Mediating magnetic interactions between lanthanide ions via radical bridges is a fruitful strategy towards stronger coupling. In this work we explore the limiting case when the role of a radical bridge is played by a single unpaired electron. We synthesize an array of air-stable Ln2@C80(CH2Ph) dimetallofullerenes  featuring a covalent lanthanide-lanthanide bond. The lanthanide spins are glued together by very strong exchange interactions between 4f moments and a single electron residing on the metal–metal bonding orbital. Tb2@C80(CH2Ph) shows a gigantic coercivity of 8.2 Tesla at 5 K and a high 100-s blocking temperature of magnetization of 25.2 K. The Ln-Ln bonding orbital in Ln2@C80(CH2Ph) is redox active, enabling electrochemical tuning of the magnetism.Engineering intramolecular exchange interactions between magnetic metal atoms is a ubiquitous strategy for designing molecular magnets. For lanthanides, the localized nature of 4 Dilanthanide complexes that possess radical bridges exhibit enhanced magnetic exchange coupling, affording molecular magnets with high blocking temperatures. Here, the authors explore a series of dilanthanide-encapsulated fullerenes where the radical bridge is taken to its limit and the role is played by a single unpaired electron. Single molecule magnets (SMMs) can be used as core elements of nanospintronic devices3, such as spin valves4, spin transistors6, or building blocks of quantum computers8. Optimization of symmetry and ligand environment led to a dramatic improvement of lanthanide SMMs during the last decade11 with the latest discovery of magnetic hysteresis at 60–80 K in Dy-metallocenium salts15. Effective barriers of magnetization reversal higher than 1000 K were reported for several lanthanide SMMs20. Single lanthanide atoms were also found to keep magnetic bistability up to 45 K on MgO|Ag(100) substrate23.Lanthanides are well-known for their large atomic moments and magnetic anisotropies, and embedding discrete lanthanide ions in molecular environments leads to nanomagnets exhibiting magnetic bistability and slow relaxation of magnetization on a single molecule level12 complex24. The temperature range at which a multinuclear magnet can be considered as a giant spin rather than a combination of weakly interacting individual spins is limited by the strength of exchange interactions. Whereas exchange interactions between transition metals can be tuned in a wide range, the localized nature of 4f electrons results in weak exchange interactions in lanthanide compounds rarely exceeding 1 cm−1. As a result, when the relaxation of magnetization in multinuclear lanthanide SMMs is driven via exchange excitations, the barriers to magnetization reversal are usually well below 100 K25.Combination of several magnetic centers within one molecule may lead to high-spin ground states and can largely suppress quantum tunneling, which is the main low-temperature relaxation mechanism for single-ion magnets in zero magnetic field. Design of multinuclear SMMs has been a viable strategy since the discovery of SMM behavior in the Mn26. The radical bridge usually features a rather diffuse singly occupied molecular orbital, which exhibits stronger interactions with the 4f electrons. The lanthanide-radical exchange coupling constants can reach values of −27 cm−1. The strongest coupling so far has been found in dilanthanide complexes with N23− radical bridges29, and the corresponding Tb complex has a blocking temperature near 30 K, which is the highest known value among multinuclear SMMs30. In this work we explore the limiting case of this concept in which the role of a radical bridge is played by a single unpaired electron, residing on the lanthanide-lanthanide bonding orbital and coupling the lanthanide spins inside a fullerene.Exchange coupling in lanthanide molecule magnets can be increased by introducing radical bridges31, such as metal dimers in dimetallofullerenes. Although Coulomb repulsion between two metal ions prevails over the covalent bonding34, the metal dimers cannot dissociate inside fullerenes. This unique situation allows for direct Ln–Ln bonding in dimetallofullerenes38, which could not be realized in any other molecular lanthanide compound so far. Of particular interest are Ln2@C80 molecules. The C80-Ih fullerene cage is unstable in the neutral state due to the presence of a four-fold degenerate orbital occupied by only two electrons. At the same time, the hexaanion of C80-Ih has a very stable closed-shell electronic structure, and hence this fullerene is the most preferable host for endohedral species acting as donors of six electrons, such as the early lanthanide dimers La2 or Ce239. However, in the Ln2@C80 molecules with heavier lanthanides (Gd to Lu), the Ln2 dimers transfer only 5 electrons to the fullerene orbitals, leaving one electron on the Ln–Ln bonding orbital40. The electronic structure of the fullerene cage in such Ln25+@C805− dimetallofullerenes can be stabilized by addition of one electron41, substitution of one carbon by a nitrogen atom (giving azafullerenes Ln2@C79N42) or by functionalization with a radical group . A formal oxidation state of the lanthanides in such dimetallofullerenes is Ln+2.5. EPR studies of Gd2@C79N revealed a ferromagnetic ground state with a giant spin of S = 15/2, and magnetization studies showed that the exchange coupling between the Gd spin and the unpaired electron residing on the Gd−Gd bonding orbital is as large as 170 cm−1 (refs. 45). Very recently we have reported that Dy2@C80(CH2Ph) shows exceptional magnetic properties with magnetic hysteresis up to 22 K and Dy-electron exchange coupling of 32 cm−138.The empty space inside carbon cages provides unmatched possibilities for stabilizing small metallic clusters in unconventional valence and spin states2@C80(CH2Ph) molecules , all featuring single-electron Ln–Ln bonding molecular orbitals (MO). This bonding situation leads to giant exchange interactions in all magnetic molecules and is very beneficial for the molecular magnetism, especially in Tb2@C80(CH2Ph) showing a giant coercivity and the highest known blocking temperature among dinuclear lanthanide complexes. Furthermore, we demonstrate that the single-electron Ln–Ln bonding MO is redox-active, which allows control of the magnetism via electron transfer.In order to investigate and understand the principles underlying the SMM behavior in encapsulated lanthanide dimers, in this work we synthesize and study an array of Ln38. Carbon soot obtained by arc-discharge of metal-oxide filled graphite rods is extracted with hot DMF, giving a mixture of fullerene anions in DMF solution. The anions are reacted with benzyl-bromide to yield neutral air-stable benzyl monoadducts, which are further separated by multistep HPLC protocol to yield pure Ln2@C80(CH2Ph) derivatives  and the presence of an unpaired valence electron  serves as a measure of a magnetic anisotropy, taking the following form in a point dipole approximation:46Ri and θi of the i-th proton in the coordinate system centered on the Ln2 dimer with polar axis along the Ln–Ln bond and is expected to be very similar for all {Ln2} compounds. The second term in Eq. 3+–e–Ln3+] system. {Dy2} and {Tb2} exhibit almost identical 1H chemical shifts, indicating a similarity of their magnetic properties, whereas the values of {Ho2} are at least twice smaller. Substitution of one Tb in {Tb2} by Y results in a 4.3-fold drop of the paramagnetic shift in {TbY}. Remarkably, 1H resonances in {Er2} are shifted into the positive direction, revealing that the sign of the magnetic anisotropy in the endohedral Er2 dimer is opposite to that in Tb2, Dy2, and Ho2 dimers.The Lnces Fig. , 33 stro1H NMR spectrum of {Gd2}). If the Gd spin in {TbGd} were behaving isotropically, the 1H chemical shifts of {TbGd} would be close to those of {TbY}. However, the measured 1H shifts in {TbGd} are two times larger than in {TbY}  effects together with the Kondo description of magnetic interactions between the lowest J-multiplet of lanthanides with the partially delocalized unpaired electron occupying the spd-hybrid Ln–Ln bonding orbital:i-th lanthanide site. Later, the LF Hamiltonian will be limited to the crystal-field shape commonly used for 4f systems. Further, j12 is the direct exchange coupling between the localized lanthanide moments t is the electron hopping amplitude between sites 1 and 2, ciσ  calculations predict two very close Ki values for {Gd2}, 181 and 184 cm−1, and much smaller direct Gd–Gd exchange coupling, j12=−1.2 cm−1.38 When j12 is that small, the Ki and j12 parameters cannot be determined from experimental data separately, leading to the approximate Hamiltonian Eq. Keff (ref. 45). Having used this single-parameter Hamiltonian in the analysis of the measured magnetization and χT curves for {Gd2} we have obtained the Keff value of 160±10 cm−1 45 and is in good agreement with the DFT results.A recent theoretical study of the  system between two lanthanide sites glues their magnetic moments together. The strong ferromagnetic coupling emerging from these interactions is responsible for the high-spin magnetic ground state in certain {Ln2} molecules. But the strong exchange coupling alone is not enough to make a good SMM. Single-ion anisotropy and collinearity of lanthanide spins play a crucial role as well, and whereas {Tb2} and {Dy2} with high-spin easy-axis single-ion ground states and collinear moments make the best SMMs in the series, {Ho2} with mixed LF states and tilted magnetic moments is only a modest SMM, and {Er2} with easy-plane single-ion anisotropy is hardly an SMM at all. The different magnetic anisotropy of these chemically very similar molecules can be understood from the element-specific shape of the Ln-4f charge density, interacting with the molecular charge distribution and being spin-orbit coupled to the 4f magnetic moments (Supplementary Note 3). Furthermore, the coupling of a single lanthanide spin to the delocalized electron spin is also not sufficient as illustrated by the soft SMM behavior of the mixed-metal {TbY} system. Thus, homonuclear lanthanide dimers with collinear magnetic moments and strongly-axial single-ion magnetic anisotropy give the best SMMs.To summarize, we showed that just like delocalization of electrons is the essence of covalent chemical bonding, delocalization of the unpaired electron spin in the [Ln2 dimer is protected by the fullerene, it is not completely isolated from the environment. The carbon cage remains transparent for electrons66, and {Ln2} compounds exhibit lanthanide-based redox-activity. In the first reduction step, the Ln–Ln bonding orbital is populated by a second electron, thus allowing to change the valence state from Ln+2.5 to Ln+2. Simultaneously, the exchange interactions between Ln spins are reduced in the anionic state. Importantly, the valence electrons are strongly coupled to the lanthanide spins. Thus, magnetic properties of {Ln2} molecules can be switched by an electron transfer, which forms a background for their possible application in spin-polarized molecular transport, as redox magnetic switches or as an electron spin detector using magnetoresistance  as shown in Supplementary Figs 2} compounds could be isolated in this work. The yield of {Ln2} depends on the metal size: the yields of {Er2}, {Ho2}, and {Dy2} are similar, the yield of {Tb2} is ca twice lower, and the yield of {Gd2} is the lowest in the series.