The short hydrogen bonds have donor–acceptor distances in the region of 2.45 Å and are formed in substituted urea and organic acid molecular complexes of N,N′-dimethylurea oxalic acid 2 : 1 (1), N,N-dimethylurea 2,4-dinitrobenzoate 1 : 1 (2) and N,N-dimethylurea 3,5-dinitrobenzoic acid 2 : 2 (3). From the combined analyses, these complexes are found to fall within the salt-cocrystal continuum and exhibit short hydrogen bonds that can be characterised as both strong and electrostatic  or very strong with a significant covalent contribution (2). An additional charge assisted component is found to be important in distinguishing the relatively uncommon O—H⋯O pseudo-covalent interaction from a typical strong hydrogen bond. The electron density is found to be sensitive to the extent of static proton transfer, presenting it as a useful parameter in the study of the salt–cocrystal continuum. From complementary calculated hydrogen atom potentials, we attribute changes in proton position to the molecular environment. Calculated potentials also show zero barrier to proton migration, forming an ‘energy slide’ between the donor and acceptor atoms. The better fundamental understanding of the short hydrogen bond in the ‘zone of fluctuation’ presented in a salt-cocrystal continuum, enabled by studies like this, provide greater insight into their related properties and can have implications in the regulation of pharmaceutical materials.In this work we use high-resolution synchrotron X-ray diffraction for electron density mapping, in conjunction with  ab initio modelling reveal how H-atom position and molecular environment tune short hydrogen bond characteristics and properties.Mapped electron density and  Hydrogen bonds involving oxygen or nitrogen donor/acceptor atoms such as O—H⋯O, N—H⋯O or N—H⋯N tend to be stronger. There is particular interest in the shorter O—H⋯O hydrogen bonds whose donor–acceptor (D–A) distances approach 2.45 Å as they are found to be on the border between strong and very strong interactions.7Hydrogen bonds are important in biology and chemistry for their roles in maintaining structure,8 in some cases this may equate to covalency in a 3-centre 2-electron system.9,10 The interactions can have hydrogen potentials approaching that of a single well with a diminished barrier to proton transfer11 with large values of hydrogen bond energy (>100 kJ mol−1).12,13 Varying proton positions are often found, either bonded to the original donor , transferred to the acceptor  or, in some cases, the donor and acceptor atoms compete for the hydrogen atom such that it may be centred14  or appear on the edge of proton transfer.15 Where proton transfer occurs, these are charge assisted hydrogen bonds (CAHBs) and are even stronger due to the additional charges increasing the electrostatic component of the interaction.16 Protonation state is particularly important in the regulation of pharmaceutical cocrystals, determining whether the final form is an approved drug or a new chemical species.17 ΔpKa of the interacting components (pKbase − pKacid) can be used to predict salt formation by proton transfer, expected if ΔpKa > 2 or 3.18Specific features of these short, strong hydrogen bonds include a sharing of electron density distribution across the hydrogen bond,19–21 when formed in organic molecular crystals between acidic and basic molecular components.22 The number of systems identified with this behaviour are still relatively few but the proton hopping can have useful applications in ferroelectrics23 or colour changing24 materials. This behaviour is only observed for systems where the D–A distance is less than 2.45 Å whilst a less negative ΔpKa (i.e. approaching zero) between components may result in larger proton shifts.19 Combined inelastic neutron scattering and molecular dynamics simulations also indicate migration across short O—H⋯O hydrogen bonds to be a result of changes in hydrogen atom (H-atom) potentials induced by thermal fluctuations of the molecular environment.25Temperature dependent proton migration behaviour can be observed across short O—H⋯O hydrogen bonds+—H⋯O− hydrogen bonds formed in a selection of known substituted urea organic acid cocrystals and salts with O⋯O D–A distances in the region of 2.45 Å, where their character and temperature dependent behaviour are varied  and 3  are neutral whilst in 2  the hydrogen bond is charge assisted. The proton position is known to be sensitive to temperature in the short hydrogen bonds of 1 and 2, gradually shifting across the hydrogen bond as a function of temperature .In this work, we explore short O—H⋯O/Oe varied .19,20 Th26 we examine the experimental electron density distribution (EDD) interpreted using Bader's quantum theory of atoms in molecules (QTAIM)27,28 from single crystal samples of 1–3. The experimental EDDs are obtained following a multipolar crystal structure refinement29 against high-resolution single crystal synchrotron X-ray diffraction data. Synchrotron X-ray facilities are less routinely used to obtain the EDD despite offering high X-ray flux and tuneable wavelengths.30 The experimental EDD analysis is combined with first principle calculations of properties including the reduced density gradient from non-covalent interaction analysis,31 the electrostatic potential,32–34 PIXEL interaction energies35,36 and H-atom potentials.37–39 A combined analysis approach is commonly used to assess interaction characteristics in terms of material properties.40–42 Here it allows a complete characterisation of the short hydrogen bonds 46 with NoSpherA2,47 was performed to obtain accurate X—H distances. The SHADE3 server48 is a recommended method to obtain ADPs for charge density analysis.49 However, in this study, HAR was implemented to obtain the H-atom ADP model, giving the better fit to the experimental data.As this study focuses on hydrogen bonds, additional methods  are used to improve on the H-atom parameters determined from the X-ray data; typically the X—H distances are underestimated whilst only isotropic displacement parameters for the H-atoms can be refined. Obtaining the best models for the H-atoms is important in charge density analysis where any deficiencies will significantly affect the derived properties involving these atoms (and thus hydrogen bond characterisation).ab initio first principles computational methods is also described  basis set  basis set used. However, this was selected as a balance between cost and accuracy, particularly for the larger cluster models.A gas-phase calculation does not fully account for the deformation of the electron density of the molecule due to the crystal environment, and therefore the conclusions from the modelling must be treated with caution. On the other hand, the use of molecular calculations allows us to use a hybrid functional, a level of theory which is not routinely accessible to solid-state calculations, and it has recently been shown that such hybrid functionals provide a more accurate description of hydrogen bonding.1 , 2  and 3  have a common structural motif of a R22(8) carboxylic acid : amide hydrogen bonded dimer formed between substituted urea and organic acid molecular units  distances of the short hydrogen bonds are ca. 2.45 Å  and thid H-atom  also has31 (31 can be used to directly compare the reduced density gradient (Laplacian ∇2(rBCP)) observed during the multipole reduction of experimental charge densities to those of a comparable molecular adduct in vacuum.58 Both analyses are performed at bond critical points of the short hydrogen bonds  where its gradient is ∼0 being a minimum in the direction of the bond and a maximum perpendicular to it).The electron density distributions (EDD) of the short hydrogen bonds are examined in the topological parameters of the experimentally obtained EDD together with isosurfaces of the calculated reduced density gradient following non covalent interaction (NCI) analysis31 . The NCIρ(r)BCP at the O—H/H⋯O BCP decreases with increasing atomic separation (2(rBCP) for the H⋯O interaction is found in 2 (−1.71(11) e Å−5) where full proton transfer has occurred across the hydrogen bond. In agreement with previous findings,9 as the electron density reflects proton position in 1–3, it therefore appears sensitive to the extent of static transfer of the H-atom across the hydrogen bond. The large values of the electron density in the H⋯O region of the short hydrogen bonds given by the topological parameter ρ(r)BCP (0.79 to 0.9 e Å−3) combined with the ring shaped deep blue/covalent bond of the respective calculated NCI isosurface BCP ≈ 1 e Å3).10 These observations agree with the short hydrogen bond O⋯O separation distance of ca. 2.45 Å often being associated with stronger hydrogen bonds. As an indicator of strength, the values of ρ(rBCP) correspond to ca. 30% of that determined in the covalent bond of O—H in H2O (ρ(rBCP) 2.63 e Å−3).27,60 In weak to moderate strength hydrogen bonds, typical values are significantly lower on the order of 0.05 to 0.23 e Å−3 (ref. ρ(r)BCP are useful in assessing hydrogen bond strength whilst ρ(r)BCP gives a better indicator of position  within the salt–cocrystal continuum.The topological parameters  show thaparation  whilst oosurface  and S7†,Å−3 ref.  and haveÅ−3 ref. . This an2(rBCP) and energy densities  across the systems  is negative (−1.71(11) e Å−5) indicating a concentration of charge in the H⋯O region and, when found alongside a negative total energy density (HBCP), is characteristic of a ‘very strong’ hydrogen bond with a shared-shell covalent character. In contrast, the values of the Laplacian are positive (∇2(rBCP) > 0) for 1 and 3 corresponding to a depletion of charge within the H⋯O atom–atom region and is characteristic of a closed shell electrostatic interaction or ionic type bonding.61 The ratio of the Lagrangian kinetic and potential energy densities (|VBCP|/GBCP),62 which can be used to further define the hydrogen bonding interactions at the short hydrogen bond BCPs )64 or partial covalent character 65 from similar experimental electron density analysis. Of these, UPA64 is a system which also exhibits temperature dependent proton migration.21 Across the systems exhibiting this type of behaviour , there does not appear to be any correlation to the electron density distribution across the short hydrogen bond and the migration behaviour observed. Therefore, despite the electron density being sensitive to the extent of static proton transfer, it does not appear to be useful in predicting the likelihood of further proton transfer events as a function of temperature.A comparison of the values of the topological parameters of the Laplacian ∇ systems  identifyond BCPs , additioQ0) was found to be important in influencing the value of the Laplacian ∇2(rBCP). A larger Q0 elongates the charge concentration of the H-atom along the interaction increasing likelihood of overlap with the H⋯O BCP and therefore affecting its value. This effect is made clear in the 2D Laplacian maps . The importance of obtaining the ‘best’ ADP model for H-atoms in short hydrogen bonds is therefore highlighted, as extracted parameters at the BCPs such as the Laplacian can vary significantly.When analysing the topological parameters at the H⋯O BCP, the O—H bond directed quadrupole  occupies a negative (red) contour region with a correspondingly low population (0.68(1)e), modelled and refined as a monopole, and has a large, positive net charge (+0.32(1)e)  and net atomic charges (ca. +0.2e) are correspondingly larger and less positive, respectively.2D deformation density maps obtained from the experimental electron density distribution (EDD) and showing bonding and lone pair features  indicate Table S7. This ev1–3, calculated using Gaussian09  indicates an extent of charge sharing to the oxygen atom in the O—H bond. The electrostatic potentials for n09 ref.   interaction energies69,70 ESI 5.5 for 69,70 ESI. They de2 is highlighted as being different having a total dimer interaction energy (ETOTAL) of −493 kJ mol−1versus those for 1 and 3 that are in the region of −60 to −80 kJ mol−1. The ETOTAL is significantly larger for 2; to compare, an O—H