Hydrological transformations induced by climate warming are causing Arctic annual fluvial energy to shift from skewed (snowmelt-dominated) to multimodal  distributions. We integrated decade-long hydrometeorological and biogeochemical data from the High Arctic to show that shifts in the timing and magnitude of annual discharge patterns and stream power budgets are causing Arctic material transfer regimes to undergo fundamental changes. Increased late summer rainfall enhanced terrestrial-aquatic connectivity for dissolved and particulate material fluxes. Permafrost disturbances  reduced watershed-scale dissolved organic carbon export, offsetting concurrent increased export in undisturbed watersheds. To overcome the watersheds’ buffering capacity for transferring particulate material (30 ± 9 Watt), rainfall events had to increase by an order of magnitude, indicating the landscape is primed for accelerated geomorphological change when future rainfall magnitudes and consequent pluvial responses exceed the current buffering capacity of the terrestrial-aquatic continuum. Climate warming is causing annual Arctic fluvial energy budgets to shift seasonality from snowmelt-dominated to snowmelt- and rainfall-dominated hydrological regimes, enhancing late summer and fall terrestrial-aquatic connectivity and higher material fluxes. As the Arctic warms and precipitation patterns change8, shifts in the timing and magnitude of fluvial energy  in response to rainfall inputs will alter landscape connectivity in watersheds by increasing the late-season delivery of organic  and inorganic  terrestrial materials into downstream aquatic ecosystems11. The impacts of shifts in the timing and magnitude of fluvial energy coupled with terrestrial ecosystem changes are scarcely documented in these environments due to a lack of integrated longer-term (≥10 years) biogeochemical records12 and the remote nature of the High Arctic13. Accurate biogeochemical budgets across full hydrological seasons are critical reference points for earth system models and are required to predict the strength and timing of climate feedback mechanisms in the Arctic, including the permafrost carbon feedback14. Therefore, quantifying the impacts of the altered timing and magnitude of hydrological connectivity in these environments is necessary to parameterize earth system models that predict global change15.Despite recent advances in our understanding of terrestrial-aquatic connectivity in permafrost-underlain watersheds5, but is not quantified for most Arctic rivers. In particular, little is known about the role late-summer rainfall plays in coupling hillslopes with stream networks. Projected increases in the magnitude and frequency of summer rainfall events8 are expected to elevate terrestrial-aquatic fluxes of organic and inorganic material in Arctic watersheds16. This is because the majority of rainfall events occur when suspended sediment (SS), dissolved and particulate organic carbon , nutrients, and major ions are readily available for transport due to active layer depths and terrestrial biological productivity being at their annual maximums19. However, few hydrological and biogeochemical records capture the full Arctic thaw season due to the logistical constraints imposed by working in remote locations12. The impact of changes in temperature and precipitation on permafrost systems is further complicated by the increasing frequency of thaw-induced geomorphological disturbance , which alter Arctic landscapes, local hydrology, biogeochemistry, and terrestrial-aquatic connectivity23. Permafrost disturbances that hydro-geomorphologically couple with fluvial systems amplify the effect of climate change through disproportionate increases in the mobilization and delivery of terrestrial material into stream networks25.The increasing importance of the under-studied and under-represented “shoulder seasons”  in annual Arctic biogeochemical budgets has been recently highlighted5.Here we use data from the hydrological and biogeochemical research program at the Cape Bounty Arctic Watershed Observatory , located in the Canadian High Arctic . Arctic streams and rivers have distinct nival hydrological regimes  and fall “freeze-up”  temperatures at our study site warmed ~ 2 °C between 1948 and 2019 . Locally, above average summer temperatures coincided with: (i) earlier first day of nival runoff and a longer summer flow season ; (ii) decreased watershed snow water equivalent10; (iii) a general reduction in maximum nival stream power from 68.0 ± 14.6 W to 42.2 ± 19.0 W; and (iv) reduced influence of the snowmelt phase on material transfer 10.The unique seasonality of Arctic hydrology varies with latitude and is closely linked to regional weather, and in particular regional air temperaturen = 8; Table 28; 86% of the annual available stream power was expended during the entire nival period (29 days) the same year.The timing and magnitude of peak nival runoff at the CBAWO is strongly controlled by snow accumulation in river channels . In years rainfall had minimal hydrological significance in summer/fall (n = 4), the timing of seasonal stream power shifted from a nival-dominated (skewed) to a nival- and pluvial-dominated  hydrological regime  and resulted in high stream powers exceeding that of the nival peak in respective years: (i) a rain-on-snow event ; (ii) consecutive low-intensity rainfall ; and (iii) consecutive high-magnitude rainfall events   Fig. . In comp2 watersheds; Supplementary Figs. It is important to note that, despite the CBAWO being the longest integrative data set from the Canadian High Arctic, we have hydrological observations for only 39% of recorded rain events . Internal regulators  and thresholds are critical for determining if materials and energy are dissipated, stored within a subsystem, or conveyed to the adjacent subsystem. Hydrology is a key driver of the watershed continuum. Terrestrial-aquatic connectivity at the CBAWO follows a temporary storage-remobilization regime along the watershed continuum. The channel slope and morphology  favor energy dissipation and sediment deposition, temporarily storing materials on channel bars and the channel bed28. This material is readily available for remobilization once the fluvial energy increases and overcomes the channel’s buffering capacity threshold.Shifts in the timing and magnitude of annual discharge patterns and stream power budgets are causing Arctic material transfer regimes to undergo fundamental changes. The term “watershed continuum” describes source-to-sink controls on material  erosion, transfer, and deposition. The watershed continuum can be defined as a “cascading system” composed of a chain of subsystems dynamically linked by a cascade of mass and energy28. Conversely, pluvial runoff couples the terrestrial-aquatic continuum at a time when: (i) the seasonal active layer is deepening (mean JJA active layer depth 0.8 ± 0.3 m), increasing subsurface drainage connectivity30; and (ii) vegetation productivity is at its peak, increasing above-ground biomass and available organic material from modern terrestrial vegetation31  and serve as an indicator for terrigenous DOM33. Concurrent higher growing degree day . Spectral slope ratios (SR) of chromophoric DOM indicated that pluvial runoff from rainfall events with higher intensities, longer durations, and/or less frequent recurrence intervals led to higher average molecular weight DOM in the stream networks35 . These data suggest that low- to intermediate hydrological events effectively couple the watershed-scale terrestrial-aquatic continuum for dissolved material and drive the majority of dissolved material export in these streams10 , significantly higher BIX values during baseflow periods . The enhanced BIX values in the main streams were less evident when stream powers exceeded ~25 W, suggesting a threshold where autochthonous DOM production is reduced and/or the autochthonous DOM signal is masked by increased allochthonous DOM input. As the hydrological season continues to lengthen and both air and river temperatures increase, in-stream productivity and increased DOM processing during baseflow periods will also likely increase, further impacting C processing potentials in this landscape38.Baseflow periods had the lowest stream powers (0.05 ± 0.02 W in headwaters and 3.0 ± 2.2 W in main streams), with limited hydrological connectivity and terrestrial to aquatic material flux34. However, the low magnitude rainfall events are unable to overcome energy thresholds needed to transport particulate material downstream to watershed outlets. This results in the temporary storage of particulate material within steam channels, where it can be processed and become available for microbial use during low energy hydrological periods. When higher magnitude pluvial events overcame our measured energy thresholds (stream powers >30 W), particulate material from eroded channel banks and disturbed hillslopes was directly coupled downstream with the watershed outlet28. At this site, rainfall magnitudes great enough to produce runoff energy to overcome stream power thresholds only occur on an 8 to 100-year recurrence interval (<12% chance of exceedance in any given year). The projected increase in the magnitude and frequency of summer rainfall events in the Arctic8 suggests these stream power thresholds will be met with greater frequency, resulting in future increased lateral particulate export.Combined, our results show lower magnitude rainfall events are important mechanisms for supplying Arctic rivers with