{Ln2} solution in toluene were measured using cw-EPR spectrometer EMX Plus (Bruker), working in X-band and Q-band regions. The EPR spectra were fitted using Easyspin, a MATLAB toolbox67. UV-vis-NIR absorption spectra were measured in toluene solution at room temperature with a Shimadzu 3100 spectrophotometer. Raman spectra were recorded at 78 K on a T 64000 triple spectrometer (Jobin Yvon) using 656 nm excitation wavelength of a tunable dye laser Matisse 2 (Sirah Lasertechnik). IR spectra were measured at room temperature with Vertex 80 FTIR spectrometer (Bruker) equipped with a Hyperion microscope. For Raman and IR measurements, the {Ln2} samples were drop-casted from toluene solution onto single-crystal KBr disks. NMR spectra were measured with an Avance 500 spectrometer (Bruker). Voltammetric experiments were performed with a sweep rate of 100 mV s−1 in o-dichlorobenzene solution with TBABF4 electrolyte salt in an oxygen-free glove box using potentiostat-galvanostat PARSTAT 4000 A. A three-electrode system with a platinum working and a counter electrode and a silver wire pseudo-reference electrode was used, potentials were calibrated by adding ferrocene as an internal standard in the end of each measurement.Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass-spectra were measured with a Bruker autoflex mass-spectrometer with 1,1,4,4-tetraphenyl-1,3-butadiene as a matrix. EPR spectra of {Gd2@C80-CH2Ph۰0.67(CH3Ph) was accomplished by layering hexane over a solution of {Dy2} in toluene. Slow diffusion of two solutions resulted in formation of small black crystals (30 × 30 × 10 μm3). X-ray diffraction data have been collected at 100, 130, 160, 190, 220, 250, 270, and 290 K on the BL14.3 beamline operated by the Joint Berlin MX Laboratory at the BESSY II electron storage ring 68 using a MAR225 CCD detector, λ = 0.89429 Å. Processing diffraction data was done with XDSAPP2.0 suite69. The structure was solved by direct methods and refined using all data (based on F2) by SHELX 201670. Hydrogen atoms were located in a difference map, added geometrically, and refined with a riding model. Crystal data and data collection parameters are summarized in Supplementary Table 1Crystal growth of Dy2 solution into a standard powder sample holder. Long magnetization relaxation times of {Tb2} were determined from the measurement of magnetization decay using a dc-SQUID. After the sample was magnetized to the saturation at 7 Tesla, the field was swept fast to zero or 0.3 T, and then the decay of magnetization was followed over hours and fitted with stretched exponential . See Supplementary Methods for further details.DC magnetic measurements were performed using a Quantum Design VSM MPMS3 magnetometer. The samples were drop-casted from CS71. Ab initio energies and wave functions of CF multiplets for the {LnY}– molecules have been calculated at the CASSCF/SO-RASSI level of theory using the quantum chemistry package MOLCAS 8.072. The single ion LF-parameters were calculated based on ab initio data with the use of SINGLE_ANISO module73. Modelling of the magnetic properties was accomplished with the PHI program60 and included powder-averaging. See Supplementary Methods for further details.VASP code, version 5.0, was employed to optimize the molecular structures at the PBE-D level using PAW pseudopotentialsSupplementary InformationTransparent Peer Review File


Plasmodium falciparum malaria frequently encounter situations in which molecular genotyping is unable to discriminate between parasitic recurrence, either new infection or recrudescence. The current WHO guideline recommends excluding these individuals with indeterminate outcomes in a complete case (CC) analysis. Data from the four artemisinin-based combination (4ABC) trial was used to compare the performance of multiple imputation (MI) and inverse probability weighting (IPW) against the standard CC analysis for dealing with indeterminate recurrences.Antimalarial clinical efficacy studies for uncomplicated 3369 study participants from the multicentre study  with molecularly defined parasitic recurrence treated with three artemisinin-based combination therapies were used to represent a complete dataset. A set proportion of recurrent infections  were reclassified as missing using two mechanisms: a completely random selection (mechanism 1); missingness weakly dependent (mechanism 2a) and strongly dependent (mechanism 2b) on treatment and transmission intensity. The performance of MI, IPW and CC approaches in estimating the Kaplan-Meier (K-M) probability of parasitic recrudescence at day 28 was then compared. In addition, the maximum likelihood estimate of the cured proportion was presented for further comparison . Performance measures  were reported as an average from 1000 simulation runs.The CC analyses resulted in absolute underestimation of K-M probability of day 28 recrudescence by up to 1.7% and were associated with reduced precision and poor coverage across all the scenarios studied. Both MI and IPW method performed better (greater consistency and greater efficiency) compared to CC analysis. In the absence of censoring, the analytical solution provided the most consistent and accurate estimate of cured proportion compared to the CC analyses.The widely used CC approach underestimates antimalarial failure; IPW and MI procedures provided efficient and consistent estimates and should be considered when reporting the results of antimalarial clinical trials, especially in areas of high transmission, where the proportion of indeterminate outcomes could be large. The analytical solution estimating the cured proportion could provide an alternative approach, in scenarios with minimal censoring due to loss to follow-up or new infections. Plasmodium falciparum malaria is the risk of recrudescence, defined as the recurrence of peripheral parasitaemia genetically identical to the parasites present before treatment. Molecular analysis of the parasite samples collected at pre-treatment and on the day of recurrence is used to discriminate homologous (recrudescent) from heterologous (new) infections For each subject ii.p) of the smallest ui as having missing outcome; p = 0.10, 0.30, 0.45Set the desired proportion  to determine the predictors associated with indeterminate outcomes i with recurrent parasitaemia, the probability of their outcome being missing (πi) was estimated using a logistic regression model based on the treatment regimen and the transmission level of the site the subject came from:δi is an indicator variable for missing outcome for individual i, and k = 1 and 2 for low and moderate transmission respectively (k = 0 for high settings as reference category), and j = 1 and 2 for antimalarial treatments ASAQ and DP respectively (j = 0 for AL as reference arm).For each subject ii)yi) for missingness for subject i based on the probability of outcome being missing (πi) estimated in step (i) as:Generate a Bernoulli outcome  used for assigning missingness for the weak scenario (mechanism 2a) and strong scenario (mechanism 2b) are detailed in Table Under mechanism 2, two different scenarios were studied representing weak and strong relationship between the covariates and missingness. The coefficients (Mechanism 2a (Weak scenario) logit(π(δi = 1)) = ψ0 + ψ whereψ0 =  − 2.10, − 0.75, − 0.11 for approximately 10, 30, and 45% missingness respectively.Mechanism 2b (Strong scenario) logit(π(δi = 1)) = ψ0 + ψ whereψ0 =  − 2.03, − 0.68, − 0.02 for approximately 10, 30, and 45% missingness respectively.ψ0 was chosen by iteration to approximately achieve the desired proportions of missing outcomes. Under this generating model, patients treated with ASAQ and DP were progressively less likely to be assigned indeterminate outcomes, reflecting the increasingly longer elimination half-lives with these drug regimens which prevents some of the recurrences to be fully observed by 28 days; and similarly, patients in the moderate and low transmission settings were progressively less likely to have indeterminate outcomes compared to those in high settings as the genotyping method is less likely to fail as clonal competition is lower due to reduced parasitic diversity in low transmission areas.For the strong scenario, the strength of the association between transmission, treatment and missingness was 2-fold higher than that of the weak scenario, to represent a more extreme case. The constant Three different approaches were used for handling missing data: complete case analysis, multiple imputation and inverse probability weighting. In addition to these three methods, the simulated datasets were also analysed using the analytical approach outlined in equation 2. The estimate of the variance of the analytical solution is presented in Additional file δi = 1), the missing values were modelled based on the covariate set outlined in the imputation model (Table Missing outcomes were imputed using a logistic regression restricted to patients with recurrent parasitaemia (81 recrudescences and 455 new infections) using the MICE algorithm in R. For each observation with simulated missing outcome (m = 50) were selected following the recommendation that m should be at least equal to the percentage of missing cases when the fraction of missing information is less than 50% [The number of imputations  was constructed to estimate the probability of a patient having an observed treatment outcome (cure/recrudescence/new infection) using a logistic regression as specified in Table θ be the true value of the “fixed” estimand of interest derived from the full dataset and θ generated from the sth simulation. The estimand of primary interest was the Kaplan-Meier estimate of 28 day parasitic recrudescence, Let entclass1pt{minimaentclass1pt{minimaθ). The bias was expressed as relative percentage bias, which is bias relative to the full data estimate Bias was defined as the difference between the average of the estimates  included the point estimate of the K-M derived from the full dataset (‘fixed’ full data estimate) before inducing missingness. For a 95% confidence interval, the theoretical coverage is expected to fall between 93.6 to 96.4% with 1000 simulated datasets. A drop in coverage below 90% is regarded as problematic . FinallyMultiple imputation was carried out using mice library and Kaplan-Meier estimates were generated using survival library in R statistical software .n = 1033) from moderate and 21% (n = 691) from the areas of low transmission settings from a total of 10 different study sites. The median baseline parasitaemia was 28,855/μL and was similar between the treatment regimens. On the recurrence day, the median parasitaemia was 6080/μL for recrudescences and 4600/μL for new infections.There were a total of 598  recurrences of which 81 were recrudescences, 455 new infections and 62 indeterminate outcomes after performing