covalent bond has a bond energy of ca. 465 kJ mol−1.73 However, this is not unusual for interactions between charged species, thought to occupy the strongly stabilising, ionic coulombic contact zone range.74 Using the Hibbert and Emsley classification,75,76 the hydrogen bonding interactions forming the dimers in 1 and 3 are considered strong (ETOT of −60 to −80 kJ mol−1) whilst they are very strong in 2 (ETOTAL is −493 kJ mol−1). The co-crystal/salt nature of 1–3 is further reflected in the dimer interaction energies where the salt has a significantly larger total interaction energy.As by the other analyses, the dimer in system ETOTAL into its contributing terms  shows that, for all dimers, the coloumbic component contributes most to the stabilisation of the dimer, where ECOULOMBIC is the largest negative energy. ECOULOMBIC defines the electrostatic interaction in terms of Coulomb interactions between charges at points ‘pixels’ in space and is not always found to be the dominating term for hydrogen bonds.77 The large repulsive contribution (EREPULSION) could be due to unfavourable secondary diagonal interactions78 in the dimer motif between the diagonally opposite carbonyl groups (in the urea and acid) oxygen lone pairs or H-atoms. These repulsive interactions are shown to exist by the yellow NCI isosurfaces  carboxylic acid : amide hydrogen bonded dimer  and therefore not likely to be the cause for the flatter potential in 2. Instead, it could be the added charge assisted component of this interaction, seen in this study to affect other properties of the hydrogen bond. pKa values are known to determine the energy barrier height in potentials for moving a H-atom between donor–acceptor wells.19,84 For systems 1 to 3, this does not seem to be the case where the ΔpKa of components are similar  whilst the ‘slide’ in the potentials occupy different regions for 1 and 3 (30–40 kJ mol−1) versus in 2 (ca. 10 kJ mol−1). One study also formed a link between the extent of temperature dependent migration and ΔpKa.19 As was found by Jones et al. (2012), the least negative (closer to zero) ΔpKa in 2 results in the greatest extent of migration observed. However, the trend is not reflected in 3, where the short hydrogen bonds show no evidence of temperature dependent proton migration yet have a less negative ΔpKa than 1, which is a migration material. If taken from the literature, ΔpKa values should be used with caution as they are often determined for the solution state and are affected by intermolecular interactions in a crystal.14 Here they are obtained from the literature19,85 and as such are used only as a guide.The donor–acceptor distances for the short hydrogen bonds are similar  and not an increasingly populated first excited state  and N,N-dimethylurea 3,5-dinitrobenzoic acid 2 : 2 (3) are characterised as strong with a more electrostatic contribution whilst in N,N-dimethylurea 2,4-dinitrobenzoate 1 : 1 (2) the short hydrogen bond is characterised as very strong with a significant covalent contribution. This donor–acceptor distance region therefore remains a zone of fluctuation in terms of the nature of the hydrogen bond. The identification of O—H⋯O hydrogen bonds with a covalent nature is still uncommon. However, a charge assisted component is seen to favour covalency in this interaction and proves to be the determining factor for this set of substituted urea organic acid molecular complexes, in terms of the strength and character of the short hydrogen bonds. Therefore, if salt formation is targeted, this presents a route to access hydrogen bonds with a covalent component.In the first part we analysed properties of the experimentally determined charge density alongside calculated properties from non-covalent interaction analysis and electrostatic potentials. These properties were also compared to the calculated total Pixel dimer interaction energies. From this, the short hydrogen bonds in The relation found between the atom-atom separation or protonation state and the topological properties of the charge density highlights how the electron density in these types of interactions remains highly sensitive to the static proton transfer process. It is therefore a useful parameter in studies of the salt-cocrystal continuum. This has implications for both pharmaceutics and the design of crystalline functional materials. However, for these systems, the electron density distribution does not appear to be useful to predict further proton transfer events as a function of temperature.1 and 2 is likely to also be due to fluctuations in the molecular environment and presents these systems as suitable cases for future inelastic neutron scattering and molecular dynamics studies. System 2  is highlighted as a model system, to add to the well-studied urea phosphoric acid 1 : 1 adduct, for learning about migration materials due to the significant perturbations exhibited by the short hydrogen bond.In the second part, hydrogen bond characteristics were examined in H-atom potentials, as calculated, and as a function of the crystal packing and temperature. The extensive studies of the H-atom potentials suggest asymmetric hydrogen bonds whose energetics are different when a charge assisted component is included. Extending the potentials to consider crystal packing neighbours, identified by the non-covalent interaction analysis, confirms that proton transfer is an effect of the local environment. Here we further identify that, for these systems, the interactions forming the extended hydrogen bonding have the biggest effect on the H-atom energetics. The similarities in the changes of the potentials as a function of temperature to well-studied migration material urea phosphoric acid suggest that the reported migration of Overall, the use of high-resolution synchrotron X-ray diffraction combined with neutron diffraction or Hirshfeld atom refinement has allowed good models of the experimental electron density to be produced in which trends and conclusions about the short hydrogen bonds could be made. Obtaining reliable H-atom models was shown to be important in these studies of short hydrogen bonds, where the H-atom exhibits atypical behaviour as a result of the strength of the hydrogen bond it is involved in. The Hirshfeld atom refinement method, in the absence of neutron data, enabled the study to be extended to additional systems for a better picture of hydrogen bonding characteristics.Contributions are defined using CRediT for standardised contribution descriptions. We highlight joint first co-authorship of L. K. Saunders (LKS) and A. R. Pallipurath (ARP) having significant shared data curation and investigation roles with LKS taking lead in conceptualisation of the study and writing the original draft and ARP for writing – review and editing. Credit LKS and ARP for formal analysis, methodology, validation and visualisation. Credit M. J. Gutmann for neutron diffraction data curation and formal analysis. Credit H. Nowell for X-ray diffraction data curation, N. Zhang for software assistance with Gaussian09 and CLP-Pixel calculations and D. R. Allan for providing resources and supervision.There are no conflicts to declare.CE-023-D1CE00355K-s001CE-023-D1CE00355K-s002


Glioblastoma is the most common and aggressive brain cancer, and there is a desperate need for new therapeutic strategies for this incurable cancer. Inhibition of Diacylglycerol kinase α provides novel direct mechanisms against glioblastoma cells, but also offers the potential to simultaneously boost immune cell activities against glioblastoma. This review provides an updated summary of the promising potential of Diacylglycerol kinase α inhibition against glioblastoma, with potential implications for other cancers as well.Diacylglycerol kinase α (DGKα) inhibition may be particularly relevant for the treatment of glioblastoma (GBM), a relatively common brain malignancy incurable with current therapies. Prior reports have shown that DGKα inhibition has multiple direct activities against GBM cells, including suppressing the oncogenic pathways mTOR and HIF-1α. It also inhibits pathways associated with the normally treatment-resistant mesenchymal phenotype, yielding preferential activity against mesenchymal GBM; this suggests possible utility in combining DGKα inhibition with radiation and other therapies for which the mesenchymal phenotype promotes resistance. The potential for DGKα inhibition to block or reverse T cell anergy also suggests the potential of DGKα inhibition to boost immunotherapy against GBM, which is generally considered an immunologically “cold” tumor. A recent report indicates that DGKα deficiency increases responsiveness of macrophages, indicating that DGKα inhibition could also have the potential to boost macrophage and microglia activity against GBM—which could be a particularly promising approach given the heavy infiltration of these cells in GBM. DGKα inhibition may therefore offer a promising multi-pronged attack on GBM, with multiple direct anti-GBM activities and also the ability to boost both adaptive and innate immune responses against GBM. However, both the direct and indirect benefits of DGKα inhibition for GBM will likely require combinations with other therapies to achieve meaningful efficacy. Furthermore, GBM offers other challenges for the application of DGKα inhibitors, including decreased accessibility from the blood-brain barrier (BBB). The ideal DGKα inhibitor for GBM will combine potency, specificity, and BBB penetrability. No existing inhibitor is known to meet all these criteria, but the strong potential of DGKα inhibition against this lethal brain cancer should help drive development and testing of agents to bring this promising strategy to the clinic for patients with GBM. Diacylglycerol kinase alpha (DGKα) is one of ten DGK family members that convert diacylglycerols (DAGs) to phosphatidic acids (PAs). Both DAG and PA strongly regulate numerous biologic processes. DAG is important for activating Protein Kinase C enzymes. PA is typically found at low levels relative to other phospholipids, in the cell membrane and elsewhere, but despite its low concentrations it regulates a number of signaling pathways. DGKα has been found to regulate signaling pathways integral to cancer, despite being only one member of the large DGK family—suggesting a general lack of redundant function across the family. PA has been found to control activity of mTOR, Akt, and Erk, while DGKα has been associated with NF-κB, HIF-1α, c-met, ALK, and VEGF ,6,7,8,9.GBM is the most common and most lethal primary brain tumor, causing 12,000–14,000 deaths each year in the U.S. alone . Median DGKα and its product PA are known to help maintain activity of several oncogenic pathways that have been linked to GBM malignancy and viability. These include Ras and Raf, Akt and mTOR, VEGF, and HIF-1, as described above. It is therefore unsurprising that DGKα knockdown and inhibition have been shown to have direct activity against GBM cells. While DGKα inhibitors are highly unlikely to block these individual targets and pathways as effectively or potently as specific inhibitors for each, the combined inhibition of multiple targets—even to a lesser degree—may be a promising approach for a genetically heterogenous cancer such as GBM with diverse signaling drivers. This seems particularly salient given the poor track record of highly specific single agents in clinical trials for GBM, as single agents or in combinations. In a prior report, we showed that mTOR and HIF-1α blockade are particularly important mediators of the direct activity of HIF-1α inhibition in GBM cells , but theNotably, DGKα inhibition may offer benefits versus single-agent or combined specific and potent inhibitors of targets such as mTOR and HIF-1α. Dedicated inhibitors of the mTORC1 complex are known to drive feedback loops leading to activation of the upstream driver Akt, which may yield even greater malignant behavior; however, DGKα inhibition has been shown to suppress Akt as well as mTOR activity in GBM cells . DGKα alNF1 lesions, and has been associated with higher Akt, TGF-β, and NF-κB activity [DGKα inhibition