Prey size reflected in tooth wear: a comparison of two wolf populations from Sweden and Alaska.

Ingesta leaves distinct patterns on mammalian teeth during mastication. However, an unresolved challenge is how to include intraspecific variability into dietary reconstruction and the biomechanical aspects of chewing. Two extant populations of the grey wolf (Canis lupus), one from Alaska and one from Sweden, were analysed with consideration to intraspecific dietary variability related to prey size depending on geographical origin, sex and individual age as well as tooth function. Occlusal enamel facets of the upper fourth premolars, first molars and the second lower molar were analysed via three-dimensional surface texture analysis. The Swedish wolves displayed facets characterized by higher peaks and deeper, more voluminous dales, featuring an overall rougher surface than the wolves from Alaska. Compared to females, the Swedish male wolves had a slightly larger dale area and hill volume on their facets. Upper fourth premolars are smoother and had higher values in texture direction compared to upper first molars. The upper first molars were rougher than the occluding lower second molars and were characterized by larger and deeper dales. We find evidence supporting intraspecific dietary segregation, and antagonistic asymmetry in occlusal wear signatures. The data offer new insights into the roles of apex predators like the grey wolf.

FEP Augmentation as a Means to Solve Data Paucity Problems for Machine Learning in Chemical Biology.

In the realm of medicinal chemistry, the primary objective is to swiftly optimize a multitude of chemical properties of a set of compounds to yield a clinical candidate poised for clinical trials. In recent years, two computational techniques, machine learning (ML) and physics-based methods, have evolved substantially and are now frequently incorporated into the medicinal chemist's toolbox to enhance the efficiency of both hit optimization and candidate design. Both computational methods come with their own set of limitations, and they are often used independently of each other. ML's capability to screen extensive compound libraries expediently is tempered by its reliance on quality data, which can be scarce especially during early-stage optimization. Contrarily, physics-based approaches like free energy perturbation (FEP) are frequently constrained by low throughput and high cost by comparison; however, physics-based methods are capable of making highly accurate binding affinity predictions. In this study, we harnessed the strength of FEP to overcome data paucity in ML by generating virtual activity data sets which then inform the training of algorithms. Here, we show that ML algorithms trained with an FEP-augmented data set could achieve comparable predictive accuracy to data sets trained on experimental data from biological assays. Throughout the paper, we emphasize key mechanistic considerations that must be taken into account when aiming to augment data sets and lay the groundwork for successful implementation. Ultimately, the study advocates for the synergy of physics-based methods and ML to expedite the lead optimization process. We believe that the physics-based augmentation of ML will significantly benefit drug discovery, as these techniques continue to evolve.

Osteo-pathological analysis provides evidence for a survived historical ship strike in a Southern Hemisphere fin whale (Balaenoptera physalus).

The life history of a fin whale (Balaenoptera physalus) caught during whaling operations in the 1950s was partly reconstructed. 3D surface models of the bones of the skeleton curated at the Zoological Museum of Hamburg were used for an osteopathological analysis. The skeleton revealed multiple healed fractures of ribs and a scapula. Moreover, the processus spinosi of several vertebrae were deformed and arthrosis was found. Together, the pathological findings provide evidence for large blunt trauma and secondary effects arising from it. Reconstruction of the likely cause of events suggests collision with a ship inflicting the fractures and leading to post traumatic posture damage as indicated by skeletal deformations. The injured bones had fully healed before the fin whale was killed by a whaler in the South Atlantic in 1952. This study is the first in-detail reconstruction of a historical whale-ship collision in the Southern Hemisphere, dating back to the 1940s, and the first documentation of a healed scapula fracture in a fin whale. The skeleton provides evidence for survival of a ship strike by a fin whale with severe injuries causing long-term impairment.

Post-mortem enamel surface texture alteration during taphonomic processes-do experimental approaches reflect natural phenomena?

