Ancient fluvial deposits typically display repetitive changes in their depositional architecture such as alternating intervals of coarse-grained highly amalgamated (HA), laterally-stacked, channel bodies, and finer-grained less amalgamated (LA), vertically-stacked, channels encased in floodplain deposits. Such patterns are usually ascribed to slower, respectively higher, rates of base level rise (accommodation). However, “upstream” factors such as water discharge and sediment flux also play a potential role in determining stratigraphic architecture, yet this possibility has never been tested despite the recent advances in the field of palaeohydraulic reconstructions from fluvial accumulations. Here, we chronicle riverbed gradient evolution within three Middle Eocene (~ 40 Ma) fluvial HA-LA sequences in the Escanilla Formation in the south-Pyrenean foreland basin. This work documents, for the first time in a fossil fluvial system, how the ancient riverbed systematically evolved from lower slopes in coarser-grained HA intervals, and higher slopes in finer-grained LA intervals, suggesting that bed slope changes were determined primarily by climate-controlled water discharge variations rather than base level changes as often hypothesized. This highlights the important connection between climate and landscape evolution and has fundamental implications for our ability to reconstruct ancient hydroclimates from the interpretation of fluvial sedimentary sequences. In theory, both “downstream” i.e., base level changes which can be relative sea-level representing the joint effect of eustasy and tectonics , or a stratigraphic reference level above which sub-aerial erosion prevails4, and “upstream” factors i.e., sediment flux (syn. Sediment transport), sediment size, and water discharge , have been recognised for their ability to determine patterns of channel-floodplain sequential arrangements10. For instance, in the downstream sectors of a fluvial system, the historical approach involving stratigraphic base level11 considers the interplay of two rates—the rate of change of accommodation space, hereafter referred to as A, i.e., the space available for sedimentation, and the rate of sediment supply, hereafter referred to as S. In this view, the resulting balance in the form of A/S is the predominant factor controlling sediment depositional architecture . In practice, changes in the stacking pattern of alluvial/fluvial conglomerates and sandstone are often interpreted as changes in A/S  while vertically stacked strata are interpreted as being deposited under high A/S  . Similarly, Wright & Marriott7 in their sequence stratigraphic model proposed the deposition of multistorey sand bodies under low A, while vertically stacked isolated channels encased into thick floodplain deposits would form during periods of increasing A. Although the role of S in sequence stratigraphy is widely acknowledged14, sequence stratigraphic interpretations are often based on the primacy of base level controlled A, due in part to independent knowledge of sea-level variations  intervals being the dominant landform deposited under high water discharge conditions, while high gradient low amalgamation (LA) intervals would be dominant under low water discharge context , flow velocity, water discharge and sediment flux, across several HA-LA stratigraphic cycles. Palaeohydraulic estimates show a systematic increase (resp. decrease) in discharge and sedimentary fluxes during HA (resp. LA) intervals, thereby pointing towards an upstream-driven climate control on water discharge in channels and stratigraphic architecture, that we discuss in relation to Earth’s orbital cycles.Yet, to our knowledge, the evolution of river slope across fluvial sequences in relation to documented cyclical changes in stratigraphic architecture has never been explored. In this work, we address this problem using the well-documented Middle Eocene Escanilla Formation in Spain36. The Escanilla Formation was mainly sourced from the Pyrenean axial zone through large valleys filled with transverse alluvial fans, such as the fan system of the Sis palaeovalley37 and the Gurb escarpment38 further east  package which can be defined as a thick (5–12 m) and laterally extensive (~ 600–2000 m) channel-body complex (90% channel to 10% floodplain) typically consisting of multiple stories 1–4), and a Low Amalgamation (LA) interval which can be defined as a floodplain dominated (30% channel to 70% floodplain) interval with isolated channel bodies that are 2 – 4 m thick and approximately 100 – 500 m laterally extensive and typically consist of a single storey, although multiple stories can be found as well. On the field, the thickness of each such fining-upward sequence is around 35 – 45 m and a HA interval typically lies above a thick (~ 5 – 7 m) floodplain from the ‘LA’ interval of the underlying sequence . It is noteworthy that the bulk grain size of LA intervals would be significantly lower if the fine-grained sediments of the floodplain were taken into consideration .Coarse grain size fraction (> 4 mm) Fig.  was meast test on bankfull depth data rejects the null hypotheses that HA and LA intervals have the same average values at the 95% confidence level .Bankfull depths based on preserved storey thickness Fig.  reveal a31 , equivalent to 0.03°, while 11 storeys within LA stratigraphic intervals (103 grain size sampling stations and 49 channel depth estimates) have a palaeoslope of 8 × 10−4 ± 6 × 10−5 [m/m] (N = 11), equivalent to 0.05°, and representing a 60% increase in slope in the LA interval. A t test on palaeoslope estimates  rejects the null hypotheses that HA and LA intervals have the same average values at the 95% confidence level. It is important to remark that the data in Fig. Palaeoslope estimates, obtained using the equation proposed by Trampush et al.31 Eq. . Palaeos44. Despite excellent outcrop conditions, fully preserved channel cross-sections required to measure width in the field, are nevertheless difficult to assess due to their size and the 3D nature of the outcrops. To circumvent this limitation, we estimate channel-belt widths using the relationship proposed by Bridge & Mackey45 based on the model of Bridge & Leeder46, and compare them to width measurements of channel plug and lateral accretion deposits where preserved  and 7 ± 1 [m2 s−1] (N = 49) in LA intervals  in HA intervals, and a discharge rate of 700 ± 200 [m3 s−1] (N = 49) in the LA intervals  while LA intervals have a total discharge of 200 ± 50 [m3 s−1] (N = 49).Unit discharge  in HA intervals is found to be 11 ± 2 [m−1 s−1] (N = 45) and 2.0 ± 0.1 [kg m−1 s−1] (N = 49) for HA and LA intervals respectively , and 200 ± 20 [kg s−1] for LA intervals (0.2 ± 0.02 [m3 s−1]). This represents a 1.5-fold increase in bedload sediment flux during HA intervals  requires a, yet to be documented, physical mechanism that would account for tectonic oscillations with a periodicity of the order of 105 years to explain the observed stratigraphic changes  it is not clear how such eustatic changes would propagate upstream from the shoreline into the alluvial realm 50, (2) a eustatic control would not account for the observed channel discharge evolution, and (3) it would also imply an opposite relationship between aggradation and slope  water discharge and bedload sediment flux during HA (resp. LA) intervals Fig. . Moreoveges Fig. c, and  and associated sediment discharge. With decreasing channel discharge, the fluvial equilibrium profile would be forced towards a greater gradient , approximately around 40 Ma near the C18r to C18n transition, which corresponds roughly to the “Olson sheet”, i.e., the HA interval of sequence 2. We indeed document an increase in water discharge and sediment flux at that time, which could also correlate well with the pulse of deltaic progradation recently documented by Peris Cabré et al.64 in coeval MECO successions of the adjacent Jaca basin to the West.The suggestion that climate has an important influence on stratigraphic architecture in the Escanilla system is consistent with the work of Armitage et al.35, suggest that this cyclic control may have persisted over an extended period of more than 2.8 Ma according to current stratigraphic constraints. Cyclical oscillations of the orbital configuration of the Earth-Sun system over so-called Milankovitch periodicities constitute a plausible mechanism to account for the observed cyclic changes in water discharge in the Escanilla fluvial succession. Milankovitch cyclicity is known to have played a major role in driving climate change during Earth’s history , in the Cenozoic , and has also been invoked in several studies in the Spanish Pyrenees as an important factor controlling depositional sequences .The repetitive pattern of the three fining-upward sequences studied here points at a recurrence in the controlling forcings at their origin. Moreover, other studies in the area have described several similar sequences above and below those studied in the present work69 with the succession in Olson by matching the base  71 and the intensity of channel-forming discharge events. This scenario is also coherent with the study of Kodama et al.72 who demonstrated a systematic link between the 400 ky eccentricity maxima and increased terrigenous supply from fluvial source to coeval shallow-marine successions near Arguis in the neighbouring Jaca basin. Finally, such orbital control on stratigraphic cyclicity has also been shown to influence depositional style through control on siliciclastic sediment supply to the deep-marine Early-Middle Eocene Ainsa basin76 and may thus have been an important factor throughout an extended period of time in the South-Pyrenean foreland despite the important tectonic activity linked with mountain building at that time.To test this hypothesis, we tentatively calibrated the eccentricity model of Laskar et al.S on the stratigraphic architecture of a fluvial system, which contrasts with more classical hypotheses invoking a dominant role of accommodation A in determining fluvial stratal patterns . Second, our results suggest higher sediment fluxes during HA intervals, which is in agreement with classical sequence stratigraphic predictions in which such amalgamated packages are associated to low A/S conditions, and thus with greater down system transport of sediments to shallow-marine and deep-sea environments. Third, this work is also fundamental for our ability to reconstruct ancient hydroclimates from the sedimentary record and compare it to numerical model predicted response of the hydrologic cycle to warming conditions. For instance, modelling studies of the Palaeocene–Eocene Thermal Maximum (PETM) have suggested an intensification of the hydrological cycle on a global scale in response to greenhouse gas levels77. Results from this study are also consistent with the findings of Barefoot et al.63 where higher seasonality during the PETM increased lateral channel mobility. During the Middle Eocene greenhouse conditions of the Escanilla succession, HA intervals therefore record periods of increased lateral channel mobility linked with channel-forming discharge events (floods) of higher magnitude than LA intervals which present lower lateral channel mobility (lower magnitude channel-forming discharge events).These findings have several fundamental implications for classical sequence stratigraphic predictions, sedimentary landscape evolution over geological timescales, the prediction of ancient hydroclimates during greenhouse forcing, and for industrial applications in resource exploration. First, our findings illustrate the dominant role of the sediment supply The framework of palaeoslope and palaeohydrological reconstruction across three fining-upward sequences in the Middle Eocene aged Escanilla Formation, for the first-time, documents lower river slope during higher water discharge and sedimentary flux with the deposition of river channels having a high degree of amalgamation, and higher slope during lower water discharge and sedimentary flux with the deposition of river channels having a low degree of amalgamation. The studied fining-upward sequences together with our finding of cyclic variations in water discharge and sediment flux represents a major paradigm shift by suggesting climate may have controlled the entire sedimentary landscape evolution during the Middle Eocene greenhouse conditions instead of eustatic variations, and with palaeohydraulic reconstructions to test different options without the need of an independent eustatic curve.Palaeohydrological field data were collected sequence-wise in a lateral spatial domain and from the respective HA and LA intervals. Data from channel fill deposits included channel basal gravels for grain size distribution and storey thicknesses as flow depth estimates. This data along with uncertainties associated with individual measurements were propagated through a quantitative framework to reconstruct hydrological parameters such as flow depths, palaeoslope, flow velocities, water discharge rates, bedload sediment flux. A Google Earth panel with the location of sampling stations has also been provided in the 78. The longest axis was measured as a proxy for the intermediate b axis on 100–200 grains per sampling station. The procedure was performed on photographs taken with a Canon EOS 2000D camera of 24.1 Mpixels resolution on an outcrop area of 1 × 1 m2. Grains were measured at the nodal intersection of a virtual grid such that a repeat count of grains is avoided. Measurements were made using ImageJ2 version 2.3.0/1.53f. The data obtained is normalized using the (psi) scale, a logarithmic scale with base two, to perform statistical analyses and obtain the 50th percentile (D50) of the grain size distribution.Grain size measurements were collected using the Wolman sampling procedure79 and Kelly48. It is important to note that while preserved thicknesses are lower than the original flow depths, preserved thicknesses do not severely underestimate the original depth80.Flow depth estimates are based on preserved storey thicknesses, channel-plug, and bar-scale clinoform heights measured using a laser range finder (TruPulse model 200) and following the procedure outlined in Mohrig et al.31 , median grain size  while widths from lateral accretion deposits was estimated using the procedure outlined in Greenberg et al.44.Channel-belt width W can be estimated using empirical scaling relations when direct measurements are not possible on the field. We estimate channel-belt widths using the relationship, W = 8.8HFlow velocity, U was calculated using Manning’s equation Eq.  where n Total water discharge sing Eq. 3\documenFor unit water discharge, W = 1.s was calculated using the Meyer-Peter and Muller equation  was estimated as ‘(nx + ny) − 2’ where ‘nx’ and ‘ny’ are the lengths of the two independent parameters. Power analysis of t tests was performed, using ‘pingouin.power_ttest2n’, to detect Type II errors. Pingouin is an open-source package written in Python 381.Uncertainty on results reported in this study consist of the standard error of the mean (SE) calculated as Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.


