Due to the large Coulomb repulsion of 4f electrons, the bandwidth-control Mott-type delocalization, commonly observed in d-electron systems, is difficult in 4f-electron systems and remains elusive in spectroscopic experiments. Here we demonstrate that the bandwidth-control orbital-selective delocalization of 4f electrons can be realized in epitaxial Ce films by thermal annealing, which results in a metastable surface phase with reduced layer spacing. The quasiparticle bands exhibit large dispersion with exclusive 4f character near f-electron systems.The 4 The mechanism of the delocalization transition of 4f electrons in closely-packed Ce metal has been debated. Here, the authors present photoemission evidence for bandwidth-controlled Mott delocalization in a previously unreported structural phase of thin epitaxial Ce films obtained by thermal annealing. Key to understand these exotic phases lies at unraveling the peculiar nature of the 4f-electron delocalization, which is often described by the periodic Anderson model (PAM) composed of localized 4f electrons and dispersive conduction bands. The 4f electrons can be delocalized by the many-body Kondo effect, giving rise to an effective hybridization with conduction electrons and a flat quasiparticle band near the Fermi energy (EF). Such a hybridized band picture is often employed to describe realistic 4f-based materials8. By contrast, in transition metal systems, d electrons typically have appreciable dispersions due to large intersite hopping and their localization/delocalization follows the well-known Mott mechanism as described in the single or multi-band Hubbard model9. It is therefore intriguing to think if in 4f-based materials, the Mott physics from 4f electrons may exist and cooperate with Kondo hybridization to cause correlated phenomena beyond the standard theoretical framework.Rare earth metals and intermetallics are prototypical strongly correlated electron systems and host a variety of interesting quantum phases, including heavy fermion, quantum criticality, unconventional superconductivity, etcf bandwidth is often negligible compared to the onsite Coulomb repulsion U, direct experimental evidence for the Mott-type delocalization is very rare (if any) for 4f electrons. The most notable 4f-electron system where the bandwidth-control orbital-selective delocalization (OSD) might occur is the pure Ce metal, where the Ce atoms are ordered in closed-packed structures with smallest possible f–f distance10. In fact, the 4f-electron Mott delocalization was proposed early on to explain the famous first-order γ–α transition of Ce14, but an alternative Kondo volume collapse (KVC) scenario was also proposed, attributing the γ–α transition to a large increase in the hybridization strength between conduction and 4f electrons (c–f hybridization)18. Some theoretical studies further pointed to the importance of the interplay between two mechanisms23. While previous photoemission studies provided compelling evidences that the Kondo effect plays an important role in the electronic structure of Ce30, no spectroscopic evidence supporting the Mott scenario has ever been reported. Unambiguous identification of the bandwidth-control 4f delocalization is experimentally challenging, as the coexisting Kondo screening could lead to somewhat similar itinerant 4f bands. Nevertheless, there are clear differences in their 4f quasiparticles: in the bandwidth-control Mott scenario, 4f quasiparticle bands can exhibit large dispersion with nearly pure 4f character, while those in the Kondo picture can acquire dispersion only through hybridization with nearby conduction bands, leading to mixed orbital characters. Here in this paper, we present direct spectroscopic evidence for the bandwidth-control OSD of 4f electrons in epitaxial Ce films, by combining high-quality thin film growth by molecular beam epitaxy (MBE) and in situ measurements from angle-resolved photoemission spectroscopy (ARPES). In particular, we uncovered a hitherto unreported metastable phase of Ce, where sharp dispersive quasiparticle bands with pure 4f character can be observed near EF, accompanied by an appreciable dispersion of the lower Hubbard band. Such spectral character, which cannot be explained by the Kondo hybridization picture, can be well accounted for by the bandwidth-control Mott physics.Unfortunately, since the 426: the lower Hubbard band (or 4f0) at −2.2 eV, weak EF and −0.25 eV, respectively. The broad peak at −0.9 eV is likely due to the unbinding 5d electrons. The Kondo resonance peaks near EF do not show any momentum dependence , implying that these resonance states remain localized at each individual Ce site.The single-crystal Ce films are grown by MBE on epitaxial graphene layers and in situ ARPES measurements were performed immediately after film growth (see “Methods”). Figure 31. Compared to the P0 phase, the lower Hubbard band shifts to slightly lower energy (−1.9 eV) and remains dispersionless, and the Kondo resonance peaks near EF become sharper and more pronounced. The quasiparticle bands near c–f hybridization expected from the hybridized band picture within PAM (see below)33.Upon annealing to intermediate temperature, an ordered single-crystal Ce film can be obtained (P1), as determined by streaky diffraction pattern in RHEED and clear dispersive conduction bands in the ARPES spectra. This phase was observed in previous ARPES studies and identified as the γ-phaseentclass1pt{minimaf electrons is suddenly turned on . This is a direct manifestation of the bandwidth-control delocalization of 4f electrons expected from the Mott–Hubbard model. Since the 4f spectral function still consists of two parts similar to P1, i.e., a lower Hubbard band that remains far from EF and the quasiparticle bands that lie very close to EF, the P2 phase is not in a strongly mixed-valent state.Further annealing to higher temperature leads to the P2 phase, which has not been reported before. It maintains a highly ordered structure with similar in-plane lattice constant as P1. Now the lower Hubbard band at ~−1.8 eV develops clear dispersion   can reproduce the experimental results well , which are shown in Fig. f bands for these two phases: the 4f band in P1 is very flat and shows only slight bending upon crossing the conduction bands, as expected for a Kondo resonance; by contrast, the 4f bands in P2 show obviously larger dispersion with very fine structures. Surprisingly, the observed 4f bands for the P2 phase can be very well described by “itinerant 4f“ calculations from DFT  leads to dramatic change in the Fermi surfaces (FSs), as shown in Fig. f character centered at the −1 due to the flatness of heavy quasiparticle bands. The FS also has elliptical pockets centered at the f bands at the f electrons participate the FS: one forms heavy bands near EF through the c–f hybridization, while the other develops coherent band-like quasiparticles well explained by DFT. Away from EF, the constant energy contours for the P1 phase are dominated by conduction bands, showing a hole pocket centered at EF contours for the P2 phase are similar to P1, except that the pockets at f band at ~−0.2 eV gives rise to an additional strong pocket at zone center. This again demonstrates that the quasiparticle bands in the Mott mechanism span a larger energy window compared to those from the Kondo physics, which is typically constrained near EF.The transition from the Kondo regime (P1) to the 4 regime P leads to regime P leads to34, which can be dependent on the annealing conditions and therefore account for the observed P1–P2 transition. However, our in-plane FS maps in Fig. 34. We mention that the similar suppression of the γ–α transition in epitaxial Ce films has also been reported35, although the percentage of the α-phase at low temperature differs due to different growth conditions.Now we address the physical origin of the P1–P2 transition. We first emphasize that this transition is caused by different annealing temperatures of Ce films, as the ARPES measurements in Figs. ∘ and ~65∘ emerge and persist in the entire temperature range  to overcome the local kinematic barrier, possibly resulting in reduced interlayer spacing38. To check if a metastable surface structure with reduced layer spacing could indeed be possible in Ce films, we performed structural relaxation calculations of a freestanding γ-phase slab  to the bandwidth-control delocalized state (P2), as illustrated in Fig. f calculation, which uses the experimental structural parameters, yields good agreement with the experimental data for the P2 phase  forms near the surface upon annealing, leading to the observed P1–P2 transition. A ~3.5% reduction in the layer spacing, considered to be large for strongly correlated 4se Figs. b, c. In EF gradually diminishes with increasing temperature , the Ce valence is always close to 3+ and shows negligible temperature dependence46. It is hence attempted to associate the observed shift with the more itinerant character of 4f electrons and their hybridization with conduction bands. We emphasize that the change is nonetheless tiny and the lower Hubbard band in the P2 phase remains sufficiently far from EF, implying that the system is unlikely in a strongly mixed-valent state.The temperature-dependent band shift in the P2 phase, absent in P1, could be a manifestation of the interplay between the Mott and Kondo physics. Detailed analysis shows that the conduction bands exhibit a slight downward shift with decreasing temperatures , although the dispersion of quasiparticle bands near EF was not available48.Our results show that the bandwidth-control delocalization from the Mott mechanism is indeed possible for 4ase Fig. c, althouase Fig. b, c, indf electrons observed here bears some analogy to the orbital-selective Mott transition (OSMT) proposed in multi-orbital ruthenates51. Nevertheless, there are obvious differences: (1) There is much stronger tendency towards localization for 4f electrons compared to 4d electrons . (2) The bands involved in OSMT in ruthenates, i.e., dxy and dxz/dyz orbitals, do not hybridize due to different wavefunction symmetries, but the 4f and conduction electrons in Ce exhibit clear hybridization, i.e., Kondo hybridization, which very likely cooperates with the bandwidth-control Mott physics.The OSD of 4f Mott physics.The bandwidth-control OSD in Ce is most likely caused by reduced layer spacing near the surface, supported by our ex situ XRD measurements and DFT calculations, although more in situ study, e.g., from surface X-ray scattering, is still needed to understand the detailed structure. Theoretically, the reduced layer spacing at the surface reduces the crystal symmetry (from fcc to hcp), which in turn changes the CEF splitting and broadening. The altered CEF states could further affect the Kondo screening and possibly the bandwidth-control delocalization. More studies are needed in the future to