Experimental approaches are often used to better understand the mechanisms behind and consequences of post-mortem alteration on proxies for diet reconstruction. Dental microwear texture analysis (DMTA) is such a dietary proxy, using dental wear features in extant and extinct taxa to reconstruct feeding behaviour and mechanical food properties. In fossil specimens especially, DMTA can be biased by post-mortem alteration caused by mechanical or chemical alteration of the enamel surface. Here we performed three different dental surface alteration experiments to assess the effect of common taphonomic processes by simplifying them: (1) tumbling in sediment suspension to simulate fluvial transport, (2) sandblasting to simulate mechanical erosion due to aeolian sediment transport, (3) acid etching to simulate chemical dissolution by stomach acid. For tumbling (1) we found alteration to be mainly dependent on sediment grain size fraction and that on specimens tumbled with sand fractions mainly post-mortem scratches formed on the dental surface, while specimens tumbled with a fine-gravel fraction showed post-mortem formed dales. Sandblasting (2) with loess caused only negligible alteration, however blasting with fine sand quartz particles resulted in significant destruction of enamel surfaces and formation of large post-mortem dales. Acid etching (3) using diluted hydrochloric acid solutions in concentrations similar to that of predator stomachs led to a complete etching of the whole dental surface, which did not resemble those of teeth recovered from owl pellets. The experiments resulted in post-mortem alteration comparable, but not identical to naturally occurring post-mortem alteration features. Nevertheless, this study serves as a first assessment and step towards further, more refined taphonomic experiments evaluating post-mortem alteration of dental microwear texture (DMT).

Dental wear proxy correlation in a long-term feeding experiment on sheep (Ovis aries).

Dietary reconstruction in vertebrates often relies on dental wear-based proxies. Although these proxies are widely applied, the contributions of physical and mechanical processes leading to meso- and microwear are still unclear. We tested their correlation using sheep (Ovis aries, n = 39) fed diets of varying abrasiveness for 17 months as a model. Volumetric crown tissue loss, mesowear change and dental microwear texture analysis (DMTA) were all applied to the same teeth. We hereby correlate: (i) 46 DMTA parameters with each other, for the maxillary molars (M1, M2, M3), and the second mandibular molar (m2); (ii) 10 mesowear variables to each other and to DMTA for M1, M2, M3 and m2; and (iii) volumetric crown tissue loss to mesowear and DMTA for M2. As expected, many DMTA parameters correlated strongly with each other, supporting the application of reduced parameter sets in future studies. Correlation results showed only few DMTA parameters correlated with volumetric tissue change and even less so with mesowear variables, with no correlation between mesowear and volumetric tissue change. These findings caution against interpreting DMTA and mesowear patterns in terms of actual tissue removal until these dental wear processes can be better understood at microscopic and macroscopic levels.

Dental microwear texture gradients in guinea pigs reveal that material properties of the diet affect chewing behaviour.

Dental microwear texture analysis (DMTA) is widely used for diet inferences in extant and extinct vertebrates. Often, a reference tooth position is analysed in extant specimens, while isolated teeth are lumped together in fossil datasets. It is therefore important to test whether dental microwear texture (DMT) is tooth position specific and, if so, what causes the differences in wear. Here, we present results from controlled feeding experiments with 72 guinea pigs, which received either fresh or dried natural plant diets of different phytolith content (lucerne, grass, bamboo) or pelleted diets with and without mineral abrasives (frequently encountered by herbivorous mammals in natural habitats). We tested for gradients in dental microwear texture along the upper cheek tooth row. Regardless of abrasive content, guinea pigs on pelleted diets displayed an increase in surface roughness along the tooth row, indicating that posterior tooth positions experience more wear compared with anterior teeth. Guinea pigs feedings on plants of low phytolith content and low abrasiveness (fresh and dry lucerne, fresh grass) showed almost no DMT differences between tooth positions, while individuals feeding on more abrasive plants (dry grass, fresh and dry bamboo) showed a gradient of decreasing surface roughness along the tooth row. We suggest that plant feeding involves continuous intake and comminution by grinding, resulting in posterior tooth positions mainly processing food already partly comminuted and moistened. Pelleted diets require crushing, which exerts higher loads, especially on posterior tooth positions, where bite forces are highest. These differences in chewing behaviour result in opposing wear gradients for plant versus pelleted diets.

Movement analysis of primate molar teeth under load using synchrotron X-ray microtomography.

