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The early Eocene Okanagan Highland fossil sites of Washington (USA) and British Columbia (Canada) contain exquisitely preserved plant and insect fossils that showcase a critical time and place in the evolution of the Northern Hemisphere temperate deciduous biome. A comprehensive understanding of the dynamics of fossil deposition and preservation at these sites is not fully resolved but is critical for reliable reconstructions of these ancient forests. To expand on previous interpretations (e.g., deep, stratified, anoxic lake bottoms) and address uncertainties about the environment of deposition (e.g., distance to shore, influence of diatoms), we analyzed sediment samples from three Okanagan Highland fossil sites—McAbee, Falkland, and Driftwood Canyon—for organic biomarkers, their stable carbon isotopic compositions, and glycerol dialkyl glycerol tetraethers (GDGTs; at McAbee only). Terpenoids suggest relative trends in gymnosperm abundance between sites that agree with prior macrofossil evidence, though absolute values may overestimate local gymnosperm abundance. A combination of biomarker evidence indicates a predominantly autochthonous aquatic source (e.g., diatoms) for organic matter in shale and mudstone samples, even contributing to long chain n-alkanes and likely to branched GDGTs, which are often assumed to be terrestrially sourced. In combination with biomarker evidence for anoxia and stratification, fossiliferous shales are interpreted to have been deposited offshore in deep and mesotrophic lakes that were thermally stratified with an anoxic hypolimnion, away from in-flowing tributaries, while a coal horizon at Driftwood Canyon was deposited in a shallower, eutrophic, anoxic wetland. Anoxic conditions likely minimized some degradation-based biases and promoted high quality fossil preservation. Deposition of sediment and fossil remains offshore and away from inflowing tributaries suggest fossil plants were locally sourced but highlights the need for careful consideration of transport-induced biases.

Multimodal neuroimaging grants a powerful window into the structure and function of the human brain at multiple scales. Recent methodological and conceptual advances have enabled investigations of the interplay between large-scale spatial trends (also referred to as gradients) in brain microstructure and connectivity, offering an integrative framework to study multiscale brain organization. Here, we share a multimodal MRI dataset for Microstructure-Informed Connectomics (MICA-MICs) acquired in 50 healthy adults (23 women; 29.54 ± 5.62 years) who underwent high-resolution T1-weighted MRI, myelin-sensitive quantitative T1 relaxometry, diffusion-weighted MRI, and resting-state functional MRI at 3 Tesla. In addition to raw anonymized MRI data, this release includes brain-wide connectomes derived from (i) resting-state functional imaging, (ii) diffusion tractography, (iii) microstructure covariance analysis, and (iv) geodesic cortical distance, gathered across multiple parcellation scales. Alongside, we share large-scale gradients estimated from each modality and parcellation scale. Our dataset will facilitate future research examining the coupling between brain microstructure, connectivity, and function. MICA-MICs is available on the Canadian Open Neuroscience Platform data portal (https://portal.conp.ca) and the Open Science Framework (https://osf.io/j532r/).

The intrinsic functional organization of the brain changes into older adulthood. Age differences are observed at multiple spatial scales, from global reductions in modularity and segregation of distributed brain systems, to network-specific patterns of dedifferentiation. Whether dedifferentiation reflects an inevitable, global shift in brain function with age, circumscribed, experience-dependent changes, or both, is uncertain. We employed a multimethod strategy to interrogate dedifferentiation at multiple spatial scales. Multi-echo (ME) resting-state fMRI was collected in younger (n = 181) and older (n = 120) healthy adults. Cortical parcellation sensitive to individual variation was implemented for precision functional mapping of each participant while preserving group-level parcel and network labels. ME-fMRI processing and gradient mapping identified global and macroscale network differences. Multivariate functional connectivity methods tested for microscale, edge-level differences. Older adults had lower BOLD signal dimensionality, consistent with global network dedifferentiation. Gradients were largely age-invariant. Edge-level analyses revealed discrete, network-specific dedifferentiation patterns in older adults. Visual and somatosensory regions were more integrated within the functional connectome; default and frontoparietal control network regions showed greater connectivity; and the dorsal attention network was more integrated with heteromodal regions. These findings highlight the importance of multiscale, multimethod approaches to characterize the architecture of functional brain aging.