resolve the fine structures in the quasiparticle bands associated with the CEF states. Finally, the possibility of stabilizing such surface phase in appreciable volume (detectable by bulk-sensitive XRD and transport measurements) offers exciting opportunities in the future to study the physical properties associated with the 4f electrons, which was studied extensively by theory but lacks spectroscopic proof. The bandwidth-control delocalization and Kondo effect conspires to give rise to an intriguing electronic phase with coherent 4f quasiparticle bands, which agree well with itinerant 4f calculation and exhibit unusual temperature dependence. Our results demonstrate that the Kondo and Mott mechanisms can coexist (and even act cooperatively), in some Kondo systems with small f–f distance, which could be important for a fundamental understanding of 4f-electron systems. Our observation in Ce thin films may also help clarify the mechanism of the γ–α transition in bulk Ce.To summarize, by combining MBE growth and in situ ARPES measurements, we were able to track the evolution of the electronic structure of epitaxial Ce films under different growth conditions and measurement temperatures. The electronic structure with sequential annealings undergoes transition from the P0 phase (described by SIAM) to the P1 phase (described by the hybridized band approach within PAM), and finally to the P2 phase (described by the Mott physics with coexisting Kondo process). Our work provides direct evidence for the bandwidth-control OSD of 4−11 mbar. The deposition was controlled with a high-temperature effusion cell to yield a rate of approximately 2 Å/min, as determined by a quartz crystal monitor mounted near the sample. Highly doped 6H-SiC(0001) substrates were used to obtain epitaxial graphene layers by heating up to ~1400 ∘C in ultrahigh vacuum. The Ce films were grown on top of these epitaxial graphene layers at a temperature of ~100 ∘C. After the deposition, the films were annealed sequentially (from ~370 up to ~540 K) to achieve different electronic phases as shown in Fig. −11 mbar, which increased to ~2.3 × 10−10 mbar during the Helium lamp operation (beam size ~1 × 1 mm2). All the ARPES data were taken with He-II photons , where the photoemission cross section for the 4f electrons is appreciable. Due to the low counts from He-II photons, we used an analyzer pass energy of 10 eV, which yielded an overall energy (momentum) resolution of ~15 meV (~0.01 Å−1). The RC-FDD, used for recovering the spectral function near EF in Figs. Thick Ce films  were grown by MBE in a growth chamber with a pressure <5 × 10f calculations, the Ce-4f electrons are included as the valence states; while for the localized 4f calculations, the Ce-4f are treated as core electrons, similar to the open-core treatment in full-potential linearized augmented plane-wave calculations. These two calculations are often carried out in Ce-based systems to represent two limiting situations of Ce-4f electrons , see for example ref. 52. An energy cutoff of 437 eV (300 eV) for itinerant  4f calculations was employed, with the k mesh being 17 × 17 × 17(16 × 16 × 4) for the fcc (hcp) phase. The localized 4f calculation for P1 adopted the bulk-like γ structure. For the P2 phase, we found that itinerant 4f calculations within DFT yield very good agreement with the ARPES data. This is perhaps not too surprising as dynamic mean-field theory calculations show reasonably good agreement with DFT calculations in the itinerant 4f-electron regime, after considering a renormalization factor54. The itinerant 4f calculations for P2 used the hcp structure with reduced interlayer spacing determined from ex situ XRD  and ABAC-stacking  hcp phases are performed and they yield overall similar results . For the itinerant 4 17 × 171 × 16 × 4Ex situ XRD and transport measurements were performed for very thick films , after the films were characterized by in situ ARPES measurements. To minimize sample oxidation, the XRD and transport measurements were done immediately after they were taken out of the vacuum chamber.Supplementary InformationPeer Review File


Capsicum was fine-mapped to a region of 0.4 Mbp, and a multidisciplinary approach has been used to study putative underlying mechanisms.A major thrips resistance QTL in Capsicum identified diterpene glycosides as metabolites that are correlated with thrips resistance. In this study, we fine-mapped a previously defined thrips resistance QTL on chromosome 6, to a region of 0.4 Mbp harbouring 15 genes. Two of these 15 candidate genes showed differences in gene expression upon thrips induction, when comparing plants carrying the resistance allele in homozygous state to plants with the susceptibility allele in homozygous state for the QTL region. Three genes, including the two genes that showed difference in gene expression, contained a SNP that was predicted to lead to changes in protein structure. Therefore, these three genes, i.e. an acid phosphatase 1 (APS1), an organic cation/carnitine transporter 7 (OCT7) and an uncharacterized locus LOC107874801, are the most likely candidates for playing a role in thrips resistance and are a first step in elucidating the genetic basis of thrips resistance in Capsicum. In addition, we show that the diterpene glycoside profiles did not differ between plants with the resistance and susceptibility allele for the chromosome 6 QTL, suggesting that these compounds do not play a role in the resistance conferred by the genes located in the major thrips resistance QTL studied.Resistance to thrips is an important trait for pepper growers. These insects can cause extensive damage to fruits, flowers and leaves on field and greenhouse grown plants worldwide. Two independent studies in The online version of this article (10.1007/s00122-021-03790-6). Orius spp. (Hemiptera: Anthocoridae) can limit thrips population growth, but predator pressure should remain high, as thrips population growth will occur when the predator population is too small  . However, in all these cases further fine mapping of the QTLs is still needed to identify the gene or genes that are involved in thrips resistance.Screening wild accessions for thrips resistance and subsequent quantitative trait loci (QTL) mapping of the resistance factor(s) are the first steps to identify candidate genes that play a role in thrips resistance and in developing thrips-resistant varieties. Previous studies have identified QTLs involved in resistance to Senecio plants. In thrips-resistant chrysanthemum, high concentrations of chlorogenic acid (caffeoyl quinic acid) and feruloyl quinic acid were found  in thrips resistance in Capsicum.Maharijaya et al.  combinedCapsicum, and to determine if diterpene glycosides can be linked to the resistance mechanism. We used plants that originated from the F2 mapping population that was developed by Maharijaya et al.  and CGN17219  were included as resistant and susceptible references, respectively. Seeds were sown in potting compost in a greenhouse of Unifarm . Plants were grown with a photoperiod of L16:D8 and 70% RH at 25 °C. No insecticides were applied. Thrips were controlled using the Orius laevigatus (Fieber) . Plants were watered three times a week. Nutrients were added two times a week.For fine mapping of the resistance QTL, F3 plant for the fine-mapped QTL region on chromosome 6 (M11 to M15) was selfed to obtain F4 plants with the “R” or “S” allele in homozygous state  at 25 Hz for 70 s. Microprep buffer was prepared as described by Fulton et al. , and 5001 that resulted from a cross between the parents of the F2-mapping population. RNA extracted from the leaves of the F1 plant was sequenced by BGI Genomics. SOAPdenovo  discovery was carried out on RNAseq data of the FCapsicum annuum CGN16975 (M13.4). DNA was extracted from CGN16975A and sent to Novogene Technology Co., Ltd (Hong Kong) for library preparation and sequencing. Reads were mapped to the Capsicum annuum Zunla-1 assembly . Additional markers in the area of interest  were tested with LightScanner® System (Idaho Technology Inc.) using the small amplicon approach  with two SNP markers , flanking the 2-LOD interval of the QTL region as determined by Maharijaya et al. , using KFrankliniella occidentalis originated from Greenhouse Horticulture of Wageningen University and Research . Frankliniella occidentalis was reared in a growth cabinet at 25 °C, L16:D8, 70% relative humidity, on Phaseolus vulgaris beans in glass jars covered with thrips-proof gauze. Synchronized first instar larvae (L1) were obtained by allowing female adult thrips to lay eggs on snack cucumbers. After 24 h, the female adults were brushed off and the cucumber was kept in the growth cabinet at 25 °C. Synchronized L1s emerged after 4 days.The population of 3 plant  it was expected that the resistance mechanism is activated in an early stage of L1 to L2 development, and (2) several differentially expressed genes upon thrips infestation are still strongly expressed after 5 h  or susceptibility (“S”) allele in homozygous state between M11 and M15 were selected for RNA sequencing in two different treatments . Fifteen plants with genotype R and fifteen plants with genotype S were randomized in the greenhouse. Leaves of the plants were collected and either infested or mock-treated, as described in the previous paragraph. Three pools of leaves from five plants with the same QTL genotype (“R” or “S”) and treatment (induced or mock) were ground to a fine powder with mortar and pestle in liquid nitrogen. Total RNA was extracted from the pooled samples using RNAeasy Plus Mini Kit (QIAGEN) and sent to Novogene Technology Co., LTD (Hong Kong), for library preparation and sequencing. STAR . The analysis of differentially expressed genes was done using SARTools in R-3.5.2 , consisting of mixed powder from different samples, were included. One of the pooled samples of group R, mock-treated, dropped out due to a technical problem of the LC–MS. Metabolites were extracted with 900 µL 99.87% MeOH containing 0.13% formic acid. LC–MS was performed using an HPLC system (Dionex Ultimate) coupled to an Q Exactive Orbitrap FTMS mass spectrometer (Thermo Scientific) using a Phenomenex Luna C18 column and a gradient of 5–75% acetonitrile in 0.1% formic acid in 45 min  before analysing the data. A Student’s t test was used to determine whether the transformed fractions L1 significantly differed between plants with the “R” or “S” allele within the same F3 line to validate the previously identified QTL region. Student’s t test was also used to determine significant differences in resistance between plants that had the recombination in homozygous state and plants with the “R” or “S” allele within each F4 or F5 line. All statistical analyses were conducted using Genstat 18th edition , and t tests were used to generate P-values. False-discovery rate correction (FDR) with α = 0.10 was used to correct for