may also provide traction against a challenging and common resistance mechanism and subtype found in GBM and other cancers as well—the mesenchymal subtype. The Cancer Genome Atlas (TCGA) and other profiling efforts have suggested three GBM subtypes ,25,26: pactivity ,25,26. Mactivity ,28 and dactivity , but indactivity ,30,31. Aactivity . MES GBMactivity ,34,35. Factivity . Taken tactivity . Our finactivity . DGKα inGBM is a highly vascularized tumor, and the anti-VEGF agent bevacizumab remains in wide use for recurrent GBM. Antiangiogenic agents such as bevacizumab have yet to show an overall survival benefit in trials for all patients with GBM. However, they improve quality-of-life and reduce edema, and one retrospective analysis did show an overall survival benefit of upfront bevacizumab in patients with proneural GBM lacking IDH1/2 mutation . AntiangWhile the effects of DGKα inhibition on the immune system will be covered in more detail elsewhere in this issue, it is important to review their potential specifically for GBM therapy. As described above, GBM is considered an immunologically “cold” tumor, with a strongly immunosuppressive local milieu as well as challenges such as the BBB and decreased communication between the immune system and brain/GBM microenvironment. The immunologic functions already ascribed to DGKα suggest that its inhibition may help counteract some of the challenges involved in immunotherapy for GBM. DGKα inhibition has been found to reverse T cell anergy, a prominent mechanism by which T cells become inactivated in tumors ,41. OtheA new report from our group indicates that DGKα also acts to regulate macrophages; DGKα knockout/knockdown increases macrophage responsiveness to diverse stimuli . This maA number of reports have suggested that the addition of DGKζ knockout to DGKα knockout markedly increases T cell-activating effects. In fact, specific knockout of DGKζ may have stronger T cell-promoting effects than specific DGKα knockout, and this translated to greater anti-tumor activity in one report testing three syngeneic tumor models in mice . HoweverIt is possible that DGKα inhibition could also have beneficial effects on the expression of certain immunosuppressive checkpoint proteins. Interestingly, the treatment-resistant MES GBM subtype has much higher expression of the immune checkpoints PD-L1 and TIM3 than PN GBM ,34. In aIt is increasingly being recognized that the heterogeneity and adaptability of GBM will likely necessitate the use of combination regimens to achieve meaningful efficacy. DGKα inhibitors may be particularly well-suited for such combinations, with potential to enhance the activity of numerous therapies with direct anti-cancer activity but also immunotherapies. The broad signaling effects of DGKα inhibition, as well as preferential activity against the treatment-resistant mesenchymal phenotype in GBM and other cancers, could translate to a host of promising anti-GBM combinations with a DGKα inhibitor. We previously reported in vitro synergistic activity combining a DGKα-inhibiting compound with radiation against GBM stem-like cells , and ourCombining DGKα inhibition with immunotherapies may be particularly promising for GBM and other immunologically cold cancers. As described above, DGKα inhibition may help break T cell anergy and potentially exhaustion as well, thus eliminating barriers that impede the efficacy of immunotherapies in GBM. DGKα inhibition has already been shown to boost the anti-cancer activity of CAR-T cells in preclinical studies , and othEffectively incorporating DGKα inhibitors into regimens for GBM will of course require the development of adequate inhibitor compounds with favorable pharmacological properties, including sufficient penetration of the BBB. The most widely-used tool compounds in the laboratory in past decades, R59022 and R59949, have modest potency and activity against DGKα and also inhibit other targets more potently . HoweverNewer DGKα-inhibiting compounds with different structural bases may be able to provide better potency and specificity than ritanserin and its analogs, as well as other potential advantages. Efforts are underway to further develop such compounds, likely driven in large part by the appeal of the immunotherapeutic potential of DGKα inhibitors in combination regimens for cancer. The compound CU-3 has been reported as a DGKα inhibitor with a novel structure , and BriDGKα inhibition thus offers the highly attractive potential for multifaceted direct action against GBM with simultaneous immune-boosting activities and potential antiangiogenic activity as well (summarized in


Here, we show that this structural transition in antiferroelectric ZrO2 gives rise to a negative capacitance, which is promising for overcoming the fundamental limits of energy efficiency in electronics. Our findings provide insight into the thermodynamically forbidden region of the antiferroelectric transition in ZrO2 and extend the concept of negative capacitance beyond ferroelectricity. This shows that negative capacitance is a more general phenomenon than previously thought and can be expected in a much broader range of materials exhibiting structural phase transitions.Crystalline materials with broken inversion symmetry can exhibit a spontaneous electric polarization, which originates from a microscopic electric dipole moment. Long-range polar or anti-polar order of such permanent dipoles gives rise to ferroelectricity or antiferroelectricity, respectively. However, the recently discovered antiferroelectrics of fluorite structure (HfO Applying an electric field to an antiferroelectric material transforms its non-polar crystal structure into a polar one. Here, the authors show that the antiferroelectric transition in zirconia causes a negative capacitance, useful for electronics. First predicted in 19516, antiferroelectricity was subsequently discovered in the archetypal perovskite oxide, lead zirconate (PbZrO3)8. Ever since, the range of materials has been expanded to two-dimensional hybrid perovskites9, interfacially engineered heterostructures and superlattices10, fluorite structure binary oxides12, and more have been predicted by first principles calculations14. However, compared to their ferroelectric counterparts, antiferroelectrics have remained less explored and understood so far, despite their intriguing properties and rich phase transition phenomena16. Therefore, antiferroelectric materials hold a large untapped potential for the discovery of emergent phases for example in antiferroelectric oxide heterostructures, which have been investigated in this work.Antiferroelectric materials are promising for diverse applications ranging from energy harvestingP vs. electric field Ea characteristics as shown in Fig. 6. From a thermodynamic perspective, such a macroscopic response can be described by a free energy (G)-polarization landscape as shown in Fig. Ea = 0, where the only stable state is the macroscopically non-polar ground state (A) at P = 0. However, when an electric field Ea = E1 is applied, see Fig. 2G/dP2 < 0) between points B and C is thermodynamically unstable15. Note that the relative energies of the non-polar and polar states in Fig. 2G/dP2)−1, which means that at the antiferroelectric transition the capacitance of the material would become negative if stabilized in a larger system18. Note that we always mean “negative differential capacitance” when we write “negative capacitance” in this context. Due to the inversion symmetry of the non-polar ground state, the same transition occurs when an opposite electric field Ea = −E1 is applied, see Fig. P/dEa < 0.Antiferroelectricity is characterized by a distinctive, double hysteresis loop in the macroscopic electric polarization P–E curves have been derived from the Landau-Ginzburg-Devonshire theory of ferroelectric phase transitions many decades ago but were previously understood to be inaccessible to experiments due to their unstable nature19. Only recently, it was suggested that one could access these forbidden thermodynamic regions in ferroelectric/dielectric heterostructures using pulsed electrical measurements21. In such a structure, the positive capacitance of the dielectric layer can stabilize the region of negative capacitance and prevent the screening of bound polarization charge by free electrons in the metal electrodes. Here, we apply a similar approach to first probe the inaccessible region of a non-polar to polar structural phase transition in an antiferroelectric oxide. While experimental insights into such thermodynamic instabilities are of fundamental interest to materials science, they are also useful for prospective applications since the resulting negative capacitance can be used to amplify voltage signals in electronic devices and circuits22.Similar ‘S’-shaped 6. For example, in the ground state of antiferroelectric PbZrO3, two adjacent columns of Pb ions point in the same direction, while the next two columns have antiparallel alignment7. The application of a sufficiently large electric field then leads to a spontaneous alignment of the anti-parallel dipoles resulting in a transition to a polar ferroelectric state23. In a similar way, first principles calculations of ferroelectric/paraelectric heterostructures have predicted antiferroelectric-like negative capacitance regions due to the transition between a striped domain pattern and a ferroelectric monodomain state24. In contrast, the newly discovered HfO2 and ZrO2 based antiferroelectrics of fluorite structure transcend the classical definition of antiferroelectricity12, since their ground state does not exhibit anti-polar order but is microscopically non-polar25. Therefore, the original Kittel-model in Fig. 21.Historically, antiferroelectricity has been related to the anti-polar alignment of microscopic electric dipoles in the unit cell2 as a model system, which is of significant technological importance due to its compatibility with semiconductor manufacturing and thickness scalability to the nanometer regime12. Additionally, no doping of ZrO2 is needed to induce antiferroelectricity in contrast to HfO2 based thin films26. At room temperature, the stable bulk phase of ZrO2 is the non-polar monoclinic P21/c phase, which can be suppressed in thin films of around 10 nm thickness and below, which favor the non-polar tetragonal P42/nmc phase12. The current understanding of the origin of antiferroelectricity in ZrO2 is that the non-polar tetragonal P42/nmc phase undergoes a first-order structural phase transition into the polar orthorhombic Pca21 phase by application of an electric field of around 2-3 MV cm−126. The polar orthorhombic Pca21 phase has been shown to be responsible for the ferroelectric behavior observed in HfO2 based thin films27. While obtaining definitive experimental proof of such a field-induced first-order phase transition has proved difficult so far28, this mechanism is consistent with first principles calculations29 as well as composition- and temperature-dependent experimental results26. While the microscopic switching pathway between the P42/nmc and Pca21 phases is still unclear, it has been suggested to include an intermediate phase of orthorhombic Pmn21 symmetry for Hf0.5Zr0.5O230.Here, we investigate this unique type of non-polar to polar antiferroelectric transition in ZrO2/TiN capacitors were fabricated as described in methods. Grazing incidence X-ray diffraction (GIXRD) measurement results presented in Supplementary Fig. 2 layer is in the non-polar tetragonal P42/nmc phase in the as fabricated capacitors. Figure 2 layer, respectively, measured using a ferroelectric tester. Since the structural data suggests that our ZrO2 films are fully tetragonal without applied voltage, the measured double hysteresis loops can only be explained by a field-induced structural transformation into a polar ferroelectric phase. While we cannot directly determine the symmetry of this polar phase at high electric field, there is substantial evidence in literature which suggests that it is of orthorhombic Pca21 symmetry26. For example, a temperature-dependent phase transition from the