Mammalian teeth have to sustain repetitive and high chewing loads without failure. Key to this capability is the periodontal ligament (PDL), a connective tissue containing a collagenous fibre network which connects the tooth roots to the alveolar bone socket and which allows the teeth to move when loaded. It has been suggested that rodent molars under load experience a screw-like downward motion but it remains unclear whether this movement also occurs in primates. Here we use synchroton micro-computed tomography paired with an axial loading setup to investigate the form-function relationship between tooth movement and the morphology of the PDL space in a non-human primate, the mouse lemur (Microcebus murinus). The loading behavior of both mandibular and maxillary molars showed a three-dimensional movement with translational and rotational components, which pushes the tooth into the alveolar socket. Moreover, we found a non-uniform PDL thickness distribution and a gradual increase in volumetric proportion of the periodontal vasculature from cervical to apical. Our results suggest that the PDL morphology may optimize the three-dimensional tooth movement to avoid high stresses under loading.

Distinct GluN1 and GluN2 Structural Determinants for Subunit-Selective Positive Allosteric Modulation of N-Methyl-d-aspartate Receptors.

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic ligand-gated glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system (CNS). Several neurological disorders may involve NMDAR hypofunction, which has driven therapeutic interest in positive allosteric modulators (PAMs) of NMDAR function. Here we describe modest changes to the tetrahydroisoquinoline scaffold of GluN2C/GluN2D-selective PAMs that expands activity to include GluN2A- and GluN2B-containing recombinant and synaptic NMDARs. These new analogues are distinct from GluN2C/GluN2D-selective compounds like (+)-(3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ) by virtue of their subunit selectivity, molecular determinants of action, and allosteric regulation of agonist potency. The (S)-enantiomers of two analogues (EU1180-55, EU1180-154) showed activity at NMDARs containing all subunits (GluN2A, GluN2B, GluN2C, GluN2D), whereas the (R)-enantiomers were primarily active at GluN2C- and GluN2D-containing NMDARs. Determination of the actions of enantiomers on triheteromeric receptors confirms their unique pharmacology, with greater activity of (S) enantiomers at GluN2A/GluN2D and GluN2B/GluN2D subunit combinations than (R) enantiomers. Evaluation of the (S)-EU1180-55 and EU1180-154 response of chimeric kainate/NMDA receptors revealed structural determinants of action within the pore-forming region and associated linkers. Scanning mutagenesis identified structural determinants within the GluN1 pre-M1 and M1 regions that alter the activity of (S)-EU1180-55 but not (R)-EU1180-55. By contrast, mutations in pre-M1 and M1 regions of GluN2D perturb the actions of only the (R)-EU1180-55 but not the (S) enantiomer. Molecular modeling supports the idea that the (S) and (R) enantiomers interact distinctly with GluN1 and GluN2 pre-M1 regions, suggesting that two distinct sites exist for these NMDAR PAMs, each of which has different functional effects.

Synthesis and Antiviral Activity of a Series of 2'-C-Methyl-4'-thionucleoside Monophosphate Prodrugs.

The NS5B RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is a validated target for nucleoside antiviral drug therapy. We endeavored to synthesize and test a series of 4'-thionucleosides with a monophosphate prodrug moiety for their antiviral activity against HCV and other related viruses in the Flaviviridae family. Nucleoside analogs were prepared via the stereoselective Vorbrüggen glycosylation of various nucleobases with per-acetylated 2-C-methyl-4-thio-d-ribose built in a 10-step synthetic sequence from the corresponding ribonolactone. Conjugation of the thionucleoside to a ProTide phosphoramidate allowed for evaluation of the prodrugs in the cellular HCV replicon assay with anti-HCV activities ranging from single-digit micromolar (µM) to >200 µM. The diminished anti-HCV potency of our best compound compared to its 4'-oxo congener is the subject of ongoing research in our lab and is proposed to stem from changes in sugar geometry imparted by the larger sulfur atom.

Shape, size, and quantity of ingested external abrasives influence dental microwear texture formation in guinea pigs.