multiple comparisons  was used to identify genes in the QTL region that might conceivably play a role in diterpene glycoside biosynthesis. The initial steps in the diterpene glycoside biosynthesis occur through the methylerythritol 4-phosphate (MEP) pathway in the plastids  or susceptibility allele (“S”) in homozygous state in the 2LOD interval region between M2 to M15 were scored for first instar larval development (Table S2). A high fraction L1 larvae indicates thrips resistance. As expected, plants with the “R” allele showed a significantly higher fraction L1 compared to plants with the “S” allele for all F3 lines, confirming the previously identified QTL  recombination between markers M2 and M15 that flanked the QTL region. In order to fine map the resistance locus, these 120 plants were phenotyped (Supplementary data Table S4). The strongest correlation between fraction L1 larvae and marker genotype  in the QTL region was found for marker M12.Among the 2000 plants from F3 plants with a recombination between M10 and M15 were selfed for one or two generation and the F4 and F5 plants with the recombination in homozygous state were selected. They were compared to plants from the same line with the “R” or “S” allele in homozygous state  based on the 4 plants from group “R” and group “S”, i.e. with the resistance allele (“R”) or the susceptibility allele (“S”) in homozygous state, was confirmed , the RNAseq data of these two groups of plants, in two different treatments, i.e. thrips-exposed or mock-treated, were used to compare differentially expressed genes (DEGs) in the fine-mapped QTL region. Two DEGs were found in the region of interest, i.e. LOC107876110, an organic cation transporter (OCT7) and LOC107874801, an uncharacterized locus , including the two genes found in the DEG analysis (OCT 7 and LOC107874801); the third gene is an acid phosphatase 1 (APS1). The allele that has a mutation that was predicted to lead to an altered protein structure was observed in the susceptible group for all three genes.4 plants that either have the R-allele or S-allele in homozygous state between M11 and M15, and the resistant and susceptible references, both exposed to L1 larvae and mock-treated leaves. In negative ionization mode, which was the most sensitive mode for detecting diterpene glycosides, a total of 1363 putative metabolites were detected. Forty of these compounds were putatively identified as diterpene glycosides, as they contained a positive ionization mode fragment with a mass to charge ratio (m/z) of 271.2410, i.e. [C20H31]+, which is an indicative fragment of diterpene glycosides  and susceptible (CGN17219A) reference, thrips-exposed and mock-treated, shows a grouping of plants into 3 different clusters Fig. . A clearA and sus4 lines, 32 metabolites differed significantly in their relative abundance for both treatments  affected the expression of carnitine-related genes and led to developmental defects , which plays a role in plant adaptation to environmental stresses such as cold, drought and salt stresses in The second candidate gene is an uncharacterized locus, LOC107874801. This candidate gene did not show significant difference in expression between mock-treated and L1 induced plants that have the resistance allele in homozygous state, but did show a significantly increased expression when plants with the susceptibility allele in homozygous state were induced with L1 larvae. This might indicate that LOC107874801 is a susceptibility gene that plays a role in increasing the compatibility between plant and insect, for instance by blocking the plant’s defence pathway upon induction , an acid phosphatase which is induced upon wounding, insect feeding, methyl jasmonate and phosphate deprivation (Mason and Mullet The third candidate gene is the predicted gene encoding a protein similar to acid phosphatase 1 (APS1). Acid phosphatase 1 is similar to vegetative storage protein 2 in As we only studied gene expression at one time point (6 h), we might have missed genes that have differential expression between group R and group S with different treatments at earlier or later time points. For instance, Sarde  showed t4 line that either had the thrips resistance or susceptibility allele of the fine-mapped QTL region on chromosome 6 in homozygous state, and leaves exposed to first instar (L1) larvae or mock-treated. We performed an untargeted LC–MS analysis of water–methanol extractable metabolites, including capsianosides (diterpene glycosides) previously reported to be associated with thrips resistance, on young leaves of 12 weeks old plants in which the phenotypic effect of the resistance QTL was clearly detectable. No significant differences in the relative abundances of any of the detected metabolites were found between the two contrasting groups of F4 plants, neither between L1-exposed and mock-treated samples within each sample group; those few mass peaks that appear to differ significantly between the treatments were most likely artefacts due to yet unexplained differential ionization issues in specific chromatographic regions. This observation suggests that at least the metabolites that could be detected using our extraction and LC–MS method do not play a direct role in the resistance mechanism. We cannot exclude the possibility that specific metabolites do play a role, but at least the 40 diterpene glycosides detectable with our method do not. This finding is not in line with the role in thrips resistance hypothesized for these metabolites in Capsicum that was based on association in completely independent studies Below is the link to the Supplementary Information.


Conversely, during highstands, the Baza lake flooded the Venta Micena area and the freshwater fauna was replaced by a saline one. Bulk-rock isotopic data indicate that the lower interval C1 of the limestone  displays general negative values, while the upper interval C2 (bone free) displays less negative values. The bones of predated mammals accumulated in the marginal areas, which were flooded and buried by recurring water-table fluctuations. Lake dynamics played a critical role in bone accumulation, which was previously considered as representing a hyena den.Despite the paleontological relevance of the terrestrial Early Pleistocene Venta Micena bonebed , it lacks a comprehensive geochemical/sedimentological study. Here, we demonstrate that the 1.5-m-thick Venta Micena limestone formed in a relatively small freshwater wetland/pond located at the periphery of the large saline Baza paleolake. Two microfacies are observed, with high and low contents of invertebrate fossils, and which originated in the centre and margin of the wetland, respectively. X-ray diffraction (XRD) mineralogy and paleohydrological characterization based on ostracod and bulk-rock geochemistry (δ The youngest part of the Early Pleistocene interval of this transition is well dated in the paleolake succession of the Guadix-Baza basin  paleontological site is a reference locality for Eurasian land mammal biostratigraphy at the Early-Middle Pleistocene transition from Eurasiasin Fig.  with com9 where fossils appear minimally weathered or abraded, are commonly articulated (or associated) and display no evidence of tractive transport. On the basis of bone taphonomy and the overrepresentation of subadults, it has been suggested that such bone accumulation took place in a hyena den12. The mammalian fossil bones at the site represent a combination of species living in several environments, such as an open shrub environment  and aquatic settings . The VM site is a cluster of several close-by localities 22, characteristic of the Guadix and Baza sub-basins, respectively. The Baza Fm has a maximum thickness of over 100 m and is widely known for hosting Plio-Pleistocene mammalian sites3.The Guadix-Baza intramontane basin is located in the Betic Chain, at the contact between the internal and external units  contains dolomitic marls and micritic dolostones locally interbedded with gravel, sand, limestone and shale, the latter in finer strata. Root bioturbation is common and sandy levels display large-scale cross-stratification and ripple cross-lamination. This unit records a period of important subaerial exposure based on (a) a relatively poor content of invertebrates, since most of them have been dissolved by weathering (some moulds are visible), and (b) the presence of pedogenic features.Cyprydeis torosa and the mollusc Cerastoderma, which live in waterbodies with salinities between 4‰ and 60‰25.Unit B (0.3 to 1.5 m) consists of bioclastic sands, marls and mudstones containing foraminifera, gastropods and ostracods. The unit is interpreted as representing a saline lake transgression episode on the basis of the presence of ostracods 16 that the bones rest on the relief of a paleosol, indicating that they accumulated during a subaerial exposure phase that was followed by micritic mud deposition.Unit C (2.3 to 3.4 m) has a lower part  with whitish sandy micritic limestone that is locally greenish and blackish  contains muddy limestones interbedded with sandy marls, dolostones and marly dolostones Fig. c. Some lUnit E (7.5 m) is a succession of calcareous-dolomitic marls and marly-sandy dolostones that alternate with levels of sands and gravels with cross-stratification.In the Galera-Orce-Fuente Nueva area (containing the VM site), up to three subunits (members) can be distinguished within the Baza Fm26, and the base of the section  contains the site VM127. This site essentially contains microvertebrates and is not considered part of the VM bonebed . Second, section VM4 in Fig. 12. The latter was a natural outcrop, while section VM4 is an artificial trench. Third, section VM2 in Fig. 12, and is also the only locality where the Venta Micena limestone has yielded significant rodent faunas3. Fourth, section VMX in Fig. 26. Finally, section VM3 in Fig. 26 , with abundant smooth-shelled Cyprideis. For unit C, no data were obtained in section CT, since no invertebrate fossils were found. Alternatively, unit C has been studied in section VM1, where coarsely reticulated and heavily noded Cyprideis indicate lower salinities . After analysing the Sr/Ca and Mg/Ca ratios of ostracods, it was concluded26 that there was no marine influence, even for the high salinity intervals.The investigation of ostracodsThe analyses performed on the samples examined in this work (see applied methods in the Supplementary Information) yielded the following results :XRD data were obtained from the lower section VM1  and from section VM4 . The whole dataset is presented in Fig. Minerals such as gypsum Fig. a, aragonXRD performed on section VM1 also demonstrated an increase in the calcite content from the base to the top. The highest contents of calcite in the equivalent levels of the carbonate of section VM1 stand out (MV 17–010 = 90.7%) in contrast to section VM4 (VM 17–230 = 64.4%). Four samples from VM3 