Our findings reveal distinctively higher seawater 234U/238U values from 15,400 to 14,000 years ago, corresponding to the period of the highest iceberg-rafted debris flux and the occurrence of the meltwater pulse 1A event. This correlation suggests a causal link between enhanced subglacial discharge, synchronous retreat of the AIS, and the rapid rise in sea levels. The enhanced subglacial discharge and subsequent AIS retreat appear to have been preconditioned by a stronger and warmer Circumpolar Deep Water, thus underscoring the critical role of oceanic heat in driving major ice-sheet retreat.Subglacial discharge from the Antarctic Ice Sheet (AIS) likely played a crucial role in the loss of the ice sheet and the subsequent rise in sea level during the last deglaciation. However, no direct proxy is currently available to document subglacial discharge from the AIS, which leaves significant gaps in our understanding of the complex interactions between subglacial discharge and ice-sheet stability. Here we present deep-sea coral  This study presents seawater uranium isotope records based on deep-sea corals from the Drake Passage to track subglacial discharge from the Antarctic Ice Sheet, demonstrating a causal link between enhanced subglacial discharge, retreat of the ice sheet, and the rapid rise in sea levels. Due to the relatively mobile nature of 234U induced by α-recoil effects3, 234U is preferentially released and transported to the ocean via riverine input5, resulting in an enrichment of 234U relative to 238U in modern seawater (δ234U = 146.8‰)6. Within debris-laden basal ice and subglacial sediments, however, recoil rejection of 234U is maintained in either basal ice or subglacial waters, thus leading to a 234U-enriched reservoir beneath the ice sheets2. For example, lake waters derived from the melting of glaciers in the McMurdo Dry Valleys, East Antarctica, have been found to exhibit δ234U values exceeding 4000‰7. High δ234U values of a similar magnitude have also been observed in chemical precipitates formed in subglacial aquatic environments in East Antarctica2. These observations collectively suggest that 234U-enrichment is likely a prevalent characteristic of subglacial water beneath the Antarctic Ice Sheet (AIS).The uranium isotopic  encircling the Antarctic continent implies that any seawater δ234U anomaly caused by subglacial discharge from the AIS would quickly disperse in the Southern Ocean9 11. However, the reliability of this method faces two primary challenges linked to the preservation of fossil coral samples. Firstly, potential post-mortem diagenetic alteration of the coral skeleton can lead to open-system behavior within U-series isotopes, resulting in biased dates and δ234Ui values12. One common approach to identify altered fossil deep-sea coral samples involves comparing their δ234Ui with the modern seawater value. This relies on the assumption that seawater δ234U remains relatively stable over deglacial timescales (~10 thousand years (kyr)) due to the long residence time of U in seawater (~400 kyr)14. However, a growing body of research suggests that seawater δ234U is not constant, exhibiting discernible glacial-interglacial cycles17, with lower δ234U during the glacial period than the interglacial period, and resolvable inter-basin difference during the last deglaciation1. It is therefore important to deconvolve the relative contributions of diagenetic overprinting versus seawater variations to obtain a reliable Southern Ocean seawater δ234U record, which requires high sample density within the target age interval. Secondly, U-Th dating of deep-sea corals is complicated by the need to correct for initial 230Th, introduced by either detrital silicates or Fe-Mn oxides with uncertain 230Th/232Th activity ratios. The initial 230Th correction, based on measured 232Th contents, has a significant impact on the final age results, although its influence on the δ234Ui values is somewhat muted  shallow Sars Seamount (647–981 m), Interim Seamount (1064–1196 m), and SFZ (806–823 m) that are located in the core region predominantly influenced by eastward ACC are most likely to record any δ234U anomaly caused by enhanced subglacial discharge originating from the Antarctic Peninsula and the Amundsen Sea; (II) deep Sars Seamount (1662–1701 m), deep Cape Horn (1214–1877 m), and deep Burdwood Bank (1419–1879 m) that are today bathed by Circumpolar Deep Water (CDW) may help to discern the δ234U signal originating from other ocean basins; (III) shallow Cape Horn (450–1012 m) and shallow Burdwood Bank (316–894 m) to the north can be used to assess whether the observed δ234U anomalies are related to discharge from the neighboring South American continent. We analyzed 38 new samples from Sars Seamount 695–981 m, and integrated these data with 335 existing U-Th measurements23 to investigate the presence of δ234U anomalies in the Southern Ocean at the highest possible temporal resolution. Benefiting from our high sample density, deep-sea coral samples that likely experienced diagenetic alternations were excluded  (Cape Horn and Burdwood Bank), one site aligning with the PF (Sars Seamount), and two sites to the south of the PF (Interim Seamount and Shackleton Fracture Zone (SFZ)), were precisely dated by isotope-dilution U-Th disequilibrium , all of which exhibit a comparable degree of variability and follow a common temporal trend  but δ234Ui differences up to 3‰ ) from different ocean basins, which exhibit a maximum difference of ~2.6‰ although their averaged values agree well with modern seawater δ234U  to a value of ~148.9‰ during the early Holocene (~10 ka), followed by a gradual decreasing trend towards a modern value of 146.8‰  , this overall deglacial trend in seawater δ234U closely aligns with previous studies26. The deglacial increase of seawater δ234U reflects the input of excess 234U relative to 238U to the global ocean, which has been linked to the intensified physical weathering resulting from the rapid retreat of ice sheets during the last deglaciation16. However, the subsequent Holocene drop of ~2‰ suggests that the decay of 234U and the gradual mixing with lower-δ234U water masses has surpassed the input flux of excess 234U, which is likely related to reduced physical weathering when ice sheets stabilized during the Holocene  within the core region primarily influenced by the eastward ACC, whereas the other sites in the Drake Passage remain unaffected 29 would imply reduced influence of North Atlantic Deep Water (NADW) on Southern Ocean seawater δ234U, despite the relatively high δ234U observed in the North Atlantic1  bathing these sites today is a mixture of Atlantic-derived deep waters and recirculating Pacific waters9. Moreover, there is also no sign of high δ234U in the Pacific Ocean during this time interval   exhibit high δ234U values from ~15.4 to 14 ka. Some samples display δ234Ui values within the deglacial trend as constrained by samples from Group II and III. This may reflect the episodic nature of the subglacial discharge events from the AIS, a phenomenon also supported by modern observations31. Consequently, some short-lived corals may not have captured these transient events.Superimposed on the general deglacial trend is a remarkable spike in δ ka Fig. . Statistals Fig. , which s234U spike at the Drake Passage provides additional constraints on the possible source regions of excess 234U. Specifically, the turbulent nature of ocean circulation means that tracer anomalies tend to be mixed away over rather short distances33. Consequently, there is a maximum distance that this δ234U anomaly could still be detectable at these specific depths. To visualize this distance in the vicinity of the Drake Passage, we conducted an analysis of backward-in-time trajectories originating from the approximate locations of the deep-sea coral sites in the north and south of the Drake Passage  , the δ234U anomaly would likely be found at both the northern and southern Drake Passage sites. While a more precise determination of the exact origins of excess 234U would require far tighter constraints on regional circulation during the last deglaciation, we can reasonably conclude that the excess 234U must have originated from a region within a few thousand kilometers upstream of the Drake Passage, such as the Antarctic Peninsula or the Amundsen Sea  located ~1000 km downstream from the coral sites in the Southern Ocean39  isotope composition of marine sediments serves as a novel proxy for ice-sheet dynamics40 and has been applied to constrain the retreat history of the AIS43. However, due to the absence of a continuous Be isotope record offshore of Antarctica that covers the last deglaciation period, it is currently unfeasible to compare the deep-sea coral δ234U anomaly with a sediment Be isotope record.To investigate the potential temporal and causal connection between enhanced subglacial discharge and the retreat of the AIS during the last deglaciation, we have compared the deep-sea coral δ39 Figs. . Despite234U anomaly recorded by deep-sea corals in the Drake Passage  stands out with the highest flux of iceberg-rafted debris. Importantly, Event 6 coincides in time with the prominent high δage Fig. . The δ23 44 Fig. . Consist234U anomaly can be further divided into two distinct time intervals  that was initially documented by coral reefs from Barbados in the Caribbean Sea48 and re-constrained by the Tahiti49 and Great Barrier Reef50 sea-level records  is also documented in the Barbados sea-level records234U record with several climate proxies: the mean ocean temperature record as constrained by noble gases from ice cores60    Fig. , and an xy) Fig. . The meaord Fig. . The rap 62 Fig.  and a co 61 Fig. . This su 61 Fig. . Once in-1A Fig. .Fig. 6Sc234U record provides compelling evidence for enhanced discharge of 234U-enriched subglacial waters from the AIS during the last deglaciation. This discharge occurred synchronously with the peak in iceberg-rafted debris originating from the Weddell Sea sector of the AIS39 and MWP-1A49. These findings suggest a connection between enhanced subglacial discharge, AIS retreat, and subsequent rapid sea-level rise. We further demonstrate that stronger upwelling of warmer CDW may have preconditioned this rapid AIS retreat and the release of meltwater. Our results therefore underscore the critical role of subsurface warming and enhanced upwelling in driving AIS retreat in the past. Considering the current centennial-scale CO2-driven warming of subsurface waters63 and southward shifts and strengthening of the westerly winds in the Southern Ocean65, there is a high risk of AIS retreat by oceanic forcing in the coming centuries.In summary, our high-resolution Southern Ocean seawater δ21  and Cape Horn (57° 10′ S 66° 06′ W) to the north of the Polar Front (PF), Sars Seamount (59° 48′ S 68° 58′ W) and Interim Seamount (60° 36′ S 66° 0′ W) on the PF, and Shackleton Fracture Zone  to the south of the PF21 Fig. . Coral s21 Fig. . Samples66. Approximately 0.3 g of the coral piece was cut and the very surface part of the sample was carefully removed using a Dremel tool. Samples were then extensively cleaned with both oxidizing and reducing chemical procedures11. A 236U-229Th mixed spike was