Food processing wears down teeth, thus affecting tooth functionality and evolutionary success. Other than intrinsic silica phytoliths, extrinsic mineral dust/grit adhering to plants causes tooth wear in mammalian herbivores. Dental microwear texture analysis (DMTA) is widely applied to infer diet from microscopic dental wear traces. The relationship between external abrasives and dental microwear texture (DMT) formation remains elusive. Feeding experiments with sheep have shown negligible effects of dust-laden grass and browse, suggesting that intrinsic properties of plants are more important. Here, we explore the effect of clay- to sand-sized mineral abrasives (quartz, volcanic ash, loess, kaolin) on DMT in a controlled feeding experiment with guinea pigs. By adding 1, 4, 5, or 8% mineral abrasives to a pelleted base diet, we test for the effect of particle size, shape, and amount on DMT. Wear by fine-grained quartz (>5/<50 µm), loess, and kaolin is not significantly different from the abrasive-free control diet. Fine silt-sized quartz (∼5 µm) results in higher surface anisotropy and lower roughness (polishing effect). Coarse-grained volcanic ash leads to significantly higher complexity, while fine sands (130 to 166 µm) result in significantly higher roughness. Complexity and roughness values exceed those from feeding experiments with guinea pigs who received plants with different phytolith content. Our results highlight that large (>95-µm) external silicate abrasives lead to distinct microscopic wear with higher roughness and complexity than caused by mineral abrasive-free herbivorous diets. Hence, high loads of mineral dust and grit in natural diets might be identified by DMTA, also in the fossil record.

Correction to "Accelerated Discovery of Novel Ponatinib Analogs with Improved Properties for the Treatment of Parkinson's Disease".

[This corrects the article DOI: 10.1021/acsmedchemlett.9b00612.].

Chewing, dental morphology and wear in tapirs (Tapirus spp.) and a comparison of free-ranging and captive specimens.

Feeding practice in herbivorous mammals can impact their dental wear, due to excessive or irregular abrasion. Previous studies indicated that browsing species display more wear when kept in zoos compared to natural habitats. Comparable analyses in tapirs do not exist, as their dental anatomy and chewing kinematics are assumed to prevent the use of macroscopic wear proxies such as mesowear. We aimed at describing tapir chewing, dental anatomy and wear, to develop a system allowing comparison of free-ranging and captive specimens even in the absence of known age. Video analyses suggest that in contrast to other perissodactyls, tapirs have an orthal (and no lateral) chewing movement. Analysing cheek teeth from 74 museum specimens, we quantified dental anatomy, determined the sequence of dental wear along the tooth row, and established several morphometric measures of wear. In doing so, we showcase that tapir maxillary teeth distinctively change their morphology during wear, developing a height differential between less worn buccal and more worn lingual cusps, and that quantitative wear corresponds to the eruption sequence. We demonstrate that mesowear scoring shows a stable signal during initial wear stages but results in a rather high mesowear score compared to other browsing herbivores. Zoo specimens had lesser or equal mesowear scores as specimens from the wild; additionally, for the same level of third molar wear, premolars and other molars of zoo specimens showed similar or less wear compared specimens from the wild. While this might be due to the traditional use of non-roughage diet items in zoo tapirs, these results indicate that in contrast to the situation in other browsers, excessive tooth wear appears to be no relevant concern in ex situ tapir management.

Accelerated Discovery of Potent Fusion Inhibitors for Respiratory Syncytial Virus.

A series of five benzimidazole-based compounds were identified using a machine learning algorithm as potential inhibitors of the respiratory syncytial virus (RSV) fusion protein. These compounds were synthesized, and compound 2 in particular exhibited excellent in vitro potency with an EC50 value of 5 nM. This new scaffold was then further refined leading to the identification of compound 44, which exhibited a 10-fold improvement in activity with an EC50 value of 0.5 nM.

Accelerated Discovery of Novel Ponatinib Analogs with Improved Properties for the Treatment of Parkinson's Disease.

Parkinson's disease (PD) is a debilitating and common neurodegenerative disease. New insights implicating c-Abl activation as a driving force in PD have opened a new drug development avenue for PD treatment beyond the symptomatic relief by L-DOPA. BCR-Abl inhibitors, which include nilotinib and ponatinib, have been found to inhibit this process, and nilotinib has shown improvement in outcomes in a 12-patient, nonrandomized trial. However, nilotinib is a potent inhibitor of hERG, a cardiac K+ channel whose inhibition increases risk of sudden death. We used our machine learning approach to predict novel molecules that would inhibit c-Abl while also having minimal liability against hERG. Of our six novel compounds tested, we identified two that had c-Abl potencies comparable to nilotinib, but with significantly improved profiles regarding the hERG channel. Our best compound exhibited a hERG IC50 of 12.1 µM (compared to nilotinib with an IC50 of 0.45 µM and ponatinib with IC50 of 0.767 µM). This work is a step forward for a machine learning enabled, multiparameter optimization of a chemical space and represents a significant advance in the development of novel Parkinson's therapies.