yielded similar results and demonstrated a mineral assemblage dominated by low-Mg calcite , along with quartz (2–17%) and minor amounts of dolomite (0.1–5%) and clay minerals (0.7–8.8%).Petrographic determinations. Thin sections from section VM4 were extracted from two slightly indurated levels observed at the base and in the middle of interval C1 , such as complete or fragmentary ostracod shells, as well as abundant charophyte gyrogonites. The matrix between these bioclasts is irregular and is built up from anhedral micritic-microsparite carbonate crystals of different sizes. Taxonomic analysis of the recorded species indicates sedimentation within a fresh or oligohaline water mass15.SEM imaging was very informative and provided straightforward determinations of two microfacies in VM limestone, one invertebrate rich and the other invertebrate poor, on the basis of aquatic skeletal remains of invertebrates and carbonate texture. Each microfacies is unrelated to C1 or C2 units, which are based on isotopic data. In addition to SEM imaging, the differentiation of these two microfacies was obvious during ostracod sieving and picking under a binocular microscope for isotope studies. The invertebrate-rich microfacies is dominant in section VM1 Fig. c,d and cInvertebrate-poor microfacies Fig. e–h were Skeletal invertebrate isotopes. Isotopic δ18O and δ13C analyses of the skeletal parts of invertebrates  are also included with the aim of obtaining a record from unit B.18O of the calcite from the ostracod valves is a function of the temperature of the water where ostracods moulted and the oxygen stable isotopic composition of the water. δ18O results from ostracod and gastropod opercula were normalized (corrected) by summing the vital fractionation and the values obtained in the analyses. According to the compilation in Ref.29, the correction factors are + 2.4‰ for Candona neglecta, + 2.2‰ for Candona angulata, + 0.8‰ for Cyprideis torosa and + 3.9‰ for Ilyocypris bradyi. For the opercula of Bithynia sp., the normalization factor would be 1.2‰, but for the δ18O data on gastropod aragonitic shells (Bithynia), no correction was applied, since it is usually observed32 that the shells reflect the oxygen conditions of the environment .The δ13C value is an approximation to the isotopic composition of the dissolved inorganic carbon in the water. The δ13C signal in each individual is directly related to the fractionation of each species, even in different ontogenetic stages. In unit B (sampled in section VMX), Cyprideis torosa yielded values ranging between -5.51‰ and -3.93‰ for δ13C and between -1.55‰ and + 1.28‰ for δ18O.The δThe general negative trend, particularly for O values, indicates that the system evolved progressively to less concentrated waters. On the other hand, C and O values in section VM1 are rather different. See Fig. C. torosa and Ammonia beccarii28 , indicating that they formed in saline waters. Samples from three beds at the top of this interval display C. torosa shells with δ13C from − 6.41‰ to − 3.42‰ and δ18O from − 1.66 to 0.13‰ . These features suggest that these levels formed in saline and isotopically more concentrated waters for δ18O.In section VM3, unit E levels located 7.5 to 10 m above the VM limestone contain 13C = − 9.64‰; δ18O = − 5.32‰). These beds are overlain by sandy carbonate mudstones (unit B) with a saline ostracod and mollusc fauna15. The isotopic signature of the C. torosa shells .At VMX (50 m to the S of VM3), the lowermost levels  contain freshwater ostracods and planorbid gastropods that exhibit depleted isotopic signatures  were targeted for isotopic δ13C and δ18O analyses of the carbonate fraction  ranging from − 7.67‰ to − 6.11‰, while δ18O values ranged from − 6.56‰ to − 5.01‰. Regression analysis of all the samples . The upper interval of the section (C2)  showed a correlation of r = 0.18. A general comparison between intervals C1 and C2 permitted the recognition of partial covariance in C1 (at least for some spans), a phenomenon not observed in interval C2.Section VM4 displayed δ13C values between a maximum of − 5.52‰ and a minimum of − 8.23‰ (VPDB), with a total range of 2.7‰. The majority of the δ13C data fell between − 6‰ and − 8‰; only two horizons (150 and 160 cm) showed slightly more enriched values  to − 4.40‰ (180 cm) and a total range of 1.6‰. The average section VM3 values for δ13C and δ18O were only weakly correlated (r = 0.48). However, when the δ13C and δ18O values of the carbonates were submitted to cluster analysis (Supplementary notes S-3), the two resulting zones defined between 0–130 cm and 140–220 cm displayed very low to low correlations . The bulk-rock samples from section VM1 (n = 4) displayed δ13C values ranging from − 5.95‰ to − 4.15‰. δ18O values ranged from − 7.72‰ to − 6.10‰, displaying a correlation of r = − 0.977. Cluster analysis provides an independent criterion to split C1 from C2 (which is consistent with bone occurrence). A significant feature of the isotopic data is that considering the overall C1 samples from VM3 and VM4 (n = 35), a weak correlation between δ13C and δ18O is observed, and a significant correlation for C2 is attained .Section VM3 samples (n = 34) Fig. a display22. This means that during these intervals, the dimensions and connectivity of Guadix-Baza lake were large enough to permit siliciclastics from these distant zones to reach the VM area. On the other hand, the occurrence of in situ-formed carbonates in C1 and C2 indicates sedimentation in isolated and low-energy conditions. Notably, minerals such as aragonite, gypsum and dolomite are only found in interval C0, suggesting that waters were more concentrated than in unit D, in agreement with the salinity determinations25 pointing to less saline conditions during the sedimentation of VM limestone. This is supported by the paleoecology of ostracods and molluscs in section VM1, which indicates that these invertebrates thrived in freshwater or slightly saline waters, while saline species (C. torosa) are found in the underlying unit B16. Salinity variations based on the ostracod paleoecology of these sections25 are in agreement with the ostracod isotopic data presented here.Mineralogical XRD data from section VM4 Fig. 19,21,22.17 seem plausible in this scenario.Petrological and SEM observations indicate that the invertebrate-rich microfacies (with well-preserved bioclasts in VM1) may have formed in an offshore position in a wetland/pond. This last setting would have no (or hardly any) aerial exposition, and persistent aquatic conditions would enhance the preservation of invertebrate shells. On the other hand, invertebrate-poor microfacies would result from episodes of emersion enhancing skeletal dissolution/recrystallization. These features indicate that they accumulated at the margin of a lake, where subaerial and subaquatic conditions coexisted. A contribution of travertine-like formation processes cannot be ruled out. In any case, the previously defined paleosols13C and δ18O values of bulk carbonate samples from sections VM3 and VM4 , could reflect the evolution of a closed system, in which evaporation through time causes enrichment to more positive values. Although these trends are comparable to those from closed lakes that have been studied33, covariance is also a common feature of pedogenic calcites formed in arid environments. In this case, the enrichment trend of δ18O is due to kinematic effects related to evaporation34. The correlated δ13C enrichment is attained through decreasing respiration rates and CO2 degassing. The low variance in δ18O, however, could indicate that the time span of these hydrological conditions was relatively short. A more detailed study could shed light on this point. Interestingly, fossil accumulations at sites VM4 and VM3 were mainly found within this C1 interval. The interval C2 showed no correlation between δ13C and δ18O, indicating a hydrologically open system34. The general variability and trend of our dataset . Therefore, C2 sediments have inherited more of their isotopic composition from the parental lacustrine carbonates. In summary, we correlate subaerial exposition, geochemistry and bone occurrence.The generally more negative δ15 and the isotopic signatures of the biogenic skeletal carbonates presented here indicate that unit A records an early freshwater wetland that was succeeded by a transgressive saline-lake phase recorded in unit B (in VMX). Unit C, in VM1, represents a freshwater wetland/pond where carbonate deposition took place. The carbonate deposits (VM limestone) in the other sections of VM area represent deposits that experienced successive subaerial exposures and pedogenesis. The overlying sediments of units D and E record a new saline lake phase and isotopically concentrated waters, as recorded by fossils and the isotopic features.The paleoenvironmental features deduced from the invertebrate fossil content28 in the nearby locality of Barranco León (BL)36, a similar sequence was found. Tentatively, the transgression unit at BL (levels A and B) could be correlated with interval B in the VM area  support the model for the formation of VM sites suggested here. Although BL is a paleontological site that underwent reworking, a long-term model for bone concentration (or preconcentration) during lowstands is envisaged. The widespread occurrence of present-day hydrothermalism at the margins of the Guadix-Baza basin was used to explain the occurrence of vertebrate fossil sites37. This last model was debated, since freshwater environments were considered to be peripherical ones in the context of interconnected subenvironments38. Our data and model  there was hardly any clastic input, (2) sedimentation occurred during a freshwater interval separated by two more saline ones, as evidenced by ostracod paleoecology and the isotope signal, (3) this freshwater period had to take place during a lowstand, when the peripheric VM source was disconnected from the main saline Baza lake. Paradoxically, high rainfall would correspond to higher salinities in the VM area , while in drier periods (lake lowstand), the saline lake would not interfere with freshwater inputs. Regarding the conditions and isotope data of C1 , it is observed that (1) the central microfacies of VM1 contain no bones, while marginal (dominant) microfacies are isotopically more diluted, and (2) the shifts and partial oxygen covariance in C1 indicate that lake bones accumulated in short-lived intervals with closed hydrological conditions.13.In terms of taphonomy, it is concluded that VM bones accumulated at the margin of a freshwater area that attracted mammals and their predators. Bones accumulated in situ in depressions that were flooded and buried by pond sediments due to water table pulsations. This paleoenvironmental setting also makes this scenario plausible for the activity of hyenas as a complementary factor for bone accumulationSupplementary Information.