added before dissolving the mixture in 2 ml 7 mol/L Optima-grade HNO3. The mixed spike was previously calibrated to the gravimetric standards (388 ppb U solution (CRM-145) and 58 ppt 232Th solution)19. This was followed by co-precipitation of U and Th with pure iron hydroxides before isolating U and Th by passing through anion-exchange columns67. U and Th isotopes were measured and corrected by bracketing standard (U112a for U and SGS for Th) on MC-ICPMS (Neptune) at the University of Bristol. A desolvating nebulizer system (Aridus I) was employed to increase the sensitivity of U and Th. A pure 236U single spike was added to the Th fraction to correct for any drift during the peak jump between 229Th and 230Th on the secondary electron multiplier. Mass bias correction was performed using the standard-sample-bracketing (SSB) method. The accuracy and precision were monitored by regularly measuring Harwell uraninite standard (HU1) and ThB standards, which give a long-term external reproducibility of ~1‰ for 234U/238U and ~2 ‰ for 229Th/230Th, respectively. We use a modern 230Th/232Th ratio of 2 ± 2 × 10−4 (2σ) as measured in unfiltered seawater collected at depths close to the dredge sites for the initial 230Th correction68. Because a relatively large uncertainty in the modern-day 230Th/232Th atomic ratio was applied to correct the initial 230Th, the final age uncertainties strongly depend on the 232Th concentrations. All errors associated with the blank correction, mass bias correction, and initial 230Th correction were propagated with a Monte Carlo method21.The protocol for isotope-dilution U-series dating of the fossil deep-sea corals follows the previously established method234U values due to the differences between individual aliquots of the secular equilibrium standard (HU1) and between HU1 and the gravimetric standards69. Since the mixed U-Th spikes used in this study were calibrated to gravimetric standards with older decay constants11, it becomes necessary to update the decay constant values for both 234U and 230Th to make comparisons with more recent dataset69. Nevertheless, it should be noted that these corrections for decay constants primarily affect the absolute δ234Ui values and do not impact the overall trends observed in the evolution of deep-sea coral δ234Ui. Given that all deep-sea coral δ234U data shown in this study were generated in the same lab and calculated with the same decay constants22, additional decay constant corrections were not performed here. For this reason, the δ234U values of modern seawater and recent deep-sea corals reported in this study exhibit a ~2‰ difference when compared to a recent compilation that did account for differences in decay constants and calibration standards16 . Samples were rejected if their δ234Ui values were >3‰ departure from the mean value, considering the upper limits of natural variability of deep-sea coral δ234U 73. They are evolved through time using a 4th order Runge–Kutta scheme, with a time-step of 180 min (results presented herein show no sensitivity to this time-step choice). To circumvent complexities arising from advective pathways in the presence of parameterized vertical mixing, trajectories were terminated upon intersecting with the ocean’s mixed layer. No additional diffusive parameterization for the trajectories was employed because the turbulent nature of the model circulation already accounts for a significant level of tracer mixing32.Trajectories were evaluated using the Ocean Parcels python packageFor the present analysis, trajectories were initialized at the approximate horizontal locations of two of the coral sites. All trajectories were initialized at 66°W; the southern (pink) trajectories were initialized between 59.5° S and 60.5° S, and the northern trajectories were initialized between 56.5° S and 57.5° S. At each site, 8000 trajectories were evenly spaced between latitudes and distributed every 10 m in the depth range of 400–2000 m, during each of the last 12 months of the simulations, for a total of 193,200 trajectories. These were run backward in time for a duration of 6 years, with their position recorded every 30 days. All trajectories were considered together in the analysis.P and Q is the square-root of the Jensen-Shannon Divergence, which is given by:M = (P + Q)/2 and KLD is the Kullback–Leiber Divergence, a measure of statistical entropy given by:The Jensen–Shannon distance between probability distributions Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Source Data


Flavivirus are characterized by a complexly folded 3′ UTR with a number of RNA structural elements that are conserved across isolates of each species. The present work provides evidence of intra- and intermolecular RNA–RNA interactions involving RNA structural elements in the 3′ UTR of the West Nile virus genome. The intermolecular interactions can be visualized in vitro by the formation of molecular dimers involving the participation of at least the SLI and 3′DB elements. Certainly, the 3′ UTR of dengue virus, which lacks the SLI element, forms molecular dimers in lower quantities via a single interaction site, probably 3′DB. The functional analysis of sequence or deletion mutants revealed an inverse relationship between 3′ UTR dimerization and viral translation efficiency in cell cultures. A network of RNA–RNA interactions involving 3′ UTR structural elements might therefore exist, helping to regulate viral translation.RNA viruses rely on genomic structural elements to accomplish the functions necessary to complete the viral cycle. These elements participate in a dynamic network of RNA–RNA interactions that determine the overall folding of the RNA genome and may be responsible for the fine regulation of viral replication and translation as well as the transition between them. The genomes of members of the genus  RNA-mediated regulatory pathways have become the subject of great interest over the past two decades. The discovery of the importance of RNA–RNA interactions in the control of gene expression has allowed the deciphering of new information-coding systems across the entire phylogenetic spectrum, from viruses to humans. The small size of viral RNA genomes renders them important reservoirs of conserved structural elements that help in the maintenance of viral persistence without impeding adaptive fitness. These elements operate via the direct recruitment of cellular and viral protein factors and via the establishment of the inter- and intramolecular RNA–RNA interactions involved in complex and dynamic networks . For example, many viral and cellular RNAs form dimers in order to enlarge their conformational space, thus improving their plasticity and functional abilities . FurtheFlavivirus is the largest in the family Flaviviridae and comprises a large group of viruses commonly referred to as flaviviruses. These are small, enveloped, positive single-stranded RNA viruses. Several are human pathogens, including dengue (DENV), Zika (ZIKV), yellow fever (YFV) and West Nile virus (WNV), all of which belong to the mosquito-borne flavivirus (MBFV) group [The genus V) group ,3,4. TheEvidence exists that specific RNA elements in the 3′ UTR help regulate essential viral processes, including translation, replication and infectivity ,6,7, as Flaviviridae, has been shown to form genomic dimeric units mediated by a palindromic sequence motif (dimer linkage sequence (DLS)) [The hepatitis C virus (HCV), which also belongs to the family e (DLS))  located e (DLS)) . We havee (DLS)) . Thus, ge (DLS)) ,16.Here, it was hypothesized that, since HCV and flaviviruses show similar genetic organization and an essential information storage system based on discrete structural and functional units, they may also share common molecular mechanisms for the regulation of viral processes. Although no DLS has been identified in the flaviviral genome, RNA–RNA assembly may occur by means other than Watson–Crick and non-Watson–Crick base pairing. For example, base stacking and helix co-stacking are commonly used to establish RNA–RNA contacts in crowded RNA environments . Thus, uThis work provides evidence of the in vitro formation of molecular dimers in WNV and DENV, a process reliant on specific structural elements of the 3′ UTR RNA, and proposes the existence of a complex network of intra- and intermolecular contacts that needs to be balanced in order to achieve the control of translation. Together, the data presented support a plausible functional role for the phenomenon of genomic RNA dimerization in flavivirus infectious cycles.Flaviviridae and that it might operate as a regulatory mechanism.The 3′ UTR of HCV is known to dimerize in vitro ,20. Here2+. Different conformations were resolved by agarose gel electrophoresis under non-denaturing conditions. Up to four different conformers were detected at the highest tested concentrations of 3′WNV  were incubated in the presence of Mgof 3′WNV a, reflecof 3′WNV a. Data wof 3′WNV a.2+ and, to a lesser extent, by monovalent cations such as Na+ [2+ or Na+  technique  was usedBriefly, the RNA construct encompassing the whole 3′WNV was modified with NMIA under denaturing conditions. RNA molecules were then renatured and subjected to dimerization assays under optimal experimental conditions. The monomeric and dimeric populations were then separated by native agarose gel electrophoresis. Modified positions were detected via stoppages in the reverse transcription reaction. HMX scores were calculated from the differential reactivity profiles between the monomeric and dimeric conformers .HMX analysis showed the need for precise nucleotide conformations in essential structural elements of the 3′ UTR for the dimeric complex to form a,c. The RNAStructure software  was thenTaken together, these results support the existence of new RNA–RNA interactions that occur in the 3′ UTR of the WNV genome, and that these might be critical for preserving the functional interactome at the 3′ end of viral RNA.′ UTR, such as 3′DB, as detected by HMX. The participation of SL-I in the formation of intra- and intermolecular interactions is also suggested from the secondary structure prediction. To confirm the role of these elements in dimer formation, a collection of mutant constructs was generated by the sequential deletion of the autonomously folded structural elements within the 3′ UTR. These deletions were generated in both the 3′ end  3′UTR) was synthesized and subjected to dimerization assays using increasing RNA concentrations. Data were fitted to a non-linear curve, as described for the (+) strand. The results showed a low yield of dimer complex formation (~50%)  strand has been suggested to be critical for enhancing the synthesis of the (+) strand . The abin (~50%) .