Broad-scale morpho-functional traits of the mandible suggest no hard food adaptation in the hominin lineage.

An on-going debate concerning the dietary adaptations of archaic hominins and early Homo has been fuelled by contradictory inferences obtained using different methodologies. This work presents an extensive comparative sample of 30 extant primate species that was assembled to perform a morpho-functional comparison of these taxa with 12 models corresponding to eight fossil hominin species. Finite Element Analysis and Geometric Morphometrics were employed to analyse chewing biomechanics and mandible morphology to, firstly, establish the variation of this clade, secondly, relate stress and shape variables, and finally, to classify fossil individuals into broad ingesta related hardness categories using a support vector machine algorithm. Our results suggest that some hominins previously assigned as hard food consumers (e.g. the members of the Paranthropus clade) in fact seem to rely more strongly on soft foods, which is consistent with most recent studies using either microwear or stable isotope analyses. By analysing morphometric and stress results in the context of the comparative framework, we conclude that in the hominin clade there were probably no hard-food specialists. Nonetheless, the biomechanical ability to comminute harder items, if required as fallback option, adds to their strategy of increased flexibility.

Dust and grit matter: abrasives of different size lead to opposing dental microwear textures in experimentally fed sheep (Ovis aries).

External abrasives ingested along with the herbivore diet are considered main contributors to dental wear, though how the different sizes and concentrations of these abrasives influence wear remains unclear. Dental microwear texture analysis (DMTA) is an established method for dietary reconstruction which describes a tooth's surface topography on a micrometre scale. The method has yielded conflicting results as to the effect of external abrasives. In the present study, a feeding experiment was performed on sheep (Ovis aries) fed seven diets of different abrasiveness. Our aim was to discern the individual effects of size (4, 50 and 130 µm) and concentration (0%, 4% and 8% of dry matter) of abrasives on dental wear, applying DMTA to four tooth positions. Microwear textures differed between individual teeth, but surprisingly, showed no gradient along the molar tooth row, and the strongest differentiation of experimental groups was achieved when combining data of all maxillary molars. Overall, a pattern of increasing height, volume and complexity of the tooth's microscopic surface appeared with increasing size of dietary abrasives, and when compared with the control, the small abrasive diets showed a polishing effect. The results indicate that the size of dietary abrasives is more important for dental microwear texture traces than their concentration, and that different sizes can have opposing effects on the dietary signal. The latter finding possibly explains conflicting evidence from previous experimental DMTA applications. Further exploration is required to understand whether and how microscopic traces created by abrasives translate quantitatively to tissue loss.

Error Tolerance of Machine Learning Algorithms across Contemporary Biological Targets.

Machine learning continues to make strident advances in the prediction of desired properties concerning drug development. Problematically, the efficacy of machine learning in these arenas is reliant upon highly accurate and abundant data. These two limitations, high accuracy and abundance, are often taken together; however, insight into the dataset accuracy limitation of contemporary machine learning algorithms may yield insight into whether non-bench experimental sources of data may be used to generate useful machine learning models where there is a paucity of experimental data. We took highly accurate data across six kinase types, one GPCR, one polymerase, a human protease, and HIV protease, and intentionally introduced error at varying population proportions in the datasets for each target. With the generated error in the data, we explored how the retrospective accuracy of a Naïve Bayes Network, a Random Forest Model, and a Probabilistic Neural Network model decayed as a function of error. Additionally, we explored the ability of a training dataset with an error profile resembling that produced by the Free Energy Perturbation method (FEP+) to generate machine learning models with useful retrospective capabilities. The categorical error tolerance was quite high for a Naïve Bayes Network algorithm averaging 39% error in the training set required to lose predictivity on the test set. Additionally, a Random Forest tolerated a significant degree of categorical error introduced into the training set with an average error of 29% required to lose predictivity. However, we found the Probabilistic Neural Network algorithm did not tolerate as much categorical error requiring an average of 20% error to lose predictivity. Finally, we found that a Naïve Bayes Network and a Random Forest could both use datasets with an error profile resembling that of FEP+. This work demonstrates that computational methods of known error distribution like FEP+ may be useful in generating machine learning models not based on extensive and expensive in vitro-generated datasets.

Dental microwear texture reflects dietary tendencies in extant Lepidosauria despite their limited use of oral food processing.