genotyping  in the missingness model provided the most consistent estimate of all approaches  resulted in SEs which were  smaller than the MI estimates. The comparison of SEs for IPW using the naïve approach and bootstrap method are presented in Additional file Like with the K-M estimates, the model based SEs and empirical SEs were progressively larger with increasing proportion of missingness for the analytical solution for estimating cured proportion Table . At 10% Figure The CC approach had the least overall accuracy of all the missing data methods followed by IPW-Exclude across all missingness mechanisms. For all the missing data methods, the overall accuracy decreased with increasing proportion of missingness. MI and IPW approach both provided similar estimates of accuracy. With MI and IPW approach, the accuracy was higher under missingness mechanism 2 compared to the mechanism 1. The overall accuracy of the estimator is presented in Fig. Missing data in clinical trials can pose analytical challenges, including undermining the validity and interpretation of the results. In antimalarial studies, indeterminate recurrences resulting from genotyping failure are frequently encountered, especially in the areas of high transmission intensity, where multiple infections are common. Principled approaches for handling missing data have proliferated the medical and statistical literature in recent years , 10, 38,Two different missingness mechanisms were investigated and differences in estimates compared for scenarios in which 10, 30 and 45% of the known recurrences were reclassified as missing. In all these scenarios, the current recommendation of excluding indeterminate cases resulted in an upwards biased estimate of day 28 probability of cure (K-M method) by up to a maximum of 1.7% -2 genes whereas Rouse et al. (2008) reported that an identical msp-2 allele could be different by up to 18 base pairs [The definition of recrudescence and new infection depends on the how different sized bands are binned and classified as being the same or different alleles. For example, Cattamanchi et al. (2003)  considerse pairs . The defse pairs ).The widely used approach of excluding indeterminate outcomes results in underestimation of antimalarial failure. In the example studied, the incorporation of missing data through correctly implemented IPW (including recurrence status as the predictor and using bootstrapping to estimate the standard error) and MI approaches and the analytical solution outlined in equation 2 greatly reduced bias. The IPW and MI approaches were associated with the smallest standard errors and provided superior coverage probability of the derived estimates of day 28 recrudescence. IPW and MI approaches are easily implementable in standard statistical software and should be considered for handling indeterminate outcomes in the derivation of antimalarial failure.Additional file 1: Section A: Estimating treatment efficacy for antimalarial drugs; Section B1: Quantifying bias in complete case estimator; Section B2: Variance of the maximum likelihood estimator; Section C: Application of Rubin’s combination rules for pooling multiply imputed Kaplan-Meier estimates; Section D: Comparison of naïve and bootstrapped standard error for inverse probability weighting approach; Section E: Additional results on performance measures for the simulation study. TableS1. Antimalarial treatment outcomes for the 4ABC Trial [Table S2. Full data estimate of cure at day 28 follow-up using the Kaplan-Meier method Table S3. Specification of the logistic regression model used to impose missingness Table S4. Outline of the imputation and missingness models Table S5. Performance measures of various methods for handling 45% missingness in recurrences for individuals treated with artemether-lumefantrine Table S6. Performance measures of complete case and maximum likelihood estimator for handling 45% missingness in recurrences for individuals treated with artemether-lumefantrine in estimating day 28 cured proportion Figure S1. Therapeutic responses post antimalarial treatment in P. falciparum malaria. Adapted from White NJ: The assessment of antimalarial drug efficacy. Trends Parasitol 2002, 18:458–464.9BC Trial  Table S2


Leaf uptake should alter plant metabolism and overall nitrogen balance and indirectly influence plant consumers; however, these consequences remain poorly understood. Here we show that direct foliar assimilation of NO2 increases levels of nitrogen-based defensive metabolites in leaves and reduces herbivore consumption and growth. These results suggest that atmospheric reactive nitrogen could have cascading negative effects on communities of herbivorous insects. We further show that herbivory induces a decrease in foliar uptake, indicating that consumers could limit the ability of vegetation to act as a sink for nitrogen pollutants . Our study suggests that the interactions of foliar uptake, plant defence and herbivory could have significant implications for understanding the environmental consequences of reactive nitrogen.Reactive nitrogen from human sources (e.g., nitrogen dioxide, NO 2 increases defensive metabolites, reduces herbivore consumption and growth, and herbivory reduces foliar NO2 uptake.Reactive nitrogen oxides can be assimilated by leaves, though the trophic and nitrogen cycling impacts of this are unclear. Here Campbell and Vallano show foliar uptake of NO For example, nitrogen oxides (NOx) have negative effects on human health5 and alter important biogeochemical processes, including carbon sequestration, N cycling and global warming9. Reactive N also poses a significant and growing threat to biodiversity11. In terrestrial ecosystems, numerous studies have demonstrated that reactive N deposition to soils is causing widespread declines in plant diversity by altering soil chemistry and disrupting competitive dynamics14. However, the cascading effects of reactive N on higher trophic levels remain poorly studied, and there are few data on the complex pathways by which reactive N could influence consumers15.As a result of human activity, inputs of reactive nitrogen (N) compounds into the atmosphere have tripled in the past 50 years, with deleterious consequences for urban and natural ecosystemsx gases can also be taken up directly by plant leaves and used as a nutrient source16. Up to 15% of a plant’s N budget can be obtained via foliar uptake, making it a significant pathway for the cycling of reactive nitrogen18. NOx enters leaves via stomatal diffusion and undergoes apoplastic disproportionation and ascorbate scavenging before downstream metabolism. Variation in these processes within the plant and among species19 suggests that the environmental consequences of foliar uptake will be more complex than root uptake, particularly for consumers. NOx deposited onto soil is taken up by roots as NO3– and should simply increase plant %N and may benefit herbivores, a prediction supported by empirical studies20. In contrast, foliar NOx assimilation could have divergent effects on consumers depending on its metabolic fate: N derived from NOx uptake could be stored as free amino acids and could benefit leaf-feeding herbivores, similar to the effects of root fertilisation. Conversely, if NOx was incorporated into defensive metabolites or caused shifts in defensive secondary metabolism, then consumers would suffer reduced growth and reproduction. Negative effects of foliar uptake of reactive N for herbivores have not yet been demonstrated. The only prior studies used high-concentration, short-term fumigation and showed positive effects on aphid colony growth22. However, aphids feed on phloem, which is severely N-limited and contains few defensive compounds23. Thus the potential for negative impacts of foliar NOx assimilation may have been underestimated.One mechanism by which reactive N could affect higher trophic levels is via foliar uptake. Most research has focussed on root uptake of reactive N from soil following deposition. However, NO2 uptake on plant metabolism and herbivorous insects and then test the reciprocal effects of herbivory on NO2 uptake, using tobacco, Nicotiana tabacum, and its natural leaf-feeding herbivore, the tobacco hornworm, Manduca sexta. We are able to accurately trace reactive N uptake in leaves and monitor how herbivory affected reactive N assimilation by using a stable isotope fumigation system with 15NO2. Plants exposed to NO2 assimilate significant quantities of atmospherically derived N, exhibit upregulation of alkaloid defensive metabolites and support lower herbivore growth. In turn, herbivore feeding causes plant-wide reductions in foliar NO2 assimilation. Our study demonstrates that, in addition to disrupting plant communities through soil deposition, anthropogenic reactive N may have extended negative consequences for higher trophic levels. Our results also indicate that insect herbivores could influence the capacity of leaves to absorb reactive N and act as a sink for these atmospheric pollutants. This feedback between foliar NO2 uptake and herbivory may have implications for predicting the fate of reactive N in terrestrial ecosystems.In this study, we test the effects of foliar NO15NO2 at a concentration (40 ppb) similar to current urban levels;24 control chambers were maintained at 0 ppb. Plant roots were suspended in a hydroponics system at either 50mM NO3– (low-N) or 500mM NO3– (moderate-N) to compare responses at two ecologically realistic root N levels; shoots and roots were segregated to allow accurate partitioning of plant N sources18. After 4 weeks, M. sexta larvae were applied to half the plants in each chamber and allowed to feed. Herbivores feeding on NO2-exposed plants showed a pronounced reduction in growth (a proxy for fitness in this species25) compared to those feeding on plants in control chambers  revealed that NO2-derived nitrogen was used by herbivores for growth . In support of this interpretation, total alkaloid content of plants under 40 ppb NO2 was positively correlated with the amount of 15NO2-derived N, while remaining independent of total N   may have been higher in the high NO3− treatment. Our results demonstrate that plant defences will be sensitive to N deposition to soil but will also be uniquely sensitive to N derived from foliar uptake, with the potential to affect a wide range of organisms that interact with a plant’s secondary metabolome, including herbivores, pollinators and microbes.The genus 15N-enriched NO2 allowed us to test whether herbivore-induced changes to plant physiology would influence foliar uptake of NO2 and its allocation among plant tissues. Plants exposed to herbivory contained 36% less NO2-derived N  and then exposed them to 40 ppb 15NO2. Prior feeding by herbivores caused significant reductions in 15N assimilation in both damaged and undamaged leaves, confirming that herbivory reduced plant-wide foliar uptake , and second, by reducing plant size through consumption . The systemic reduction in 15N assimilation under relatively low damage (<10%) suggests that herbivores, which are ubiquitous even in urban environments, could