′ UTR regulate viral translation [′UTR-dependent protein synthesis mediated by the 3′ UTR, a new set of constructs was designed containing the 5′ UTR of WNV fused to the reporter gene encoding the FLuc protein  were attached to the 3′ end of the FLuc coding sequence to generate WNV-Mut_X constructs .As has been mentioned, evidence exists that specific RNA elements in the 3nslation . In an a protein a. Differnstructs a. All ofInterestingly, mutations in SL-I or CS1 induced a significant increase in FLuc synthesis in both cell types, an effect that was even stronger for WNV-Mut_CS1 in mosquito cells b. More i′DB element in WNV translation. Its deletion reduced WNV translation efficiency by up to 50%  . From aynthesis . Taken tnd 3′DB) , leading′ UTR of certain MBFVs. In WNV, this occurs at two different anchoring sites with different affinities and could overlap with the establishment of previously unreported, theoretical intramolecular interactions predicted with RNAStructure  was generated by PCR using the primers T7p3′WNV and as3′WNV-BamHI from the modified NY99-flamingo382–99 strain [′WNV oligonucleotide contained the promoter sequence of T7 RNA polymerase after the EcoRI site. The resulting amplification product was cloned into EcoRI-BamHI sites of the pUC19 vector to generate pUC3′WNV. The latter was digested using the endonuclease BamHI to generate the template for subsequent 3′WNV RNA synthesis.The plasmid pUC39 strain , a kind ′ UTRs of DENV (NC_001474.2) and YFV (KF769016.1), fused to the T7 promoter sequence, were purchased from IDT . These constructs were introduced into the plasmid pUC19, which was first linearized by BamHI using the In-Fusion® HD Cloning Kit , to generate pUC3′DENV and pUC3′YFV. These plasmids were linearized with the enzyme SalI to produce templates for transcription.The DNA fragments coding for the 3′WNV were generated by PCR amplification from the pUC3′WNV vector. For 3′ deletions, the T7p3′WNV oligonucleotide was used as a 5′ primer for all constructs, while the 3′ primers were as follows: asWNV-10950 to generate DNA T7pMut1_3′, asWNV-10904 for DNA T7pMut2_3′, asWNV-10830 for DNA T7pMut3_3′, asWNV-10740 for DNA T7pMut4_3′, and asWNV-10660 for DNA T7pMut5_3′. To generate different DNA constructs bearing 5′ deletions, the 3′ primer asWNV-BamH1 was always used, while the 5′ oligonucleotides used were as follows: T7pWNV-10500 to yield DNA T7pMut1_5′, T7pWNV-10586 for DNA T7pMut2_5′, T7pWNV-10659 for DNA T7pMut3_5′, T7pWNV-10743 for DNA T7pMut4_5′, and T7pWNV-10831 for DNA T7pMut1_5′. The resulting amplicons were used as DNA templates for RNA synthesis (see below).DNAs encoding sequential deletions in the 3′WNV was generated by PCR using the oligonucleotides rc3′ WNV and T7 GGG- asrc 3′WNV, with pUC3′WNV as a DNA template. The amplification product carried the T7 promoter sequence fused to the 5′ end of the first 625 nt of the negative strand of the WNV genome, i.e., the complementary sequence of the 3′ UTR. This DNA was used for subsequent in vitro transcription (see below).The DNA coding for (-) 3All of the above-mentioned DNAs were used as templates for the synthesis of their respective constructs by in vitro transcription using T7 RNA polymerase ver.2.0 (Takara). RNAs were purified, and their quality was analyzed as previously described .′WNV (pUC3′WNV-Flip series) or pGLWNV (pGLWNV-Flip series) [′ UTR of the NY99-flamingo382–99 WNV strain and the 3′ UTR of WNV with the indicated Flip mutations. These constructs were designed to allow fluc translation from the AUG of the 5′ UTR of WNV.DNA plasmids carrying “Flip” mutations derived from the parental pUC3 series)  vectors ′WNV was used as a template for site-directed mutagenesis to generate two constructs: Mut-SLI_GNRA, which substitutes the apical loop of SL-I for a stable GNRA tetraloop, and Mut_delSt1, which does not have the apical portion of the stem of SL-I. Mutations were introduced using the appropriate primers and employing the Phusion™ Site-Directed Mutagenesis Kit (Thermo Fisher Scientific).pUC3′WNV were synthesized by in vitro transcription from the corresponding BamH1-linearized plasmids, as described above. The RNAs derived from the pGLWNV-Flip series bearing the 5′ cap were obtained using the HighYield T7 RNA Synthesis Kit  following the manufacturer’s instructions [RNA constructs derived from pUC3ructions . The synructions . RNAs we2) [w/v) in TBM buffer  [max1)/(Kd1 + x)) + ((Bmax2)/(Kd2 + x)), where y is the percentage of dimeric conformers, Bmaxn is the maximum yield of the reaction, x is the concentration of 3′WNV RNA, and Kdn the concentration of RNA required to achieve the maximum dimer percentage.RNA dimer formation was performed essentially as previously described . Briefly2)  for 30 mM MgCl2)  at 4 °C 2+ requirements, the reaction buffer included EDTA  and increasing concentrations of Mg2+ , while for monitoring Na+ requirements, monovalent cations were added to the reaction buffer  in the range 2–200 mM .Ionic requirements were studied via dimerization assays performed as above but with appropriate reaction buffers. Thus, to test Mg260). Modified RNAs were subjected to dimerization assays as described above. Complexes were also resolved as described above. The monomeric and dimeric conformers were gel-purified and used as templates in a primer extension reaction with the appropriate fluorescently labeled oligonucleotide to detect NMIA modifications by capillary electrophoresis, as previously described [HMX analyses were performed as previously described with minor modifications . Brieflyescribed . Electroescribed . HMX scoescribed ,22.® by LifeTechnologies™, Invitrogen, Waltham, MA, USA), 2 mM L-Glutamine  and 1 mM sodium pyruvate (Sigma). Cells were grown at 37 °C in a 5% CO2 atmosphere.Vero cells, derived from African green monkey kidney cells, were cultured as previously described . Briefly2 atmosphere.Aedes albopictus larva C6/36 cells were cultured in M3 insect medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine and 0.1 mM non-essential amino acids, as previously described , and mai3 cells were seeded into 24-well plates 24 h before transfection in order to reach ~80% confluence. Then, 1.5 µg of the RNA under study and 0.3 µg of cap-RLuc RNA were mixed with the transfection reagent  in Opti-Mem®  according to the manufacturer’s instructions. This mix was added to cells followed by incubation for 4 h in order to achieve maximum translational efficiency. The cells were then lysed and subjected to firefly and Renilla luciferase assays using the Dual-Luciferase Reporter Assay Kit . Relative translation efficiency was calculated as the FLuc/RLuc ratio compared to that obtained for wt WNV RNA.Cells were transfected essentially as previously described ,39. Brie


By contrast, N-rich compounds were released at higher temperatures from samples with 2 : 1 clays and short-range ordered (SRO) amorphous minerals. Temperatures of release overlapped for SRO minerals and crystalline oxides, although the mean age of OC released was older for the SRO. In soils with more mixed mineralogy, the added presence of older OC released at temperatures greater than 450°C from clays resulted in a broader distribution of OC ages within the sample, especially for soils rich in 2 : 1 layer expandable clays such as smectite. While pedogenic setting affects mineral stability and absolute OC age, mineralogy controls the structure of OC age distribution within a sample, which may provide insight into model structures and OC dynamics under changing conditions.Organic carbon (OC) association with soil minerals stabilizes OC on timescales reflecting the strength of mineral–C interactions. We applied ramped thermal oxidation to subsoil B horizons with different mineral–C associations to separate OC according to increasing temperature of oxidation, i.e. thermal activation energy. Generally, OC released at lower temperatures was richer in bioavailable forms like polysaccharides, while OC released at higher temperatures was more aromatic. Organic carbon associated with pedogenic oxides was released at lower temperatures and had a narrow range of This article is part of the Theo Murphy meeting issue ‘Radiocarbon in the Anthropocene’. For example, high-surface area 2 : 1 clays can adsorb large molecules through cation bridging (e.g. Ca groups) . Aromati groups) . In addi groups) ,23. 1 :  groups) . They ca groups) ,26. Thus groups) .Reactive soil mineral surfaces and OC input to soil are spatially heterogeneous. Notably, physical occlusion mechanisms can play a role in long-term persistence by isolating SOM from decomposers and extracellular enzymes ,28. StabIn addition to mineral stabilization, the persistence of OM in soil has been attributed to chemical ‘recalcitrance’, or the difficulty with which soil microbes break bonds within an organic molecule, e.g. differentiating low molecular weight fresh sugars from more stable biopolymers such as lignin or poorly recognizable charred aromatic OM. The influence of chemical complexity along with the potential for one or more mineral stabilization mechanisms to select for organic molecules with specific properties likely results in quantifiable differences in the distribution of activation energies that control the time until a given organic molecule is made available for decomposition and its C released from soil back to the atmosphere ,30.14C distribution for bulk SOM sampled at a given point in time [14C observations directly against either time series of samples from the same site or the age distribution of a single sample determined using laboratory physical or chemical fractionation. Thermal fractionation, which measures SOM decomposition into CO2 under increasing energy inputs (i.e. heat) can now be used to isolate the 14C signal of quantitatively and qualitatively distinct parts of SOM that differ in thermal stability, molecular structure and bonding mechanisms [14C distributions of OC can be compared with other fractionation approaches [The OC in SOM is known to have a range of ages that are related to soil mineralogy. Recent advances in multi-pool SOM models allow the calculation of the probabilistic  in time . This prchanisms ,33. By uproaches , time seproaches  or modelproaches . Thus, tproaches  showed tGiven the wide range of potential physical and chemical interactions, SOM associated with various soil minerals differs greatly in quality, quantity and mean age, represented by the time OC has persisted in the soil. In order to predict SOM dynamics and parameterize SOM turnover in models, it is crucial to accurately represent different timescales of SOM stabilization and their patterns across soil types. Most models of SOM dynamics –39 requi14C analyses provide one of the only tools to quantify the age and rate of cycling of soil OC. However, relatively few studies have linked 14C to mineral stabilization mechanisms directly [14C measurements of bulk SOM are increasingly common, mean values insufficiently describe SOM dynamics. This is evidenced by the rapid incorporation of recent bomb 14C into SOM with mean 14C ages of hundreds to thousands of years, or the ability of various chemical and physical fractionation methods to separate bulk SOM into fractions with different mean ages  for geochemically and pedogenetically distinct soils with a wide range of mineral stabilization mechanisms. To address this, we proposed the following questions and hypotheses: 1.How does mineralogy control MOM age structure and thermal stability? We hypothesize that variation in bonding mechanisms between SOM and minerals in different soils corresponds to variation in activation energy required to mineralize SOM and this will be reflected in the mean age of the OC oxidized at different temperatures. We