non-polar P42/nmc phase to the polar Pca21 phase in similar ZrO2 thin films has been experimentally observed with in situ high-temperature X-ray diffraction31. Furthermore, ZrO2 can be stabilized in the Pca21 phase even at room temperature under certain processing conditions32. Lastly, the field-induced transition from the P42/nmc phase to the Pca21 phase has been directly observed in Hf0.5Zr0.5O2 thin films33. From these experimental data and previous first principles calculations29, it seems reasonable to conclude that the polar phase observed at high electric field in Fig. 1 symmetry. Furthermore, using high-resolution transmission electron microscopy (HRTEM) with in situ voltage biasing, it was directly shown that antiferroelectric ZrO2 always returns to its initial non-polar P42/nmc structure after the applied voltage is removed28. Since the ZrO2 film investigated in ref. 28 was fabricated in the exact same way as the ones shown here, it is reasonable to assume that they also return to the initial non-polar P42/nmc phase after each field-induced phase transition.For basic antiferroelectric characterization, TiN/ZrO21. The dielectric layer has two important functions: It prevents the injection of compensating charge which could screen the bound polarization P and it creates a depolarization field in the antiferroelectric which is antiparallel to P, such that Ea can decrease while P is increasing. When the antiferroelectric enters the unstable region in such a heterostructure, the resulting negative capacitance would then increase the total capacitance of the dielectric/antiferroelectric stack beyond than that of the dielectric layer alone. Such a capacitance enhancement would thus be a clear signature of the structural transition itself. To test this prediction, we fabricated heterostructure capacitors consisting of the same ZrO2 layer and a stack of Al2O3/HfO2 dielectrics with TiN used as top and bottom electrodes using atomic layer deposition as described in methods. The intermediate Al2O3 layer was introduced to prevent the crystallization of the top HfO2 layer due to templating effects from the underlying crystallized ZrO2. Scanning transmission electron microscopy (STEM) analysis of a representative TiN/ZrO2/Al2O3/HfO2/TiN heterostructure in Fig. 2 layer was stabilized in its non-polar tetragonal phase while the HfO2 layer remained amorphous. The combined results of our GIXRD measurements together with STEM and nanobeam electron diffraction data  than the time scale for charge injection, breakdown and related mechanisms20.To probe the capacitance of the heterostructure under high applied fields, we adopt a pulsed capacitance measurement techniqueVin(t) (t ≡ time) of T = 1.1 μs duration, different amplitudes Va were applied to the capacitors in series to an external resistor R. Waveforms of Vin, measured current I, and integrated charge (=∫Idt) for different amplitudes Va for a HfO2(8 nm)/Al2O3(∼1 nm)/ZrO2(10 nm) capacitor are shown in Fig. Va, Qmax is the total charge supplied by the voltage source  is larger than the capacitance of the constituent dielectric stack CDE which is shown as a slope in Fig. 2(8 nm)/Al2O3(∼1 nm) capacitor fabricated under the same processing conditions .Figure Q−Va curves in Fig. Qres–Va behavior one can conclude that the capacitive behavior is unaffected by leakage currents or charge injection. Furthermore, a symmetric ΔQ–Va curve is expected only for an ideal antiferroelectric/dielectric stack with negligible trapped charges at the interface between both layers. This contrasts with recent reports of similar ferroelectric/dielectric capacitors, where large negative trapped charge densities (comparable to the spontaneous polarization) were found at the interface21. This indicates that the initial ferroelectric polarization during fabrication in such stacks causes the trapping of charges at the interface, which is not the case for the antiferroelectric heterostructure due to the non-polar antiferroelectric ground state. Therefore, having a non-polar ground state seems beneficial for avoiding trapped interface charges, which can be detrimental when building negative capacitance devices.From the inversion symmetry of the measured ΔVa, the electric field in the antiferroelectric layer Ea can be calculated as Ea = (Va − VDE)/tAFE, where tAFE is the ZrO2 thickness and VDE = ΔQ(Va)/CDE (see Methods for details). The corresponding P − Ea curve (P = ΔQ/A with A being the area of the capacitor) of the ZrO2 layer in Fig. Q − Va curve in Fig. P-Ea characteristics of an equivalent, single-layer ZrO2 capacitor measured using a ferroelectric tester is also plotted in Fig. 2 layer, in agreement with the theoretical prediction  = ∫EadP) which is shown in Fig. G′′ < 0) of thermodynamic instability at the non-polar to polar structural phase transition can be accessed in the antiferroelectric/dielectric heterostructure. It is interesting to note here, that previous first principles calculations for the antiferroelectric transition in ZrO2 found that G” is always positive along the direct switching pathway between the P42/nmc and Pca21 phase, corresponding to a cusp in the energy landscape25. This is in contrast to our experimental findings, where negative G” regions are clearly observed . For all different HfO2 thicknesses, the extracted P − Ea characteristics of the ZrO2 layer have the same quantitative shape as shown in Supplementary Fig. r = CDE-AFE/CDE > 1) in these samples obeys the ideal capacitance matching equation r=|CAFE | /( | CAFE | − CDE) with a best fit antiferroelectric negative capacitance CAFE° = −4.75 μF cm−2, extracted at the polarization value of P = 16 µC cm−2 for all samples. Figure CDE-AFE) at P = 16 µC cm−2 as a function of the inverse dielectric capacitance (CDE) with and excellent linear fit (R2 = 0.9986). Two important conclusions can be drawn from Fig. CDE = 0 and the consistent capacitance enhancement shows that the ZrO2 capacitance must indeed be negative for all samples. Second, the ZrO2 and Al2O3/HfO2 layers do act as expected for two capacitors in series, i.e., the antiferroelectric negative capacitance is independent of the thickness of the HfO2 layer. Previous results for multi-domain ferroelectric/dielectric superlattices showed a similar behavior35. However, theory suggests that multi-domain ferroelectric negative capacitance can strongly depend on the domain configuration and lateral domain wall motion in the ferroelectric and thus changes, e.g. with the ferroelectric film thickness39. On the other hand, the constant CAFE < 0 of our antiferroelectric ZrO2 films with both HfO2 and ZrO2 thickness  based on our experimental results to investigate what performance improvements might be expected compared to current device technologies. The details can be found in the 2 using pulsed electrical measurements. In contrast to previous reports in multi-domain ferroelectrics, we report a different type of antiferroelectric negative capacitance which seems to be an intrinsic property of the structural instability at the boundary between P42/nmc and Pca21 phases in fluorite-type oxides. This is of great interest for applications in energy-efficient electronics since mixtures of these phases have been reported in the widely used HfO2 and ZrO2 based ultrathin films, which are present in most semiconductor products today. The observation of negative capacitance in ZrO2 indicates that the switching pathway between the P42/nmc and Pca21 might include an intermediate saddle phase of different symmetry. Furthermore, our findings suggest that the phenomenon of negative capacitance is not limited to polar ferroelectric order but can be expected at any transition with a polarization instability. This includes phase transitions between non-polar and polar phases as shown here, but which should also include transitions between different polar phases or even between different non-polar phases44. This opens up opportunities for characterizing structural phase transitions and for building negative capacitance devices from a much broader range of possible materials.In summary, we have explored the forbidden thermodynamic region of the non-polar to polar antiferroelectric transition in ZrO2 and bottom TiN layers were conducted in a 300 mm Tokyo Electron thin film formation tool. The ZrO2 and TiN films were deposited by ALD at 350 °C and ~ 430 °C respectively by previously described processes45. The as-grown ZrO2 layer was amorphous. With regards to the thickness of ZrO2 in the ZrO2/TiN heterostructure of these samples, there were two differently processed stacks: ZrO2/TiN with 10 nm ZrO2 and 5 nm ZrO2, respectively. A post-ZrO2 deposition annealing was performed on both stacks at 450 °C for 30 s in nitrogen atmosphere to stabilize the ZrO2 layer in the antiferroelectric tetragonal phase. The ZrO2/TiN heterostructure was cleaned in standard clean-2 (SC-2) solution. Afterwards, HfO2 /Al2O3(~ 1 nm) heterostructure was deposited on the ZrO2/TiN heterostructure in a Cambridge NanoTech Fiji G2 Plasma Enhanced ALD (PEALD) system at Georgia Tech. Subsequently, a top TiN layer was deposited without breaking the vacuum in the same ALD reactor. The deposition was carried out at 250 °C using tetrakis (dimethylamido) hafnium, tetrakis (dimethylamido) aluminum and tetrakis (dimethylamido) titanium precursors for HfO2, Al2O3 and TiN, respectively. For oxides, water was used as the oxygen source and for TiN, nitrogen (plasma) was used as the nitride source. After the ALD of TiN/HfO2/Al2O3/ZrO2/TiN and TiN/HfO2/ZrO2/TiN heterostructures, Al (~ 100 nm) was evaporated and patterned into rectangular electrodes using standard microfabrication techniques. Top TiN layer was wet etched afterwards in H2O2:H2O solution at 50 °C using the patterned Al layer as a hard mask.The atomic layer deposition (ALD) of ZrOR = 5.6 kΩ.Electrical measurements were conducted on a Cascade Microtech Summit 1200 K Semi-automated Probe Station. Polarization versus electric field curves were measured using an aixACCT TF-3000 ferroelectric parameter analyzer at 1 kHz with dynamic leakage current compensation (DLCC) turned off. The capacitance versus electric field curves were measured by a Keysight E4990A impedance analyzer with a small-signal amplitude of 25 mV and a frequency of 100 kHz. The pulsed measurements on dielectric/antiferroelectric heterostructure capacitors were conducted using a Keysight 81150 A Pulse Function Arbitrary Noise Generator and a Keysight DSOS104A Oscilloscope. The series resistor had a resistance of ω = 0.6°. Measured diffraction data were fitted using whole powder pattern procedure - Rietveld method. A computer program MStruct was used for the fitting. The investigated samples contain tetragonal ZrO2 phase . Some minor fractions of other ZrO2 phases might be present, but in fractions below the detection limit of GIXRD. Scanning transmission electron microscopy (STEM) imaging was performed using a Hitachi aberration corrected STEM HD2700 operating at 200 kV at Georgia Tech. The convergent angle is 35 mrad, and the inner angle of the annular dark field (ADF) detector is 50 mrad. Nano beam electron diffraction (NBED) was performed using a FEI transmission electron microscope (TEM) Technai F30 working at 300 kV. The determination of the phase of ZrO2 was accomplished by analyzing the NBED patterns.X-ray diffraction (XRD) measurements were performed using SmartLab diffractometer (Rigaku) equipped with 9 kW cooper rotating anode X-ray source (wavelength CuKα = 0.15418 nm). A standard co-planar diffraction geometry with constant angle of incidence (grazing incidence X-ray diffraction: GIXRD) was used for the studies. To perform the GIXRD measurements the diffractometer was equipped with a parabolic multilayered X-ray mirror and a set of axial divergence eliminating soller slits with divergence of 5° in incident and diffracted beam, and parallel plate collimator with acceptance of 0.5° in diffracted beam. Diffracted intensity was acquired with hybrid-pixel single photon counting 2D detector HyPix 3000. The incidence angle of the X-rays, with respect to the sample surface, was set up slightly above the