represent a significant control on how readily plants assimilate and sequester reactive N pollutants.The use of ake Fig. . The eff2 caused increases in N-based plant defences and reductions in herbivore performance, suggesting that increasing levels of NO2 could have profound consequences for consumers, particularly in urban ecosystems. In turn, herbivore-induced reductions in foliar uptake may impose a limit on the effects of NO2 on defensive chemistry. However, the stability of this feedback remains unclear: while low herbivory caused pronounced reductions in NO2 uptake, the dose–response relationship between NO2 and defence remains unknown. In one scenario, plants in high NOx environments would exhibit chronically elevated defences, with negative consequences for consumers, while the presence of even diminished herbivore populations could reduce the beneficial sequestration of NOx and foliar cycling of reactive N. Models of ecosystem functioning under global change rarely consider interactions with herbivores. However, in a recent study of CO2 enrichment, herbivory significantly reduced plant carbon sequestration30, which, together with our results, indicates a need to better understand how consumers will influence ecosystem and plant-level responses to different atmospheric changes.We have identified a feedback between atmospheric reactive N, plants and consumer fitness that has implications for our understanding of plant responses to global change Fig. . First, x emissions will interact with other global change drivers with synergistic negative effects on consumers. For example, elevated carbon-to-nitrogen (C:N) ratios in plants growing under rising CO2 are predicted to challenge herbivores to increase consumption rates in order to maintain a balance of limiting nitrogen32. However, herbivory will also depend critically on interactions among other anthropogenic changes, including the effects of NOx and CO2 on plant defences, and effects of N deposition on plant %N and responses to CO235. Moreover, in our experiments herbivore performance was not related to the effects of NOx on leaf C:N or %N . Our data suggest that there will be significant consequences of these interactions for plant-feeding insects, including agricultural pests and rare species already vulnerable to the effects of soil N deposition on host plant abundance11. Recent demonstration of pronounced declines in arthropod diversity and abundance in regions with long histories of industrialisation37 further underscores the need to understand the wide-ranging environmental impacts of atmospheric reactive N on plant and insect communities.Our study also suggests that NON. tabacum L., (Solanaceae) and its natural leaf feeding herbivore, the tobacco hornworm, M. sexta L. (Lepidoptera: Sphingidae). Tobacco was selected because it is a fast-growing herbaceous species that grows well in hydroponic systems, exhibits significant N reduction in leaf tissue38 and has a well-characterised suite of defence-related secondary metabolites39. Tobacco hornworm was chosen because it is a native Solanaceae specialist that readily feeds on tobacco and rarely leaves a tobacco host plant with sufficient leaf material; experimental larvae were obtained from a large in-house colony. Seeds (N. tabacum x. sandera) were purchased from a commercial producer  and germinated and grown in perlite  in a climate-controlled growth chamber  at day and night temperatures of 27 and 21 °C, respectively, under moderate light (700 μmol m−2 s−1) and a 16-h photoperiod. Plants were watered daily to saturation for 2 weeks and fertilised with a Hoagland’s solution containing NO3– of known δ15N. Forty-eight 2-week-old plants were transplanted into the hydroponics–fumigation system and used for the experiment. The remaining 12 plants were harvested and measured for biomass, leaf area and isotopic composition to provide baseline data for the experimental plants.We used a model plant–herbivore system consisting of tobacco, 3) each fitted with three airtight Plexiglas fumigation chambers (36 × 25 × 43 cm3) with an opaque base. The system was located in a greenhouse with day and night temperatures of 27 and 21 °C, respectively, relative humidity of 60–70% and moderate light conditions (800 ± 75 μmol m−2 s−1) using natural and supplemental metal halide lighting  under a 16-h photoperiod. Roots were suspended in nutrient solution (20 °C) in the hydroponics tank via small holes in the chamber base, and shoots were enclosed in fumigation chambers (ambient temperature). Plants were stabilised and fitted with modelling clay  at the root–shoot junction at the base of each chamber to ensure an impermeable seal between the fumigation and nutrient solution system components.We used a factorial design in which we manipulated foliar exposure to atmospheric reactive N (simulating pristine and urban environments), together with the availability of root N (low vs. moderate), using a coupled hydroponics–fumigation system. The system consisted of 4, 50-L polyethylene nutrient tanks , which was diluted into the filtered air of randomly selected chambers using high-precision rotometers and mass flow controllers  at a fixed partial pressure (40 ppb). NO2 was selected as the atmospheric N source because it is a common atmospheric reactive N compound. The two fumigation treatments were selected to simulate pristine (0 ppb) and realistic 1-h urban (40 ppb) atmospheric NO2 concentrations44. The δ15N of the enriched 15NO2 was 1720 ± 17‰, providing a large signal separation from the NO3– source. NO2 and nitric oxide (NO) concentrations were monitored using a chemiluminescence NO-NO2-NOx analyser . Exhaust air from each chamber was filtered using activated charcoal and exited the system through an output line extending outside the greenhouse. Temperature and relative humidity within the chambers were monitored using humidity and temperature probes  connected to a datalogger .Fumigation chambers were supplied with activated charcoal-filtered, ambient air using a reciprocating air compressor  at a flow rate of 15 L min3 regimes, simulating low (50 μM) and moderate (500 μM) N availability, were applied to roots using nutrient solutions with fixed concentrations of NO3− as the sole root N source in a modified quarter-strength Hoagland’s solution45. The δ15N value of the NO3− nutrient solutions was −0.56 ± 0.1‰. The nutrient solutions were vigorously aerated at all times to provide adequate oxygenation and ensure complete mixing and pH was maintained at 5.8–6.2 by addition of either KOH or H2SO4. A 130-L reservoir of stock solution was used for each experimental N treatment. Nutrient solutions were replaced weekly to minimise microbial activity and prevent N depletion, and NO3– concentrations were measured weekly using an auto-analyser .Two NO2 and 50 μM NO3–, [2] 40 ppb NO2 and 50 μM NO3–, [3] 0 ppb NO2 and 500 μM NO3–, and [4] 40 ppb NO2 and 500 μM NO3–) was replicated in three chambers (N = 12) with four plants/chamber. After 4 weeks of growth under these treatment conditions, 3 neonate (freshly hatched) M. sexta larvae were placed on two plants in each chamber (6 larvae per chamber). We began the herbivory treatments at 4 weeks to maximise the period of NO2 exposure while ensuring that plants had sufficient space within the chambers and did not initiate reproduction . To minimise herbivore movement and ensure that feeding was confined to the selected plants, we used the pair of plants at a randomly selected end of the rectangular chamber and visually inspected plants several times per day. Two small cups filled with a wheat-germ diet46 and a single neonate larva were also placed in each chamber to test for effects of unforeseen environmental variation and any direct toxic effects of NO2 exposure. Larvae on both plants and diet were allowed to feed for 10 days and were then removed, allowed to purge their gut contents and peritrophic membranes (gut lining) and weighed as a proxy for performance/fitness25. Individual larvae were then killed by freezing and freeze-dried for subsequent stable isotope analysis; allowing natural purging of the gut lining allowed us to infer insect 15N assimilation without plant contaminants (see next). Plants were harvested for analysis following removal of the herbivores.Each of the four treatment combinations . Tissue N and C contents were measured using a CHN elemental analyser . Tissue δ15N and δ13C were measured using a continuous flow isotope ratio mass spectrometer . All analyses were conducted at the Cornell Stable Isotope Laboratory.Whole-plant samples were separated into leaf, shoot and root tissue. Shoot length and leaf number were measured for each individual and leaf area was estimated using a leaf area meter . Plant samples were then dried, weighed and sub-samples analysed for tissue N and C content and δ2 and nutrient solution NO3–) was calculated using a two-ended linear mixing model47 and published fractionation factors for root NH4+/NO3– assimilation48. Because we used an artificially high enrichment of 15N in the NO2 fumigation, fractionation events associated with foliar uptake were likely not detectable . Using this model, we estimated the total amount of NO2 incorporated via direct foliar uptake during the fumigation period for leaf, stem and root tissues in each experiment. In testing for larval uptake of NO2-derived N, we used the δ15N values directly due to unknown fractionation factors associated with sequential larval instars49 and since larvae differed in their developmental stage due to the treatments.Partitioning of plant N among sources , and variation in herbivory leads to differentially induced metabolite expression. Fresh tissue from a fully expanded leaf was excised (avoiding the midvein), weighed (ca. 100 mg), flash frozen, ground to a fine powder in liquid N2 using a pestle and stored at –80 °C. We homogenised samples using a FastPrep® tissue homogeniser  at 6 m s−1 for 90 s using 0.9 g of grinding beads  with 1 mL of ice-cold 40% methanol and 0.5% acetic acid solvent. Samples were centrifuged and a 15-µL aliquot of supernatant was analysed by high-performance liquid chromatography (HPLC) using an Agilent® 1100 series HPLC-DAD equipped with a Gemini C18 reverse-phase column  and a standard method50. Alkaloid, phenolic and diterpene glycoside analytes with identifiable ultraviolet spectra were selected and initially quantitated by peak area. Individual compound identification of nicotine, anatabine and chlorogenic acid was based on comparison with authentic standards, and peak areas were converted to µg gFW−1 using standard curves. A third, unidentified pyridine alkaloid  and two unidentified caffeic acid derivatives (Caffeic acid 2 and Caffeic acid 3) were converted to mass equivalents of nicotine and chlorogenic acid, respectively. All analyte quantities were normalised by the fresh sample masses prior to statistical analysis.We estimated the levels of defence-related secondary metabolites in control (undamaged) plants using protocols developed for the major classes of tobacco defensive compounds, specifically pyridine alkaloids  phenolic compounds (caffeic acids and flavonoids) and terpenoid glycosides15N under herbivory in our main experiment, suggesting the potential for herbivores to induce changes to foliar NO2 assimilation. To test this hypothesis and distinguish effects of herbivore-induced plant responses on uptake from effects of induced responses on N allocation (see Results), we conducted a second experiment: We grew a new group of 24 N. tabacum plants in the hydroponics–fumigation system at 50 µM NO3– and 0 ppb 15NO2 for 2 weeks, at which point we applied four neonate M. sexta on each of two plants per chamber, as in the main experiment. Larvae fed for 2 days, at which point damage levels were ca. 5–7%, which is sufficient to cause an induced response in Nicotiana26. Half the chambers then received 15NO2 fumigation at 40 ppb for a subsequent 5 days, and half served as controls (0 ppb). During this period, plants were inspected and 5–6 additional larvae were added to a few plants to maintain similar total damage levels (ca. 10% on each of four leaves) across treatments. Leaf tissue was harvested from control plants and from the damaged and undamaged leaves of the herbivore-treated plants and analysed for incorporation of 15N from NO2 uptake in the four treatments . Analysis of the damaged  and undamaged leaves on damaged plants allowed us to test whether herbivory had caused a systemic (plant-wide) induced change in foliar NO2 uptake.We observed indications of reduced incorporation of 15N uptake and plant metabolite data were analysed by two factor models, with nutrient solution (NO3– level) and NO2 fumigation level as fixed factors and their interaction. For consistency, the data for larval performance on diet were analysed in the same way, though there was no predicted effect of NO3–. Morphological data were analysed by three factor models with NO3, NO2 and herbivory as fixed factors and their interactions. Data were averaged within individual chambers; analyses with plant average as the observational unit did not qualitatively change the results, indicating that our experiment had sufficient statistical power for the observed effect sizes for these response variables. Plant 15N uptake, %N and C:N data were analysed by three-factor models that included NO3–, NO2 and herbivory levels. We used plant averages for these analyses to compensate for the reduced power available in higher-order models; however, plant-level and chamber-level averages again gave similar results. We analysed foliar uptake data using separate models for (a) damaged leaves, (b) undamaged leaves and (c) pooled leaf data from plants exposed to herbivores, in order to verify that plant responses were consistent at the local and systemic (plant-wide) scale. Means and standard errors for uptake rates for all treatments and tissue types are provided in Supplementary Table 2 exposure on secondary metabolites prompted us to determine whether there was a quantitative relationship between e.g. alkaloid content and the amount of NO2-derived N taken up by leaves. For this, we used plants in the 40 ppb NO2 treatment and tested for correlations (restricted maximum likelihood estimation) between total amounts of each metabolite class in each plant and the amount of 15NO2-derived N and total percentage of nitrogen in leaves.Data were confirmed for normality and analysed using standard general linear models in a maximum likelihood framework (JMP® v.13). Larval performance data, larval Supplementary InformationPeer Review File


Tableting had a significant impact on the self-assembly of lipids in redispersed milk tablets whereas no effect was seen for infant formula tablets. Incorporation of the disintegrant poly(vinylpolypyrrolidone) to reduce tablet dispersion times promoted the formation of hexagonal liquid crystalline phases upon digestion but had minimal effect on drug solubilisation. These findings show that similar to the use of liquid milk, the formulation of milk-drug tablets can be used to improve solubilisation of poorly water-soluble drugs.Oral delivery of dispersible tablets is a preferred route of administration for paediatrics due to ease of administration and dose control. Milk has gained interest as a drug delivery system due to its ability to dissolve poorly water-soluble drugs. There are no reports of milk tablet formulations being assessed in the context of lipid digestion, which is critical in influencing orally administered drug solubility and bioavailability. Milk-drug tablets were formulated by blending freeze-dried bovine milk or infant formula with the poorly water-soluble drug cinnarizine, which were directly compressed. Tablet strength, friability and dispersibility were quantified and synchrotron X-ray scattering was used to determine the lipid liquid crystalline phases formed during  This includes swallowing a tablet as a whole, which might pose as an obstacle for toddlers and infants or liquid oral formulations that could lead to variability in doses given by the caretaker . ManipulOral delivery of dispersible tablets is regarded as a preferred route of administration for paediatrics. The variability involved in breaking the tablet into smaller pieces, increasing the likelihood of affecting the dose, would be averted as the desired dose can be controlled by the reconstituted volume for administration. Although the excipients in dispersible tablet formulations are regarded as safe for consumption by adults, this does not necessarily translate to paediatrics .w/v% fat, of which the triacylglycerols (TAGs) comprise around 98 wt% of the total milk fat content  both in the presence and absence of cinnarizine and added disintegrant. Finally, SAXS was used to determine the scattering intensity of diffraction peaks from crystalline cinnarizine to track its solubilisation during digestion.To address these problems, blending the drug with locally available milk would minimise issues relating to accessibility and furthermore freeze-drying the milk could potentially eliminate the issue of maintaining cold-chain storage for liquid milk. Infant formula was also used in this study to remove the issue of temporal variability in milk fat composition. The use of direct compression (DC) to generate these tablets is less complex than other tableting techniques enabling easier manufacturing and accessibility in developing countries. Therefore, this study aims to add to the understanding of the role of milk and infant formula as a lipid-based formulation by investigating its behaviour in formulation as dispersible freeze-dried milk/infant formula-drug tablets, illustrated schematically in 22.1w/v% fat) was purchased from Coles supermarkets  and powdered infant formula was kindly donated by Medicines for Malaria Venture . The nutritional information for milk and the infant formula are provided in Full fat bovine milk (3.8 2.2Freeze-dried milk Bovine milk (5 mL) was added into a 20 mL glass scintillation vial and freeze-dried for two nights. The freeze-dried milk (585 mg) was weighed into a 20 mL glass scintillation vial using an analytical balance. Typically, 30 × 5 mL portions of milk were freeze-dried at a time to produce 30 tablets from 150 mL of milk.Freeze-dried milk-cinnarizine Bovine milk (5 mL) was added into a 20 mL glass scintillation vial and freeze-dried for two nights. Freeze-dried milk (585 mg) and cinnarizine powder (13 mg) were weighed separately using an analytical balance before being added into a 20 mL glass scintillation vial. The powders were then mixed thoroughly for 5 min with a vortex mixer. Typically, 30 × 5 mL portions of milk were freeze-dried at a time to produce 30 tablets from 150 mL of milk.Freeze-dried milk-cinnarizine-PVPP Bovine milk (5 mL) was added into a 20 mL glass scintillation vial and freeze-dried for two nights. Freeze-dried milk (585 mg), cinnarizine powder (13 mg) and PVPP (179 mg) were weighed separately using an analytical balance before being added into a single glass vial. The powders were then mixed thoroughly for 5 min with a vortex mixer. Typically, 30 × 5 mL portions of milk were freeze-dried at a time to produce 30 tablets from 150 mL of milk.Infant formula Using an analytical balance, the infant formula powder (701 mg) was weighed into a 20 mL glass scintillation vial.Infant formula-cinnarizine Using an analytical balance, infant formula powder (701 mg) and cinnarizine powder (13 mg) were weighed into a 20 mL glass scintillation vial and thoroughly mixed for 5 min using a vortex mixer.Infant formula-cinnarizine-PVPP Using an analytical balance, infant formula powder (701 mg) , cinnarizine powder (13 mg)and PVPP (214 mg) were weighed into a 20 mL glass scintillation vial and thoroughly mixed for 5 min using a vortex mixer.Tablet compression The blended formulations were tableted directly after vortex mixing. Magnesium stearate (0.5 w/w% to tablet) was added to lubricate the tablet press  before compressing the tablets with a compression force of 60 kg at 5 mm/min fitted with a 14 mm diameter flat-faced bevelled edge.2.32.3.1According to 2.3.2Breaking force of the tablets was performed (n = 10) using a hardness tester  as described in 2.3.3iW) were placed into the friability apparatus and rotated at 25 rpm for 4 min. The tablets were then de-dusted and re-weighed  and if any of the tablets were cracked, cleaved or broken, the test was considered as failed. The friability was calculated from Eq. Similarly, the friability test  was carr2.3.4Disintegration tests were conducted using a disintegration apparatus  in accordance with 2.3.5The release rate of cinnarizine from the compressed tablets (n = 3 for each type of tablets) was determined using the paddle apparatus USP type II . The dissolution test was conducted in 900 mL of Milli-Q water as the dissolution medium at 37 ± 0.5 °C at 75 rpm in accordance with 2.43 and the refractive indexes of milk and water were taken to be 1.462 and 1.330, respectively , equipped with a He-Ne laser of wavelength 633 nm and a 300-RF lens for the detection of size ranging from 0.05 to 880 μm. The particle density of the milk fat globules was taken to be 0.92 g/cmectively . Water  fatty acids was determined following the subtraction of volume of NaOH from a blank digestion (digestion buffer only with no added lipids). After an hour of digestion, the pH of the digested milk lipids was increased to pH 9.0 using NaOH (‘back titration’), for which the molar amount of NaOH required corresponds to the amount of unionised fatty acids at the end of digestion. Together with the amount of ionised fatty acids determined earlier, the total amount of fatty acids released during digestion was calculated. Subsequently, the extent of digestion was calculated using Eq. 2.6in vitro digestion apparatus described above was coupled to the SAXS/WAXS beamline as described previously   to detereviously .q range of 0.01 < q < 0.67 Å−1) and 0.6 m (approximate q range of 0.04 < q < 2.00 Å−1) were used to monitor the liquid crystalline structures in milk/infant formula and the intensity of the drug diffraction peaks, respectively. q refers to the length of the scattering vector or momentum transfer and expressed as in Eq. 2 