further predict that the oldest OC would be associated with minerals with the strongest stabilization pathways—i.e. soils rich in amorphous short-range ordered (SRO) minerals require the highest amount of activation energy  followed by soils rich in expandable 2 : 1 clay minerals and pedogenic oxides. Mineralizing SOM in soils rich in end-member minerals such as non-expandable 1 : 1 clays, quartz and highly crystalline pedogenic oxides should require only a low amount of activation energy . Similarly, soils rich in primary minerals where soil development has not yet created strong organo-mineral bonds with secondary minerals will show comparatively low activation energy and younger C. 2.How does mineralogy control MOM chemistry? We hypothesize that the relationship between thermal stability and mean age is driven primarily by the ability of soil minerals to stabilize organic molecules, and only secondarily by their biochemical molecular structure. As such, soils with the strongest mineral-related stabilization mechanisms will also have the ability to stabilize potentially bioavailable organic molecules, whereas soils with weak mineral-related stabilization mechanisms can be dominated by molecules that show inherent resistance to decomposition.To test these hypotheses, we selected mineral fractions from soil B horizons, where mineral–organic interactions should dominate SOM dynamics. We chose soil B horizons because mineral–OM interactions likely dominate there, and because of recent studies suggesting that a portion of B horizon OM is cycling on timescales of decades [2 oxidized in different temperature ranges to determine patterns in quantity, 14C, and 13C of released C. The same samples also underwent Rock-Eval pyrolysis, an assessment of SOM evolution and maturity, and pyrolysis gas chromatography and mass spectrometry (py-GC/MS) to characterize the chemical composition of the MOM in each soil. By linking chemistry and 14C thermal analysis to distinct thermal fractions of MOM, we provide new insights on characterizing MOM composition and move beyond bulk 14C ages to better link soil minerals to the age structure of SOM.In this study, we explore the potential of thermal analysis to improve the description of  decades  and is v decades . To maxi decades , and soi decades ,53, as w decades ,55. We s. 2 (a)14C-free OC inherited from sedimentary mineral–OC associations. A previous publication demonstrated the potential for inherited OC from sedimentary parent material to influence the thermal analysis [Samples were selected from well-characterized B horizons of soils representing a variety of mineralogies, including those with single mineral classes and combined mineralogies to test endpoints and mixtures . The infanalysis ,59.Tabl(i)We selected a volcanic soil rich in amorphous minerals, with a high OC content. Site ‘SRO’ is dominated by reactive secondary SRO minerals. The ‘SRO’ soil was collected from site Laupahoehoe of the LSAG parent material weathering chronosequence in Hawaii  ,60. ‘SRO(ii)Soils that represent an array of clay mineral compositions were selected from previous studies in Kruger National Park in South Africa ,56,61,62(iii)To contrast soils rich in secondary minerals with those in early development stages, we included soils with varying parent material from a cooler climate transect site in the Sierra Nevada mountains in California, USA ,53. Thes(iv)As a measure of OC stabilization and turnover in soils nearly devoid of reactive minerals (including clays), we selected soil developed on post-glacial dune sands located in northern Michigan, USA. ‘Quartz’ is classified as a Spodosol (Entic Haplorthod) where the only mineral-derived stabilization mechanism is (oxy)hydroxides precipitated on sand particles. Although classified as a Spodosol, the concentrations of extractable metals from this mineral B horizon are low ( (b)(i)−2 sodium polytungstate (SPT) solution) for thermal analysis to remove low-density particulate OM. Note that samples ‘1 : 1 Clay + Quartz’, ‘2 : 1 Clay + CO’ and ‘Mixed Clay + Quartz’ were not density fractionated, but were determined to have low (less than 5% of soil OC) FPOM concentrations in prior work [For our analysis, we selected only the MOM fraction in the B horizons of the investigated soils after density separation  are selected by producing an initial profile of OC release (thermogram) which is deconvolved and transformed to a probability distribution of activation energy (Ea) based on the time-temperature relationship of sample collection . Using this methodology, a total of five thermal fractions were collected for each sample. Briefly, a sample is heated at a constant rate (12°C min−1) from 40°C to 900°C under carrier gas flow composed of 75% N2 and 25% O2 . Any C released from the sample is fully oxidized to CO2 by a platinum catalyst held at 800°C. The produced CO2 is then quantified by a non-dispersive infrared (NDIR) detector. Carrier gas and CO2 then flow through a manifold consisting of parallel glass U-traps submerged in liquid nitrogen (LN2) under vacuum (10−2 mBar) and CO2 is cryogenically frozen and removed from the carrier gas. Once the sample reaches a desired upper-limit temperature, the first trap is sealed to trap CO2 produced at this temperature interval before a second parallel trap is opened and the next aliquot of CO2 is collected. The process is repeated for each subsequent temperature range.Methods for thermal fractionation are described in detail by Stoner et al.  and elseet al. ,33. Firs2 trapped at LN2 temperatures (−190°C) is then purified and quantified on a vacuum line using an isopropanol and dry ice trap to remove water and additional LN2 traps. A small CO2 subsample is collected for 13C measurement with a syringe and transferred to an He-flushed vial. The remainder is frozen into borosilicate glass tubes containing Ag and CuO wire and flame-sealed. Sample tubes are baked at 525°C for 1 h, during which the Ag and CuO remove additional contaminant gases (mostly N oxides that form from the reaction of N2 and O2 in carrier gases at high temperatures that freeze in the LN2 trap), necessary to avoid interferences with sample graphitization before 14C analyses (see §2b(iv)).The CO(ii)Ea) distributions were calculated using the ‘rampedpyrox’ Python package [Ea. In this study, Ea distributions and means (Eμ) are used to describe the average bond strength of OC released, and standard deviations (Eσ) to describe the heterogeneity of the bonding environment, where greater Eσ indicates more diverse types and strengths of bonds. While these values should not be compared with Ea metrics determined via other methods, they can be used to compare samples measured under the same conditions.Activation energy The molecular structure of SOM was studied by analytical pyrolysis (pyrolysis gas chromatography-mass spectrometry: py-GC/MS) using a double-shot micro-furnace pyrolyzer  attached to a GC/MS system . First, a general overview of the sample pyrolyzate was obtained by direct pyrolysis of the samples at 400°C . Then, aBriefly, soil samples  were placed in stainless-steel capsules (Frontier Laboratories Ltd. Eco-Cup LF) and introduced in a pre-heated pyrolysis micro-furnace at the starting temperature for 1 min. For the direct pyrolysis, the furnace starting temperature was set at 400°C and the pyrolysis was performed once in single shot mode. For sequential pyrolysis analysis, the furnace was set at the lowest temperature for each fraction, before a sample was introduced into the pre-heated furnace and thermal desorption was performed for 1 min. The resulting gases were directly injected into the GC/MS system. The sample in processing was immediately moved to a cold area of the pyrolyzer while the furnace temperature increased to the next temperature and the sample was reintroduced to the pyrolyzer hot area for 1 min. Following this procedure, the treatment was repeated for each temperature increment using the same sample. Thus, in total and using aliquots, six chromatograms per sample were analysed; one for the direct pyrolysis at 400°C and five for the sequential analysis, corresponding to each of the selected temperatures.−1. The oven temperature was held at 50°C for 1 min, increased to 100°C at 30°C min−1, from 100°C to 300°C at 10°C min−1 and stabilized at 300°C for 10 min. Mass spectra were acquired at 70 eV ionizing energy. The compound assignment was achieved via single-ion monitoring for various homologous series, low-resolution mass spectrometry and by comparison with published and stored (NIST and Wiley libraries) data. The relative abundance of each pyrolysis product was calculated as a percentage of the chromatographic area of all identified compounds.The GC was equipped with a low polar-fused silica (5% phenyl-methylpolysiloxane) capillary column (Agilent J&W HP-5 ms UI), of 30 m × 250 µm × 0.25 µm film thickness. The carrier gas was He with constant flow at 1 ml min(iv)14C in each sample, collected CO2 (see 2b(i)) was graphitized following the method of Steinhof et al. [et al. [14C (Fm) [14C data for the year of sample collection to a one-pool model as described by Khomo et al. [In order to quantify the f et al.  and meas [et al. , and are14C (Fm) . For theo et al. .δ13C was performed on an aliquot collected via syringe during purification (§2b(i)) using a modified gasbench inlet to a continuous flow isotope ratio mass spectrometer (IRMS) [Analysis of r (IRMS) .(v)To assess the relative degree of decomposition and thermal lability versus stability of MOM and implied biogeochemical stability in soil , we appl−1; under N2 atmosphere) then in a combustion oven . At the beginning of pyrolysis, samples underwent an isothermal step at 300°C for 180 s, during which the free hydrocarbons (HC) were vaporized (S1 peak), before proceeding to higher temperatures as described above. The pyrolysis effluents (mostly HC) were detected and quantified with flame ionization detection, while CO and CO2 were quantified by infrared detection during both the pyrolysis and oxidation stages.Briefly, approximately 60 mg of powder-ground sample was added to a Rock-Eval 6 Turbo  and underwent two consecutive heating phases, first in a pyrolysis oven  and R (refractory) that better describe SOM were developed by Sebag et al. . These Ihorizons . The R-i(vi)Ea. The primary advantage of oxidation in our study is in minimizing artefacts due to charring, or the conversion of OC released at low temperatures to high-stability molecules rather than being released as a COX gas, potentially misrepresenting SOM thermal stability. Although the mechanisms of decomposition can vary between methods, previous studies have observed no significant difference in the Ea or the 14C measured in thermal fractions collected under ramped pyrolysis and oxidation [Ea gradients between both methods.Thermal analysis techniques commonly measure soil characteristics with or without the presence of oxygen as an oxidizing agent, depending on the theory and goals of the research . In thisxidation ,72. Thus. 3 (a)Ramped thermal oxidation yielded distinct trends and differences between soils according to their dominant minerals . All samT450–550, T450–550) there was significantly less  high-temperature OC