Lepidosauria show a large diversity in dietary adaptations, both among extant and extinct tetrapods. Unlike mammals, Lepidosauria do not engage in sophisticated mastication of their food and most species have continuous tooth replacement, further reducing the wear of individual teeth. However, dietary tendency estimation of extinct lepidosaurs usually rely on tooth shape and body size, which allows only for broad distinction between faunivores and herbivores. Microscopic wear features on teeth have long been successfully applied to reconstruct the diet of mammals and allow for subtle discrimination of feeding strategies and food abrasiveness. Here, we present, to our knowledge, the first detailed analysis of dental microwear texture on extant lepidosaurs using a combination of 46 surface texture parameters to establish a framework for dietary tendency estimation of fossil reptilian taxa. We measured dental surface textures of 77 specimens, belonging to herbivorous, algaevorous, frugivorous, carnivorous, ovivorous, insectivorous, molluscivorous, as well as omnivorous species. Carnivores show low density and shallow depth of furrows, whereas frugivores are characterized by the highest density of furrows. Molluscivores show the deepest wear features and highest roughness, herbivores have lower surface roughness and shallower furrows compared to insectivores and omnivores, which overlap in all parameters. Our study shows that despite short food-tooth interaction, dental surface texture parameters enable discrimination of several feeding strategies in lepidosaurs. This result opens new research avenues to assess diet in a broad variety of extant and extinct non-mammalian taxa including dinosaurs and early synapsids.

Why ruminating ungulates chew sloppily: Biomechanics discern a phylogenetic pattern.

There is considerable debate regarding whether mandibular morphology in ungulates primarily reflects phylogenetic affinities or adaptation to specific diet. In an effort to help resolve this debate, we use three-dimensional finite element analysis (FEA) to assess the biomechanical performance of mandibles in eleven ungulate taxa with well-established but distinct dietary preferences. We found notable differences in the magnitude and the distribution of von Mises stress between Artiodactyla and Perissodactyla, with the latter displaying lower overall stress values. Additionally, within the order Artiodactyla the suborders Ruminantia and Tylopoda showed further distinctive stress patterns. Our data suggest that a strong phylogenetic signal can be detected in biomechanical performance of the ungulate mandible. In general, Perissodactyla have stiffer mandibles than Artiodactyla. This difference is more evident between Perissodactyla and ruminant species. Perissodactyla likely rely more heavily on thoroughly chewing their food upon initial ingestion, which demands higher bite forces and greater stress resistance, while ruminants shift comminution to a later state (rumination) where less mechanical effort is required by the jaw to obtain sufficient disintegration. We therefore suggest that ruminants can afford to chew sloppily regardless of ingesta, while hindgut fermenters cannot. Additionally, our data support a secondary degree of adaptation towards specific diet. We find that mandibular morphologies reflect the masticatory demands of specific ingesta within the orders Artiodactyla and Perissodactyla. Of particular note, stress patterns in the white rhinoceros (C. simum) look more like those of a general grazer than like other rhinoceros' taxa. Similarly, the camelids (Tylopoda) appear to occupy an intermediate position in the stress patterns, which reflects the more ancestral ruminating system of the Tylopoda.

Forage silica and water content control dental surface texture in guinea pigs and provide implications for dietary reconstruction.

Recent studies have shown that phytoliths are softer than dental enamel but still act as abrasive agents. Thus, phytolith content should be reflected in dental wear. Because native phytoliths show lower indentation hardness than phytoliths extracted by dry ashing, we propose that the hydration state of plant tissue will also affect dental abrasion. To assess this, we performed a controlled feeding experiment with 36 adult guinea pigs, fed exclusively with three different natural forages: lucerne, timothy grass, and bamboo with distinct phytolith/silica contents (lucerne < grass < bamboo). Each forage was fed in fresh or dried state for 3 weeks. We then performed 3D surface texture analysis (3DST) on the upper fourth premolar. Generally, enamel surface roughness increased with higher forage phytolith/silica content. Additionally, fresh and dry grass feeders displayed differences in wear patterns, with those of fresh grass feeders being similar to fresh and dry lucerne (phytolith-poor) feeders, supporting previous reports that "fresh grass grazers" show less abrasion than unspecialized grazers. Our results demonstrate that not only phytolith content but also properties such as water content can significantly affect plant abrasiveness, even to such an extent that wear patterns characteristic for dietary traits (browser-grazer differences) become indistinguishable.