with a pixel size of 172 µm), with a 5 s acquisition time and 15 s delay between each measurement. The phases of the liquid crystalline structures were identified from their characteristic diffraction peaks and the presence of crystalline drug was monitored using the intensity of characteristic diffraction peaks as a function of time. 2D diffraction patterns recorded were converted into a 1D plot of intensity of scattered X-rays against scattering vector (q) using the in-house developed software Scatterbrain. In the 1D profiles, the presence of lamellar, hexagonal and cubic liquid crystalline phases were determined based on the spacings of the diffraction peaks  using Eq. hkl are the Miller indices of each lattice plane.Subsequently, the lattice parameter or physical dimension of unit cells in the crystal lattice, a, were calculated based on the interplanar spacing between the lattice planes . Friability of this value indicates that the tablets are able to endure the rigours of handling in manufacture, transportation, dispensing and usage.3.1.4Initially, three compression forces of 60, 250 and 440 kg were used to produce freeze-dried milk and infant formula tablets. Tablets produced with 250 and 440 kg compression forces displayed disintegration times greater than two hours and thus all subsequent batches of tablets were made with a compression force of 60 kg. As shown in 4,3) of homogenised milk is around 0.56–0.63 μm  and 80.31 μm (infant formula-cinnarizine tablets) on dispersion. In situ monitoring of the lipid liquid crystalline structures formed during the digestion of the dispersed tablets revealed that tableting induced only minor changes in structure formation, where an additional cubic phase was evident on tableting of the freeze-dried milk that was not evident on digestion of the non-tableted milk formulation. This is likely due to compression of the freeze-dried milk matrix but further studies are required to elucidate the processes occurring with the self-assembly of lipids. The lipophilic drug, cinnarizine, was solubilised as a result of the formation of the digestion products from the milk fat. This highlights that dispersible freeze-dried milk/infant formula-drug tablets can be formulated and once redispersed, the lipids digest similar to off-the-shelf milk. Future studies will focus on improving the dispersibility of such tablets and measuring the influence of altering the milk fat content and type on the performance of milk-drug tablets for oral drug delivery.Dispersible milk-drug tablets were formulated using freeze-dried milk or infant formula and their tablet properties such as uniformity of weight, thickness, tensile strength, friability and disintegration time were evaluated. While minor differences in the tensile strength and friability of the freeze-dried milk and infant formula tablets were observed, the most crucial differences were in the disintegration times. Although none of the tablets disintegrated within 3 min as required of a dispersible tablet, the disintegration times were significantly reduced by the inclusion of the PVPP excipient suggesting that further improvements are possible. Tablets containing 30


The amplitudes of these wave packet motions report on the magnitude of the excited-state structural changes, in particular, the formation of a bent and elongated octahedral PbI64− geometry. We have predicted the excited-state geometry and structural changes between the neutral and polaron states using a normal-mode projection method, which supports and rationalizes the experimental results. This study reveals the polaron formation via nuclear dynamics that may be important for efficient charge separation.Hybrid organic–inorganic perovskites have attractive optoelectronic properties including exceptional solar cell performance. The improved properties of perovskites have been attributed to polaronic effects involving stabilization of localized charge character by structural deformations and polarizations. Here we examine the Pb–I structural dynamics leading to polaron formation in methylammonium lead iodide perovskite by transient absorption, time-domain Raman spectroscopy, and density functional theory. Methylammonium lead iodide perovskite exhibits excited-state coherent nuclear wave packets oscillating at ~20, ~43, and ~75 cm Elucidating electron-phonon coupling in hybrid organic-inorganic perovskites may help us to understand the high photovoltaic efficiency. Here, the authors observe low-frequency Raman modes and related nuclear displacements of the Pb–I framework, indicating how these vibrational motions lead to polaron formation in perovskites. These materials are exciting because of their high power conversion efficiency (PCE) as well as their low-cost and facile fabrication11. Their efficiency has reached ~23% which is now comparable with single crystalline silicon-based solar cells12. However, understanding the underlying mechanisms of this high PCE is still a significant challenge.Hybrid organic-inorganic perovskites (HOIP) composed of an organic cation and inorganic framework are one of the most promising new solar cell materials20. This inhomogeneity is surprising because most other inorganic solar cell materials require pure crystalline composition for high PCEs22. Altogether these observations suggest that the inhomogeneity in the polycrystalline HOIP does not greatly influence the performance by protecting free charge carriers from ultrafast thermal relaxation, although these defects do accumulate in humidity-degraded materials25. Associated with the high PCE, polycrystalline HOIP exhibits a long lifetime (τ~1−3 μs) and a long diffusion length (LD~1−3 μm), as well as modest mobility (μ~30−100 cm2 V−1 S−1) for the free charge-carriers28. These characteristics indicate the efficient suppression of unfavorable ultrafast charge-recombination and suggest that unique excited-state dynamics may enable the longer charge-carrier lifetime and their impunity to thermal relaxation29.Polycrystalline HOIP fabricated at low temperatures (~100 °C) has significant crystalline inhomogeneity: defects and grain boundaries typically introduce multiple energy-loss trap sites that may significantly lower the PCE31. In principle, the charge-localized polaron state is energetically unstable without further stabilization. Once it is formed in a higher energy state, it should quickly relax via high energy phonon scattering32. However, polaron formation in HOIP can be favorable; electric dipole reorientation of the nearby organic cations not only minimizes the Gibbs free energy of the polaron but also protects the charges from other ultrafast thermal relaxations according to Zhu and coworkers33. They have successfully measured femtosecond time-resolved fluorescence of the hot electron–polaron in methylammonium lead iodide (MAPbI3) and shown that after a few hundred femtosecond relaxation, which is related to the methylammonium (MA) cation reorientation time, the polaron lives as long as ~100 ps24.The formation of a polaron, a charged quasi-particle, may account for the unique excited-state photo-physical properties in HOIP3, which exhibits not only MA dipole reorientations toward the charge but also the structural distortion of the inorganic Pb–I framework to stabilize the isolated charge34. In particular, the structural distortion associated with a polaron suggests that ground and excited-state potential energy surfaces (PES) have different energy minima along nuclear coordinates that are related to the distortion. Thus, coupled electron–phonon dynamics can be coherently excited on the polaron PES by impulsive excitation, where the associated reorganization energies (λ) and displacements (Δ) determine the polaron formation pathways36. These coherent dynamics have been detected by using femtosecond time-resolved spectroscopies38. For example, Wang et al.39 recently observed a single coherent wave packet oscillating at ~23 cm−1 in a MAPbI3 film using femtosecond transient absorption. However, additional experimental studies of excited-state dynamics in HOIP are necessary to resolve higher frequency vibrations disclosing the structural dynamics of polaron formation.We have recently reported a theoretical prediction on the structure of the small electron polaron of MAPbI3 that oscillate at low frequencies ( < 100 cm−1) as measured by femtosecond impulsive stimulated-Raman spectroscopy (ISRS). We also present first principles density functional theory (DFT) simulations of the structural distortions associated with polaron state. The PbI64− octahedral charge-state geometry changes based on the experimental excited-state Raman displacements and the theoretical simulation are then compared. These results reveal coherent Pb–I vibrational normal mode dynamics that lead to the stabilized polaron state.Herein, we report the production and analysis of coherent nuclear wave packets in MAPbI3 perovskite in the near infrared (NIR) wavelength (830−940 nm) region which is distinct from the ground-state absorption (GSA), stimulated emission (SE) and other bleach bands (see Methods). Fig. We probed the transient excited-state absorption (ESA) band of MAPbI−1. The FFT spectral intensities exhibit a bi-lobed pattern with strong components below 860 nm and above 880 nm but weak amplitudes around 865 nm near the ESA maximum  and linear prediction with singular value decomposition (LPSVD) analyses were performed to quantitate the oscillatory components in the NIR ESA band. The FFT spectra are presented from the residuals extracted from the exponential fittings at each wavelength Fig. , which m−1 modes from the LPSVD results. The most intense wave packet at ~20 cm−1 has the largest phase shift from 150° to 0° which is nearly out of phase as expected. The 45 and 75 cm−1 modes exhibit relative phases of 240/170 and 140/80 on the red/blue sides that are roughly 60–70° out of phase. The deviation from the expected 180° phase differences might result from the somewhat larger errors in these phase determinations than for the 20 cm−1 mode. Especially, the ~180° out of phase features of the strongest ~20 cm−1 mode together with the amplitude pattern in Fig. To examine whether these oscillatory signals are indeed due to band position oscillation of the ESA band we examined the relative phase of these oscillations. In principle, for the band position shift model they should be ~180° out of phase. However, it is sometimes difficult to extract the ideal phases and frequencies when there are highly damped and mixed oscillations with other frequencies. This ambiguity may result in serious overestimation using LPSVD analysis. In order to avoid this error, we cross-checked both the FFT and LPSVD results, as shown in Fig. I4/mcm) phase of MAPbI3 were performed using periodic boundary conditions (see Methods)42. Figure −1 modes of ground state MAPbI3 perovskite energetically lying in the region of interest. These vibrational modes are mostly composed of I−Pb−I bond bending and stretching motions unlike other higher frequency Raman modes above ~100 cm−1 which involve MA motion42. Figure −1 which can be described as a B1g