release in ‘Quartz’, and ‘SRO’  likely contain OC that may be strongly associated with clay minerals, which is described by a thermal index ( (b)Ea, as ‘SRO’ and ‘Quartz’ yielded significantly lower mean activation energies (Eμ) than other samples , a proxy measure for the diversity of OC bonding strengths, was also much lower in these samples , suggesting the most heterogeneous bonds. Samples containing expandable 2 : 1 minerals and variable abundances of SRO and CO  had significantly higher Eμ (148.9–152.1 kJ mol−1) and high Eσ (17.2–19.1 kJ mol−1).Similar grouping trends were observed in  (c)14C data  released at the lowest temperatures, and the lowest Fm 14C (oldest C) released at highest temperatures  of 420–460 years  than all other samples (−0.0018 to −0.0027 Fm per kJ mol−1).Despite the large range in mean MOM eratures . For ‘SR60 years . By conteased Ea . However (d)Pyrolysis gas chromatography and mass spectrometry (py-GC/MS) revealed divergent trends in SOM quality and composition across mineralogies and temperatures  released N-containing OM with lower Ea than non-SRO soils (132–150 kJ mol−1). Polysaccharides, bioavailable compounds of potentially microbial origin, were detected in all samples except for ‘2 : 1 Clay + CO’. No soil released polysaccharides above approximately 178 kJ mol−1. Alkane/alkene chains were detected at low temperatures in ‘2 : 1 Clay + CO’ and ‘SRO’, while ‘Int. PM + SRO’ and ‘Mafic PM + CO’  released alkanes with Ea > 175 kJ mol−1. The only strong lipid signals were detected in ‘Mafic PM + CO’ and a small amount in ‘Int. PM + SRO’, all below Ea of 178 kJ mol−1.With the exception of the ‘SRO’ sample, aromatic molecules were the most prominent group in all soils. Nitrogen-bearing compounds were detected in the ‘SRO’ soil, and soils with expandable 2 : 1 clay minerals. These compounds were generally released in thermal fractions of approximately 150 kJ mol (e)Rock-Eval SOM indices for all samples were in the expected range for B horizons  and foll. 4 (a)14C values of MOM can differ greatly depending on mineralogy and degree of mineral weathering. However, despite a variety of soil settings in our samples, we find distinct relationships between mineral reactivity, patterns of 14C distribution, and the type and number of chemical species in MOM. Following the hypothesis that greater activation energy (Ea) corresponds to stability in soil, ‘labile’ forms of OC should be oxidized at lower temperature, decompose more easily, and be younger than the mean MOM 14C age, while high Ea OC should be older and more aromatic in nature [Ea OC was associated with pedogenic oxide minerals (SRO and CO), while high Ea OC was associated with clay minerals, especially 2 : 1 clays. Thus, fractionating MOM along a continuous Ea gradient allows us to describe which minerals tend to be associated with faster cycling (younger) or slower cycling (older) OC. This helps define SOM model pool structures and may have broad implications for assessing patterns of OC persistence across soil systems.Our data confirm that the abundance, biochemical characteristics and mean n nature . This waMOM forms through numerous types of interactions . These i(i)Ea in mostly bioavailable forms, it contained very old OC 14C distribution , with o figures . The sim figures  in this (iii)Ea decreased the most after soil incubation [Compared to clay-poor samples ‘SRO’ and ‘Quartz’, clay minerals, even in low abundance, increase the range of activation energies and ages  detectedcubation ,91. In ocubation –94 that cubation ,55, withcubation ,95. Notacubation ,89,96,97cubation ) on claycubation  and (ii)cubation ,4.(iv)X) that may be positively charged, thus forming strong bonds with negatively charged mineral surfaces, e.g. oxides and 2 : 1 clay minerals [Ea and in the oldest thermal fraction within each sample  were also able to stabilize bioavailable OC species against decomposition (i)14C over the last decades has required models to represent SOM using pools with different timescales of turnover, ranging from years to millennia. Thermal analysis approaches help determine which mineral stabilization mechanisms may give rise to different model structures. For example, in subsoils with mixed mineralogy Ea in combination with describing the chemical quality of MOM from distinct thermal fractions proved to be a useful tool for separating SOM according to the type of mineral with which it is associated and its relative age in distinct soils. While the directional changes of processes in complex soil systems are not well understood and are difficult to predict [Ea, and age dependent on mineral composition 14C ages within a given sample, we were not able to predict the mean 14C age of a sample from mineralogy or OM chemistry alone. For example, polysaccharides released at low Ea (<160 kJ mol−1) make up the majority of OC release in most soils (14C content of SOM arriving in subsoil (e.g. Spodosol B horizons) reflects the mean time required for OC to arrive in that horizon and is not necessarily a good measure for the in situ stability or decomposition rate of B horizon SOM [While thermal oxidation clearly provided a distribution of st soils  but had izon SOM . Thus, tFuture climates may de-stabilize OC through enhanced microbial activity ,112, enh. 514C and energy distributions and, excluding soils rich in amorphous SRO minerals, stored generally less chemically diverse compounds with younger 14C ages. Soils containing a larger variety of minerals offered multiple stabilization mechanisms leading to a much wider range of activation energies, SOM ages, and generally greater diversity of organic compounds. Through the combination of several thermal stability analyses and characterization of MOM molecular structure, we were able to identify distinct timescales for MOM turnover and the biochemical structure of MOM associated with different types of soil minerals. MOM in soils containing 2 : 1 clays had overall higher thermal stability and released older OC at higher temperatures than soils dominated by non-expandable 1 : 1 clay minerals or highly crystalline minerals. In all soils, the majority of MOM appears to be associated with (pedogenic) (oxy)hydroxides and amorphous SRO minerals or weakly stabilizing OM–OM bonds. These minerals were able to store abundant and diverse OC compounds, including potentially bioavailable and more accessible SOM. Thus, our results support previous studies showing the role of oxides as dominant drivers of total SOM storage, but also show a smaller but significant role of clays in strongly stabilizing relatively old and chemically distinct C. Thermal oxidation demonstrated that it can successfully distinguish OC associated with different minerals and provide predictable timescales and chemical characteristics. We conclude that while the directional changes of processes in complex soil systems are not well understood and remain difficult to predict, thermal fractionation may have the potential to identify SOM that is more weakly stabilized and more likely to be vulnerable to future change on multi-decadal to multi-centennial timescales. The mineralogical control on the age structure of MOM has important implications for how soil SOM models need to account for differences in soil mineralogy, and how specific portions of SOM may have distinct reactions to changing environmental conditions that affect plant C input, microbial C turnover and mineral OC stabilization to varying degrees and at varying timescales.A direct comparison of diverse MOM using a variety of thermal oxidation and pyrolysis methods successfully highlighted the distinct effects of soil minerals on MOM persistence. Soils with singular mineral stabilization pathways and without clay minerals had narrower


Gestational diabetes mellitus (GDM) is the most common type of hyperglycaemia in pregnancy. GDM is a risk factor of adverse perinatal outcomes, with the incidence rate increasing proportionally to the level of maternal dysglycaemia. Therefore, glycaemic control plays an important role in management of GDM. The aim of this study was to assess the efficacy of flash glucose monitoring (FGM) in GDM.n = 50) or control group . Clinical and laboratory results were assessed at four follow-up visits. The primary outcome was mean fasting and postprandial glycaemia. The secondary outcomes were perinatal outcomes.This was a non-blinded, randomised controlled trial, that recruited 100 pregnant women diagnosed with GDM between 24 and 28 weeks of gestation at the 1st Department of Obstetrics and Gynaecology, Medical University of Warsaw. After meeting the inclusion criteria patients were randomly allocated to the study group  Compared to the control group, the study group significantly reduced their fasting (p = 0.027) and postprandial glycaemia (p = 0.034) during the first 4 weeks following GDM diagnosis, with no significant difference in progression to insulin therapy . Incidence of fetal macrosomia was significantly higher in SMBG as compared to FGM group . There was no significant difference in mean glycaemia between the groups (Study results indicate that FGM has an impact on glycaemic control, dietary habits and incidence of fetal macrosomia in patients with GDM.Trial registration clinicaltrials.gov ID: NCT04422821.The online version contains supplementary material available at 10.1007/s00592-023-02091-2. Gestational diabetes mellitus (GDM) is the most common type of hyperglycaemia in pregnancy, with the incidence rate of 14% varying worldwide , 3. It iWe performed a non-blinded, randomised controlled trial between March 2020 and October 2022, that recruited 100 pregnant women diagnosed with GDM between 24 and 28 weeks of gestation at the 1st Department of Obstetrics and Gynaecology, Medical University of Warsaw. A diagnosis of GDM was based on 2013 World Health Organisation criteria: (1) fasting plasma glucose 92–125 mg/dl (2) 1-h glucose concentration ≥ 180 mg/dl or (3) 2-h glucose concentration 153–199 mg/dl . The incn = 50) or control group (n = 50). Simple randomisation with the computer-generated list was used for patient’s randomisation process by the non-member of the trial research staff. During the first 4 weeks following the diagnosis of GDM participants in the study group measured glucose concentrations using the FGM system , whereas the control group used the SMBG . In the following weeks until birth, glucose concentrations were measured exclusively with SMBG method in both groups.After meeting the inclusion criteria patients were randomly allocated with a nested qualitative evaluation and 1:1 allocation ratio to the study  clinical and laboratory results were assessed, including: fasting and 1 h-postprandial glucose concentrations , qualification to insulin therapy and dosage, diet control, physical activity and gestational weight gain. The qualification to insulin therapy was decided in case of hyperglycaemia, defined as fasting glycaemia ≥ 90 mg/dl or 1 h-postprandial glycaemia ≥ 140 mg/dl. At the second and third follow up visits Haemoglobin A1c (HbA1c) concentration and ultrasound estimated fetal weight (EFW) were measured. For the assessment of fetal birthweight percentile INTERGROWTH-21st Chart was implemented. At the last follow up visit  