dissolved and particulate terrestrial material39. Thaw-induced geomorphological disturbances are anticipated to occur in over 20% of the permafrost zone, thawing an additional 80 ± 19 Pg C by 2300 compared to gradual thaw processes alone  and greatly enhancing the permafrost carbon feedback20. These disturbances increase the delivery of terrestrial materials to aquatic systems25, but the longer-term persistence (>2–5 years) of these impacts remain relatively unknown. The paired watersheds in this study were impacted by localized ALDs in 2007–2008, disturbing 1.2–2.7% of the watersheds’ areal extent .In addition to shifts in the timing and magnitude of fluvial energy, climate warming is causing thaw-induced geomorphological disturbance across the Arctic−1 NDVI between 1985–201540) as a result of longer growing seasons leading to increased vegetation biomass41 and warmer summer temperatures causing higher concentrations of DOM to be released to aquatic ecosystems42  enhanced DOC processing within the stream network48; and (iii) preferential sorption of available DOC to newly exposed mineral soils50.We show that despite disturbances covering a relatively small areal extent (<2.7%) of the watershed, processes that reduce DOC availability and transport following permafrost disturbances were strong enough to offset concurrent watershed-scale processes that increased DOC export from vegetated geomorphologically undisturbed headwater-slope streams. DOC concentrations from these undisturbed slopes increased during our study period  to trigger this change in fluvial C export from DOC- to POC-dominated in High Arctic systems. Despite this shift in the form of C export, localized ALDs did not increase the downstream, multiyear watershed-scale flux of particulate material due to fluvial energy limitations causing temporary channel storage10. This temporarily stored POC may be made accessible for additional processing during summer baseflow as discussed in the above section.At all watershed scales the ALDs led to a shift in the primary form of C export from a DOC- to a POC-dominated flux, with the magnitude and persistence of impact increasing with the areal extent of watershed disturbance Fig. . Our res28, but are an important mechanism resupplying channel stores with newly mobilized/eroded terrestrial material9. This suggests that, at the watershed-scale, the effect of thaw-induced geomorphological disturbance on particulate material export is smaller than the shift toward a multimodal hydrological regime in this environment. Furthermore, disturbed sites are more vulnerable to rain-induced surface thaw51, which could further accelerate the additional release of terrestrial materials following future rainfall. Hence, we conclude terrestrial disturbance primes Arctic watersheds for accelerated geomorphological change when future rainfall magnitudes and consequent pluvial responses exceed the current buffering capacity of the terrestrial-aquatic continuum (30 ± 9 W).The lack of multiyear increase in the watershed-scale particulate flux post ALDs was primarily due to insufficient fluvial energy to overcome the thresholds for terrestrial-aquatic connectivity at a time when the additional POC sources are thawed and readily available for mobilization . In this setting, nival runoff is ineffective at mobilizing additional POC exposed by localized ALDs due to limited access to terrestrial material at times of high fluvial energy. To fully couple the POC continuum from disturbed sources to downstream at the catchment outlet, summer rainfall events had to result in stream powers >30 W in the main streams. Pluvial responses with less energy rapidly mobilize and exhaust temporary in-channel particulate stores10  or lead to the formation of a talik (former permafrost remaining thawed year-round), geomorphological disturbance itself may play a stronger role in altering C export potentials56. Our field observations show that the timing and magnitude of available fluvial energy is the key mechanism determining the role stream networks play in transporting and cycling terrigenous C and OM in High Arctic watersheds underlain by continuous permafrost. Our identified energy threshold (30 ± 9 W in the CBAWO main streams), which was necessary to fully connect the terrestrial-aquatic continuum at the watershed-scale, may explain disparities in the literature concerning the role inland waters play in cycling terrestrial C. Arctic aquatic systems where energy thresholds exceed the watersheds’ buffering capacity, including the continuum of headwaters to larger river networks, are heavily influenced by allochthonous input and terrestrial connectivity1. Lower-energy systems such as small lowland ponds, which represent ~25% of northern surface water area, lack the energy needed to hydrologically connect the terrestrial-aquatic continuum, and therefore primarily internally cycle autochthonous C and play a minor to negligible role in the mineralization of terrestrial C57.Given that watershed attributes in low-order headwater catchments like those at the CBAWO dictate stream chemistry within larger river systems, it is critical to examine changes at the headwater-scale to better integrate and predict changes at larger watershed and pan-Arctic scales8. Given that the seasonality of Arctic hydrology is closely linked to regional weather, we can expect the timing and magnitude of this fluvial energy shift to vary among sites. While the spring freshet has been previously recognized as a strong driver of terrestrial-aquatic material export in Arctic inland waters59, we identify the increasing role of rainfall events to enhance terrestrial-aquatic connectivity during periods with the highest annual terrestrial vegetation biomass and deepest active layer depths , enabling our understanding of these rainfall events and the late season baseflow we miss each year5.In this study, we found evidence of shifts in timing of fluvial energy from a skewed to a multimodal distribution in both large and small circum-arctic watersheds, which aligns with global climate model projections of amplified Arctic warming, decreased winter precipitation, and increased magnitude and frequency of summer rainfallths Fig. . Rain ev2) to trigger a fundamental shift in C export regimes from DOC- to POC-dominated. We conclude that ALDs have primed the watershed for accelerated geomorphological change when future rainfall magnitudes and consequent pluvial responses exceed the current buffering capacity of the terrestrial-aquatic continuum in this energy-limited system. It is important to note impacts from ALDs and other localized geomorphologic disturbances might be asynchronous and not additive as the watershed contributing area increases, further highlighting the importance of examining low-order headwater catchments to accurately upscale to larger watershed and pan-Arctic scales.Localized ALDs offset the increased DOC export from undisturbed vegetated hillslopes during our study despite covering <2.7% of the watershed areal extent. This indicates changes in C export following permafrost thaw-induced geomorphological disturbance, which occurs in a relatively small area of the watershed, outpaces and offsets changes in C export due to watershed-wide processes such as greening. We identified a threshold where hydrologically-coupled ALDs need to geomorphologically disturb as little as 1.2–2.7% of the watershed area (≤12 km−1). Mean summer  air temperature was 2.7 ± 2.5 °C (2003–2019). Temperature and rainfall data were obtained from local (2003–2019) and regional (1948–2019) meteorological stations proximal to the CBAWO , located on the south-central coast of Melville Island in the Canadian High Arctic , and the East watershed is 11.6 km2 . The West watershed is 8.0 kmR2016a and 2020a software. Correlations were calculated using Pearson’s r linear correlation coefficients .Daily JJA active layer depths Fig.  were est−2 s−3) is the product of water density calculated from measured water temperatures , acceleration due to gravity , discharge , and channel slope (s). We applied a 5 m buffer on either side of the middle of the channel in ArcGIS® to represent the channel area, from which the watershed’s average slope was calculated (Supplementary Table Stream power represents the rate of energy expenditure along a riverbed and banks:mentclass2pt{minimSupplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Description of Additional Supplementary Files