critical angle of investigated material, Vin of duration T = 1.1 µs with different amplitudes were applied, and the voltage across the dielectric/antiferroelectric capacitor, VDE-AFE and Vin were measured using an oscilloscope. The input pulse amplitude Vin was changed in 200 mV steps to span the full P–Ea curves. At a given applied voltage amplitude Va, voltages across the dielectric layers and the antiferroelectric layer was calculated as VDE = ΔQ/CDE and VAFE = Va–VDE, respectively. For each of the dielectric/antiferroelectric samples, an equivalent dielectric only stack with the same thicknesses of HfO2 and Al2O3 (~ 1 nm) layers was deposited under the same process conditions to measure CDE independently  flowing through the resistor R in series with the dielectric/antiferroelectric heterostructure is calculated from measured Vin(t) and VDE-AFE(t) waveforms. The amount of charge Q on the heterostructure capacitor is calculated byCpara is the parasitic capacitance that appears in parallel to the dielectric/antiferroelectric capacitor, which was experimentally determined as ~ 20 pF. Three important charges are extracted for each value of Va: the maximum stored charge on the capacitor, Qmax = Q(t=T), the residual charge on the capacitor after the applied voltage is zero again, Qres = Q(t=5 µs»T), and the charge that is reversibly stored and released from the capacitor, ΔQ = Qmax − Qres. The electric field Ea in the antiferroelectric layer can be calculated byQ is the amount of charges reversed and CDE is the capacitance of the dielectric layer. The relative permittivity εr of the dielectric layers was extracted from capacitance versus voltage measurements on samples fabricated without the antiferroelectric layer (see Supplementary Fig. A is the area of the dielectric/antiferroelectric capacitor. Using Eqs. (P versus Ea curve can be calculated. The experimental free energy density G can be obtained fromThe voltage Vres = Qt= µs»T, anFurther information on research design is available in the Supplementary InformationPeer review fileDescription of Additional Supplementary FilesReporting Summary


OpA), global shape (VS) and presence of abnormal angiogenesis (Gradα) were computed. In cross-sectional analysis, individuals with PDR had a lower Gradα indicating a vasculature that is more complex, less spread (i.e. dense) and the presence of numerous small vessels. The results show that α-shape parameters characterise vascular abnormalities predictive of PDR  and have therefore potential to reveal changes in retinal microvascular morphology.The use of 2D alpha-shapes (α-shapes) to quantify morphological features of the retinal microvasculature could lead to imaging biomarkers for proliferative diabetic retinopathy (PDR). We tested our approach using the MESSIDOR dataset that consists of colour fundus photographs from 547 healthy individuals, 149 with mild diabetic retinopathy (DR), 239 with moderate DR, 199 pre-PDR and 53 PDR. The skeleton (centrelines) of the automatically segmented retinal vasculature was represented as an α-shape and the proposed parameters, complexity ( DR is classified as non-proliferative, if there are vascular abnormalities such as microaneurysms, or PDR if damage to the retina has become sufficient to stimulate growth of abnormal retinal blood vessels (neovascularisation). New vessels are weak and have a high risk of causing irreversible visual loss through leakage, bleeding, or retinal detachment. Identifying patients at high risk of progression to PDR is critical for providing timely sight-preserving treatment. PDR risk assessment is conducted through manual grading of fundus photographs obtained through nationwide diabetic retinopathy screening programmes2. The identification of parameters obtainable automatically for quantification of pathological retinal microvascular remodelling, including the presence of neovascularisation, may permit early identification of PDR and improve patient outcomes.DR is a leading cause of preventable blindness and a major complication of diabetes mellitus3. FD is a unitless measure of the complexity of a pattern, describing how a self-similar pattern fills the space in which it is contained5. FD has previously been investigated as a imaging biomarker in neurodegenerative diseases and in conditions with abnormal angiogenesis such as diabetic retinopathy (DR)12.Retinal vasculature morphology can be objectively quantified from fundus images through automated detection of retinal blood vessels and subsequent analysis of their geometric properties, including width, tortuosity, branching angles, and branching complexity via calculation of the fractal dimension (FD)14, whilst a lower FD, indicating a decreased vascular complexity, has been reported when comparing PDR to patients with Type I diabetes15 and when comparing PDR to healthy controls16. Huang et al.17 reported no evidence of a significant difference in FD between DR grades and Orlando et al. reported a higher FD in PDR when compared to other DR grades13. Such variation in previously reported FD have been attributed to lack of standardisation in FD calculation18. FD and it’s calculation can be affected by: (1) the accuracy of vessel segmentation; (2) skeletonization, locating the centreline of each vessel; (3) camera resolution and image quality, which directly affects (1) and (2)17; (4) the FD algorithm and the choice of its parameters, such as the random sampling of points on the vessel map; and (5) the computational cost19, which may be excessive in a clinical setting.For PDR, conflicting findings with FD have been reported. A higher FD, indicating increased vascular complexity, has been reported in PDR than in healthy controls20. It has been applied previously to molecular shape characterisation21, brain tumour measurement in magnetic resonance imaging (MRI)22, cosmic web topology23, surface reconstruction24 and predicting species distributions25. Unlike FD, α-shapes are independent from sampling space, do not make assumptions of self-similarity, are computationally inexpensive and unaffected by parameter choice as no random sampling is conducted and there is only one α-shape for a point set20. We applied α-shapes to the task of characterising PDR, with a focus on deriving objective measurements that have a clinically interpretable output.In this study, we explore an alternative method of quantifying the global morphology of the retinal vasculature, using α-shapes. The α-shape is a well described geometric concept used to represent the boundary of a shape in 2D or 3D26. Of these 547 are grade 0 (no DR), 149 grade 1 (mild DR), 239 grade 2 (moderate), and 252 grade 3 . Images are stored in TIF format and have three sizes: 1140 × 960 pixels, 2240 × 1488 pixels and 2304 × 1536 pixels. Segmentations and PDR image labels from Orlando et al.13 were accessed at https://github.com/ignaciorlando/fundus-fractal-analysis. In13, the authors standardised all images to 909 × 909 pixels to approximately match the calibre of the vessels appearing in images in the Digital Retinal Image for Vessel Extraction (DRIVE) dataset27. Images were then segmented using a fully connected conditional random field model28. Vessel skeletonization was performed using an iterative thinning algorithm that extracts the centre line of the vessels , resulting in a vessel skeleton29. PDR labels were obtained by relabelling of DR grade 3 by two ophthalmologists whereby 53 images of grade 3 were assigned to the PDR grade (with neovessels)13. It should be noted that the relative number of images per DR group differ from13 due to the corrections outlined on the MESSIDOR project website which involved removal of 13 duplicate images and corrections of DR grades. We calculated FD using a multifractal approach and a generalised sandbox method, reported elsewhere5.MESSIDOR is a publicly available dataset commonly used to evaluate performance of automatic DR detection systems and consists of 1187 fundus images acquired by a 45° FOV Topcon TRC NW6 non-mydriatic camera13.For assessing accuracy of α-shape descriptors and FD we used the publicly available high resolution fundus (HRF) dataset (available here: High-Resolution Fundus (HRF) Image Database (fau.de)). The HRF dataset contains 15 images of healthy people and 15 images of people with diabetic retinopathy (DR grades for these images are unavailable) with manual annotations of blood vessels. Images are 3504 × 2336 stored in JPEG format. Images were standardised to 1525 × 1017 pixels to approximately match the calibre of the DRIVE dataset. Images were segmented using the same procedure as the MESSIDOR datasetDelaunay simplices—i.e., triangles, edges, vertices—are removed to refine the boundary of the shape. Refinement is achieved by rolling a disc with radius α along each point in collected. All the Delaunay simplices that meet this criterion forms the α-shape. For α = 15 (pixels)  applied to the vessel skeleton. We define complexity as the property of a vessel skeleton being composed of many parts ; a complex vasculature would require more refinement  than a less complex vasculature. Therefore, we extracted the minimum value of α that encompassed all points in the skeleton giving only a single region, termed OpA, was computed. Using complexity and spread, we can define vessel shape (VS),For each image in the dataset the α-shape was computed (bwmorph with parameter ‘spur’) to remove the ends of the vessels (20 iterations)29  to assess the level of significance of the differences between all grades of DR (grades 1 to 3 and PDR); (2) evaluate the contribution of each α-shape descriptor and FD as features for PDR classification; and (3) assess the accuracy of α-shape descriptors and FD values.Statistical analysis was conducted in R (version 3.6.0) and results were considered statistically significant at the significance level 0.05. In experiment (1), all groups (Grade 0 to 3 and PDR) were compared using Kruskall-Wallis test and p-values adjusted for multiple comparisons using Benjamani-Hochberg . Dunn post-hoc analysis was performed to assess which independent variable differed and p-values corrected for multiple comparisons using Bonferroni.glmnet (version 3.6.0) that performs model estimation and feature selection simultaneously31. We experimented with different strengths of regularisation (λ) and a choice of regularisation , ridge regularisation  and a mixture of the two, elastic-net (α = 0.5). Lasso tends to discard correlated variables and select a single feature; ridge tends to equally weight features and shrink coefficients towards each other and elastic-net will exclude groups of highly corelated variables. As the α-shape descriptors are expected to be highly correlated, we applied lasso and elastic-net to perform feature selection.In experiment (2), α-shape descriptors as features for PDR characterisation were modelled using regularised logistic regression by grouping the data into PDR vs all other DR grades (grades 0 to 3). This was to identify features that could be used to distinguish neovascularisation (a defining feature of PDR) compared to other DR grades. Implementation of regularised logistic regression was performed using the R package λ where the highest AUC on the validation set is achieved (λmin) and the highest AUC on the validation set within one standard error of λmin (λ1SE). Feature importance was assessed by counting the number of times each coefficient reached a non-zero weight in each bootstrap. As λmin and λ1SE will return different numbers of features (i.e. λ1SE will contain less features) we assessed feature importance for both values of λ32.Given that PDR cases consisted of 4.4% of the dataset and different sampling of the training data will give rise to different features being selected, we performed bootstrap analysis. A total of 500 bootstrap trials was performed and for each bootstrap trial the data was divided to keep proportions of classes equal  and model fitting was performed using fivefold cross-validation. Area Under the Curve (AUC) was used to assess model performance in each fold where the average AUC over all bootstrap trials is reported. fivefold cross