mode in I4/mcm) space group, while Fig. −1  due to the shallow potential energy surface and the harmonic approximation used for analysis. For example, a recent study compared IR absorption and DFT results showing ~20% deviations in < 100 cm−1 region43. Thus, to reduce the discrepancy, we previously considered several additional effects that need to be taken into account to properly compare calculations with experiments which improved the reliability of our assignments42.To identify the vibrational modes associated with the observed wave packets, DFT calculations of vibrational normal modes in the room temperature tetragonal . Specifically, we obtained geometries of a MAPbI3 neutral cluster and a cluster with an extra electron mimicking negative polaron generation. The computationally determined ground state structure was previously verified by XRD experiments, and is therefore more reliable compared to the geometry of charged states34. Figure 3 perovskite. In the polaron structure, the methylammonium counterions reorient their electric dipoles (indicated by red arrows) toward the center Pb atom to minimize the free energy of the localized electron density in the center Pb atom34. In addition, the Pb–I bonds, especially in the central PbI64− octahedra, also experience substantial structural changes, when an electron is added. Figure 64− octahedron structure of the neutral  and the polaron . The most unique structural difference between Sneu and Spol is the octahedron height along z-axis; the polaron PbI64− has a Pb–I(5) distance of 4.2 Å which is considerable longer than 3.3 Å for the neutral one. In addition, the neutral PbI64− has a nearly right angle (89°) for the I(5)–Pb–I(4) bond, while in the polaron the bond angle is 84°. Thus, the polaron has a distorted and bent octahedral structure, while the neutral geometry forms a nearly regular octahedron. These differences are indicated by the displacement vectors (green arrows) in Fig. To theoretically simulate the structural changes of excited PbIThe structural differences predicted for the excited polaron state geometry should be related to the vibrational relaxation toward the charge state minimum that occurs after Franck–Condon (FC) excitation of the neutral form. Thus, the measured vibrational modes oscillating in the ESA should be the modes that trigger the nuclear reorganization that forms the excited polaron state. In order to test this hypothesis, we quantitatively investigated the relation between the experimental excited-state vibrational oscillation intensities Figs. –3 and thσR) are approximately proportional to ∆2ω2 in the short-time limit, where ∆ and ω are the ground-to-excited state displacement of the potential minima and the vibration frequency, respectively46, the relative displacement ratio for each Raman peak can be determined. Using this relationship, the relative displacement ratio (∆Exp) is found to be ±4.3, ±1.4, and ±1.0 for the ~20, ~45, and ~75 cm−1 modes in the impulsive stimulated Raman spectra (ISRS) in Fig. ∆2 relationship. The ∆Exp result shows that the I–Pb–I bending motion at ~20 cm−1 is the most highly displaced vibrational normal mode, and thus, the most responsible for the excited-state PES deformation that we have probed in the TA. The other bending and stretching modes provide lower displacement contributions to the excited-state PES. Thus, using the ∆Exp, we can experimentally estimate a polaron state structure (SExp) given byp is a proportional factor and Spol for excited state structure. Figure Sneu (red), Spol (blue), and SExp (green) with p = 1.6 from the best nonlinear square fit toward Spol (SExp move toward the blue (Spol) from the red (Sneu) locations, thus affirming the successful simulation of the theoretically observed polaron state structure using the relative displacement ratio, ∆Exp.The key part of our analysis is relating the oscillatory signals in the ESA in Figs. Sneu to Spol by relying solely on theoretical predictions47. The theoretical displacement (∆Theo) is given byl is a diagonal matrix containing ith vibration frequency. ΔTheo are determined to be +2.8, +0.3, and −5.1 for the 20, 45, and 75 cm−1 vibration modes, respectively. In addition, the excited-state reorganization energy (λ) is calculated to be 145 meV (~1170 cm−1) given by h is Planck constant. This value is very consistent with the band gap stabilization energy (~160 meV) found in a periodic boundary condition (PBC) DFT calculation34, indicating that our observed vibration modes represent the majority of polaron stabilization dynamics.A complementary approach for analyzing these geometric changes is to perform a normal mode projection from the Exp and ∆Theo results are in good qualitative agreement; both experimental and theoretical displacements show that the 20 cm−1 displacement is larger than the 45 cm−1 displacement, both confirming that the whole octahedral I–Pb–I bending motion at ~20 cm−1 provides strong displacements toward the polaron state. However, the displacement −5.1 of 75 cm−1 mode of ΔTheo is larger than the experimentally observed ΔExp at 75 cm−1. This inconsistency principally results from the different heights of Spol (4.2 Å) and SExp (3.6 Å) which result from an overestimation of Spol due to the use of limited atom numbers in the calculation and an underestimation of Sexp due to the limited time-resolution of the experiment which damps the oscillatory signal strength for the higher frequency mode. However, both our methods  of MAPbI3 produces the coherences as determined in the current study, ultimately leading to the separation of an initial excitation into free charges in view of weak excitonic effects in MAPbI349. The observed dynamic vibrational excitations facilitate this relaxation toward different structures, where a localized charge is further stabilized through the structural deformation of the Pb–I framework  of the solvated MA was measured to be ~150 fs by using optical Kerr-gate spectroscopy and ultrafast IR spectroscopy; this time is most relevant for ultrafast coherent solvation in solutions50. In this context, we have found small FFT peaks at ~150 cm−1  solvent-like movement stabilizing the localized polaron50, while the measured Pb–I vibrations are coherent processes which trigger the Jahn–Teller distortion.Our nuclear wave packet results and theoretical simulations on MAPbIand gap ~.65 eV of51 and the transition is facilitated by Pb–I structural distortions shown as the bending and elongations of the Pb–I bonds mirrored in our theoretical studies  should be the principal nuclear distortion motion for polaron formation, in good agreement with the strong electron–phonon coupling at 0.65 THz (22 cm−1) probed by femtosecond terahertz emission51 and the light-induced I–Pb–I angle displacement measured by electron scattering52.Pb and I atoms are covalently bonded to form the sturdy perovskite framework. However, once more charge character is introduced , this Pb–I framework becomes more ionicies Fig. . Our keySneu and Spol, respectively. The geometry changes are determined predominantly by an I–Pb–I bending normal mode at 21 cm−1 , the displacements cause the excited-state nuclear wave packets to evolve along these multi-dimensional I–Pb–I vibration coordinates coherently propagating excited-state population toward the polaron state minima Spol in the first few picoseconds. Thus, we suggest that coherent polaron formation dynamics is linked to the efficient charge-separation of MAPbI3 perovskite by directing the phase–space evolution to the polaron state faster than competing relaxation mechanisms. Other reports have suggested the subsequent dynamics of charge carriers as large polarons, where limited charge–carrier mobility was linked to coupling to LO phonon branches with frequency of 88 cm−154.Figure m−1 Fig.  and an Im−1 Fig. . After e3 and found evidence for coherent skeletal vibration dynamics that lead to formation of a polaron state underscoring charge separation. The low frequency wave packet modes are observed principally for the Pb–I bending and stretching vibrations resulting from the different geometry of the polaron state compared to the neutral state. We analyzed the experimental and theoretical displacements and confirmed that the theoretical negative polaron state structure is supported by the experimental Raman intensity analysis results. Thus, the distorted octahedral geometry of the PbI64−polaron state structure is characterized, and its initial formation dynamics are demonstrated to be dominated by the coherent nuclear wave packet motion. The high efficiency of MAPbI3 perovskite solar cells may be yet another example of the importance of vibrational coherence in efficient photochemical dynamics57.In summary, we performed femtosecond TA and DFT simulations to probe the excited-state dynamics of MAPbI2  and 2.28 mmol CH3NH3I (Dyesol Inc.) in 1 mL anhydrous dimethylformamide , and stirred overnight. The prepared solution was spun on O2-plasma treated glass substrates at 1500 rpm for 60 s, and then annealed at 120 °C for 60 min in a N2-filled glovebox. Poly(methyl methacrylate) (PMMA) was coated on the fresh MAPbI3 layer to protect it from the atmosphere. We have checked powder X-ray diffraction (XRD) measured by using a Bruker AXS D8 Advance diffractometer with a Cu Kα source. , a laser-synchronized CCD , and an optical chopper  were used to measure the optical density (OD) differences in every pump/probe cycle  seeded a Ti:sapphire regenerative amplifier  pumped by a Q-switched Nd:YLF . The fundamental at 790 nm was used to generate visible pump pule  by a non-collinear optical parametric amplifier (NOPA). A negatively chirped mirror pair (Layertec GmbH) was used to compress the pump pulse. A small portion of the fundamental is focused onto a 3 mm thick sapphire plate to generate a broadband continuum probe pulse . The probe pulse was temporally compressed in the 830–940 nm region with a pair of BK7 dispersion prisms 59, and projected augmented-wave pseudopotentials61. Structural and electronic properties were computed using a plane-wave energy cut-off of 500 eV and a 4 × 4 × 4 Monkhorst–Pack k-point grid62, including spin–orbit interactions (SOI). Lattice constants and internal atomic positions were relaxed starting from the experimental structure, without symmetry constrains, until forces were smaller than 1 meV/Å. Phonon eigenfrequencies and eigenvectors were calculated in the harmonic approximation, neglecting SOI. Our phonon calculations use density functional perturbation theory and a finer 6 × 6 × 6 k-point grid. Selected phonon modes and electronic orbitals were plotted using VESTA. The room temperature tetragonal (I4/mcm) phase of (CH3NH3)PbI3 is simulated in a 6 octahedral rotation around the z-axis (a0a0c− in Glazer notation) and an antiparallel orientation of the CH3NH3 (MA) molecules64.First-principles density functional theory (DFT) calculations were performed using VASP65. In addition, we include a polar solvent (ε = 78) via the conductor-like polarizable continuum model66.All calculations of HOIP isolated clusters were performed using the CAM-B3LYP functional combined