ST266 is the biological secretome of cultured Amnion-derived Multipotent Progenitor cells containing multiple growth factors and cytokines. While intranasally-administered ST266 improves the phenotype in experimental optic neuritis, specific ST266 components mediating these effects are not known. We compared the effects of ST266 with and without removal of large molecular weight proteins both in vitro and in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) in C57BL/6J mice. Mice were treated daily with intranasal vehicle, ST266 or lower molecular weight fraction of ST266. Retinal ganglion cells were counted in isolated retinas, and optic nerves were assessed for inflammation and demyelination. ST266 treatment significantly improved retinal ganglion cell survival and reduced optic nerve demyelination in EAE mice. The lower molecular weight ST266 fraction significantly improved optic nerve demyelination, but only showed a trend towards improved retinal ganglion cell survival. ST266 fractions below 50kDa increased Schwann cell proliferation in vitro, but were less effective than non-fractionated ST266. Demyelination attenuation was partially associated with the lower molecular weight ST266 fraction, but removal of higher molecular weight biomolecules from ST266 diminishes its neuroprotective effects, suggesting at least some high molecular weight proteins play a role in ST266-mediated neuroprotection. Acute demyelinating optic neuritis is the most common presenting symptom of multiple sclerosis (MS), a debilitating demyelinating disease of the central nervous system and leading cause of neurologic disability in young adults . CorticoRecent work has shown therapeutic potential for ST266  –7, a bioIntranasally-delivered ST266 protects against vision loss, preserves retinal ganglion cells (RGCs), and decreases inflammation and demyelination of the optic nerve in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS , 6. ThesThe mechanism by which ST266 decreases demyelination and promotes neuronal survival is not fully understood but is likely multifactorial and is being actively investigated. While the total ST266 secreted protein concentration from the AMP cells is in the microgram per ml range, individual components are only present in pg to ng per ml quantities . Liquid To examine the relative role of large and presumably inert proteins such as albumin as well as higher molecular weight growth factors in the neuroprotective and myelin-protective effects mediated by ST266 in optic neuritis, we generated a lower molecular weight fraction of ST266 in which proteins larger than 50 kDa were excluded. Effects of this less than 50 kDa ST266 fraction were compared to full composition ST266 in a standardized in vitro cell proliferation assay as well as the mouse EAE model of optic neuritis.Female C57BL/6J mice were purchased from the Jackson Laboratory . All experiments in this study adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were compliant with the University of Pennsylvania Institutional Animal Care and Use Committee guidelines and policies. All animal studies and procedures reported in this manuscript were reviewed by the University of Pennsylvania Institutional Animal Care and Use Committee, who specifically approved these studies as the University of Pennsylvania Institutional Animal Care and Use Committee protocol #804701.Filtrates of ST266 were prepared using Amicon Ultra-15 filters . The centrifuge  was pre-cooled for 4°C and set to 4,000 g. For each filter, depending on the molecular weight cut off, spin time was optimized following manufacturer instructions. Filters were first washed with 15 mL Water for Injection (WFI) to remove potential residual compounds following manufacture. Water was removed, and samples loaded into the filter.2. When acclimated, NGM media was replaced by minimal starvation medium for 24-hours and subsequently replaced by one of the treatment medias: NGM, Iscove’s Modified Dulbecco’s Media , STM100 , ST266 or a filtrate fraction of ST266 below the molecular weight cut-off of either 50 kDa or 30 kDa.For Schwann cell proliferation assay, SW10 Mouse Schwann Cells  were seeded in 96-well tissue culture treated plates and cultured in normal growth media (NGM), containing Dulbecco’s Modified Eagle Medium , GlutaMAX (Cat# 35050061) and 10% FBS , for a fixed amount of time at 37°C and 5% COAfter 24-hours, proliferation of Schwan cells was determined using a commercial viable cell counting kit  per the manufacturer’s protocol. This colorimetric assay measures the water-soluble formazan dye formed by NADH-mediated reductions in extracellular tetrazolium salt WST-8. ST266 reference control and unconditioned media reference control were also run in each assay. Absorbance was read at 450nm using Synergy 2 plate reader  and either the STM100 or NGM blank media signal was subtracted. For each plate, proliferation was normalized to NGM treatment and data reported as a ratio of sample absorbance to the NGM value. Experiments were repeated three times, each with freshly made ST266 filtrate fractions.Chronic experimental autoimmune encephalomyelitis was induced in mice as previously described . BrieflyAliquots of ST266 prepared as described above for in vitro cell proliferation assays were provided by Noveome Biotherapeutics, Inc.,  and stored at 4°C. For intranasal dosing, unanesthetized mice were secured by the scruff and 20 μl of the test agent or vehicle control were instilled in the nares once daily, similar to prior studies [Optokinetic nystagmus reflex was used to estimate visual performance in mice using the OptoMotry apparatus and software . As described in prior studies , mice weRGCs were quantified by Brn3a immunolabeling using previously described methods . In summOptic nerves were isolated at the time of sacrifice, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick longitudinal sections. For assessment of inflammation, sections were stained with H&E and examined by light microscopy. Scores were assigned to each sample by a masked observer per prior studies  as folloEvaluation of EAE severity scores and OKR thresholds over time were compared using ANOVA of repeated measures followed by Tukey post-hoc comparisons between each group. RGC counts, final OKR scores, optic nerve inflammation scores, optic nerve demyelination scores, and Schwann cell proliferation levels were compared by one-way ANOVA with Tukey post-hoc comparisons between treatment groups. Where indicated, pairwise comparisons were made using the two tailed Student t-test. For the grouped analysis isolating mice with moderate demyelination phenotype, data from three separate experiments were pooled. In order to avoid potential bias from possibly variable RGC counts in different experiments, mice of each treatment group were compared to untreated controls from their corresponding individual experiment, and this is reported as a ratio, which was then combined between all three experiments in an effort to decease noise. All computations were done using Graph Pad Prism . P values less than 0.05 were considered significant.One-way ANOVA with six groups revealed significant differences in Schwann cell proliferation between treatments  and control (non-EAE) mice (n = 6) were monitored daily for development of ascending paralysis and weekly for OKR responses, prior to sacrifice at day 42 post-immunization. EAE induction produced only mild EAE disease which was not altered by daily intranasal treatment with ST266 or <50 kDa ST266 , and faiTo assess effects of ST266 and <50 kDa ST266 in mice with moderately higher levels of EAE disease, the experiment above was repeated two additional times, and all EAE mice that developed mild to moderate clinical EAE disease were used to assess optic neuritis outcomes. All mice that developed a cumulative EAE paralysis score between 0.5–60 (EAE score summed over entire 42 days of experiment) were included in this study. Mice that failed to develop any clinical signs of EAE paralysis (EAE score of 0) and mice who developed severe EAE disease defined as either a cumulative EAE score >60, or a single day EAE score of 4  or higher, were excluded. In all, 44 eyes of 22 control (non-EAE mice) were examined and compared to 23 eyes from EAE mice treated with PBS, 26 eyes from EAE mice treated with ST266, and 24 eyes from mice treated with <50 kDa ST266.Over the course of 42 days, progressive decline in OKR responses were observed in EAE mice, with a trend toward improved OKR in EAE mice treated with ST266 and <50 kDa ST266 that was not significant by ANOVA of repeated measures. On Day 42 post-immunization, OKR responses were significantly decreased in PBS-treated EAE mice as compared with control, non-EAE mice, and daily ST266 treatment significantly improved OKR responses whereas <50 kDa ST266 treatment led to a non-significant trend in improved OKR responses . ST266 aResults demonstrating the ability of ST266 to reduce RGC loss and preserve functional visual responses measured by OKR in EAE mice are consistent with prior studies showing similar neuroprotective effects of intranasal ST266 in EAE optic neuritis , 5. RemoCurrent results build on previous work showing decreased myelin loss and protection against RGC death in optic neuritis and traumatic optic neuropathy , 6 as weKnown components of ST266 itself reveal hundreds of cytokines and growth factors which have been implicated in anti-inflammatory and neuro protective pathways. Of these, the majority are less than 50 kDa in size, including platelet derived growth factor BB—which has been shown to protect against RGC loss along with Igf1 via Akt —as well As discussed in prior reports, the effect of ST266 likely relies on a multitude of simultaneous paracrine signals—as each component is present in physiologic or sub-physiologic concentrations . In contCompared to other pleiotropic strategies for neuroprotection, the non-invasive, intranasal dosing of ST266 offers pharmacokinetic advantages which highlight the importance for optimization of this route. Local diffusion along the olfactory nerves or lymphatic channels is suspected to play a role in the accumulation of ST266 to the vitreous and optic nerve based on radiotracer studies showing optic nerve accumulation as soon as 30 minutes after nasal dosing in the rat . FurtherIn this study, the initial optic neuritis experiment Figs –4 showedOverall, results here further support intranasal ST266 as a candidate neuroprotective therapy for optic neuritis, and suggest that development of strategies to deliver the full complement of biomolecules present in ST266 will be important for future clinical applications.


