mediators of insulin signaling, they have been suggested as potential therapeutic targets for the treatment of diabetes.All four FOXO isoforms are negatively regulated by insulin and insulin like growth factor (IGF) , 80, 124The FOXO1 isoform regulates hepatic glucose production by decreasing both glycogenolysis and gluconeogenesis. Decreased expression of hepatic FOXO1 is associated with lower levels of glucose in mice, both at birth and in adulthood. FOXO1 plays an important role in the induction of hepatic gene expression and on the levels of glucose production and the major differences when comparing with the control mice are observed in animals showing temporary and not chronically decreased levels of FOXO1. This indicates that FOXO1 is required for the control and promotion of the hepatic glucose production, even in the presence of FOXO3 isoform, being FOXO3 able to compensate for chronic but not acute FOXO1 ablation . MoreoveSimilarly, FOXO6 is also involved in insulin signaling and glucose production. Increased FOXO6 activity in the liver is associated with insulin resistance and insulin deficiency in mice (in fasting hyperglycemia and diabetic mice). FOXO6 transcription is induced by glucagon via cAMP and inhibited by insulin signaling. The same study also demonstrated the association between Foxo6 expression and induced hepatic gluconeogenesis, without changes verified in the transcriptional levels of the other isoforms  . AccordiThe relevance of FOXO3 and FOXO4 in hepatic glucose production is not very clear yet, however, they contribute and play a synergistic role with FOXO1 and FOXO6 in the regulation of this process . In factFOXO isoforms, in response to insulin signaling, are, therefore, important regulators of the hepatic glucose production and the pancreatic β-cells, which demonstrates the importance of these transcription factors in the regulation of insulin sensitivity and diabetes. FOXO1 is the most studied isoform in this disease, but there is also evidence for a role of FOXO3 and FOXO6. However, the manipulation of all the isoforms leads to similar results in this context. While basal levels of FOXOs expression are beneficial and necessary for the regulation of some hepatic processes, their upregulation is associated with pathological phenotypes and the worst prognosis.FOXO transcription factors have been shown to play a role in neural cell fate and function, in both physiological and pathological conditions. Neurodegenerative diseases, such as Huntington’s, Parkinson’s, and Alzheimer’s diseases are associated with impaired neuronal and motor function, impacting the individual’s life over the years , 132.In aging brain tissues, FOXO1 expression and activation seem to be significantly increased when compared with younger tissue, which was not verified for FOXO3 and FOXO4. However, simultaneous deletion of Foxo1, 3, and 4 in neurons seemed to be sufficient for initiating axonal degeneration, suggesting an important role of FOXO proteins in neuronal protection. Intriguingly, specific genes found overexpressed in aged brains, are also upregulated in adult mice lacking Foxo1, 3, and 4 expression . This daIn Huntington’s disease, FOXO3 is overexpressed and seems to regulate its own expression in a positive autoregulation loop. FOXO3 overactivity might trigger a protective response to the elevated cellular stress . In miceIn dopaminergic neurons, FOXO3 activity is related with neuronal death and ROS detoxification in oxidative stress conditions. When α-synuclein is overexpressed in these neurons, which happens in Parkinson’s disease, moderate FOXO3 expression seems to regulate α-synuclein accumulation, possibly via autophagic degradation. In this condition, but with increased levels of FOXO3 it was observed an accumulation of α-synuclein aggregates, contributed to the opposite effect and promoted cell death . OverallA recent study regarding FOXO3 expression revealed a cell- and age-dependent function of FOXO3 in the brain of mice. Loss of FOXO3 was associated with altered metabolism and function of astrocytes, increased β-amyloid plaques, a pathological hallmark of Alzheimer’s disease, and synapse loss. Overall, FOXO3 expression in astrocytes seems to protect the brain from aging and from Alzheimer’s disease .Additionally, it was observed that FOXO1 and FOXO3 could be potential targets in anxiety and mood disorders. Treatments in mice with imipramine, an antidepressant compound, were correlated with increased levels of FOXO1 and FOXO3 phosphorylation and consequent inactivation. In the same way, decreased FOXO1 levels in the brain were associated with reduced levels of anxiety in mice, being suggested as a regulator of the anxiety-like phenotypes .In conclusion, normal expression and activity of FOXO proteins, specially FOXO1 and FOXO3, have a protective role in neuronal tissue and consequently in preventing neurodegenerative diseases in mice.pS253 phosphopeptide displays a distinct mode of interaction with 14-3-3 compared to FOXO1pS256 [Non-ligand transcription factors including FOXO proteins have been considered difficult to target due to significant structural disorder and lack of defined small-molecule binding pockets . While sXO1pS256  might alXO1pS256 . FurtherXO1pS256 . DifferemiRNAs that bind to stabilizing sequences in the 3′ end of FOXO isoforms either stabilizing them or targeting them for degradation could be another approach. The availability of isoform-specific binding partners or small molecules could enable targeted protein degradation using proteolysis targeting chimaeras (PROTACs). But, maybe the most important issue to be addressed by these future studies is the feasibility and therapeutic usefulness of isoform-specific FOXO modulating drugs.Figure S1legend Figure s1