Early Eocene climates were globally warm, with ice-free conditions at both poles. Early Eocene polar landmasses supported extensive forest ecosystems of a primarily temperate biota but also with abundant thermophilic elements, such as crocodilians, and mesothermic taxodioid conifers and angiosperms. The globally warm early Eocene was punctuated by geologically brief hyperthermals such as the Paleocene–Eocene Thermal Maximum (PETM), culminating in the Early Eocene Climatic Optimum (EECO), during which the range of thermophilic plants such as palms extended into the Arctic. Climate models have struggled to reproduce early Eocene Arctic warm winters and high precipitation, with models invoking a variety of mechanisms, from atmospheric CO2 levels that are unsupported by proxy evidence to the role of an enhanced hydrological cycle, to reproduce winters that experienced no direct solar energy input yet remained wet and above freezing. Here, we provide new estimates of climate and compile existing paleobotanical proxy data for upland and lowland midlatitude sites in British Columbia, Canada, and northern Washington, USA, and from high-latitude lowland sites in Alaska and the Canadian Arctic to compare climatic regimes between the middle and high latitudes of the early Eocene – spanning the PETM to the EECO – in the northern half of North America. In addition, these data are used to reevaluate the latitudinal temperature gradient in North America during the early Eocene and to provide refined biome interpretations of these ancient forests based on climate and physiognomic data.

Objective: Juvenile myoclonic epilepsy (JME) is the most common genetic generalized epilepsy syndrome. Myoclonus may relate to motor system hyperexcitability and can be provoked by cognitive activities. To aid genetic mapping in complex neuropsychiatric disorders, recent research has utilized imaging intermediate phenotypes (endophenotypes). Here, we aimed to (a) characterize activation profiles of the motor system during different cognitive tasks in patients with JME and their unaffected siblings, and (b) validate those as endophenotypes of JME. / Methods: This prospective cross‐sectional investigation included 32 patients with JME, 12 unaffected siblings, and 26 controls, comparable for age, sex, handedness, language laterality, neuropsychological performance, and anxiety and depression scores. We investigated patterns of motor system activation during episodic memory encoding and verb generation functional magnetic resonance imaging (fMRI) tasks. / Results: During both tasks, patients and unaffected siblings showed increased activation of motor system areas compared to controls. Effects were more prominent during memory encoding, which entailed hand motion via joystick responses. Subgroup analyses identified stronger activation of the motor cortex in JME patients with ongoing seizures compared to seizure‐free patients. Receiver‐operating characteristic curves, based on measures of motor activation, accurately discriminated both patients with JME and their siblings from healthy controls (area under the curve: 0.75 and 0.77, for JME and a combined patient‐sibling group against controls, respectively; P < .005). / Significance: Motor system hyperactivation represents a cognitive, domain‐independent endophenotype of JME. We propose measures of motor system activation as quantitative traits for future genetic imaging studies in this syndrome.

Leaf carbon isotope fractionation (Δleaf) is sensitive to environmental conditions and can provide insights into the state and evolution of leaf gas-exchange in response to climate and environment factors. In modern plants, water availability is the strongest environmental predictor of Δleaf across sites that experience relatively uniform and low concentrations of CO2 in the atmosphere (pCO2). Growth chamber experiments show Δleaf of modern plants can also be sensitive to changing pCO2. However, over geologic time, it is uncertain how Δleaf has responded to shifts in pCO2 and precipitation. To address this problem, we collected sediment (rock) samples from fossil leaf sites that represent a range of pCO2 values from ∼200 to 900 ppmV, over 40 degrees of latitude from New Mexico to the High Arctic, and 40 million years spanning the Late Cretaceous to the Oligocene. For each site, the carbon isotope composition of atmospheric CO2 (δ13Catm), pCO2, mean annual precipitation, and mean annual temperature were constrained from independent proxies. From sediment samples, we extracted long-chain n-alkanes (biomarkers derived from plant wax). We then measured the carbon isotope ratios of sediment-derived n-C29 and n-C31 alkanes to calculate Δleaf. Results show a negative correlation between Δleaf and pCO2 even after controlling for mean annual precipitation. The Δleaf response to pCO2 is small (−0.3 ± 0.09‰/100 ppmV), suggesting plants are adjusting internal leaf CO2 concentrations to atmospheric pCO2 concentrations, likely by optimizing leaf gas-exchange to maximize carbon intake and minimize water loss in response to environmental conditions. Similar to previous studies of geologic sediments and living plants, Δleaf was also positively correlated with water availability and, to a lesser extent, sensitive to plant type and possibly altitude. As a result, the Δleaf – pCO2 relationship in the geologic past may be more complex than observed in modern studies and therefore, precludes its use as a pCO2 proxy.