Aligned fiber scaffolds with fiber diameters ranging from 335 ± 154 nm to 3013 ± 531 nm are characterized for their bioactivities in supporting growth and differentiation of myoblasts. During in vitro culture, polymeric scaffolds with larger fiber diameter support enhanced alignment, growth, and differentiation of myoblasts associated with phosphorylation of p38 MAPK and upregulated expression of myogenin and myosin heavy chain. In vivo studies using a dystrophin-deficient mdx mouse model show that optimized fiber scaffolds seeded with primary myoblasts result in formation of dystrophin-positive myofibers network in tibialis anterior muscles. Collectively, these experiments provide critical insights on harnessing interactions between muscle cells and engineered fiber matrices to develop effective biomaterials for accelerated muscle regeneration.Regeneration of skeletal muscles is limited in cases of volumetric muscle loss and muscle degenerative diseases. Therefore, there is a critical need for developing strategies that provide cellular and structural support for skeletal muscle regeneration. In the present work, a bioengineered cell niche composed of mechanically competent aligned polyester fiber scaffolds is developed to mimic the oriented muscle fiber microenvironment by electrospinning poly(lactide- Skeletal muscles are composed of oriented muscle fibers that are formed by the fusion of myoblasts. This orientation in fiber alignment enables an anisotropic organization of extracellular matrix (ECM) for functional contraction, which leads to bodily movement. In response to minor injuries, skeletal muscles exhibit a remarkable regenerative capacity empowered by a unique stem cell population, satellite cells. Satellite cells are localized along the surface of muscle fibers under the basal lamina, offering a niche microenvironment that regulates the cell functions  , was specifically selected to produce fiber scaffolds due to its biodegradability, biocompatibility and desirable mechanical properties for skeletal muscle engineering  (PLGA) 85:15 (94 kDa) was purchased from Lakeshore Biomaterials. Tetrahydrofuran (THF), N, N-Dimethylformamide (DMF), Triton X-100 and 4% paraformaldehyde was obtained from Fischer Scientific. High Glucose DMEM with L-Glutamine and sodium pyruvate, and Penicillin/Streptomycin was purchased from Gibco, Life Technologies. Fetal bovine serum (FBS) and horse serum were acquired from Atlanta Biologicals. FAK-100 actin cytoskeleton kit (EMD Millipore) was employed for actin staining. Fluorescent mounting medium was obtained from Dako, Agilent Technologies and Pico green assay kit was purchased from Life Technologies.Poly(lactide-PLGA [20-40% (w/v)] solution in THF: DMF (3:1) were loaded into a 10-mL syringe fitted with an 18G blunt needle. The needle was connected to a power supply of 20 kV. The polymer solution was fed at a rate of 2 mL/h. A flat plate collector was used for the fabrication of random fibers. A custom-designed drum roller with interdigitated blades was employed for the fabrication of aligned fibers . The dign = 90 fibers; n = 3 images per sample).Polymer solutions with different concentrations were electrospun for 30 min, the fibers were mounted directly onto the SEM stubs using carbon tape. The samples were coated with platinum using a Cressington turbo-pumped sputter coater for 30 s and analyzed using FEI NOVA nanoSEM FESEM. The surface morphology of the fiber samples was imaged at various magnifications using 5 kV accelerating voltage. The fiber diameters were measured, and their alignment was characterized based on the angle of individual fibers with respect to the average alignment  of the fiber samples using Image J software  were immobilized using Scaffadex 48-well plate inserts as previously reported  were characterized as previously reported using Image J software  before being permeabilized with 1% Triton X-100. The fixed cells were blocked using 1% bovine serum albumin (BSA) followed by staining for F-actin  and nuclei (DAPI). The scaffolds with stained cells were mounted on a glass slide with anti-fade mounting media and imaged using EVOS FL imaging system. The cell elongation ratio, cell spreading area, and cell alignment . The assay involves the quantification of double-stranded nucleic acids using an ultra-sensitive fluorescent probe. The intracellular content was extracted from each scaffold sample using 1% Triton X-100 in 1 × PBS. To completely rupture the cell membrane and extract the nucleic acid content, samples were freeze-thawed three times. The obtained solution was used for the quantification of the double stranded nucleic acid content  . The samples were incubated overnight at 4°C followed by washing with 1 × PBS three times before the addition of the FITC labeled secondary antibody. The samples were washed three times with 1 × PBS to remove excess secondary antibody. The stained samples were mounted on glass slides along with fluorescent mounting media. The samples were imaged using an Olympus FV10i confocal microscope at different magnifications.n = 3) were combined to extract enough total protein using RIPA buffer. The protein concentration was quantified with Pierce BCA protein reagent . SDS-polyacrylamide gel electrophoresis was performed to the separate proteins, after which they were transferred to polyvinylidene fluoride (PVDF) membranes . Membranes were blocked with 5% BSA in Tris-buffered saline—Tween 20 (TBST) buffer. Antibodies used were the following: Phospho-p38 MAPK (Thr180/Tyr182) , Phospho-FAK (Tyr397)  and MyoG F5D . The secondary antibody  conjugated with horseradish peroxidase was applied (1:5000). ChemiDoc™ Touch Imaging System (Bio-Rad) was utilized to detect the protein bands.Samples . Mice were housed in the animal facility with free access to water and standard rodent chow. All procedures involving the use of animals were performed in accordance with the guidelines presented by Purdue University's Animal Care and Use Committee. Without special mentioning, we used 2-month-old male mice for experiments. Cardiotoxin (CTX) injection (10 μM) was administered in the tibialis anterior (TA) muscle of mdx mice 24 h prior scaffold implantation. 2 × 105 primary myoblasts were seeded on Matrix 3 fibers and cultured overnight in growth media. Scaffolds (2 mm in diameter) were implanted at the site of CTX administration. Myoblasts cells (2 × 105 cells in 500 μL primary myoblast growth medium) were injected at the site of CTX administration and were used as controls. The TA muscle, for both control and scaffold group, was harvested by cryo-fixing the samples in OCT 3 weeks after implantation. Cryosections of the TA muscles were stained for dystrophin  for further analysis. The stained cross-sections (230 × 230 μm) were imaged in Nikon A1R MP confocal microscopy and quantified to evaluate dystrophin positive myofibers per cross section image obtained (n = 3 per group).post-hoc test with a minimum confidence level of p < 0.05 for statistical significance. Student t-test was used to compare experimental groups for the in vivo dystrophin staining analysis with confidence interval for p < 0.05 for statistical significance.Quantitative data were reported as mean ± standard deviation (SD). Statistical analysis was performed using a one-way analysis of variance (one-way ANOVA). Comparison between means was determined using the Tukey In the skeletal muscles, ECM protein molecules have a well-defined structural organization creating tightly packed and highly oriented fibers to facilitate specific skeletal muscle functions. Previous investigations have suggested that topography plays a critical role in modulating functional and structural properties of the cells  controls at 2 and 24 h post-seeding . InteresQuantitatively, Matrix 2 and Matrix 3 supported greater cell alignment as compared to Matrix 1 as evidenced from the alignment angle analysis after 2 h of cell seeding . FurtherIn vitro differentiation studies of C2C12 cells on aligned fibers were performed to assess the effects of fiber diameter on myoblast differentiation. In specific, differentiation of myoblasts into myotubes was characterized by immunofluorescent staining for the myotube maturation marker, myosin heavy chain (MHC). As shown in mdx mouse model was used to evaluate the in vivo regeneration efficacy of aligned fiber scaffolds. These mice lack native dystrophin, a structure protein in the cell membrane, making dystrophin an appropriate biomarker for in situ regenerative efficacy of transplanted cells through immunostaining of dystrophin-positive myofibers at the injury site. Matrix 3 was selected for the in vivo study due to enhanced myoblast proliferation and differentiation in vitro. As shown in mdx mice with cardiotoxin (CTX) treatment followed by implantation of scaffolds seeded with primary myoblasts. To determine the regeneration improvements by scaffold-mediated cell grafting, myoblast transplantation alone was used as controls. After 21 days of transplantation, presence of dystrophin-positive myofibers was scarce in the myoblast transplantation group  resemble those on planar surfaces  often result in higher necrosis due to limited diffusion of oxygen and nutrients supplied by the host. The encouraging in vivo evidence presented in this study for our scaffold-based transplantation approach attests to the promise in developing bioengineered transplantable systems that foster the survival and function of myogenic cells to promote myogenesis for in situ skeletal muscle regeneration. In particular, combining a versatile electrospinning process with FDA-approved PLGA will facilitate the clinical translation of biodegradable and biomimetic scaffolds for skeletal muscle repair and regeneration applications. Current ongoing studies focus on efficacy evaluation of this fiber scaffold platform for skeletal muscle regeneration using a volumetric muscle loss model.Myoblast transplantation therapy has been proposed as a potential treatment for Duchene muscular dystrophy and other muscle related