Aneurysm wall enhancement (AWE) after the administration of contrast gadolinium is a potential biomarker of unstable intracranial aneurysms. While most studies determine AWE subjectively, this study comprehensively quantified AWE in 3D imaging using a semi-automated method. Thirty patients with 33 unruptured intracranial aneurysms prospectively underwent high-resolution imaging with 7T-MRI. The signal intensity (SI) of the aneurysm wall was mapped and normalized to the pituitary stalk (PS) and corpus callosum (CC). The CC proved to be a more reliable normalizing structure in detecting contrast enhancement (p < 0.0001). 3D-heatmaps and histogram analysis of AWE were used to generate the following metrics: specific aneurysm wall enhancement (SAWE), general aneurysm wall enhancement (GAWE) and focal aneurysm wall enhancement (FAWE). GAWE was more accurate in detecting known morphological determinants of aneurysm instability such as size ≥ 7 mm (p = 0.049), size ratio (p = 0.01) and aspect ratio (p = 0.002). SAWE and FAWE were aneurysm specific metrics used to characterize enhancement patterns within the aneurysm wall and the distribution of enhancement along the aneurysm. Blebs were easily identified on 3D-heatmaps and were more enhancing than aneurysm sacs (p = 0.0017). 3D-AWE mapping may be a powerful objective tool in characterizing different biological processes of the aneurysm wall. The characterization and understanding of AWE as a surrogate marker of changes in aneurysm wall biology is a nascent field. Histological analysis of aneurysm walls shows endothelial disruption as a potential marker of aneurysm instability2. Formation of atherosclerotic lesions as a result of lipid accumulation is one example of the complex remodeling mechanisms that affect the aneurysm walls3. Histological analysis has linked Gd uptake in the aneurysm wall to intimal disruptions in the internal elastic lamina and endothelial cells5. AWE may be used as a biomarker of instability once the dynamic contrast uptake by different layers of the aneurysm wall is better characterized  provides a high contrast-to-noise ratio and spatial resolution for better visualization of intracranial arterial walls. Biological processes such as microhemorrhages, wall thickening, atherosclerotic changes or indirect signs of inflammation may be visualized with HR-MRI. Within this spectrum of radiological findings, aneurysmal wall enhancement (AWE) after the administration of gadolinium (Gd) has been described as a marker of aneurysm instability6. The definitions of AWE vary broadly, although recently there has been a step forward in determining AWE objectively instead of subjectively9. One of the major limitations in the current method of assessing AWE is that images are processed and analyzed in multiplanar 2D views and in the best-case scenario, include different segments of the aneurysm. This approach is limited by the manual sampling performed by the investigator in determining which areas of the aneurysm have increased AWE.Meta-analysis and several reports have consistently shown that AWE is significantly correlated with higher risk of aneurysm growth and rupture11. Other groups determined a higher risk of rupture when circumferential wall enhancement (CAWE) is present, instead of FAWE13. Intramural thrombosis  has determined that the colocalization of these areas with low wall shear stress, such as blebs Fig.  is assocIn this study we describe a semi-automated method of generating 3D-AWE maps of the entire aneurysm. The main aim of this approach is to characterize different biological processes that affect the aneurysm wall and may manifest with different patterns of AWE. We believe that a comprehensive 3D analysis of the aneurysm will lead to a better estimation of the risk of rupture in prospective clinical studies.2. An average of 62 spokes per mm2 were generated. Only saccular aneurysms were included in the morphological analysis  were included in the analysis: 28 saccular and 5 fusiform aneurysms. The average aneurysm spoke length based on wall thickness was 0.55 ± 0.09 mm and the average surface area/aneurysm was 311.8 ± 680 mm7, the CC appears to be a more reliable normalization structure in determining AWE (∆ T1 + Gd − T1) within the same aneurysm . Histogram analysis showed that normalization to the CC provided a more reliable shift of the curve from lower AWE in T1 sequences towards higher AWE in T1 + Gd sequences. In some cases, avid enhancement of the PS introduced artifact and the curve shifted to the left Fig. , Table 2ysm Fig. .Figure 2For each aneurysm, the distribution of spokes was plotted according to SI ratios of T1 and T1 + Gd images. Three metrics of AWE were determined: (1) Specific AWE (SAWE), defined as the change in enhancement between T1 and T1 + Gd for each aneurysm (T1 + Gd µ ≥ T1 µ + 2σ). (2) General AWE (GAWE), defined as the average CC ratio of all SAWE + aneurysms (µ = 1.0023) or CC Ratio ≥ 1. GAWE was used to compare AWE between different aneurysms. (3) Focal AWE (FAWE), defined as areas of high AWE with two SDs above the mean of the T1 + Gd distribution (≥ T1 + Gd µ + 2σ) Fig. . SurvivaEleven aneurysms were SAWE+ and had a higher percentage increase in AWE from T1 to T1 + Gd (μ = 88%), when compared to non-enhancing aneurysms . Moreover, enhancing aneurysms had a higher increase in CC ratio (μ = 0.46) from T1 to T1 + Gd vs non-enhancing aneurysms . The average CC ratio for enhancing SAWE+ aneurysms was 1.002 (p = 0.001). Seven aneurysms were GAWE+ and had a higher percentage increase in CC ratio from T1 to T1 + Gd (μ = 96.3%) vs non-enhancing aneurysms . Enhancing GAWE+ aneurysms also had a higher CC ratio increase (μ =  + 0.56) between T1 and T1 + Gd when compared to non-enhancing aneurysms  , bivariate analysis showed significance for aspect  and size ratios . In ROC analysis, aspect ratio ≥ 3.56  and size ratio ≥ 2.89  best predicted GAWE + aneurysms  and aspect ratios ≥ 2.31 . These morphological parameters have also been identified by other groups as reliable indicators of instabilityThis study performed a detailed topographic analysis of AWE using images acquired with 7T HR-MRI. We developed a protocol to generate and analyze 3D-AWE maps of the entire aneurysm. With this tool new metrics could be objectively analyzed and used to better understand the distribution of AWE along the aneurysm wall.22. Isonormals were used to measure AWE normalized to the nominal intensity of the image volume. This innovative approach fell short of adding objective parameters of determining AWE. In this study we generated thousands of datapoints (µ = 4490 spokes/aneurysm) from orthogonal spokes projected into the aneurysm wall axis. The measurement of AWE was optimized with 7T HR-MRI by tailoring the spoke length to the thickness of the aneurysm wall for each aneurysm (range 0.38–0.78 mm). We believe that this customized sampling of AWE decreases artifact introduced by low and high enhancing structures such as the cerebrospinal fluid and the cavernous sinus, respectively. Kleinloog et al. demonstrated on 7T MRI that the radiological aneurysm wall thickness can vary between 0.2 and 1.6 mm23. A tailored spoke length generates exceptionally reliable 3D maps of AWE. This approach has several advantages over previously described methods of AWE analysis: 3D data allows identification of areas of aneurysm instability such as blebs, the aneurysm is visualized and analyzed in its whole magnitude, and histograms generated through this protocol provide a new tool in studying the biology of brain aneurysms and in identifying possible markers of instability. Meta-analysis and preliminary prospective data have shown that AWE is a potential predictor of aneurysms instability25. However, there is no consensus on how to determine thresholds of AWE or what part of the aneurysm should be sampled to analyze enhancement. 3D maps may also provide further information about the aneurysm biology as it has been shown that aneurysm compartments have different patterns of enhancement directly influenced by local flow conditions10. 7T MRI studies have demonstrated a linear correlation between wall thickness and SI. Histological analysis of aneurysm walls has shown an eight-fold variation in thickness23. Different biological processes encompass this wide variation in thickness and structure of the aneurysm wall2. Atherosclerotic and non-atherosclerotic calcification26, inflammation27, wall remodeling with mural necrosis28, proliferation of vasa-vasorum29, among other changes, will determine the “health” of the aneurysm wall and risk of rupture30 . Our previous studies suggested that the PS is a reliable normalizing structure. However, in this study the PS exhibited an avid uptake of Gd in T1 + Gd images, which is suboptimal when analyzing specific AWE (SAWE). Other studies have successfully used white matter structures for normalization of enhancement through pre/post Gd indices32. After multiple comparisons and detailed histogram analysis, normalization to the CC resulted in a more reliable parameter in determining AWE.  in identifying aneurysms that ruptured over time. This appeared to be a more reliable metric than the analysis of enhancement determined by T1 + Gd images only33. SAWE may be a better tool in detecting “leaky” aneurysms when compared to GAWE. SAWE provides a map of enhancement for each aneurysm, which is an individualized metric inherent to Gd uptake in each segment of the wall. Moreover, SAWE has the potential of controlling for enhancement artifacts such as slow-flow34, as histogram analysis of T1 and T1 + Gd images has the potential of identifying pseudoenhancement , specific AWE (SAWE), and focal AWE (FAWE). Coupling this data to 3D reconstructions allowed qualitative and quantitative assessments of AWE Fig. . Interesent Fig. a–d.11. 3D-AWE maps ease identification of these areas of focal enhancement which on conventional 2D imaging may not be detected. An objective threshold for FAWE was determined by detailed histogram and 3D map analysis. The highest SI region in the histogram could identify both the threshold for FAWE and whether there are multiple areas of FAWE in the aneurysm sac . Aneurysms with larger areas of FAWE also had histograms with positive skew, suggesting that these highly enhancing focal areas can affect the shape of the histogram. FAWE has been linked to intraluminal thrombus and sites of rupture35, this analysis could further identify and quantify these areas. 3D-AWE maps showed that on average, blebs exhibited 8% higher enhancement than the aneurysm sac. Further topographic analysis may lead to the identification of bleb-prone areas in the aneurysm sac.The generation of AWE heatmaps allowed the identification of areas of increased enhancement including blebs. Larsen et al. have shown that areas of focal enhancement have low wall-shear stress conditions that may favor growth and ruptureThis study is limited by the sample size and the heterogeneity of aneurysms included (15% fusiform). However, the study was aimed at developing a protocol for 3D-AWE mapping and not at determining specific differences between aneurysms subtypes. With this aim in mind, we used a 7T platform that eased the identification and analysis of the aneurysm wall. We are cognizant of the limited clinical use of 7T-MRI and we plan in optimizing this protocol for 3T MRIs. The nature of the small and heterogenous sample limits the predictive value of AWE metrics studied. A larger analysis using 3T-MRI is needed to determine the clinical relevance of such methods. 