with the LANL2dz (for Pb and I) and 6-31 G*  basis sets using Gaussian 09 software package67. In order to minimize dangling bonds, the cluster was terminated on all sides with MAI. Equations I1 corresponds to an atom on the surface and is only bonded to one Pb atom, while I2 corresponds to an I atom that is bonded to two Pb atoms. The cluster needs to be large enough to include at least 8 MA cations surrounding the central Pb atom. To fulfill all conditions, we end up with a cluster having the following stoichiometry: MA54Pb27I108 (I1=I2=54). A cube fully terminated by MA and I would require 64 MA molecules. In order to realize charge balance between the ions and maximize symmetry, MA’s were removed from the 8 corners and on two opposite edges.Several considerations were taken into account when designing the isolated HOIP structures. We started with the low temperature cubic lattice parameters that were derived from variable temperature powder x-ray diffraction experimentsc-axis elongation) still holds although the amplitude changes slightly.In addition, the polaron geometry in HSE hybrid potential was checked. Supplementary Fig. Δ) between the ground and excited states in MAPbI3, we considered Rashba band splitting and polaron formation for the reason of the excited-state displacements. The former one is related to IR-active vibration modes, because the splitting is promoted with directional structural strains. Meanwhile the latter, the polaronic PbI64− is rather induced by symmetric structural changes from the neutral PbI64− of the ground state, because a negative charge (−1) is localized in 6p orbital of the center Pb. Thus, this polaronic structure shows the Pb-centered symmetric distortion shown in Fig. 64− Raman modes in the D4h Raman mode pool43. Thus, the Pb-centered Raman modes are easily discriminated from other IR-active and Pb-moving Raman modes (Supplementary Fig. Since we measured the excited-state Raman signals originated from the structural displacements (All data supporting the findings of this study are available from the corresponding author upon reasonable request.Supplementary Information


Focal Adhesion Kinase (FAK) is a major cancer drug target that is involved in numerous aspects of tumor progression and survival. While multiple research groups have developed ATP-competitive small molecule inhibitors that target the kinase enzyme, recent attention has been focused on the FAK FERM  domain that contains key residue Y397 and contributes to many protein-protein interactions. Previous x-ray crystal structures of the FAK FERM domain gave conflicting results on the structure of the Y397 region and therefore the overall druggability.Here, we report the identification of a higher resolution crystal structure of the avian FAK FERM domain that shows conformational differences in Y397 and surrounding residues in the F1 lobe. In addition, we resolve the residues of the Src SH3 binding site, an area of the FERM domain that has previously shown limited electron density.These crystallographic data suggest that the Y397 region is highly dynamic and question the druggability of a putative pocket on the F1 lobe. In addition, new electron density data around the Src SH3 binding site provide structural insight on the FAK-Src activation cascade through a putative auto-inhibitory conformation. Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor tyrosine kinase that has multiple implications in cancer cell signaling, invasion, proliferation, and metastasis –5. ActivAs an alternative approach to target FAK, several groups have attempted targeting the FAK FERM domain at a binding site proximal to key residue Y397 as a means to target the FAK activation site directly and inhibit compensatory FAK phosphorylation via transphosphorylation –24. Howe363QKEGERALPSIPK375) that may have implications in FAK activation via Src. Finally, our crystal structure confirms a previous report identifying the site of dimerization (W266) between two FERM monomers [Here, we report an x-ray crystal structure of the avian FAK FERM domain at a higher resolution (1.97 Å) than previously reported , elucidaization W6 betweenwork of 17.3 and Rfree of 21.2, is more ordered than that of the search model [2 and 77 Å2, respectively. These values are relatively close to an average B factor value for all protein atoms of the model is  of the avian FAK was solved to a resolution of 1.97 Å by molecular replacement method, using the structure of the FAK FERM (PDB code 2AL6) as search model  and homology (99%) between human and avian sequences  to previously published crystal structures at 2.35 Å (PDB 2AL6) and 2.8 Å (PDB 4NY0). We first started with sequence alignment, structural superposition, and RMSD calculation using PyMOL software  whereas similar analysis of our 1.97 Å structure resulted in the inability to predict any druggable site near Y397  and therefore have found a previously unidentified conformation of this area. As shown in Fig. The previous crystal structure of the FAK FERM domain 4NY0) did not have adequate electron density for the critical Src SH3 binding motif (residues 363–375) to be visible. In our structure we have better resolution of this region . An explanation for the disconnect between biological activity and structural validation of FAK FERM inhibitors could be that FAK FERM x-ray crystallography studies do not correctly sample the pharmacologically-active conformation required for small molecule binding. In addition, putative FAK FERM inhibitors may be indirectly acting on the FAK pathway through direct binding of an upstream molecule such as a transmembrane receptor. As such, our data bring concerns to the druggability of the Y397 region and suggest that structural confirmation of binding and alternative drug discovery/structural biology strategies  rather than a sole computational approach based on x-ray should be employed to find small molecule inhibitors of the Y397 region.It is important to note that although previous FERM Y397 drug discovery efforts have identified candidate molecules with interesting cell-based data –24, ther363QKEGERALPSIPK375), which was not found in previous FAK FERM structures [Another important finding of our FAK FERM structure was the visibility of the Src SH3 binding site . Lysate was subjected to one freeze-thaw cycle followed by sonication for 2 min on ice. Supernatant was collected after centrifugation at 12,000 rpm for 15 min and incubated with 3 mL Ni-NTA resin (Thermo) overnight. Resin was then washed with lysis buffer containing 25 mM imidazole and protein was eluted with lysis buffer containing 200 mM imidazole.Avian FERM domain protein (residues 31–405) was expressed and purified similarly as previously reported . The pETThe His-tag from the purified FERM-domain sample was removed by AcTEV-protease according to the manufacture protocol (Invitrogen). Briefly, 25 units of AcTEV-protease sample was enough to complete the cleavage of the His-tag from 1 mg of the protein during overnight incubation at 40 C. The cleaved product was removed by filtration of the sample through Ni-NTA Superflow cartridge  pre-equilibrated in 20 mM Tris-HCL, pH 8.0, 100 mM NaCl, 1 mM DTT and 20 mM imidazole. The filtrated sample was loaded on a Q-Sepharose column  equilibrated in the same buffer except the imidazole and the protein was eluted using a linear gradient of NaCl from 0.1 to 1 M. The fractions of the His-off protein were pooled and applied on a Superdex 200 column  for further protein purification. The FERM–domain sample eluted from the gel filtration column was 95% pure as assessed by SDS-PAGE analysis and used for the crystallization experiments.Sitting drops in 96-round bottom well crystallization plates  were set up using a Mosquito Robotic System . Avian FAK FERM domain (residues 31–405) protein sample at concentration of 10 mg/ml in 10 mM Tris-HCl buffer, pH 8.0, 50 mM NaCl and 1 mM DTT was used for the crystallization experiments.Diffraction quality FERM protein crystals were obtained at 291 K from drops containing 0.5 μl of the protein sample and 0.5 μl of reservoir solution . Diffraction data were collected at 100 K from a single, flash frozen, crystal , cryoproThe PyMOL molecular graphics program  was utilized for general structural representation of the FERM domain and publication-quality images were made using the ray-trace command. PyMOL was also used for sequence alignment and structural superposition using the align command. Multiple sequence alignment was performed using the Schrödinger Multiple Sequence Viewer application. For druggability prediction analysis of our crystal structure compared to previously published structures, we used the program SiteMap  as described . SiteMap


Despite the significant recent achievements in the diagnosis and treatment of colorectal cancer (CRC), the prognosis of these patients has currently plateaued. During the past few years, the opportunity to consider multiple treatment modalities  led to the research of novel prognostic and predictive biomarkers in CRC liver metastases (CRCLM) patients. In this review, we seek to describe the current state of knowledge of CRCLM biomarkers and to outline impending clinical perspectives, in particular focusing on the cutting-edge tools available for their characterization. Colorectal cancer (CRC) is the third most common cancer in men and the second in women across all regions of the world, with approximately 1.3 million newly diagnosed cases every year ,3. This Given its extremely abundant blood supply from the colon and rectum, the liver is the dominant metastatic site for patients with CRC . Hence, During the past few years, the opportunity to consider multiple treatment modalities  lead to the research of novel prognostic and predictive biomarkers in CRCLM patients. In this review, we seek to describe the current state of knowledge of CRCLM biomarkers and to outline impending clinical perspectives, in particular focusing on the cutting-edge tools available for their characterization.TP53 and CDKN1A), Ki67 proliferation index, and markers of angiogenesis  have been found to predict tumor recurrence and survival, as confirmed by the preliminary data of ongoing clinical trials  OR predictive[All Fields]) AND (“biomarkers”[MeSH Terms] OR “biomarkers”[All Fields])) AND )) AND ((“liver”[MeSH Terms] OR “liver”[All Fields]) AND (“neoplasm metastasis”[MeSH Terms] OR “neoplasm metastases”[All Fields] OR (“neoplasm”[All Fields] AND (“metastasis”[All Fields] OR “metastases”[All Fields])) OR “neoplasm metastasis”[All Fields] OR “metastases”[All Fields] OR “metastasis”[All Fields])) AND (“2008/07/30”[PDat]: “2018/07/27”[PDat] AND English[lang]).A total of 394 articles were identified using these criteria.A primary screening of the 394 studies was carried out in order to exclude irrelevant articles. Exclusion criteria included: pure morphological studies and studies without prognostic and/or predictive significance. A total of 227 articles endured this initial screening.Out of the 227 remaining articles, 117 were excluded due to lack of relevant data. In the end, 110 articles were included in this review.