disorders (Gussoni et al., In vivo study using an mdx mouse model demonstrated the promise of using optimized fiber scaffolds to improve myogenic potential of transplanted cells. Taken together, our studies demonstrated that biomimetic fiber scaffolds through topographical contact guidance enhanced myogenic potential of myoblasts; hence, providing critical insights into harnessing scaffold physical properties for accelerated muscle healing.In the present work, biomimetic structures composed of aligned electrospun polyester fibers were fabricated and characterized to optimize fiber diameter effects on myoblasts for skeletal muscle regeneration. We demonstrated that alignment of the scaffold fibers promoted FAK phosphorylation and reorientation of the actin cytoskeleton leading to elongated cell morphology. Interestingly, increase in fiber diameter from 335 ± 154 nm to 3013 ± 531 nm resulted in enhanced myoblast proliferation and differentiation as well as myoblast fusion into mature myotubes indicating the ability of cells to respond to fiber topography. All datasets generated for this study are included in the article/The animal study was reviewed and approved by Purdue University's Animal Care and Use Committee.in vitro cell culture experiments. DC and OJ contributed to the nanomechanical characterization of the fibers. NN, CW, GU, and SK contributed to the in vivo experimental design and analysis. All authors discussed the results and commented on the manuscript.NN and MD conceived and designed the experiments, and prepared the manuscript. NN, CJ, and NW performed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although exposure to cigarette smoking during pregnancy has been associated with alterations of DNA methylation in the cord blood or placental cells, whether such exposure before pregnancy could induce epigenetic alterations in the placenta of former smokers has never been investigated.Our approach combined the analysis of placenta epigenomic (ENCODE) data with newly generated DNA methylation data obtained from 568 pregnant women, the largest cohort to date, either actively smoking during their pregnancy or formerly exposed to tobacco smoking.LINE-1 sequences in the placentas of both current (beta-coefficient (β) ), − 0.004 ) and former smokers (β (95% CI), − 0.006 ) compared to nonsmokers. Second, the 203 DMRs were enriched in epigenetic marks corresponding to enhancer regions, including monomethylation of lysine 4 and acetylation of lysine 27 of histone H3 (respectively H3K4me1 and H3K27ac). Third, smoking-associated DMRs were also found near and/or overlapping 10 imprinted genes containing regions (corresponding to 16 genes), notably including the NNAT, SGCE/PEG10, and H19/MIR675 loci.This strategy resulted in several major findings. First, among the 203 differentially methylated regions (DMRs) identified by the epigenome-wide association study, 152 showed “reversible” alterations of DNA methylation, only present in the placenta of current smokers, whereas 26 were also found altered in former smokers, whose placenta had not been exposed directly to cigarette smoking. Although the absolute methylation changes were smaller than those observed in other contexts, such as in some congenital diseases, the observed alterations were consistent within each DMR. This observation was further supported by a demethylation of Our results pointing towards genomic regions containing the imprinted genes as well as enhancers as preferential targets suggest mechanisms by which tobacco could directly impact the fetus and future child. The persistence of significant DNA methylation changes in the placenta of former smokers supports the hypothesis of an “epigenetic memory” of exposure to cigarette smoking before pregnancy. This observation not only is conceptually revolutionary, but these results also bring crucial information in terms of public health concerning potential long-term detrimental effects of smoking in women. Despite increasing awareness of smoking-associated risks in pregnancy and although smoking cessation is recognized as one of the most effective actions for improving mothers’ and children’s health , betweenIntrauterine life is a critical period of plasticity during which environmental insults can alter the developmental programming via epigenetic phenomena, with immediate effects visible at birth or delayed effects that appear in childhood, puberty, or adulthood . The mosA few studies have investigated the association between placental DNA methylation and maternal smoking during pregnancy –33. MostThis work aimed at identifying cigarette-induced alterations in placental DNA methylation in women currently smoking during pregnancy and in women who quit in anticipation of pregnancy.3 were excised a few centimeters from the insertion of the umbilical cord under the chorio-amniotic membrane, washed in a saline solution, and were immediately frozen at − 80 °C. The protocol was similar for both centers. The EDEN cohort received approval from the ethics committee (CCPPRB) of Kremlin Bicêtre and from the French data privacy institution Commission Nationale de l’Informatique et des Libertés (CNIL). Written consent was obtained from the mother for herself and for the offspring.Study participants included in this analysis are a subset of the participants enrolled in the EDEN Mother-Child Cohort between 2003 and 2006 . EDEN isNon-smokers were defined as women who did not smoke during the 3 months before and during pregnancy. Current smokers were defined as mothers smoking ≥ 1 cigarette per day throughout the duration of the pregnancy. All current smokers during pregnancy also smoked during the 3 months before pregnancy. Former smokers were defined as women who reported smoking during the 3 months preceding the pregnancy and declared not smoking throughout the duration of the pregnancy. Women who quit smoking during pregnancy or cases with missing information regarding their smoking status at some point during the 3 months preceding the pregnancy or during the pregnancy were excluded, leaving 568 participants for this analysis. The choice of a 3-month exposure time period prior to pregnancy was based on a compromise between a realistic and accurate assessment of the smoking status of women around conception time  and a duration that would allow enough time for a large contingent of lung epithelial cells to renew ) years old, with a median pre-pregnancy body mass index of 22  kg/m2 Table . The med2 Table .Table 1LINE-1 and 0.16 (± 0.01) for Alu. Women who smoked had significantly lower methylation levels for LINE-1 compared to non-smoking women, with a slightly larger association for former smokers ) than for current smokers )  for 1)) Fig. a. No sighip Fig. b.Fig. 3p value distribution was close to the theoretical distribution as indicated by the BIF value of 1.06 which was substantially smaller compared to the lambda  value of 1.61  in the EWAS, Additional file p value < 0.02). We focused our subsequent analyses on these 203 DMRs and their associated 1023 CpGs. There were 54% (former smokers versus non-smokers), 84% (current smokers versus former smokers), and 87% (current smokers versus non-smokers) of CpGs whose change of DNA methylation (relative to the mean of each CpG) was higher than 1% in absolute value  and 68 DMRs (including 358 CpGs), respectively. Among these DMRs, 89% when adjusting for paternal smoking status during pregnancy (181 DMRs among 203) and 90% when adjusting for maternal passive smoking exposure (61 DMRs among 68) overlapped with the initial 203 DMRs , showing the robustness of the results  in genome regions where smoking-induced altered methylation events were observed.The 203 smoking-associated DMRs were found near and/or overlapping with 10 imprinted genes containing loci (including 16 imprinted genes) or their imprinting control regions (ICR). As shown by the volcano plots  and igcgo.ac.nz ) databasgo.ac.nz  and Hamago.ac.nz ), which NNAT/BLCAP (20q11.23) and SGCE/PEG10 (7q21.3), were both found associated with a reversible alteration of DNA methylation, which was respectively decreased or increased for all CpGs (but 2 for NNAT/BLCAP) in women currently smoking during pregnancy, compared to both former and non-smokers. NNAT/BLCAP was the longest DMR identified with 35 CpGs. Interestingly, the third locus, H19/MIR675 (11p15.5), was affected by a decreased methylation not only in the placenta of currently smoking pregnant women, but methylation alterations were also detectable in the placenta from former smokers that had never been directly exposed to cigarette smoking. The 7 other DMRs were also overlapping imprinted gene loci  locus was not present in the 68 DMRs identified when adjusting for maternal passive smoking  (not shown). Of note, the LINE-1 sequences in the placentas of former smokers. Exploration of the epigenetic status of all DMRs revealed that genomic regions bearing “enhancer-like” epigenetic marks, namely histone post-translational modifications H3K4me1 and H3K27ac, are enriched among our 203 DMRs, suggesting that placenta enhancer genomic regions could be particularly sensitive to tobacco exposure. Furthermore, tobacco-associated DMRs also overlap with genomic regions controlling imprinted genes, known to have an important role in fetal and placental development.The present study identified 203 genomic regions that were significantly differentially methylated in the placenta according to cigarette smoking during pregnancy. Interestingly, 26 of these DMRs were characterized by persistent methylation changes in the placentas of former smokers, despite an absence of direct exposure of these placentas to tobacco, suggesting the possibility of an “epigenetic memory” of exposure to cigarette smoking before pregnancy. This result is also supported by the observation of a significant demethylation of Potential limitations of our study relate to the characterization of the smoking phenotype. Indeed, maternal smoking was evaluated by questionnaires administered by midwives involved in the