7T HR-MRI poses its own challenges limiting patient enrollment. Additionally, a lack of advanced blood suppression techniques may have resulted in flow artifact as a potential source of noise. While the goal of this technique is to map the aneurysm wall, the contributions of flow artifact to AWE cannot be ruled out. However, as demonstrated on Fig. To summarize, we believe that 3D-AWE mapping may be a powerful tool in studying the biology of brain aneurysms and in identifying aneurysms that may grow and rupture. This method generates a new set of metrics which could potentially be correlated with different biological processes of the aneurysm wall.36 and the FMRIB Software Library37, as well as custom scripts. DICOM datasets were converted to NIfTI-1 format for pre-processing. All images were denoised using a Rician distribution model38. T1 images were aligned to a template brain to approximate ACPC alignment. T2 and T1 post-Gd were then co-registered using rigid, affine, and symmetric normalization to the aligned T1 pre contrast image. Intensity non-homogeneity in T1 was corrected using three sequential procedures: the T1/T2 method40, the N4 method41, and the 3dUnifize function in the AFNI toolbox42 as well as custom scripts.After approval by the University of Iowa Institutional Review Board, patients with UIAs underwent HR-MRI on a GE 7T MRI (Discovery MR950) from August 2018 to December 2020. All patients provided an informed consent to participate in the study. All methods were completed in accordance with relevant guidelines and regulations. T1-weighted  and T2-weighted images  were acquired. T1-weighted images were acquired before and five minutes after administering Gd. All image processing was completed with Advanced Normalization Tools43 on post-processed T1 and T1 + Gd images. Binary label maps were then created and resampled to the base image. Additionally, the genu of the corpus callosum (CC) was marked as a 3 mm spherical region of interest (ROI). Another ROI was selected in the pituitary stalk (PS). Stray voxels were isolated and removed from all labels. Average aneurysm wall thickness was measured on source imaging using the Picture Archiving Communication System . The aneurysm wall thickness was conservatively measured on T1 + Gd imaging in the sagittal, coronal, and axial planes using a slice that cut through the largest cross section of the aneurysm that allowed for the best visualization of the wall. Measurements were taken at the thinnest and thickest sections of the wall that could be visualized in each view, and subsequently averaged. . Base images were isotropically resampled using a cubic interpolation method. A bounding box was created with a 25 mm radius from the aneurysm and vessel labels. Labels were smoothed using a 3 mm gaussian kernel with a 1 mm standard deviation (SD). Larger aneurysms were smoothed with a 5 mm or 7 mm kernel depending on size. Isosurfaces were created from the smoothed and interpolated labels. Surface complexity was reduced to 1 or 10% of the original surface for medium and large aneurysms. Small aneurysms (< 5 mm) maintained their original surface complexities.For each vertex in the aneurysm surface model, isonormals were calculated, creating spokes extending outward from the previously generated masks at a distance equivalent to the average aneurysm wall thickness. Parent artery labels were used to exclude spokes created at the neck of the aneurysm. Along each spoke, the SI from the base image was calculated using a cubic spline interpolation. The maximal value of each spoke was used to determine the SI of the wall and to eliminate luminal contamination and potential errors from radiological wall thickness measurements. The raw SI values for each spoke were normalized to the max SI values of the CC and PS. 3D heatmaps were created by superimposing the SI data on the outer aneurysm surface as determined by the outermost vertex of each spoke. AWE was calculated as the mean value of each spoke Fig. .Figure 844 Average bleb wall thickness was calculated as the average of five measurements in the cardinal planes. Blebs were then manually labeled on the processed T1 + Gd images according to the same protocol described for aneurysms. Bleb raw values were also normalized to the CC and PS.Blebs were identified based on criteria defined by Ashkezari et al.8. Wall enhancement of each aneurysm was measured with PACS. AWE was quantified by creating ROIs of the aneurysm wall in each plane on source T1 + Gd sequences as previously described45. ROIs were sampled in three different planes and after co-registration of the T1 + Gd and T1 sequences. Normalization to the PS or CC was calculated as follows: /(Mean or Max SIPS or SICC). Similarly, bleb ROIs were used to manually determine wall enhancement, and normalized to the PS and CC.Manual SI ROIs were sampled to compare the semiautomated 3D mapping method with the manual method of determining AWE. The manual method has been used extensively by our group and other groups to quantify AWE46 Aneurysm size, size and aspect ratios were manually calculated from subtraction angiograms, computed angiograms or magnetic resonance angiograms as described by Dhar et al.16.Planar-isolated aneurysm segmentations were created on T1 + Gd aneurysm labels using 3D Slicer. These labels were processed with source images using MATLAB to find label volume, surface area, convex hull volume, and convex hull surface area. Ellipticity index (EI), non-sphericity index (NSI), and undulation index (UI) were subsequently calculated for each aneurysm according to Raghavan et al.47. Logistic regression analysis was used to determine the association of aneurysm instability based on size > 7 mm18 with morphological and AWE predictors. Odds ratios (ORs) and 95% confidence intervals were constructed for each predictor. A receiver operating characteristic (ROC) analysis was performed with cutoffs calculated according to the Youden Index49. All statistical tests used two-tail alternatives and assessed significance at α = 0.05.All statistical analysis was conducted using SAS  and SPSS Statistics 25 . Categorical variables are presented as frequency and percentage, and continuous variables as mean ± SD. A Student t-test was used to compare continuous data, and Pearson chi-squared test for the relationship between categorical data. The differences of manual measurements of aneurysm size, aspect and size ratios, and wall thickness were analyzed with Bland–Altman plots. The manual and semi-automated methods for measuring SI were compared for agreement using Pearson’s correlation. Bleb SI was compared to aneurysm SI using the Wilcoxon Signed Rank TestSupplementary Figures.


However, in lipid oxidation process induced by a Fenton reaction, TUDCA reveals substantial antioxidant activity significantly decreasing the rate of oxygen consumption in the system studied. In addition, TUDCA induces slight, but noticeable changes in the polarity and fluidity of the investigated model membranes. The results of performed MD simulation correspond very well with the experimental results.Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid containing taurine conjugated with the ursodeoxycholic acid (UDCA), has been known and used from ancient times as a therapeutic compound in traditional Chinese medicine. TUDCA has recently been gaining significant interest as a neuroprotective agent, also exploited in the visual disorders. Among several mechanisms of TUDCA’s protective action, its antioxidant activity and stabilizing effect on mitochondrial and plasma membranes are considered. In this work we investigated antioxidant activity of TUDCA and its impact on structural properties of model membranes of different composition using electron paramagnetic resonance spectroscopy and the spin labeling technique. Localization of TUDCA molecules in a pure POPC bilayer has been studied using a molecular dynamics simulation (MD). The obtained results indicate that TUDCA is not an efficient singlet oxygen ( Tauroursodeoxycholic acid (TUDCA) composed of taurine conjugated with the ursodeoxycholic acid (UDCA), together with its precursor UDCA, belong to the most hydrophilic bile acids ,2 Figur.2O2 in the presence of TUDCA, the bile acid increased cells viability and enhanced their antioxidant capacity by increasing the level of glutathione and upregulating the expression of antioxidant genes [Although present in the biliary bile acids of a number of vertebrates in a small amount below 1–5%), these bile acids constitute major components of a bear bile, in which their level reach up to 75% ,4. In re%, these nt genes . Earliernt genes ,27,28. ILight absorption and first stages of the visual signal transduction take place in photoreceptor outer segments (POSs) . In case1-palmitoyl–2–oleoyl–sn–glycerol–3–phosphocholine (POPC), 1,2–dioleoyl–sn–glycerol–3–phosphocholine (DOPC), 1,2–dimyristoyl–sn–glycerol–3–phosphocholine (DMPC), 1,2–dimyristoyl–sn–glycerol–3–phosphoethanolamine (DMPE), 1–palmitoyl–2–docosahexaenoyl–sn–glycerol–3–phosphocholine ((16:0)(22:6) PC)), 1–palmitoyl–2–oleoyl–sn–glycerol–3–phosphoethanolamine (POPE), egg-yolk sphingomyeline (SM), cholesterol and spin labels 1–palmitoyl–2–stearoyl–(5–doxyl)–sn–glycerol–3–phosphocholine, 1–palmitoyl–2–stearoyl–(7–doxyl)–sn–glycerol–3–phosphocholine, 1–palmitoyl–2–stearoyl–(10–doxyl)–sn–glycerol–3–phosphocholine, and 1–palmitoyl–2–stearoyl–(16–doxyl)–sn–glycerol–3–phosphocholine were purchased from Avanti Polar Lipids . mHCTPO was a gift from Prof. H. J. Halpern .M) and vortexed vigorously. Then, the multilamellar liposome suspension for structural measurements underwent five freeze–thaw cycles, after which it was centrifuged at 14,000× g for 15 min at 4 °C, and the resulting pellet was used for EPR measurements. In case of oxygen consumption measurements, multilamellar liposome suspension was not centrifuged and the final lipid concentration was 10 mM.Two models of liposomes were prepared. For structural measurements involvbruing electron paramagnetic resonance (EPR) technique simpler models were used consisting of POPC and DMPE (6:1 molar ratio) with two different concentration of cholesterol (5 or 40 mol%). For oxygen consumption measurements more complex liposome models were employed mimicking the membranes of POS. In this case, POPE instead of DMPE was used. Apart from POPC and POPE, a peroxidizable phospholipid containing docosahexaenoic acid ((16:0)(22:6) PC) was added in the concentration of 35 mol%. At the end, the molar ratio between POPC and POPE was 1:1. Additionally, cholesterol was present in the concentration of 5 mol %. The lipid composition was therefore similar to that of the old disks in POS . The lip1O2, 1Δg) generation up to 0.63 in benzene [1O2 (1Δg) phosphorescence decay fitted by the Levenberg–Marquardt algorithm, was performed by custom-written software.To determine rate constants of the interactions of singlet oxygen with the bile acids studied, time-resolved singlet oxygen phosphorescence at 1270 nm was measured as a function of the quencher concentration. Tetraphenylporphyrine (TPP) was used as an efficient sensitizer with quantum yield of singlet oxygen , electron paramagnetic resonance oximetry with the mHCTPO (0.1 mM) spin probe was utilized. Samples  for oxygen uptake rate measurements consisted of previously prepared liposomes (150 µL), spin probe , ferrous citrate , and ascorbic acid  solutions. Measurements were carried out in a flat quartz cell placed in the EPR resonant cavity as previously described ,36. EPR ZZ), samples were frozen to 120 K using liquid nitrogen vapor.T–PC, 5–PC, 7–PC, 10–PC, and 16–PC are phospholipid spin labels that have a nitroxide free radical moiety responsible for the EPR signal attached to the polar headgroup, or to the 5th, 7th, 10th, or 16th carbon atom in the acyl chain, respectively. Therefore, information is obtained from different regions of the membrane: the water-membrane interphase (T-PC), the region close to the polar headgroups (5- and 7-PC) and the membrane center (10- and 16-PC). The EPR measurements were performed with Bruker EMX spectrometer  equipped with a temperature control unit (EMX ER 4141 VT). The suspension of spin labeled liposomes was placed in a gas permeable capillary (i.d. 0.7 mm) made of TPX and located inside the EPR dewar insert in the resonant cavity of the spectrometer. The sample was deoxygenated with nitrogen gas (about 10 min), which was also used for temperature control. For measurements of lipid order (S parameter), the EPR spectra were recorded at 310 K whereas for polarity measurements  vector linking n − 1 and n + 1 carbon (C) atoms in the acyl chain) and the bilayer normal with corrections for sp2 hybridized carbon atoms linked by the double bond [mol are standard errors and were calculated over 5 blocks, with each being a 40 ns fragment of the productive trajectory.The orientational disorder of acyl chains in the membrane was calculated using molecular order