Magnetic resonance spectroscopy (MRS) is a non-invasive method of exploring cerebral metabolism. In Huntington’s disease, altered MRS-determined concentrations of several metabolites have been described; however, findings are often discrepant and longitudinal studies of metabolite trajectory are lacking. MRS metabolites may represent a valuable source of biomarkers, thus their relationship with established biofluid and structural imaging markers of disease progression require further exploration to assess prognostic value and elucidate biochemical pathways associated with neurodegeneration. In a prospective single-site controlled cohort study with standardised collection of CSF, blood, phenotypic and imaging data, we used MRS to evaluate metabolic profiles in the putamen of 56 participants at baseline (15 healthy controls, 15 premanifest and 26 manifest gene expansion carriers) and at 2-year follow-up. Intergroup differences and associations with established measures were assessed cross-sectionally using generalized linear models and partial correlation, controlling for age and CAG repeat length. We report no significant groupwise differences in metabolite concentration but found several metabolites to be associated with measures of disease progression; however, only two relationships were replicated across both time points, with total Creatine (creatine + phosphocreatine) and myo-inositol displaying significant associations with reduced caudate volume. Although relationships were observed between MRS metabolites and biofluid measures, these were not consistent across time points. To further assess prognostic value of the metabolites, we examined whether baseline MRS values, or rate of change, predicted subsequent change in established measures of disease progression. Several associations were found but were inconsistent across known indicators of disease progression. Finally, longitudinal mixed effects models, controlling for age, revealed no significant change in metabolite concentration over time in gene expansion carriers. Altogether, our findings show some interesting cross-sectional associations between select metabolites, namely total creatine and myo-inositol, and markers of disease progression, potentially highlighting the proposed roles of neuroinflammation and metabolic dysfunction in disease pathogenesis. However, the absence of group differences, inconsistency between baseline and follow-up, and lack of clear longitudinal change over two years suggests that MRS metabolites have limited potential as biomarkers in Huntington’s disease.

Proton magnetic resonance spectroscopy is a non-invasive method of exploring cerebral metabolism. In Huntington’s disease, altered proton magnetic resonance spectroscopy-determined concentrations of several metabolites have been described; however, findings are often discrepant and longitudinal studies are lacking. Proton magnetic resonance spectroscopy metabolites may represent a source of biomarkers, thus their relationship with established markers of disease progression require further exploration to assess prognostic value and elucidate pathways associated with neurodegeneration. In a prospective single-site controlled cohort study with standardized collection of CSF, blood, phenotypic and volumetric imaging data, we used 3 T proton magnetic resonance spectroscopy in conjunction with the linear combination of model spectra method to quantify seven metabolites (total n-acetylaspartate, total creatine, total choline, myo-inositol, GABA, glutamate and glutathione) in the putamen of 59 participants at baseline (15 healthy controls, 15 premanifest and 29 manifest Huntington’s disease gene expansion carriers) and 48 participants at 2-year follow-up (12 healthy controls, 13 premanifest and 23 manifest Huntington’s disease gene expansion carriers). Intergroup differences in concentration and associations with CSF and plasma biomarkers; including neurofilament light chain and mutant Huntingtin, volumetric imaging markers; namely whole brain, caudate, grey matter and white matter volume, measures of disease progression and cognitive decline, were assessed cross-sectionally using generalized linear models and partial correlation. We report no significant groupwise differences in metabolite concentration at baseline but found total creatine and total n-acetylaspartate to be significantly reduced in manifest compared with premanifest participants at follow-up. Additionally, total creatine and myo-inositol displayed significant associations with reduced caudate volume across both time points in gene expansion carriers. Although relationships were observed between proton magnetic resonance spectroscopy metabolites and biofluid measures, these were not consistent across time points. To further assess prognostic value, we examined whether baseline proton magnetic resonance spectroscopy values, or rate of change, predicted subsequent change in established measures of disease progression. Several associations were found but were inconsistent across known indicators of disease progression. Finally, longitudinal mixed-effects models revealed glutamine + glutamate to display a slow linear decrease over time in gene expansion carriers. Altogether, our findings show some evidence of reduced total n-acetylaspartate and total creatine as the disease progresses and cross-sectional associations between select metabolites, namely total creatine and myo-inositol, and markers of disease progression, potentially highlighting the proposed roles of neuroinflammation and metabolic dysfunction in disease pathogenesis. However, the absence of consistent group differences, inconsistency between baseline and follow-up, and lack of clear longitudinal change suggests that proton magnetic resonance spectroscopy metabolites have limited potential as Huntington’s disease biomarkers.