study, which might lead to an underestimation of the number of smoking women and the effect of smoking, due to under-reporting of tobacco consumption. However, on a subsample of 100 women whose cotinine levels were measured in the urine between 24 and 28 gestational weeks , we founp values in an EWAS is not unexpected and cannot be compared with inflation commonly seen in GWAS studies. One likely explanation for the inflation is the somewhat small but multiple effects of tobacco smoking on DNA methylation throughout the epigenome giving rise to a large number of sites impacted by smoking. Using the recently developed Bayesian method, the inflation value was substantially smaller (1.06) and consistent with little evidence of inflation.Our results showed a high lambda value of 1.61. Such inflation of low The placenta is a complex transient organ composed of different layers containing different cell types. In the absence of cytological data on our placenta samples, we used a reference-free method, based on latent factor models, to estimate cellular heterogeneity from DNA methylation array data. To account for the potential effect of cell types on DNA methylation levels, we adjusted for cellular heterogeneity using a latent factor method. Although this approach enabled us to account for the impact of cellular heterogeneity on DNA methylation, it is likely not sufficient to fully account for cell-type composition differences. Indeed, since this approach is data-driven, it is possible that residual confounding by cellular heterogeneity may have influenced some of our results. In particular, the real number of cell types in the placenta is expected to be higher than the 6 latent variables captured by the RefFreeEWAS algorithm, and therefore, the residual effect of cell types on placental methylation levels cannot be ruled out.Data from the literature show that for some loci, DNA methylation variations could be related to the nearby genetic variations –59, whicAssumptions of linear regression are often violated in EWAS, especially the normality of the distribution of the residuals. Nonetheless, a study  conducteThe present study includes the largest sample size of the placentas investigated to date, which were collected in the context of a longitudinal cohort with repeated data on maternal smoking before and during pregnancy. The placenta is considered as an accurate “record” of children’s in utero exposures  and reprp value < 0.05) with maternal smoking by Morales et al.  was reported to interact with benzo(a) pyrenes, resulting in a decreased gene expression [TINAGL1  and PDGFB (platelet-derived growth factor subunit B) are broadly expressed in the placenta and have been identified as pro-angiogenic factors [PDGFB is likely to play an important role in the maintenance of utero-placental homeostasis [TINAGL1 showed lower methylation and PDGFB higher methylation in smokers compared to non-smokers. In a recent study, TINAGL1 was found downregulated in the placenta of pre-eclamptic pregnant women compared to normotensive women [Only one study previously investigated placenta DNA methylation in relation to maternal smoking using a high-density genome-wide approach . Out of pression , which c factors . Angiogeeostasis . Nicotineostasis . In our ve women . Althougve women .Several studies identified CpGs altered in cord blood samples following exposure to tobacco smoking during pregnancy –25. AlthLINE-1 and Alu methylation [LINE-1 analysis was performed to measure methylation levels of Alu regions, which are also interspersed repeats, the latter did not vary significantly as a function of tobacco exposure. Furthermore, the mean/median methylation levels of the 425,878 CpGs did not show any significant association with the smoking status. Therefore, although methylation levels of LINE-1 and Alu are both considered as surrogate markers for global methylation levels, the present study does not suggest that tobacco exposure induces global changes in methylation levels but rather highlights that the methylation levels of these repeats are differentially affected by tobacco exposure. Furthermore, this observation of a differential sensitivity to tobacco exposure between different genomic repeats regions is paralleled by the different sensitivities to tobacco between unique genomic regions measured by the high-resolution genome-wide methylome Illumina 450K array. Only a few studies had so far investigated global methylation in the placenta. One study of approximately 40 first trimester placentas did not show any effect of maternal smoking on neither AluYb8 nor LINE-1 methylation levels [AluYb8 methylation level was significantly higher in smoking women compared to non-smokers, while no evidence of significant association was found for LINE-1 [LINE-1 methylation and maternal smoking [LINE-1 elements in terms of demethylation levels as suggested by previous observations in cancer [An interesting observation is the demethylating effect of exposure to tobacco smoking on LINE-1 sequences. Compared to other tissues, the human placenta is known to have lower levels of hylation , 71. Intn levels . In anotr LINE-1 . Another smoking . These an cancer .LINE-1 sequences observed in the placenta of former smokers, suggests that the placentas of former smokers bear epigenetic marks reflecting a past exposure to smoking, prior to the development of the placenta. It implies that somehow the memory of past smoking is transmitted to the placentas that have not been directly exposed to smoking. This observation raises new fundamental questions such as the mechanisms and molecular basis involved in such a transmission, which are presently totally unknown. Indeed, although the memory of past exposure to tobacco could involve alterations of epigenetic marks somehow transmitted to the oocyte, which would remain in the zygote and through the differentiation of placenta cells, it is possible and likely that non-epigenetic mechanisms would be involved. For instance, past smoking could affect the histological features of both the maternal and the fetal components of the placenta through the influence of maternal factors either via direct cellular contact or through endocrine signaling.Although a few studies have investigated the effect of time since quitting smoking on blood DNA methylation , 77, theSince DNA methylation is only one of the many chemical marks involved in shaping the epigenome, we wished to explore other elements of the epigenetic landscape of the DMRs identified as associated with maternal smoking status. The question was whether some other elements of the epigenetic context of these DMRs could be associated with the particular sensitivity of these regions to smoking exposure. A meta-analysis of ENCODE data from the placenta, obtained from independent external experimental settings, enabled us to explore the epigenome landscape normally associated with the most affected genomic regions. This analysis compared our tobacco-sensitive DMRs to genome regions of similar sizes that we randomly selected by using exactly the same EWAS and DMR pipelines but starting from samples which were randomly distributed between smoking status groups. The pattern of association with ENCODE epigenetic marks of these random regions showed a predominant association with H3K4me3, a gene promoter-associated hallmark, which is expected since our analysis was performed on CpG-rich regions. In contrast, our smoking-associated DMRs were depleted in histone post-translational modification (PTM) marks normally associated with poised gene promoter regions, such as H3K4me3, but were rather enriched in PTM generally associated with “enhancer” regions, H3K4me1 and H3K27ac. This observation suggests that this epigenetic context is somehow related to a higher sensitivity of these regions to tobacco exposure. Besides its biological meaning, this evidence demonstrates the biological relevance of our data and therefore could be considered as a biological validation of the identified DMRs.NNAT/BLCAP (20q11.23) and SGCE/PEG10 (7q21.3), were both associated with altered  DNA methylation in women currently smoking during pregnancy and classified as “reversible” regions. Increased expression of NNAT/BLCAP was associated with an increased risk of large or small for gestational age infants [SGCE/PEG10, its methylation in the cord blood was associated with birth weight [H19/MIR675 (11p15.5), was observed not only in the placenta of currently smoking women but also detectable in the placenta of former smokers suggesting that this important imprinted locus could be part of those bearing the memory of past exposure to tobacco. Lower methylation of this gene has been found associated with increased expression in fetal growth-restricted placentas [H19 in the cord blood was found significantly associated with maternal smoking during pregnancy [H19 was associated with large for gestational age infants [H19/MIR675 locus in the placenta exposed to cigarette smoking, combined with previous observations, strongly supports the hypothesis that the H19/MIR675 locus is a major determinant of fetal growth and that cord blood and placental DNA methylation are both sensitive to direct smoking exposure.Another major observation from this work is that tobacco-induced DNA methylation alterations also affect imprinting control regions (ICR), whose allelic differential methylation normally controls the allele-specific expression of clusters of “imprinted” genes. A recent work highlighted the incomplete erasure of germline DNA methylation in the human placenta and the effect of some allele-specific DNA methylation pattern on the expression of imprinted genes in the placenta . Interes infants . As for h weight , 80 and h weight . The demlacentas . In a reregnancy . Further infants , and gen infants , 85. OurThis study investigating the effect of smoking on human placental DNA methylation at high resolution in the largest sample size published to date has led to the identification of DMRs in the placentas of current smokers