patients were reviewed for perinatal outcomes: weeks of gestation and route of birth, newborn weight and neonatal hypoglycaemic events.As part of our study we analysed physical activity by daily footsteps measure with step counter app. After calculating daily steps, we divided participants into 4 groups–sedentary: < 5000; low active: 5000–7500; somewhat active: 7500–10,000 or active: > 10,000 steps.We also evaluated patient’s dietary habits and its modifications throughout pregnancy by using Eating Assessment Test (EAT) prepared by the Polish National Institute of Public Health–National Institute of Hygiene (Supplementary File 1). EAT contained a short questionnaire, with 20 items for diet assessment. Based on EAT, participants were assigned to one of four diet groups: good: 39–42; satisfactory: 30–38; demanding diet modification: 12–29 and not satisfactory: < 12 points.The primary outcome of the study was mean fasting and 1 h postprandial glycaemia during the first 28 days following GDM diagnosis. Additionally, we analysed variability pattern of glucose concentration inside the groups by using delta mean fasting and postprandial glucose concentration for each group, defined as difference between 3rd to 4th week mean glycaemia and 1st to 2nd week mean glycaemia. The secondary outcomes were maternal and neonatal outcomes, including: qualification to insulin therapy, insulin dosage, hypoglycaemic episodes (defined as glycaemia < 70 mg/dl), HbA1c concentration, pregnancy weight gain, physical activity and diet modifications, fetal birthweight and percentile, incidence of fetal macrosomia and neonatal hypoglycaemia.The detailed study protocol was already published in 2020 . The stuWe performed a two-sided power analysis  and estimated a sample size of 80 patients (40 patients for each group). The sample size was further increased to 100 patients based on estimated drop-out rate of 10% participants. For the estimation, we used results of a previous study comparing target glycaemic range between FGM and SMBG .p-value of less than 0.05 was a cut-off for significant difference.The statistical analysis was carried out using STATA, version 17.0  and GraphPad Prism v9 . Continuous variables were presented as means ± standard deviation (SD) or medians ± interquartile range (IQR) and categorical variables were presented as frequencies (%) by treatment group. Normal distribution of continuous variables was assessed using the Shapiro–Wilk’s test. For the analysis of continuous variables either Mann–Whitney U test or t-test were used and for categorical variables Fisher exact test was applied. For the correlation analysis, we used Pearson correlation coefficient. To estimate correlation between method of glycaemic control and perinatal outcomes we performed binary logistic regression and presented results as odds ratio (OR) and 95% confidence interval (95% CI). We performed linear regression to analyse relationship between glucose concentration and continuous perinatal outcomes. All statistical tests applied were two-sided. A Between March 2020 and October 2022, we recruited 100 patients that met the inclusion criteria. 50 patients were recruited to the study group, out of which 49 completed the study (one patient was excluded after recruitment process at the first follow up visit due to diagnosis of placenta praevia). 50 patients were allocated to the control group, with 50 women included in the statistical analysis. Maternal baseline characteristics did not differ significantly between the groups (Table p = 0.437), whereas the mean postprandial glycaemia differed significantly, with lower concentration in the control group (p = 0.011) (Table There was no significant difference in mean (SD) fasting glucose concentration during the first 4 weeks following GDM diagnosis between the groups (1) Table .Table 2Gp = 0.027)) and delta postprandial mean glycaemia (−1.01 mg/dl (7.62) vs. 1.93 mg/dl ) in the FGM group in the third and fourth week following the inclusion to the study  vs. 2.52 mg/dl . In 6 out of 49 patients (12.24%) we recorded one episode of hypoglycaemic event level 2 (glucose concentration less than 54 mg/dl). We were not able to compare it with the control group, as participants from the control group measured glucose concentration only once between 2:00 and 4:00 at night. Therefore, in the control group the mean incidence of nocturnal hypoglycaemic events was 2 events per month, but the result was not comparable with the FGM group.p-value 0.546). There were also no significant differences between the groups regarding qualification to insulin therapy . Long-acting insulin dosage at the follow up visit between 34–36 weeks of gestation did not differ between the groups (p = 0.199) (Supplementary Appendix 1). One participant from the control group (2%), whereas three participants from the study group (6.12%) demanded short-acting insulin therapy.We analysed HbA1c concentration at the 1st, 3rd and 4th visit and found no significant difference between the groups (Supplementary Appendix 1). Delta HbA1c concentration, defined as difference between HbA1c measured between 34–36 weeks of gestation and HbA1c measured at the recruitment visit also did not differ significantly between the groups (ΔHbA1c for SMBG group was 0.1% (−0.1–0.2) and for FGM group 0.05% (−0.2–0.2), p = 0.872). There was significantly higher incidence of fetal macrosomia ≥ 4000 g in the SMBG group ; large for gestational age neonates (defined as birthweight > 90 percentile) and neonatal hypoglycaemia also appeared more often in the control group, but the differences were not statistically significant . Median birthweight percentiles (INTERGROWTH-21st standards) did not differ significantly between the groups (p = 0.697).We found no significant difference in incidence of caesarean section  and weeks of gestation at birth between the groups  for fasting mean glycaemia and OR 0.99 (95% CI 0.91–1.08) for postprandial mean glycaemia, respectively. However, postprandial glucose concentration was correlated with the incidence of fetal macrosomia   or postprandial mean glycaemia  and newborn weight. A linear regression established, that both fasting and postprandial mean glycaemia could not significantly predict the fetal birthweight  = 0.01, p = 0.307 and  F = 0.01, p = 0.93, respectively)  Fig. B.p = 0.682) (Supplementary Appendix 1). We also analysed physical activity based on daily steps and allocation to the four groups. At the follow up visit between 34–36 weeks of gestation 22 participants from the control group (44%) and 16 participants from the study group (32.65%) were allocated to the sedentary group (< 5000 steps/day), whereas 13 patients from the SMBG group and 7 from the FGM group to the somewhat active or active group (at least 7500steps/day). There was no significant difference in allocation to the physical activity groups between the study and the control group (p = 0.302).Gestational weight gain, defined as difference between weight at the 3rd follow up visit and weight at the recruitment visit, did not differ significantly between the groups (p = 0.372), but between 34 and 36 weeks of gestation was significantly higher in the study group (37 points (34–39) vs. 34 points (33–37), p = 0.017). In FGM group we collected additional data about time in range (TIR) (marker showing glycaemic variability), with the median (IQR) of 96% (92–98%).The study group more frequently modified their diet habits, when compared to the control group. Median (IQR) EAT score did not differ at the recruitment visit (34 points (28–36) for FGM compared to 33 points (28–35) for SMBG, Our study revealed, that FGM might improve glycaemic control, when compared to SMBG. Although mean glycaemia did not differ significantly between the groups, and when separately analysed, postprandial glycaemia was significantly lower in the control group, glycaemia through the first month of the study improved more significantly in the study group (delta mean glucose concentration showed a pattern of decreasing changes in glycaemia in FGM group). As in previous studies on pre-gestational diabetes population, we found that FGM system led to strict glycaemic control, that consequently decreased mean fasting and postprandial glycaemias at follow up visits.FLAMINGO revealed, that FGM had an impact on decreasing incidence of fetal macrosomia (birthweight > 4000 g) in GDM patients. It is consistent with the randomised controlled trial (RCT) by Murphy et al., evaluating effectiveness of CGM in pregnant women with type 1 and 2 diabetes . LGA incThe CONCEPTT trial provided evidence that CGM led to clinically significant reduction in neonatal hypoglycaemia and NICU  admission incidence . HoweverWe found higher detection rate of hypoglycaemia in FGM group; however, these hypoglycaemic events were in most cases qualified as mild and none of them were symptomatic. As presented in previous studies continuous glucose monitoring detects masked hypoglycaemic events in pregnancy, that if qualified as mild, are clinically non-significant.Interestingly, we found, that the study group was more prone to modify their diet habits, compared to the control group; however, it had no impact on gestational weight gain and qualification to insulin therapy. Our data differ from the previous studies, in which these outcomes were improved, when CGM systems were used , 23.Our study has several strengths. To our knowledge, this is the first study with such a long assessment of glycaemia with FGM system in gestational diabetes mellitus. The data were derived from a randomised controlled trial, that diminishes the risk of bias that might be the consequence of the recruitment process. Furthermore, the baseline characteristics of the participants did not differ significantly between the groups and therefore adjustments had no significant impact on the analysed outcomes. All the additional results, including diet modifications, physical activity, were assessed using standardised tools. For the control group, we used one type of glucose meters to diminish the influence of different types of devices on primary outcome .We also acknowledge some limitations. In FLAMINGO trial, the analysed time-period of glycaemic control was only 4 weeks, and therefore did not include glycaemic fluctuations occurring after the first month from GDM diagnosis till birth. Additionally, the EAT questionnaire was filled in by the patients and the diet scheme was not unified for all participants, that might produce the risk of bias.In summary, FGM application resulted in significantly better improvement in glycaemic control in the 3rd and 4th week of the study. FGM led to higher EAT score, that might indicate better diet modifications after GDM diagnosis; however, it had no impact on lifestyle interventions including gestational weight gain, qualification to insulin therapy or dosage of insulin. FGM significantly decreased incidence of fetal macrosomia, but had no significant impact on birthweight percentile or neonatal hypoglycaemia incidence. Therefore, further studies are needed to analyse the impact of FGM on improving perinatal outcomes in GDM-complicated pregnancies.Supplementary file1 (DOCX 95 KB)Below is the link to the electronic supplementary material.