parameter Sble bond ,48. The g). Then lipids were extracted from DSM and DRM fractions using the Folch method [g. The lower chloroform portion was dried under nitrogen and then lipids left after drying were analyzed for the phospholipid, cholesterol, and TUDCA content by Raman spectroscopy.For isolation of detergent-resistant lipid rafts  fraction) we used a simplified procedure similar to that described previously . Multilah method . DSM  was performed before collection of spectra with use of Raman scattering line produced by a silicon plate (520.5 cm−1). Samples were transferred on a CaF2 slide and Raman spectra were collected from ten randomly chosen spots. Estimated acquisition time of a single spectrum was 5 s . Laser power in the focus spot was equal to 10 mW. Data collection and analysis were performed using WITec (WITec Project Plus 5), Opus 7.2 and OriginPro 2018 programs.RS measurements of the TUDCA standard, DSM and DRM samples were conducted on a WITec confocal CRM alpha 300 Raman microscope with use of 488 nm excitation wavelength. The laser was coupled to the microscope via an optical fiber (50 μm diameter). A dry Olympus MPLAN objective (100×/0.90NA) was used. Spectral resolution was equal to 3 cm1O2, 1Δg) is one of the most apparent manifestations of antioxidant activity, understood as stopping or slowing down the oxidation process. Highly reactive singlet oxygen can initiate a chain lipid peroxidation reaction and oxidize other key cell components—proteins and nucleic acids [Quenching of singlet oxygen . Excitation of TPP with laser pulse at 590 nm generated singlet oxygen, which decayed with time constant (8.82 ± 0.43) × 103 s−1. In the absence of bile acids, lifetime of singlet oxygen generated by TPP in the mixture of solvents used was 113.69 ± 5.34 µs. Representative results acquired in air-equilibrated solution of TPP excited with 590 nm in the absence and in the presence of increasing concentrations of TUDCA (5.2–19.1 mM) are shown in 1O2 phosphorescence decay at 1270 nm was observed in the presence of rising concentrations of both studied bile acids  quenching by TUDCA and UDCA were (1.99 ± 0.32) × 105 M−1s−1 and (0.39 ± 0.13) × 105 M−1s−1, respectively (The determined rate constant of singlet oxygen (ectively . Table 13 M−1s−1 [5 M−1s−1 [1O2 (1Δg), it makes a significant contribution to the total singlet oxygen quenching rate of TUDCA. Even though the rate constant of singlet oxygen quenching by TUDCA is rather low, it should be noted that it is one of the highest interaction rates with singlet oxygen among sterols and comparable to that of phospholipids [The difference between singlet oxygen quenching rates of both bile acids cannot be explained by the presence of taurine residue in the TUDCA molecule. Taurine itself interacts with singlet oxygen with very low efficiency, and the rate constant of singlet oxygen quenching by taurine does not exceed 1 × 103 M−1s−1  (Table 15 M−1s−1 . Althougholipids  (Table 12/dt) in liposomes containing none of bile acids studied (control liposomes) was 9.02 ± 0.70 μM/min. In the presence of TUDCA, oxygen consumption rate was reduced by nearly 25% to 6.77 ± 0.83 μM/min (* p < 0.05) and the difference was statistically significant (22:6)PC, the lipid highly susceptible to oxidation, were exposed to free radical flux generated in the Fenton reaction in the presence and absence of TUDCA or UDCA. The rate of oxygen uptake , however this difference was not statistically significant. Oxygen uptake rate in samples, in which the Fenton reaction could not occur due to the lack of ferrous ions did not exceed 1.48 ± 1.21 µM/min (data not shown). Significant decrease in free radical-induced oxygen uptake in peroxidizable liposomes was observed only in the presence of TUDCA. UDCA was not as effective, although its ability to scavenge superoxide anion and hydroxyl radical has been demonstrated previously [2O2-induced oxidative stress in neonatal rat cardiomyocytes [3+-complexes [In case of UDCA enriched liposomes dOeviously ,27. The eviously . Taurineeviously , activiteviously  and to reviously ,70. Neveeviously . In thiseviously . In our myocytes  and ARPEmyocytes . Accordiomplexes . UnfortuTo get a more detailed picture of TUDCA behavior in the membrane, the number of hydrogen bonds between different groups of TUDCA and POPC and water was calculated. The results ae presented in The sulphate group of TUDCA forms about five times more hydrogen bonds with water compared to hydroxyl groups (OH3 and OH7) and carbonyl oxygen (OE1) and the amine group of TUDCA forms about five times less hydrogen bonds with water compared to hydroxyl groups. This means that TUDCA molecules lie horizontally at the inner side of the interphase. Radial distribution functions (RDFs) of water oxygen atoms relative to TUDCA oxygen atoms are presented in the To make the overall picture of TUDCA localization in the POPC membrane much clearer, mass density profiles along the bilayer normal for the atoms of water, POPC, phosphorous (P), and TUDCA have been provided as a Another interesting aspect of the TUDCA location in the membrane, especially taking into consideration the POS membranes, is its preference for the liquid-disordered phase . Membran−1 [–1 [2 or CH3 groups. Bands at 2881 cm–1 and 2974 cm−1 were previously assigned predominantly to C–H stretches in CH3 groups, while bands at 3013 cm−1 and 3064 cm−1 were previously found to be associated with C–H stretch in H–C=C groups [−1, 1296 cm−1 and the characteristic triplet of bands at 1063, 1088 and 1129 cm−1. On the other hand, the presence of the shoulder at around 705 cm−1 and bands at 1660 and 1735 cm−1 suggests the presence of a cholesterol moiety [−1 confirms the presence of the mixture of SM and cholesterol.The analysis of the Raman spectrum of DRM suggests that the sample contains mainly saturated lipids including sphingomyelin (SM) and cholesterol groups. As previously reported, the RS technique can easily provide the information about the general lipid unsaturation level, for example, based on the RS intensity ratio of the bands located at around 1266/1300 cm−1 . In the −1 [–1 , which iC groups . The anaC groups ,84, has l moiety ,85. The −1, originated from the bending vibration of =CH group and stretching ν(C=C) modes, respectively, which suggests the presence of unsaturated lipids in this sample. Moreover, the presence of band at 3064 cm−1 confirms the presence of C–H stretch in H–C=C groups [−1, 870 cm−1, broad band at 1100 cm−1 and bands at 1611 and 1655 cm−1 suggests the majority of phosphatidylcholine (PC) moiety [−1 also suggests the presence of PC and the unsaturated lipids [Contrary to DRM, the DSM contains visible bands at around 1245 and 1655 cmC groups . The pre) moiety . The CH d lipids ,82.−1, which correspond to the deformation vibration of SO3, stretching modes of C–S and C–N groups, respectively [−1 corresponding vibrations to the SO3 group [−1 related to the presence of a taurine moiety. Unfortunately, although the band at 1039 cm−1 was the strongest marker band of TUDCA in the fingerprint region, its intensity was very low in the measured sample of DSM and was invisible in DRM as it overlapped with other bands in this range. Therefore, the semiquantitative analysis cannot be performed on the basis of the 1039 cm−1 band. In order to carry out the semiquantitative analysis of the presence of TUDCA in measured samples we focused on the analysis of the most intensive Raman bands in the region of 2800–3050 cm−1. As explained above, such a region differs between DRM and DSM, and TUDCA has also a distinct shape of such a region including the intensive band at 2937 cm−1. In order to determine which of these samples has a higher concentration of TUDCA we calculated the ratio of the relative integral intensity of the band at 2937 cm−1 to the relative integral intensity of the band at 1453 cm−1. The band at 1453 cm−1 originated from the C–H bending modes, which constitute the similar contribution in both samples. As presented in −1 for DSM and DRM provides the semiquantitative level of TUDCA inside measured samples and shows statistically significant higher level of TUDCA in DSM than in DRM. This confirms that TUDCA preferentially accumulates in the fraction rich in unsaturated lipids.It was previously reported that the main bands for taurine in the solid state are observed at around 530, 735 and 1050 cmectively ,87. On tO3 group . The RamSimilar preference for liquid-disordered phase rich in unsaturated lipids in the case of a model of POS membranes was observed for macular xanthophylls lutein and zeaxanthin, which are known antioxidants protecting retina against oxidative stress ,89. It w2Azz, which is a z–component of the hyperfine interaction tensor of the nitroxide spin label, can be measured directly from the EPR spectra of immobilized spin labels (such as in frozen liposome samples) as a distance between the outermost spectral extrema . This paBoth polarity profiles presented in mol for the POPC membrane [To study the effect of TUDCA on the order of lipid acyl chains in both simple models of POS membranes, the order parameter S was calculated based on the EPR spectra of 5–, 7–, 10– and 16–PC spin labels according to Marsh . Additiomembrane . In the As expected, in both membranes the values of S parameter decreased in the direction from the region close to the membrane surface toward the membrane center. Additionally, the difference can be seen between both membranes, which results from different cholesterol content. The values of the S parameter were higher in the membrane containing 40 mol% cholesterol. This effect is observed at all positions along the acyl chain and reflects a well known rigidifying effect of cholesterol on membranes ,102. Themol parameter calculated based on MD simulations are presented in mol was calculated separately for palmitic and oleic chains of POPC . In both cases TUDCA slightly decreased the values of Smol but the effect was stronger for the saturated chain. In the oleic chain the decrease of lipid order was noticeable only for the fragment below the 10th carbon atom, which corresponded to the position of a double bond (C9–C10). Generally, the Smol values were lower than the S values obtained by EPR, which could be explained by the fact that MD simulations were performed for a membrane without cholesterol. Nevertheless, the results obtained by both techniques were in agreement showing slight fluidizing effect of TUDCA on membranes, especially in their central part.The values of the SIncrease in polarity and fluidity of membranes rich in cholesterol caused by the presence of TUDCA at its critical micelle concentration (CMC) has been reported previously . HoweverThe main goal of our study was to analyze whether the protective effect of TUDCA against photoreceptor photodamage could be facilitated by its effects on membrane structural properties. The results of MD simulations show that TUDCA molecules did not penetrate deeply the lipid bilayer of model membranes but located mostly parallel to the membrane surface with their sulphate groups directed towards the water phase. This may explain rather weak effects of TUDCA on membrane properties such as lipid order and polarity observed by EPR spin labelling technique. However, this location may be beneficial for TUDCA’s antioxidant action against water soluble free radicals. Such an activity was demonstrated in our experiments with oxygen consumption in liposomes containing highly unsaturated lipids. Although no significant differences were observed in TUDCA’s effects on membranes with different cholesterol content, cholesterol presence may play a role in the antioxidant activity of TUDCA in the context of membrane domain structure. Our results obtained with Raman spectroscopy suggest that in domain-forming membranes composed of saturated phospholipids, unsaturated phospholipids and cholesterol, TUDCA partitions preferentially into detergent soluble domains, which are rich in unsaturated lipids while in detergent-resistant fraction, which is enriched in cholesterol, it is present in a smaller amount. By accumulating in the domain containing lipids susceptible to peroxidation, TUDCA can more clearly manifest its antioxidant properties against free radicals.