The adult functional connectome is well characterized by a macroscale spatial gradient of connectivity traversing from unimodal toward higher-order transmodal cortices that recapitulates known principles of hierarchical organization and myelination patterns. Despite an emerging literature assessing connectome properties in neonates, the presence of connectome gradients and particularly their correspondence to microstructure remains largely unknown. We derived connectome gradients using unsupervised techniques applied to functional connectivity data from 40 term-born neonates. A series of cortex-wide analysis examined associations to magnetic resonance imaging-derived morphological parameters (cortical thickness, sulcal depth, curvature), measures of tissue microstructure (intracortical T1w/T2w intensity, superficial white matter diffusion parameters), and subcortico-cortical functional connectivity. Our findings indicate that the primary neonatal connectome gradient runs between sensorimotor and visual anchors and captures specific associations to cortical and superficial white matter microstructure as well as thalamo-cortical connectivity. A second gradient indicated an anterior-to-posterior asymmetry in macroscale connectivity alongside an immature differentiation between unimodal and transmodal areas, indicating a connectome-level circuitry en route to an adult-like organization. Our findings reveal an important coordination of structural and functional interactions in the neonatal connectome across spatial scales. Observed associations were replicable across individual neonates, suggesting consistency and generalizability.

Epilepsy is among the most common chronic neurologic disorders, with 30%-40% of patients having seizures despite antiepileptic drug treatment. The advent of brain imaging and network analyses has greatly improved the understanding of this condition. In particular, developments in magnetic resonance imaging (MRI) have provided measures for the noninvasive characterization and detection of lesions causing epilepsy. MRI techniques can probe structural and functional connectivity, and network analyses have shaped our understanding of whole-brain anomalies associated with focal epilepsies. This review considers the progress made by neuroimaging and connectomics in the study of drug-resistant epilepsies due to focal substrates, particularly temporal lobe epilepsy related to mesiotemporal sclerosis and extratemporal lobe epilepsies associated with malformations of cortical development. In these disorders, there is evidence of widespread disturbances of structural and functional connectivity that may contribute to the clinical and cognitive prognosis of individual patients. It is hoped that studying the interplay between macroscale network anomalies and lesional profiles will improve our understanding of focal epilepsies and assist treatment choices.

The lower Eocene McAbee fossil beds (∼53 Ma), in south-central British Columbia, Canada, represent a lacustrine sequence deposited during a time of pervasive regional volcanism. Previous studies on fossil assemblages at the McAbee fossil beds consist of amalgamated collections of plants from several disjunct and stratigraphically unconstrained exposures and horizons, with limited knowledge of the spatio-temporal variation in depositional and taphonomic setting. This study presents a high-resolution lithostratigraphic analysis of the McAbee main site to provide stratigraphic, paleoenvironmental, and taphonomic context to fossil collections. A lithostratigraphic framework was developed for the McAbee main site by correlating tuff marker beds. The sequence was divided into eight lithostratigraphic units on the basis of systematic lithologic trends, a result of varying degrees of volcanic influences. An absence of shallow water indicators, bioturbation, and evidence for fluvio–deltaic influence, and the nonrestricted presence of highly abundant and diverse well-preserved plant fossils indicates a deep water, yet relatively near shore facies, suggesting steep sided lake margins. This taphonomic regime imparts minimal transport- and degradation-induced biases in fossil plant assemblages and suggests plant fossils represent local vegetation growing near the point of deposition. The new lithostratigraphic framework coupled with a refined understanding of depositional setting and taphonomic regime demonstrates the opportunity to document conditions of forest ecology within a dynamic volcanic environment over millennial and multi-millennial time scales.