Bone quality is commonly used to diagnose bone diseases such as osteoporosis, with many studies focusing on microarchitecture for fracture prediction. In this study a bovine distal femur was imaged using both micro-computed tomography (µCT) and tomosynthesis using focal construct geometry (FCG) for comparison of microarchitectural parameters. Six regions of interest (ROIs) were compared between the two imaging modalities, with both global and adaptive methods used to binarize the images. FCG images were downsampled to the same pixel size as the µCT images. Bone morphometrics were determined using BoneJ, for each imaging modality, binarization technique and ROI. Bone area/total area was found to have few significant differences between FCG and µCT (p < 0.05 for two of six ROIs). Fractal Dimension had only one significant difference (p < 0.05 for one of six ROIs) between µCT and downsampled FCG . Trabecular thickness and trabecular spacing were observed to follow trends as observed for the corresponding µCT images, although many absolute values were significantly different (p < 0.05 for between one and six ROIs depending on image types used). This study demonstrates the utility of tomosynthesis for measurement of microarchitectural morphometrics. Currently, dual energy X-ray absorptiometry (DXA) is used as the ‘gold standard’ for diagnosis of osteoporosis3 by measuring bone mineral density (BMD) at the lumbar spine and hip. DXA is often used in conjunction with additional tools such as FRAX®4 for improved fracture prediction. Bone strength is thought to be a better predictor for fracture than BMD alone, encompassing both microarchitectural and physicochemical ‘bone quality’7. In an effort to better quantify bone quality, through several different imaging techniques, a number of studies have shown that microarchitectural parameters change significantly in diseased bone compared to healthy bone, including trabecular thickness, trabecular separation, and fractal dimension12.Osteoporosis is a sweeping condition which currently affects over 3.7 million people in the UK14, cone beam CT (CBCT)15, and magnetic resonance imaging (MRI)16, often compared to micro-CT (µCT). Currently, HR-pQCT can achieve the highest resolution, at an isotropic resolution of either 61 µm or 82 µm, while in practice it achieves a spatial resolution of 100 µm or 142 µm14. However, a considerable limitation of HR-pQCT remains that it can only be used to examine peripheral sites. Relatively recent studies have used MRI to image trabecular bone and achieved an in-plane resolution of 156 µm with a slice thickness of 0.5 mm at the distal radius17, and an in-plane resolution of 234 µm with a slice thickness of 1.5 mm at the hip16. In comparison, CBCT can achieve a voxel size between 75 µm and 500 µm15. Additionally, opportunistic screening using clinical CT is a relatively common topic within literature18, as is the use of more traditional digital tomosynthesis for fracture prediction19.Recently studies have investigated the use of high resolution peripheral quantitative computed tomography (HR-pQCT) for fracture prediction21 and X-ray diffraction (XRD) in energy dispersive22 and angular dispersive modes25, notably on bone26. Recently this approach has been expanded to benefit from sporadic collection to further reduce dose and scanning time21. Additionally, a high-resolution technique using interleaved sampling has been applied to decrease pixel size in the xy plane27 by an order of magnitude.Focal construct geometry (FCG) is an alternative X-ray beam geometry which uses a conical shell X-ray beam, designed to reduce exposure time for collection. It also provides unique access to the coherent scatter signals, thus providing crystallographic detail of the inspected material. FCG has been applied to X-ray absorption imaging27, this work compares FCG to µCT imaging quantified through measurement of trabecular microarchitecture morphometrics in a distal bovine femur. To evaluate the accuracy of microarchitectural parameters measured from absorption FCG imaging, six regions of interest (ROIs) were selected, and four parameters were measured for each imaging modality. This analysis demonstrates the ability of FCG to measure microarchitectural properties, potentially useful for clinical diagnostics in the future.To evaluate the application of absorption FCG imaging within a clinical setting, and further the analytical methodology introduced previouslyA distal bovine femur was sourced from a local butcher (18–24 months), approximately 115 mm × 144 mm × 99 mm.The bone sample was scanned using a Nikon CT H225  cone beam µ-CT scanner operated at 125 kV and 101 µA, with a voxel size of 125 µm. Noise correction and beam hardening corrections were applied.27. A Hamamatsu L1261-07 microfocus X-ray source was used with an accelerating voltage and current of 150 kV and 67 µA, respectively. Conical projections were collected on a Siemens HIDEQ 33-4 ISX image intensifier with a CsI phosphor input screen (~ 160 mm diameter useful entrance field). The intensifier is optically coupled to a Videomed-XR2 GigE camera (1024 × 1024). The distance between the detector surface and the X-ray point source was fixed at 409 mm, with the bone sample and translation stage fixed 139 mm from the X-ray point source, as shown in Fig. An FCG raster scan was performed similarly to that described previouslyr = 7.89 mm to 75 mm, giving each a mean half opening from 1.11° to 10.39°, according to tan-1(r/z) where z = 409 mm.The rectilinear raster scan was performed using 501 × 501 points with a 0.3 mm step size giving a 150 mm × 150 mm field of view for the digital tomosynthesis. A series of 18 concentric conical shell projections were measured at each scan step, with the mean annular ring radii linearly spaced from 27. Applying this method produced an image stack with 75 µm pixels in both x and y axes, with a slice thickness of 1 mm in z.A digital tomogram or sectional image was reconstructed from the ring projections using sparse interleaved sampling with 4× upscaling described in detail elsewhereTo enable comparison of µCT and FCG images, a single slice of the FCG image stack was chosen for registration and three unique features were chosen within this slice  using the Matlab29 and is readily apparent within the FCG images seen here. When thresholding is attempted  poor results are seen where many trabeculae were not present, while large areas of cartilage and blur from out-of-focus planes are represented in the foreground  transforms have been used in the past for clinical imaging purposes, for example for whole body sintigraphy30 and for examination of pathological calcifications32. To ensure consistent processing, both FCG and µCT images underwent the same processing procedure. All processing steps were carried out in Matlab28 and are given below:r = 5 pixels (625µm) for µCT and downsampled FCG or r = 9 pixels (675µm) for FCGA disk structuring element was created with either imtophat was used to morphologically open the image (erosion followed by dilation using the specified structuring element) then subtract the opened image from the original imageimadjust was used to adjust image intensity values, saturating the upper 1% and lower 1% of the image.While there are several deblurring algorithms and alternative reconstruction algorithms used within clinical tomosynthesis applications previously37. It should be noted though that most studies observe the femoral head or proximal tibia rather than the distal femur, and TbTh and TbSp have been seen to vary significantly based on the skeletal site and model used for calculation33.The structuring element size was chosen to be larger than the features being targeted (trabecular thickness and separation). In this case, trabecular thickness in bovine trabecular bone is commonly reported between 110 and 600 µm, while trabecular separation is between 60 and 600 µmThe initial FCG image and processed FCG image are shown in Fig. 38 with a neighbourhood size of either 151 × 151 pixels (µCT and downsampled FCG) or 251 × 251 pixels (FCG); the sensitivity value s is user specified based on visual inspection. For global thresholding, the threshold value t is specified by the user based on visual inspection. Figure Two binarization methods were used, adaptive and global, for each of the three images . Adaptive thresholding was applied using Bradley’s methodTo interrogate accuracy of microarchitectural morphometrics, six regions of interest (ROIs) were selected in areas where trabecular bone was clearly visible for both global and adaptive binarizations of µCT and FCG images. Each region of interest was set as either 50 × 50 pixels (for µCT & downsampled FCG) or 83 × 83 (for FCG), seen in Fig. To test repeatability, each ROI was shifted to eight positions around the selected ROI (± 20% of ROI size), to produce a total of nine images which contributed to the morphometric measurement for each ROI , fractal dimension (FD), trabecular thickness (TbTh) and trabecular separation (TbSp). Calculation of both TbTh and TbSp is not dependent upon either a plate or rod model but fits maximal circles to the structures in a model-independent method42. For each ROI in each of the six image types , each parameter measured was tested for normality using either a Shapiro–Wilk or a Shapiro-Francia test, depending on the kurtosis of the sample43. To determine significant differences between image type, if both samples being compared are normal, a two-sample T-test is performed assuming unequal variances; if one or both samples being compared are non-normal a Wilcoxon rank sum test was performed. In both cases, significance was set at p = 0.05.Several morphometric parameters were determined using BoneJz resolution between the FCG image stack and µCT volume complicates the precise registration of individual trabeculae, consistent patterns within the trabecular structure can be observed. Implications of adaptive and global thresholding are considered further in the discussion.Images from each ROI were compared for each imaging modality and each binarization method, shown in Fig. Microarchitectural morphometrics were compared for both global and adaptive binarizations for each imaging modality. Results for all morphometric characteristics are given in Fig. BA/TA for each ROI is shown in Fig. Fractal dimension (FD) shows much larger discrepancies between images with different pixel sizes Fig. b. FD forWhen considering mean trabecular thickness Fig. c, genera45, and is also apparent here. Differences resulting from adaptive or global thresholding were apparent in some parameters  while they did not affect others (such as FD). Also, some image types and binarizations are clear outliers , and often explained by the visual inspection of the ROI; for global binarizations of FCG at ROI 2 a large void is present near the lower part of the region, most likely due to the drastic change in background intensity in the original image, resulting in a relatively rapid intensity change of the trabeculae images after processing. This can also be seen in Fig. Thresholding has been seen to affect morphometric measurements in previous studiesThis disagreement is perhaps demonstrated better in ROI 2, while the trabeculae near the growth plate are visually apparent in processed FCG images, the difference in intensity clearly introduces issues for global binarization. Close inspection of areas such as ROI 2 and ROI 5 with a high background intensity  shows that while adaptive binarization technique accommodates this change in intensity better than the global binarization technique, it still does not ideally threshold. There is potential if the size of the neighbourhood used in the adaptive binarization is reduced, that this may allow more accurate thresholding of trabeculae network images with large changes in intensity, but it would potentially include more noise elsewhere in the image. This can be seen already for adaptive binarizations of both µCT and FCG images in Fig. 44, and parameters can also be affected by the observer46. Thresholding remains one of the most important considerations for the microarchitectural characterisation of trabecular bone.It is readily apparent that both global and adaptive binarizations can either over- or under-estimate trabeculae throughout the thresholding process if the thresholded region is sufficiently large. Choice of thresholding technique has been the focus of several studies in the past and has shown that different thresholding techniques affect different parameters to different degrees, as much as ~ 25%50. Van Dessel et al. and Klintström et al. showed many parameters were significantly different from those measured by µCT, perhaps most importantly those parameters often used in bone disease research . Several studies reported morphometric values from either CBCT or MRI that were significantly different to corresponding µCT values, and consequently, both studies relied on the correlation of parameters between imaging techniques rather than comparison of absolute values51.Several previous studies have compared morphometrics from µCT to other imaging modalities, including cone beam CT (CBCT), multislice CT and MRI. In most cases, comparison was simply by correlation of parameters for each of the two imaging modalities37. A previous study reported highly variable FD for different regions of bovine rib52 but FD observed within this study is consistent with those seen in both human and non-human studies previously52. Treating morphometrics measured by the µCT as a ground truth, it is apparent that FCG can precisely determine BA/TA and FD in most cases. Calculated FD has been seen to vary with different box size53 and different signal to noise ratios54, which may explain the different values seen between different pixel sizes .In this study, BA/TA values are consistent with literature values for the bovine femur and tibia, as are mean TbTh and TbSp valuesz (1 mm).It can be seen that FCG can accurately measure FD, for an equivalent pixel size. Additionally, while FCG (particularly downsampled) accurately measures TbSp compared to µCT, it does not have the same accuracy measuring TbTh. This is potentially due to the circle fitting method imageJ uses, and potentially convoluted further by the relatively large step in z axis, the potential for future exploitation of the geometry is profound. Simultaneous absorption and diffraction imaging using FCG has tremendous implications for the future of fracture prediction once both structural and physicochemical information is available.While clearly the ability of FCG to image all microarchitectural characteristics accurately is affected by the decreased resolution available in the vBMD. Microarchitectural characteristics measured are also limited to features within the 2D images investigated, and in the future a better average may be achieved by use of the 3D volume. This study has made every effort to follow guidelines accepted by the community for measurement of microarchitectural characteristics45.A potential limitation of this study is the resolution of the µCT images; if a smaller voxel size could be achieved , morphometric results may be more precise. Though all efforts have been made to allow easy comparison with other imaging techniques used for morphometric analysis, some limitations are present. The presence of out-of-plane objects within the FCG image preclude this analysis from including traditional parameters commonly used in the clinical sphere such as TMD and 55. The combination of absorption and diffraction FCG would provide the simultaneous interrogation of both morphometric and physicochemical bone quality via a single scan using the same hollow beam. Thus, given that such quality measures are known to be significantly altered with bone disease56, a combined absorption/diffraction probe promises significant clinical diagnostic utility.This study has shown that tomosynthesis using FCG presents an accurate measurement of microarchitectural morphometric parameters. FCG tomography allows for collection of both absorption and diffraction simultaneouslySupplementary Information 1.Supplementary Figures.Supplementary Information 2.