validation simultaneously selects a value of In experiment (3), the Dice similarity coefficient (DSC) was used to evaluate performance of the automatic segmentation. To investigate variation in α-shape descriptors and FD measures obtained from the manual and automatic segmentation we use relative error . Relative error is obtained by |AV-MV|/AV, where AV is the value of α-shape descriptors or FD obtained from the manual segmentation and MV is the value of α-shape descriptors or FD obtained from the automatic segmentation. To test whether measures obtained manually and automatically were correlated we used Pearson correlation coefficient test. Healthy and DR α-shape descriptors and FD measures were not normally distributed and therefore compared using Wilcoxon rank sum.OpA when compared to Grade 0 (p = 0.001), Grade 1 (p = 0.01), Grade 2 (p = 0.02) and Grade 3 (p = 0.05), indicating a vasculature with less spread. A significantly lower Gradα was observed in PDR when compared to Grade 0 (p = 0.002), Grade 2 (p = 0.02) and Grade 3 (p = 0.02) suggesting that PDR contained numerous small vessels. For VS there was no evidence of significant difference between groups following post-hoc testing and p-value adjustment. PDR exhibited a lower FD when compared to all other groups, indicating a vasculature that is less space filling (p < 0.001)  who had large number of microaneurysms, haemorrhages and exudates that obstructed the vasculature and resulted in a sparse and fragmented segmentation. DR exhibited a higher Opαmin, lower OpA  compared to Healthy, indicating a vasculature that was less space filling, but was not significant between groups in measures derived from the manual or automatic segmentation. Gradα was lower in DR compared to healthy individuals indicating the presence of small vessels but was not significant in measures derived from the manual or automatic segmentation. FD exhibited a 0.9% relative error in Healthy and a 1.4% error in DR individuals and was correlated to FD derived from manual segmentations. FD was significantly lower in Healthy (p = 0.01) and DR (p = 0.01), indicating a less space filling vasculature.Table 13, likely due to the removal of duplicates, alteration of DR grades (as specified on the MESSIDOR project website) and parameter choice (e.g. random sampling on the vessel map), highlighting a challenge with the repeatability of FD analysis. The α-shape from which the features proposed here are extracted would not be affected by parameter choice as no random sampling is conducted and there is only one α-shape for a point set20.We implemented α-shapes as a method to quantify retinal microvasculature morphology from fundus imaging and investigated its application to the task of PDR characterisation. A significantly lower FD was observed in PDR compared to all other DR grades suggesting that the vasculature is less space filling in PDR. We did not observe the increase in FD of PDR eyes as reported inOpαmin as a measure of complexity and OpA as a measure of vessel spread and observed that PDR had a significantly lower OpA compared to all other DR grades and a lower Opαmin compared to grade 0. This suggests that this the vasculature was dense . OpA was determined to be the most distinguishable feature of PDR and was the most important feature for PDR classification for lasso and elastic-net (when Gradα and FD were not included as features for classification), suggesting that a decreased vessel spread could translate to a predictive marker of progression to PDR.We experimented with Gradα was introduced into the model (without FD) this became the most important feature. PDR had a lower mean Gradα compared to all other grades suggesting the vasculature contains numerous small vessels indicating the presence of neovascularisation. However, the limitation to this parameter is that the presence of numerous small erroneous segmentations of PDR related pathology  could be contributing to the lower Gradα observed. Indeed, Gradα was significantly lower in PDR when comparing to grade 0, 2 and 3 but was not observed in grade 1. Individuals with grade 1 DR contain a small number of microaneurysms and is therefore expected to be vastly different from PDR. Grade 1 was the second smallest group in the MESSIDOR dataset and may be underpowered to detect a significant difference between PDR and grade 1. However, assuming that the dataset was sufficiently powered other reasons could include image quality such as the presence of artefact that may be contributing to a similar distribution of Gradα measured from PDR cases. To address such limitations, manual correction of the vessel segmentation or improvements to automatic vessel segmentation would be required. Nevertheless, Gradα could be a potential metric to evaluate segmentation quality in datasets that are several orders of magnitude greater than MESSIDOR where visual assessment and manual correction of the vessel skeleton prior to quantification of morphology is not feasible.When 13. Orlando et al. further improved their performance to AUC 0.91 when red lesion features were included as features for PDR classification13. We used fivefold cross validation and bootstrap to obtain performance measures and feature importance, when larger PDR datasets become available a held out experimental set-up should be implemented along with the addition of red lesion features if available.We achieved good classification performance for PDR (AUC 0.67 to 0.76) that was improved with the addition of FD as a feature into the model and was comparable to the performance achieved by Orlando et al.17. We showed that α-shape parameters are also affected by segmentation accuracy. We therefore recommend careful selection of segmentation algorithms  to ensure α-shape and FD measures are as reliable as possible. Experimental results did not show a significant difference between healthy and DR that could be due to low sample size and presence of a broad range of DR severities in the DR group. FD measures obtained from manual and automatic segmentations showed a significant difference between groups, suggesting that it may be more sensitive than α-shape descriptors.We conducted a comparison of α-shape descriptors and FD measured from manual and automatic segmentations to assess measurement accuracy. The main differences between the manual and automatic segmentations are the presence or absence of small vessels. Additionally, manual annotations in the HRF are meticulously drawn, such that vessels are annotated to the complete length of the path. As vessels taper towards the end, they can lose contrast and may not be detected by the algorithm. In previous work that compared FD measured from automatic segmentations to manual annotations from two human observers (DRIVE dataset) reported mean relative errors between 7.1 and 9.3% and attributed this to the accuracy of the segmentation method34.Interpretation of objective measures derived from the automatic segmentation of the retinal vasculature requires assessment of statistical significance of difference between groups and their clinical importance. To establish a threshold that identifies a change to be clinically important requires group-level between patient contrasts and within-patient longitudinal contrasts and therefore could not be established here. Nevertheless, providing careful segmentation procedures are followed, α-shapes appear to be a promising measure of vascular geometry. Future applications of α-shapes for describing retinal vasculature morphology could be applied to research involving the assessment of vascular changes in other ocular diseases as well as characterising vascular changes related to systemic comorbidities or neurodegenerative diseases that are increasingly reported to manifest in the retina


Our results show a low variation overall within sheep and goats in terms of pasture type that may have been composed of seasonal halophyte plant communities, which have higher δ13C values due to the saline rich growing environments. Cattle feeding strategies were more variable and provided with supplementary forage, such as cut branches or leafy hay, during winter. The sharp distinction in the management of cattle and sheep/goat may be associated with the development of herding strategies that sought to balance livestock feeding behaviours with available forage or, more provocatively, the emergence of household-based control over cattle–an animal that held a central economic and symbolic role in Vinča societies.Late Neolithic Vinča communities, spread over much of central and northern Balkans during the late sixth to mid-fifth millennium BC and characterised by unusually large and densely population centres, would have required highly organised food production systems. Zooarchaeological analysis indicates that domesticate livestock were herded, but little is known about the seasonal husbandry practices that helped ensure a steady supply of animal products to Vinča farming communities. Here, we present new stable carbon (δ Porčić [During the Late Neolithic , much of the central Balkans and southeast parts of the Vojvodina and Transylvania regions were occupied by Vinča communities which produced dark burnished ceramics and figurines often bearing unique triangular faces ,2. Compa. Porčić ). The in. Porčić . Crop grTriticum monococcum), emmer (Triticum dicoccum) and barley , as well as pulses such as lentils (Lens culinaris) and pea (Pisum sativum) [Bos taurus) and pigs (Sus scrofa) increased in socio-economic importance over time within Vinča communities, likely due to intensification in meat and dairy production associated with a diachronic increase in settlement size and populations [Vinča communities exploited a wide spectrum of domesticated plants including einkorn (sativum) . Domestiulations . The higulations ,13, whilulations ,14–19. Pulations  collecteulations ,22. Wateulations ,21.13C signatures of animal tissues reflect the carbon component of protein (bone) and carbohydrate (fats) intake, and total diet (teeth) and, for herbivores, tissue carbon isotope ratios directly reflect plant stable isotopic composition [13C values indicative of open and closed forest pasturing [18O values expressed during the winter months and higher values during the summer [13C values that may be related to seasonal variation in pasture and fodder sources.Carbon and nitrogen stable isotopes can be used to investigate husbandry practices, including forage and pasture management strategies –27. The position . Thus caposition –25. The position ,23,26,27asturing . Howeverasturing ,30. At me summer ,31. ConsWe present isotopic results measured from the bioapatite fraction of incrementally sampled teeth from domesticated and wild herbivores to further explore the Late Vinča fodder and pasture management strategies. These results are further contextualized within published stable isotopic results from bone collagen samples .i.e. Vinčа D/Vinča–Pločnik II phase . On 0 cal BC –39 and s0 cal BC , and spe0 cal BC ,41. The 0 cal BC –44, as w0 cal BC ,45. Over0 cal BC , complexc. 