as well as former smokers. These tobacco-induced DMRs are enriched in epigenetic marks corresponding to enhancer regions, and some of them overlap regions containing imprinted genes or controlling their monoallelic expression, suggesting mechanisms by which tobacco could impact the epigenome and affect placental development and fetal growth. Additionally, altered DNA methylation patterns were not only observed in the placenta directly exposed to cigarette smoking, but some alterations of DNA methylation patterns were also observed in the placenta of women who had smoked but quit smoking in anticipation to pregnancy, suggesting the establishment of a “memory” of exposure to tobacco and transmission of epigenetic marks to placentas that had never been directly exposed to smoking. Hence, this work brings new concepts and questions in the field of human epigenetics and suggests mechanisms by which an exposure to environmental cues could have not only direct but also long-term effects on human health. In addition to the important potential impact on our current knowledge of epigenetic transmission, our results bring essential information in terms of public health concerning potential long-term detrimental effects of smoking in young women, which could affect their offspring even after smoking cessation. This scientific report should support the primary and secondary prevention of smoking-associated health effects.Additional file 1: Table S1. Metadata of analysed ENCODE files. Table S2. Results from the Epigenome Wide Association Study (EWAS): 1,800 CpGs differentially methylated between the three groups of women   <0.05). Table S3. Results from the regional analysis using comb-p : 203 Differentially Methylated Regions including 1023 CpGs. Table S4. Comparison of the number of CpGs found significantly associated with smoking between the present EDEN study and previous studies conducted on placenta and cord blood.Additional file 2: Supplementary Figure S1. cellular heterogeneity estimated by the RefFreeEWAS method on 668 placenta samples from the EDEN cohort (A) contribution (%) of each latent variable (B) Pearson correlation between the 6 latent variables. Supplementary Figure S2. Q-Q plot, genomic inflation factor (lambda) and Bayesian inflation factor (BIF) for the association between each methylation site of the EWAS  and tobacco smoking during pregnancy . Results were adjusted for child sex, parity, education level, season of conception, study center, maternal body mass index before pregnancy, maternal age at delivery, gestational duration, paternal smoking status at conception, batch, plate and chip and estimated cellular heterogeneity. Supplementary Figure S3. H3K4me3 (A), H3K4me1 (B) and H3K27ac (C) ChIPSeq signal of 3 placenta replicates. The top panels represent the mean signal values centered on our 203 DMRs +/-2kb (blue) and the mean signal values centered on the 420 random regions +/-2kb (green). The center panels represent heatmaps of the corresponding ChIP-Seq signals centered on our 203 DMRs +/-2kb (upper heatmaps) or on the 420 random regions+/-2kb (lower heatmaps). Supplementary Figure S4. Expression of genes in normal human tissues from RNA-seq data. The barplots show average expression levels of the 16 imprinted genes overlapping our DMRs in normal tissues and development stages . For the sake of clarity, the plots are separated in two panels of 8 genes. Vertical lines on the top of the barplots represent standard deviations. Barplots representing expressions in placenta are in green. RNA-seq data in normal tissues were provided by GTEx portal and NCBI Sequence Read Archive . The expression levels are represented in log-transformed RPKM (Read per Kilobase Million) values after addition of a pseudo count of 1, i.e. log-transformed RPKM = log2(1+RPKM). MIR675 has no expression in any of the analyzed samples. Figure S5. The methylation levels of 7 imprinted loci are consistently modified following exposure to cigarette smoking. The left, center and right panels are respectively centered on the DMRs associated with the NNAT locus +/- 2kb, the SGCE/PEG10 locus +/- 2kb and the H19/MIR675 locus +/- 2kb. The top panels show heatmaps respectively corresponding to H3K4me3, H3K4me1 and H3k27ac enrichments in placenta around the center of the region of interest +/- 2 kilobases. For each mark we downloaded triplicates from ENCODE data (see data see . We compAdditional file 3: Table S5. Imprinted genes whose ICR [hose ICR , 51, 86


Hydrothermal processes in impact environments on water-rich bodies such as Mars and Earth are relevant to the origins of life. Dawn mapping of dwarf planet (1) Ceres has identified similar deposits within Occator crater. Here we show using Dawn high-resolution stereo imaging and topography that Ceres’ unique composition has resulted in widespread mantling by solidified water- and salt-rich mud-like impact melts with scattered endogenic pits, troughs, and bright mounds indicative of outgassing of volatiles and periglacial-style activity during solidification. These features are distinct from and less extensive than on Mars, indicating that Occator melts may be less gas-rich or volatiles partially inhibited from reaching the surface. Bright salts at Vinalia Faculae form thin surficial precipitates sourced from hydrothermal brine effusion at many individual sites, coalescing in several larger centers, but their ages are statistically indistinguishable from floor materials, allowing for but not requiring migration of brines from deep crustal source(s). Dawn mission’s second extended phase provided high resolution observations of Occator crater of the dwarf planet Ceres. Here, the authors show stereo imaging and topographic maps of this crater revealing the influence of crustal composition on impact related melt and hydrothermal processes, and compare features to those on Mars, Earth and the Moon. Global imaging at ~35 m pixel scales revealed that Occator, the Tycho of the Asteroid Belt, is unique among craters >50 km on Ceres in that original formation morphologies are relatively intact relative to older degraded craters6. An extensive planar lobate floor deposit (LFD) in southeast Occator covers ~40% of the otherwise hummocky and ridged floor units9. LFD morphologies at these scales9 and modeling of impact heating10 suggested formation either as impact-derived deposits reminiscent of those observed in large craters on the Moon11  and Earth , or as post-impact volcanic outflows from deep subsurface water-rich reservoirs16, with important implications for Ceres’ internal thermal and geochemical structure. Similarly, bright salt-rich deposits (including Na-carbonates) in the central pit region at Cerealia Facula6 and within the LFD at Vinalia Faculae2 indicated that salt- and carbonate-rich fluids were exposed on the surface in a process involving either impact-derived or deep-rooted hydrothermal fluids, or both. The physical mechanisms by which any of these materials were deposited were not resolved at these scales, however. Key objectives of Dawn high-resolution observations across Occator17  outer layers of Ceres23, with those on the mostly anhydrous basaltic and anorthositic lunar crust, the ice-enriched basaltic crust of Mars27, and with the more eroded hydrothermal deposits in the water-enriched silicic crust of Earth. Combined Dawn observations indicate a mixed crustal composition for the dwarf planet Ceres. The low density and high strength crust inferred from crater morphologies and topography relaxation has been interpreted as a mixture of ice (up to 40 vol.%), hydrated salts and silicates (<20 vol.%), and clathrate hydrates22. Available constraints indicate that the martian crust is dominantly anhydrous basaltic with ~5–15 wt% water in the near-surface regolith and a few percent water in the form of hydrated minerals and water ice down to at least 10 km depth27, although concentrations could be much higher locally. Despite dust mantling and recent gully formation on steep slopes in even young craters28, original textures can be discerned on recent well-preserved Mars craters  volcanic deposits, as recently demonstrated experimentally in a Mars context34.Impact-generated melt deposits on Ceres are likely to be very similar in composition to the bulk composition of its outer layers9. Low arcuate ridges are now resolved on most of these flow lobes, confirming widespread lateral flow of mobile material on the crater floor9, particularly in the northeast and northwest quadrants. A few flow lobes are observed merging into broader flow units at lower elevation or overlapping with other flow lobes, indicating overlapping deposition (or outbreak) flow events as these larger units were emplaced. These types of thicker large-scale flows lobes are significantly more common within Occator than on the terrestrial planets, where impact melt flows have very low relief and smaller dimensions35. This difference in morphology suggests that mud-like impact melts at Occator could have had effective viscosities an order of magnitude higher than melted basalt or anorthosite, depending on compositions and emplacement conditions that are poorly constrained at present.Lobate flow features several kilometers wide and with relief of 10’s to ~350 m Fig.  and 8 ar3 of melted material . Mantling materials observed elsewhere are much thinner, ~10 m or less . Most irregular-shaped neutral-colored floor mounds thus resemble the floor mounds observed on fresh larger craters on the Moon11 (where ice does not occur in significant quantities) and Mars  Figs.  are brig10 was predicted owing to the high volatile content of Ceres outer layers22 and derived impact deposits. Large arcuate troughs crossing Occator37 are now resolved as sharply defined V-shaped troughs that crosscut all features and pinch-and swell from near 0 to ~250–350 m width over similar distances, forming deep elongate oval pits in some areas. Dawn XM2 imaging resolves initial unmodified narrow troughs ~10–20 m wide connecting the sharp-rimmed wider trough segments  