I.AbstractAging is characterised by accumulation of structural and metabolic changes in the brain. Recent studies suggest transmodal brain networks are especially sensitive to aging, which, we hypothesise, may be due to their apical position in the cortical hierarchy. Studying an open-access healthy cohort (n=102, age range = 30-89 years) with MRI and Aβ PET data, we estimated age-related cortical thinning, hippocampal atrophy and Aβ deposition. In addition to carrying out surface-based morphological and metabolic mapping, we stratified effects along neocortical and hippocampal resting-state functional connectome gradients derived from independent datasets. The cortical gradient depicts an axis of functional differentiation from sensory-motor regions to transmodal regions, whereas the hippocampal gradient recapitulates its long-axis. While age-related thinning and increased Aβ deposition occurred across the entire cortical topography, increased Aβ deposition was especially pronounced towards higher-order transmodal regions. Age-related atrophy was greater towards the posterior end of the hippocampal long-axis. No significant effect of age on Aβ deposition in the hippocampus was observed. Imaging markers correlated with behavioural measures of fluid intelligence and episodic memory in a topography-specific manner. Our results strengthen existing evidence of structural and metabolic change in the aging brain and support the use of connectivity gradients as a compact framework to analyse and conceptualize brain-based biomarkers of aging.

Plant wax n-alkanes are valuable paleoclimate proxies because their carbon (δ13C) and hydrogen (δ2H) isotopes track biological and environmental processes. Angiosperms produce higher concentrations of n-alkanes than conifers, with some exceptions. Vegetation source is significant because in similar climates, both taxa produce n-alkanes with unique δ13C and δ2H values due to different physiological strategies. To test whether conifers contribute significantly to sediment n-alkanes and result in distinctive isotopic signatures, we collected sediment samples from a suite of Paleogene paleobotanical sites in North America with high and low conifer abundances. To disentangle the source of sediment n-alkanes, we measured the δ13C values of nonsteroidal triterpenoids (angiosperm biomarkers) and tricyclic diterpenoids (conifer biomarkers) to determine angiosperm and conifer end member δ13C values. We then compared these end member values to n-alkane δ13C values for each site to estimate their major taxon sources. At sites dominated by conifer macrofossils, δ13C values of n-alkanes indicate a conifer source. At mixed conifer and angiosperm sites, conifer contributions increased with increasing n-alkane chain length. At sites where conifers were not as abundant as angiosperms, the δ13C values of n-alkanes indicate a predominant angiosperm source with some sites showing a conifer contribution to n-C33 and n-C35 alkanes. This suggests that conifers in the Paleogene contributed to longer chain n-alkanes (n-C33 and n-C35) even when not the dominant taxa, but this likely differs for other geographic locations and taxa. This new approach allows unique floral information to be extracted when chain length is carefully considered in the absence of other paleobotanical data and necessitates having some paleovegetation constraints when interpreting carbon and hydrogen isotopes of plant wax-derived n-alkanes.

Molecular markers derived from cerebrospinal fluid (CSF) represent an accessible means of exploring the pathobiology of Huntington’s disease (HD) in vivo. The endo-lysosomal/autophagy system is dysfunctional in HD, potentially contributing to disease pathogenesis and representing a potential target for therapeutic intervention. Several endo-lysosomal proteins have shown promise as biomarkers in other neurodegenerative diseases; however, they have yet to be fully explored in HD. We performed parallel reaction monitoring mass spectrometry analysis (PRM-MS) of multiple endo-lysosomal proteins in the CSF of 60 HD mutation carriers and 20 healthy controls. Using generalised linear models controlling for age and CAG, none of the 18 proteins measured displayed significant differences in concentration between HD patients and controls. This was affirmed by principal component analysis, in which no significant difference across disease stage was found in any of the three components representing lysosomal hydrolases, binding/transfer proteins and innate immune system/peripheral proteins. However, several proteins were associated with measures of disease severity and cognition: most notably amyloid precursor protein, which displayed strong correlations with composite Unified Huntington’s Disease Rating Scale, UHDRS Total Functional Capacity, UHDRS Total Motor Score, Symbol Digit Modalities Test and Stroop Word Reading. We conclude that although endo-lysosomal proteins are unlikely to have value as disease state CSF biomarkers for Huntington’s disease, several proteins demonstrate associations with clinical severity, thus warranting further, targeted exploration and validation in larger, longitudinal samples.