Three clusters were identified, “Western”, “Convenience” and “Local/hawker”, none of which were defined by more prudent dietary choices. The “Convenience” cluster group had the lowest total energy intake (mean 85.8 ± SD 25.3% of Average Requirement for Energy) compared to the other groups  but also had the lowest calcium intake (66.3 ± 34.7% of Recommended Dietary Allowance), similar to intake in the “Local/hawker” group (69.5 ± 38.9%) but less than the “Western” group . These findings highlight the need for longitudinal analysis of dietary habit in younger Singaporeans in order to better define public health messaging targeted at reducing risk of major noncommunicable disease.Pattern analysis of children’s diet may provide insights into chronic disease risk in adolescence and adulthood. This study aimed to assess dietary patterns of young Singaporean children using cluster analysis. An existing dataset included 15,820 items consumed by 561 participants (aged 6–12 years) over 2 days of dietary recall. Thirty-seven food groups were defined and expressed as a percentage contribution of total energy. Dietary patterns were identified using  The study of diet–disease relationships has been previously conducted through the examination of individual nutrients or food groups and their relationship with risk factors or outcomes of chronic diseases ,2. HowevWhile a posteriori dietary patterns have been widely investigated in Western and European countries ,10,11, rThe prevalence of overweight and obesity among Singaporean children has increased over the past three decades ,23, highData used in this project was obtained from a study conducted by Neo et al. in 2016 involving a nationally representative cross-section of 561 6–12 year olds , further detail of which is available in the original study . This crThe cluster analysis method uses multivariate statistical techniques, where large datasets representing total food intake are aggregated and reduced to smaller datasets to summarise estimated total dietary exposure . ClusterA total of 15,820 food items were recorded from the multiple 24 h dietary recalls in the original dataset. All food items consumed were sorted into one of 65 food groups which were determined according to the Health Promotion Board’s food group classification through their online nutrient analysis tool, “Energy and Nutrient Composition of Foods” which provides the energy and nutrient content of popular local and western foods and beverages, as well as popular fast foods . The 65 Dietary data were standardized against recommended intake values defined by age, sex and physical activity using a similar approach applied to evaluation of infant dietary habits in a previous study . WhereveData processing was carried out using SPSS v25 software . Original dietary recall data was collected on one weekday and one weekend day.The mean daily nutrient intake of each food group (grams per day) was calculated for each individual. This first involved defining estimated daily intake data for all nutrients from each food group for each participant. The sum of the daily intake of each nutrient for the weekday and weekend day was divided by 2  to calculate the mean daily nutrient intake per participant.Estimates of energy and nutrient intake were made as previously described outcomes of dietary intake analysis ,35. The p < 0.05), further comparisons of the means were evaluated using post hoc Bonferroni tests. The confidence level of statistical analyses was set at 95%, where p < 0.05 was considered to show significant differences in mean values.The SPSS® statistical software package v25  was used for data manipulation, k-means cluster analysis and basic statistical analyses of the data sets. Chi-Square tests were used to compare the distribution of participants by sex, age and BMI in relation to the different dietary patterns among the entire cohort and the three dietary patterns derived from cluster analysis, as well as the proportion of individuals meeting national recommendations for nutrient intake. Differences in the mean percentage contribution of each food group across clusters, mean nutrient intake across clusters, and cohort characteristics across clusters were evaluated using one-way analysis of variance (ANOVA). For statistically significant results  from the equal ratio (50:50) in the “Local/Hawker food” cluster. The proportion of kindergarten  children, lower primary children  was not statistically different across all dietary clusters . The proportion of children who were underweight, acceptable weight and overweight by Health Promotion Board classifications were also similar across the three food clusters .There was a slightly higher percentage of girls to boys in the “Western food” (51:49) and “Convenience food” clusters (55:45), although this was not statistically different (p < 0.001) mean ± SD % of recommended energy intake  compared to the “Convenience” cluster group (85.8 ± 25.3% Average Requirement for Energy).The average nutrient and energy intake in relation to recommendations or proportion of total energy within each of the three clustered groups is presented in p < 0.001) mean intake of calcium (82.8 ± 36.1% of age-specific Recommended Dietary Allowance) than the “Local/Hawker” and “Convenience” groups . The % of energy consumed as fat and saturated fat was lowest (p < 0.001) in the “Convenience” group (28.3 ± 5.4% and 7.6 ± 2.8%) in comparison to the “Western” (30.7 ± 5.9% and 8.3 ± 2.9%) and “Local/Hawker” groups (31.7 ± 7.1% and 9.1 ± 3.4%). The mean percentage of dietary energy consumed as protein was different across all groups , with around 95% of all participants consuming adequate amounts of dietary protein by age and sex (data not shown). Although one-way ANOVA tests presented p-values suggestive of differences between groups in relation to the % of energy from carbohydrates and added sugars , targeted post hoc tests suggested no statistical difference between each group (p ≥ 0.056 and for %E from carbohydrates and p ≥ 0.074 %E from added sugars for comparisons across cluster groups).The “Western” cluster group also tended to have statistically higher (p = 0.479), with only 6.25% of participants meeting recommendations for their age and sex). Average calcium intake tended to align more closely with recommendations in the “Western” group  in comparison to “Convenience” (66.3 ± 34.7%) and “Local-Hawker” (69.5 ± 38.9%) groups.Vitamin D intake appeared to be frequently low across dietary pattern groups  and a data collection method originally designed to limit the potential for recall bias, as previously discussed ,35. The The coding of dietary items into food groups is based on a single researcher’s (M. J. Y. C.) perspective on food categorisation. Some commonly consumed Southeast Asian foods were challenging to code as they often contain more than one type of food group in a single food item or meal. Therefore, to reduce subjectivity bias, all food items were determined according to the Health Promotion Board’s food group classification through their online nutrient analysis tool . This toPrevious studies that have compared variables presented in grams and percentage of energy found that dietary patterns derived from grams of food per day were more difficult to interpret and often did not result in defining a major dietary pattern ,38. As sThe nomenclature attached to the final pattern solutions in the cluster analysis was also subjective and was agreed on by all authors following the analysis. These terms may not directly align with other dietary patterns that have been similarly named . For exaThe current findings and associated research highlight the need for more careful evaluation of dietary habit in young Singaporeans. Such future research could increase the age range of Singaporean children studied and ideally consider longitudinal data collection to better understand the relationship between health outcomes in a population based in a global food hub, where there is a high potential to see rapid, population-level changes in dietary habits over time .Future studies on a posteriori dietary pattern in Singaporeans or other multiethnic populations could also reduce participant and researcher burden through use of more rapid dietary data collection tools dietary collection methods, like food frequency questionnaires. While such approaches tend to be less accurate than dietary recalls, they are usually developed with a specific population in mind  and exisCluster analysis supported definition of dietary patterns in this multi-ethnic cross-section of children. Of the three dietary patterns defined within the current study, all tended to provide an inappropriate balance of nutrients and none tended to be dominated by more prudent food item choices. Future public health efforts should continue to prioritise improving dietary habits in Singaporean children.