5700–5300 cal BC) and followed by Vinča culture groups until c. 4550 cal BC when occupation ceased [sp.  and reeds (Phragmites communis). Thermophilous taxa, particularly oak, grew on rich chernozem soils and dominated forests that spread over hilltops and slopes [We analysed material from two settlements, Vinča-Belo brdo and Stubline, both located on the alluvial plains and lower terraces of the Danube and Sava respectively, and their tributaries in northern Serbia . The pron ceased ,52. Recen ceased . Damp ald slopes .circa 50 km east of Vinča–Belo Brdo (The site of Crkvine at Stubline (herein Stubline) is located elo Brdo . In a brThe climate today is characterised as humid sub-tropical (Cfa) according to the Köppen climate classification . The warAll animal bone and teeth from Vinča-Belo brdo and Stubline were sampled by REG, assisted by JB and KP, with the permission of the local authorities and excavators . The collections are housed in City Museum, Belgrade. Details of the sampling protocol for wild and domesticated faunal bones for stable isotopic analysis of bone collagen are outlined in Gillis, Bulatović . The comBos taurus; Vinča-Belo brdo n = 5, Stubline n = 1) and sheep/goats  were selected for analysis. The third molar in both species begins development around 10 months and erupts around 24 months [To examine the seasonality of husbandry practices, we selected teeth from cattle, sheep/goat, and domesticated pig, based on state of occlusal wear and overall specimen preservation. The third mandibular molars of domestic cattle , 3 individuals from both sites were sampled with both the mandibular M2 and M3 selected. The bunodont teeth of pigs display short crowns in comparison to ruminants. Enamel mineralisation of the lower M2 in modern pigs is initiated at around 3 months and finishes around 11 months, while mineralization of the mandibular M3 begins at approximately 11 months and extends through ca. 18 months [For domesticated pigs  individuals  from Vinča-Belo brdo, with two teeth from a single individual (7237-M2/M3). Red deer mandibular M2 mineralisation begins around 4 months, around when weaning occurs, with the crown complete at 9 months. For the M3, mineralisation begins around 13 months with completion of the crown at 26 months based on a study on modern red deer from Richmond Park, London [To provide a reference for a ruminant grazing/browsing within a forest environment we sampled two red deer  and for those of δ18O was 0.04‰ (range: 0.03–0.06‰). The red deer samples were analysed at the Leibniz labor at Kiel University (Germany). The analytical precision was for δ13C analyses was 0.02 (range: 0.01 to 0.03) and δ18O was 0.03‰ (range: 0.01 to 0.04).Prior to sequential enamel sampling, the tooth surface was cleaned using an abrasive drill bit. A sequence of enamel samples was then drilled on the buccal surface from the enamel-root junction (ERJ) to the apex of the crown. Purification protocol for stable isotopic analysis followed . Purifie13C values between diet and bioapatite have shown that it can vary between species depending on dietary composition, physiology, and body size [3 or C4 plants) of an animals’ diet [Studies concerning the enrichment of δody size ,66–69. Tls’ diet –69, and ls’ diet  for the 2. We applied a correction factor of 1.7‰ to carbon isotope data obtained from modern tissues. This calculation is based on the δ13C value of CO2 at present (−8‰) and the given studied period. The δ13C value of CO2 during the Late Vinča period was −6.3‰ based on interpolated data from Antarctic ice-cores [The burning of fossil fuels and to a lesser extent, the removal of large stretches of forests, has contributed to changes in the carbon isotope composition of atmospheric COce-cores .(δxxXMax+ δxxXMin)/2, and then an average and standard deviation was calculated per species. The amplitude was calculated by subtracting the δxxXMin from the δxxXMax, an average was then calculated per species. The maximum and minimum value per species is true range and not an average.The summary statistics used were the average midpoint value, range, and amplitude per species. The midpoint was calculated 18O values of Vinča-Belo brdo cattle average mid-point was −7.2‰ (range: −9.6 to −4.2‰)  . In comp the M2s . Red dee13C values from bioapatite samples of domesticated animals ranged from −14.1‰ and −7.9‰ with the samples from Vinča-Belo brdo cattle exhibiting the greatest range in values observed from all species . The M2s from domestic pigs at Vinča-Belo brdo exhibit values that were on average 0.3‰ higher midpoints values than M3s. At Stubline, there was little difference between the average midpoints from M2s and M3s of domesticated pigs .The δ species . Cattle 13C values ranging from −15.6‰ to −10.9‰, with the lowest midpoint of the entire dataset of −13.3‰ while the M3s from the same individual and 7238, exhibited a smaller range in values (−13.7‰ to −11.9‰) and an average midpoint of −12.7‰.The two red deer individuals exhibited a large range in values  exhibiting an amplitude of intra-tooth isotopic change of less than 1‰ to 2.4‰ . In comparison, the single goat identified in the Vinča-Belo brdo assemblage (O/C 7245) exhibited a low amplitude of intra-tooth change (1.4‰). Two sheep/goat individuals (7267 and 7287) had the largest intro-tooth amplitude of 4.7‰ and 4.9‰ respectively. Overall, sheep/goat samples from Stubline exhibit the largest average amplitude of change for domesticated animals in the dataset (3.4‰). Pig intra-tooth amplitude is greatest at Vinča-Belo Brdo (0.7–1.3‰) compared to those from Stubline (0.3–0.4‰). Red deer M3s exhibit an average amplitude of 1.6‰, while the single M2 has the largest amplitude in the dataset of 4.6‰.Amplitude of intra-tooth carbon isotope change provides insight into isotopic variation in forage ingested by animals. The average amplitude for cattle from Vinča-Belo brdo was 1.7‰ (range: 0.7 to 2.4‰) with some individuals  coincide with high summer season δ18O values.Within our study, sheep/goat individuals from both sites exhibited a strong correspondence between δ a versa  except f a versa . In this13C values from bone collagen from domestic cattle at Vinča-Belo brdo suggests they may have been grazed across different environments . While the relative low bone collagen δ13C values at Stubline may indicate that they were kept mainly within wooded or waterlogged environments. At both sites, caprines and domestic pigs consumed a diet enriched in 15N, which strongly suggests these animals were raised on either crop residue from cultivars raised on manured soils or pastures repeatedly visited by livestock [13C and δ18O values from incremental bioapatite samples presented here provides us with the opportunity to test these hypotheses through integration of seasonal short-term stable isotopic records with those from bone collagen.Previous stable carbon and nitrogen isotopic analysis of bone collagen samples from the Late Vinča phases (4800 to 4600 BC) at Vinča-Belo brdo and Stubline pasture and foddering practices for sheep, goat and cattle . The larivestock . We previvestock , with acivestock . The δ1318O values, which is influenced by altitude, local humidity, and temperature [18O average, while rivers will vary seasonally depending on the input of melt water. Leaf water is enriched in 18O in comparison to the local average due to evapotranspiration [18O values between species. These will provide us an insight into water consumption habits of domesticated animals.There were clear differences between ruminants and omnivores in their oxygen isotope observed at both sites. Sheep/goat samples from Vinča-Belo brdo and Stubline exhibit the largest average intra-tooth amplitude observed in domestic livestock, averaging 5.6‰ and 6.1‰, respectively for Vinča-Belo brdo and Stubline. The average amplitude per tooth for cattle from both sites were similar , and close to that observed in deer , with domestic pigs exhibiting the lowest amplitude <1 ‰; . The oxyperature ,73. Drinpiration . Here we18O relative to precipitation [18O record of semi-obligate species, which include caprines, is more sensitive to aridity [th millennium BC, Romania) located on the Danube [The large amplitude of intra-tooth oxygen isotopic change observed in sheep/goat may be due to ingestion of 18O enriched leaf water which comprises a large proportion of total body water in caprines . Leaf wapitation . Consequ aridity . Serbia  aridity . Our rese Danube .18O values expressed in these species [18O values of water sources is dependent on the frequency of a body of water is fed by sources, such as melt water or tributaries [18O values of standing water, such as lakes and wells, is dampened in comparison to local δ18O precipitation because of the effects of evaporation and influx water cancel each other out [18O values was less than 2‰ [18O values within single cattle and deer teeth was greater than 3‰, which is greater than observed by Jasechko, Gibson [Cattle, deer, and pigs are obligate drinkers, and it has been shown that leaf-water has limited effect on the δ species . The combutaries . The seather out . In Laur than 2‰ . The amp, Gibson . It may 18O values, perhaps due to a thinner enamel layers than is seen in molars. Future studies should incorporate incisors to directly investigate pig watering habits. This could potentially open-up new research avenue into analysing differences between species in terms of δ18O values to understand how communities may have managed water sources for livestock.The geometry of teeth between species will influence the stable isotopic record. Pig molars have different internal and external dental geometry in comparison to cattle and deer. The mineralisation process is faster in pigs and as with most species the timing of the process is not uniform across a crown , which m13C values from sheep/goat enamel bioapatite samples ranged from −28.6‰ to −23‰, considerably greater than the range previously observed in collagen samples (−26.4‰ to −23.4‰) [13C and δ18O values, which indicates that that caprines consumed fodder sources that varied considerably in 13C over the seasonal cycle. Environmental factors, such as light levels and soil/atmospheric humidity, impact photosynthesis efficiency and respiration in C3 plants impact the isotopic fractionation of 12C/13C [13C values, 1) grazing in marsh land environments; 2) winter grazing in dense non-deciduous forests; 3) supplementary foddering during winter with leafy hay collected from either closed canopy forests during summer when the ‘canopy effect’ would be greatest, or collected from waterlogged galley forests, which also may exhibit low δ13C values [Predicted dietary δ −23.4‰) . A stron 12C/13C –84. We pC values .13C values ., which is greater than the values observed in hair samples from modern herbivores grazing on open pastures throughout the year (~1.5‰) [13C values [2 intake by plants coupled with increased photosynthesis activity results in 12C being discriminated over 13C [13C, resulting in higher carbon isotopes values in plant tissues. For example, in semi-arid regions where the mean annual precipitation is less than 500mm, the mean value of C3 plants, adjusted for the Suess effect, were between −24.7‰ to −23.9‰ [The seasonally flooded landscape surrounding the sites may explain the large amplitude of intra-tooth change in δ (~1.5‰) . Plants C values  with lowover 13C . In geneo −23.9‰ . We prop13C values. These environments contribute to osmotic stress in plants, thus reducing stomatal activity leading to an increase in the uptake of 13C increasing overall the foliar δ13C values [13C values with strong seasonal variation with relatively high values in the summer.The Danube and Sava are active meandering rivers, with paleo-channels that are visible from satellite images. Focusing on Vinča-Belo Brdo, at present the Danube runs close to the site, however in the past it may have flowed further east. There is no evidence of flooding during the occupation levels, which suggests that the site was not as close to the river as today . Areas oC values . Therefoth and 19th century have led to loss of these environments [13C values due to the high salinity and low soil moisture, which can affect stomatal activity [3 plants  native to highly saline marsh environments of Western Canada exhibit high δ13C values around −21.8‰ (adjusted for Suess effect), and vary between 6.5‰ and 4‰ across a 30m transect [13C and δ18O values may be a consequence of sheep/goat grazing on halophyte and/or vernal plant communities within the alluvial plain under a continental climatic regime.Stubline and Vinča-Belo brdo lies on the edge of the Vojvodina region, which is part of the Pannonian Basin known for its salt steppe, pans, marshes, and lakes ,90. Mostronments . Halophyactivity . Experimtransect . Halophytransect . We prop15N values of plants are also influenced by saline environments due to enrichment in soil 15N due to higher rates of denitrification that lead to 15N enriched soil nitrates [15N values (8.1±1‰) were found in caprine collagen in comparison to cattle (7.1±0.9‰). Enriched δ15N may be an indication of sheep/goat individuals grazing within saline soils, as has been seen in