Insular cortex is a core hub involved in multiple cognitive and socio-affective processes. Yet, the anatomical mechanisms that explain how it is involved in such a diverse array of functions remain incompletely understood. Here, we tested the hypothesis that changes in myeloarchitecture across the insular cortex explain how it can be involved in many different facets of cognitive function. Detailed intracortical profiling, performed across hundreds of insular locations on the basis of myelin-sensitive magnetic resonance imaging (MRI), was compressed into a lower-dimensional space uncovering principal axes of myeloarchitectonic variation. Leveraging two datasets with different high-resolution MRI contrasts, we obtained robust support for two principal dimensions of insular myeloarchitectonic differentiation in vivo, one running from ventral anterior to posterior banks and one radiating from dorsal anterior towards both ventral anterior and posterior subregions. Analyses of post mortem 3D histological data showed that the antero-posterior axis was mirrored in cytoarchitectural markers, even when controlling for sulco-gyral folding. Resting-state functional connectomics in the same individuals and ad hoc meta-analyses showed that myelin gradients in the insula relate to diverse affiliation to macroscale intrinsic functional systems, showing differential shifts in functional network embedding across each myelin-derived gradient. Collectively, our findings offer a novel approach to capture structure-function interactions of a key node of the limbic system, and suggest a multidimensional structural basis underlying the diverse functional roles of the insula.

The uppermost Eocene Florissant Formation of central Colorado, U.S.A. contains a diverse flora and fauna preserved in lacustrine facies and represents a key episode in Earth history immediately preceding the Eocene-Oligocene boundary. Laminated shales contain impressions of non-monocot angiosperm leaves that were used to estimate paleoecological and paleoclimatic parameters using leaf physiognomic methods including: leaf mass per area (MA), digital leaf physiognomy (DiLP), leaf margin analysis (LMA), and leaf area analysis (LAA). The majority (58%) of the morphotypes analyzed for MA suggested a semi-evergreen leaf lifespan, whereas another 27% indicated a deciduous habit and 15% an evergreen habit. There was no significant relationship between MA and insect damage based on a small subset of Florissant's leaves. Higher MA values (~73% of leaves ≥ one-year lifespan), coupled with a tendency toward long and narrow leaf shapes and small leaf areas, indicate the existence of sclerophyllous vegetation. Using the global regression for mean annual temperature (MAT), the DiLP estimate of MAT was anomalously cold: 5.5 ± 4 °C. However, using a Northern Hemisphere regression the DiLP MAT estimate of 11.6 ± 3.3 °C was more plausible. Using DiLP, mean annual precipitation (MAP) was estimated at 740 + 608/−334 mm∙yr−1, which supports dry conditions. Estimates for MAT and MAP using the univariate LMA and LAA methods overlapped within uncertainty of the DiLP results. In addition, those taxa classified as growing in wet areas (riparian) had significantly more teeth than non-riparian taxa. These paleoclimatic and paleoecological results suggest that outside the riparian forest, the Florissant flora sampled a seasonally dry temperate sclerophyllous shrubland to woodland, perhaps similar to modern chaparral forests, in the western interior of the U.S.A. just before the transition into the cooler Oligocene.

Ammonium nitrate (AN) based fertilizers are inexpensive and easily obtained, characteristics that often lead to their use in homemade explosive devices. The stable nitrogen and oxygen isotope ratios ( 15 N/ 14 N and 18 O/ 16 O, expressed as δ 15 N and δ 18 O) of AN have the potential to aid in forensic investigations by providing supplemental properties for sample-to-sample comparison in materials which are otherwise chemically identical. The forensic utility of stable isotope analyses depends on demonstrated variation between different sources and minimal variation within a source. To test the variability within a single manufacturer (here considered a source), a total of 26 samples representing two production time periods and two product lines were analyzed for bulk δ 15 N and δ 18 O. Additionally, because AN is known to have a modest isotopic range, a potassium nitrate precipitation method was developed to separate the component ions (NO 3 - and NH 4 + ) for individual δ 15 N analysis and increased discriminatory power. The average δ 15 N and δ 18 O of bulk AN (− 0.10‰ and + 22.8‰, respectively) is similar to the isotopic signature of atmospheric N 2 and O 2 , the starting reactants in AN production. The bulk δ 15 N, δ 18 O, and NO 3 - δ 15 N show average values from both product lines that differ by 1.5‰, 2.0‰, and 2.6‰, respectively, between the production periods of June and November 2015. Conversely, the NH 4 + δ 15 N remained relatively consistent over time. Furthermore, whereas samples in the two product lines produced on the same day in June are isotopically similar, there are isotopic differences between samples in the two product lines manufactured within 6 h of each other in November. The observed variability could be useful in comparing AN from two or more bombs, or a bomb and a stash of AN in a suspect's possession, but the observed lot-to-lot differences within one manufacturer could complicate attribution efforts. In contrast, the NH 4 + δ 15 N values, which appear to be the most consistent over time within this factory, need to be further explored as a potentially reliable signal.