other studies [15N enrichment of bone collagen reflects animals visiting long-term pastures or grazing on crop by products under a manuring regime. Future studies are necessary to test this by determining the δ15N and δ13C values of remains of cereals and crop by-products.The δnitrates . Moderat studies . On the 13C values between −28.6‰ to −27.1‰, except for individual 7245 from Vinča-Belo Brdo. These values are comparable to ruminants dwelling in deciduous forested environments (−29.5‰ to −25.1‰) [13C values can occur in forest with dense leaf cover caused by the canopy effect, which is caused by 13C depletion of atmospheric CO2 under the canopy due to uptake of recycled CO2 respired by 13C depleted organic matter [13C [13C values in by 1 to 6‰ relative to floral growth in open environments, depending on canopy density which is dependent on tree species present in forests, the timing of the bud break, and leaf expansion [During winter, the caprines from both sites exhibited low dietary δ −25.1‰)  and deerc matter , a decreter [13C ,84. The xpansion ,95,96. I13C values registering in caprine tissues. Another possibility is that leafy hay was collected from arboreal species native to riverine environments, such as ash (Fraxinus excelsior). Waterlogging caused by seasonal flooding has a similar impact on leaf carbon isotopes values as the canopy effect,  of amino acids (AA) extracted from dentine can test for the consumption woody plants. This is due to the difference in δ15N values of glutamine (Glx) and phenylamine (Phe) AA, which has been found to be significantly different between herbaceous and woody plants [Charcoal analyses of Vinča-Belo brdo material indicate two main types of deciduous woodland environments were present near the settlement: riverine and hill slopes, with no large stands of non-deciduous trees . The losy plants . This mey plants ,99.13C values exhibit a wider ranger greater than values previously observed for collagen (−26.4‰ to −23.0‰). This may reflect the attenuation of bone collagen stable isotope values due to bone turnover. The results from the sequential analysis indicate strong inter-individual variation in cattle from Vinča-Belo brdo, unlike that previously observed in sheep teeth.Overall, the results from the sequential analysis indicate strong inter-individual variation in cattle, unlike that previously observed in sheep teeth. Previous analysis proposed that domestic cattle from Vinča-Belo brdo grazed across different types of pastures while animals at Stubline were kept mainly on wooded or waterlogged pastures . At Vinč13C values −26.1 to −24.5‰ with limited seasonal variation. However, three cattle individuals from Vinča-Belo brdo  exhibit δ13C values (>−24.1‰) as observed in caprine teeth. For Bos 7242 and 7244, these values occur in summer, as observed in caprine teeth. We propose this is the result of cattle grazing pastures supporting halophyte/vernal plant communities close to the sites, similar as proposed for caprines. For Bos 7230, high δ13C values reaching −21.1‰ during the period of winter season tooth formation may be evidence provisioning with hay collected from these vernal pastures in summer. There appears to be evidence for winter leaf hay provisioning in a single cattle, Bos 7240. This animal exhibits δ13C values as low as −28.2‰ during winter. Previous analyses of cattle from the LBK site of Bischoffsheim , this lack of evidence for supplementary fodder is surprising given that during winter the snow cover as well as spring floods may have restricted access to fresh pastures.Bos 7241 and Bos 7268 exhibit low δ (Alsace ) and the (France ) found c13C values for red deer between −29.7‰ and −25.0‰ are similar to the range of values for modern forest dwelling ruminants [Our study benefits from paleoenvironment evidence from the teeth of deer, which are typically forest dwelling. Dietary δuminants  and mid-uminants . Carbon uminants  highlighThe results from incremental bioapatite samples from domesticated and wild animals of Vinča-Belo brdo and Stubline allow for nuanced investigation of the land use by late Vinča communities. Previous paleoenvironmental and zooarchaeological studies have proposed that these communities exploited a wide range of niches for wild resources and to raise their herds, particularly cattle ,11,21,2213C values due to water stress [13C values observed in sheep/goat individuals. The relatively high δ15N values found in sheep/goat bone collagen could reflect a foddering strategy where cereals were enriched with 15N due to manuring regimes [The use of crop by-products and stubble for animal pasture and fodder may have also occurred. Archaeobotanical analysis of carbonised cereals and weed assemblages from Opovo, a contemporaneous site north of Vinča-Belo brdo, suggests that the majority of crops were sowed in the winter and harvested in the summer . Allowinr stress , which m regimes ,105.13C and δ18O values similar to suggesting multi-species herding for all or part of the year . It is possible some of the individuals analysed here were male and this variation in δ13C values indicates animals being fattened for meat production.Stable isotopic analysis of cattle teeth indicate several different husbandry strategies were applied. Some cattle exhibit δthe year , while oAnother source of carbon isotopic variation in cattle may be the emergence of extensive herding strategies where animals were able to roam between different habitats within the vicinity of the sites. This may represent a flexible and sustainable herding strategy where cattle were move naturally between pasture, reducing overgrazing. This type of management may only require one or two herdsmen that corrals individual household herds daily and moves them to pastures within the vicinity of the settlement. Collective herding strategies supports social cohesion and cooperation between households. Similar strategies are evident in traditional African societies, where village herds are graze together and returned to their owners in the evening for milking . The simContinual herd growth likely took place alongside increased intensity of settlement and population size in during the Late Vinca phases and would have placed significant pressure on local pasture resources. Due to the proximity of Vinča with other settlements and coupled with the increase in specialisation of animal products , herders13C isotope values from different animal tissues has provided a means to reveal multi-faceted perspectives on herding strategies at different temporal scales  thus highlighting the importance of multi-tissue analysis. Our study has uncovered the use by sheep flocks of seasonally available halophyte pastures and leafy hay, providing a new perspective on the sheep/goat management by Vinča communities. Future studies should consider including isotope analyses of botanical material to generate an integrated and detailed picture of herding and agricultural practices. There is also a need for detailed organic residues analysis of ceramic sherds to provide more information about processing of animal products.Livestock herds were an important social and economic component of Vinča world and here we have uncovered the complex herding strategies employed for animal pasture and fodder. The combination of stable δThe low variation in herding strategies for sheep/goat indicates a pooling of resources carried out a community level, while in contrast, the diversity in cattle pasturing strategies may be an indication of the emergence of household economies associated with the growing social and economic importance of cattle. This contrast in herding strategies between the two species highlights a level of complexity within the organisation of food procurement strategies for individual households and for the community in general. This study is limited to two sites, however, there is enormous potential for future stable isotopic studies considering the excellent preservation of faunal material from Vinča culture sites. More detailed analysis at other sites would enable a deeper understanding of Vinča culture herding practices as well as the landscape inhabited by these communities and their herds. Integrated with existing archaeological studies would provide a greater insight into the cultural evolution and the development of complex societies in Europe.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Mono-therapeutics is rarely effective as a treatment option, which limits the survival of patients in advanced grade aggressive cancers. Combinational therapeutics (multiple drugs for multiple targets) to combat cancer is gaining momentum in recent years.Hence, it is of interest to document known data for combinational therapeutics in cancer treatment. An amalgamation of therapeutic agents enhances the efficacy and potency of the therapy. Combinational therapy can potentially target multiple pathways that arenecessary for the cancer cells to proliferate, and/or target molecules, which may help cancer to become more aggressive and metastasize. In this review, we discuss combinational therapeutics, which include human γδ T cells in combinations withbiologically active anti-cancer molecules, which synergistically may produce promising combinational therapeutics. As per patients . Eliminapatients .Biologipatients ,11-13. Bpatients ,7,10.Medicinal chemistry is unique in generating molecules with specific structural activity relationships to target specific biological functions . The divSeveral research studies focus on new organic synthetic methods for developing high yielding organic compounds and drugs under environmentally benign conditions with advantages over traditional methods . New metThe global oncology trend report (2018) revealed that the global spending on cancer medication and therapeutic and supportive care rose from $96 billion in 2013 to $133 billion globally in 2017 . OverallUtilizing current medicinal chemistry approaches is possible in the synthesis of various complex molecules in comparatively short periods of time and with efficient use of energy . There hImmune cells play an integral part in tumor cell control (immunosurveillance), and immune defects are frequently associated with cancer development and disease progression . Consequγδ T cells are unique unconventional T cells, which are distinguished from the major T cell subset i.e., αβ T cells, by the T cell antigen receptor (Vγ9Vδ2-TCR), which they express on their cell surface ,12. γδ in vivo on gaining access to tumor cells.The above finding revealed that large numbers of γδ T cells were well tolerated although clinical benefits have not been fully ascertained . γδ T ceCancer cell progression is not easy to understand and possess complex multiple pathways, which include numerous interconnected molecules. Single drug or vaccine has limitations in countering the complexity of tumor pathogenesis .Combinain vivo γδ T cells.The use of potential methodologies to develop small biological active molecules with standardized reactions has been discussed in this review. We document known data for combinational therapeutics in combating cancer. These molecules exhibit potentialanti-tumor activities and remain stable at room temperature. Biologically active molecules have inherent advantages over adaptive immunotherapies, as these molecules can reach a wide spectrum of molecular targets, including intracellular targets or those presentdeep in the tumor milieu. Human γδ T cells exhibit expression for cell surface markers for antigen presentation, co-stimulation, cell adhesion, cell activation and effector state, all together with tumor killing activity. Thus, the combination ofboth small molecules and γδ T cell are an important consideration for the future development of cost-effective combinational therapeutics. Interesting outcomes would be expected due to the concomitant synergistic effects of biological active smallmolecules and γδ T cell, driving efficient tumor cytotoxicity. In future small molecules can be engineered to perform dual functions; first as we mentioned in this review to inhibit cancer cell proliferation and second to activate