no more than a few 10’s of meters deep. The formation of secondary craters back onto Occator itself has been demonstrated numerically39 and documented in crater counts40 . From this, we argue that many of these shallow ring depressions are secondary craters that formed rapidly, i.e., before thicker LFD materials could completely crystalize into solidified formations and thereby contaminating crater statistics. The shallow ring morphologies of these unusual craters are also consistent with impact experiments into viscous clay-water slurries41.Abnormally shallow ring-shaped depressions up to ~1 km across Fig.  common i40 is a few to as much as ~20 myr. Even so, all floor units are extensively pitted with small roughly circular depressions <500 m across . Most of these depressions are broadly circular in shape, ubiquitously distributed and follow steep power-law size distributions and are inferred to be post-impact general cratering, though we admit that circular explosive pits could be difficult to distinguish from regular impact cratering at these scales.The inferred age of Occatorss Figs.  that cou10.Limited occurrences of definitive irregularly shaped non-impact endogenic pitting are now resolved across Occator Figs. –4. These4. Other sinuous troughs lie within the planar LFD units  brine seepage through innumerable small  vents9, (2) energetic fountaining and ballistic emplacement3, and/or (3) precipitation and sedimentation of mineral deposits in static if transient lacustrine environments. Duration of emplacement is also important as durations of ~107 yr or more would require deep reservoirs17, but shorter periods would imply shallow reservoirs such as the LFD itself, owing to the limited timescales over which residual impact heat would remain49.The 10 or so distinct concentrations or centers of bright deposits at Vinalia FaculaeLFD Fig. –18 are tVinalia Faculae bright deposits have complex topographic relationships Figs. –18 with 48. In central Vinalia Faculae, the two largest bright centers are resolved as broader contiguous deposits 2–3 km across (center (e) in Figs. The brightest western and southern bright centers form radiating lobate or amoeboid patterns ~1 km across that are strongly controlled by the local topography (centers (a–d) in Figs. 15 at innumerable submeter vents within the LFD, and broadly consistent with occurrences of hydrothermal deposits on Earth50. Those surface sites with low seepage rates or volumes formed isolated small bright spots or downslope ribbons where flow persisted, those with higher rates or volumes resulted in more extended and coalesced carbonate surface formations as fluid flow and deposition spread laterally. The central regions could also be more contiguous if there were more vents in these regions.The radially outward decrease in brightness of these deposits suggests that active deposition at each bright center contracted inward with time, with the outermost deposits fading in brightness owing to exposure to space. The numerous circular spots smaller than ~10 m across indicate that the later stages of deposition were dominated by point sources. Although the cores of the western and southern bright centers are confined in closed topographic depressions (centers (a–d) in Figs. Constructional features from hydrothermal activity  are lacking or not resolved. The unusual dark ring in the middle of bright center (f) Figs.  and 7 is16 is not excluded by XM2 observations but there is little if any observational evidence that it occurred extensively. The distribution of small bright spots and surface coatings on floors of closed depressions, the flanks of some ridges and the tops of others, and the formation of meter-scale ribbon-like deposits is not consistent with directional ballistic sources  greater than a few meters in scale. Although lacustrine sedimentation within closed depressions is also suggested by the formation of some deposits on the floors of inter-ridge LFD depressions , the formation of smaller spots on slopes and ridge tops   linear segments of these troughs, but are crosscut by the wider pinch-and-swell segments that have been locally enlarged to ≤300 m by mass wasting . These take the form of ridged (or ropey), and knobby/hummocky and smooth plains, each of which find analogs at Occator , within crater floor materials (including melts), and along the faults defining the outer terrace blocks1. Mineralization is dependent on target lithologies and with depth below the surface29, and higher temperature precipitates occur at depth on planetary bodies, including probably Ceres. The Vinalia Faculae deposits  confirms that the zone of hydrothermal activity beneath Occator was much more centrally restricted, or rim wall sources were shut off early and any bright carbonate deposits there have faded. The concentration of the densest carbonate deposition into the dozen or so major bright centers at VF suggests that brine volumes may have been limited or originated deep in the crust17, leading to focused flow centers. The potential role of deeper hydrothermal crustal reservoirs48 rather than mobilization of fluids in rapidly cooling subsurface impact melt reservoirs relies in large part on the ages of these deposits. Unfortunately the uncertainties currently associated with age determinations from crater counting on the deposits as described above , which result in lower heating rates for a given size projectile on Ceres10 relative to Mars or Earth. These lower impact heating levels preclude melting of silicates on Ceres, resulting in muddy deposits that behave as volcanic flows34, and may result in a more centrally concentrated distribution of hydrothermal fluids and salt/carbonate surface precipitation compared with those observed on impact-derived materials and structures on the Moon, Earth, and Mars29. The irregular pits and pit clusters, thin lava-like mud flows, and sinuous troughs observed within the thick LFDs and other floor materials at Occator are both less common and are different in expression to those within lunar and Mars craters . The rapid formation of extensive cliff-forming . If chondritic materials55, including hydrated silicates32, are abundant Callisto’s surface, the higher impact velocities may also result in hydrothermal fluid redistribution and formation of precipitates of salts comparable to Mars or Ceres within larger fresh central pit craters on large icy satellites as well.The differences in surface morphology of impact deposits on Ceres compared with the Moon and Mars shown here must be related to the higher fraction of salts and water ice in Ceres’ outer layers46 Figs. –26 is cuHere, we show that impact-related deposits on the floor of the very young Occator crater on Ceres exhibit distinctive morphologies indicative of volatile release owing to the melting and refreezing of water ice during and after impact. These features include ovoid and sigmoidal pits and pit clusters, sinuous troughs, low bright mounds. Carbonate deposits at Vinalia Faculae are revealed to be only a few meters thick and controlled by local topography of the ridged floor materials they formed on. The complex relationship of these deposits with topography is consistent with hydrothermal brine effusion and carbonate precipitation at innumerable small vents, coalescing at several larger centers. These morphologies are substantially different from those observed in large martian craters, indicating that while hydrothermal processes previously identified on Mars and Earth are also prevalent on Ceres the composition of Ceres’ outer layers strongly controls how these processes operate on each planetary body.56. We also use the stereo images to determine the stratigraphic and topographic relationships of LFD and salt-carbonate deposits to the terrains they formed on and to test hypotheses for their formation17. Dawn FC image registration and construction of XM2 stereo mosaics, tied to Dawn FC LAMO mosaics, was performed using USGS-ISIS (version 3.5.2.0) FC calibration and control net bundle adjustment software (https://isis.astrogeology.usgs.gov). Digital Elevation Models (DEM) were produced from registered Dawn stereo images using stereogrammetry software designed in the MATLAB toolbox, identical to that used to produce DEMs for the surface of Pluto57. Crater counts were completed from the FC mosaics and interpreted using the asteroid-based crater chronology59. Cratering ages were derived using hard-rock cratering scaling law with a low value of strength, 1e6 dyne/cm2 . The target and impactor density were 1.3 and 2.6 g/cm3, respectively, and assumed an average impact speed of 5.1 km/s59.Dawn Framing Camera (FC) stereo imaging of most of Occator floor acquired during XM2 at 3.5–8 m pixel scales Fig. –5 are usSupplementary Information


The data presented in this data paper describe the early growth of cover crop cultivated in growth chamber under non-limiting conditions. Seventeen species of four botanical groups were described after one month of growth. Traits related to plant growth and leaf area development were measured (five traits) and calculated (eight traits). This data set is made available to enable comparisons between dataset, extended analysis and meta-analysis on cover crop traits. The data presented in this article were used on the research article entitled “Leaf area development strategies of cover plants used in banana plantations identified from a set of plant traits’ [1]. It is composed of 17 rows and 18 columns. The first five columns are species names and abbreviation, botanical group and the times of the two plant harvest. The last thirteen columns are aboveground traits: five raw traits  and eight calculated traits , aboveground absolute and relative growth rates on a biomass basis and on a leaf area basis. The dataset presented in this article .−2 s−1 of photosynthetic active radiation [see Seventeen cover crop species  were groinitial leaf area (LA0) was measured with WinRhizo Pro analytical software (Regent Instruments) and the initial aboveground biomass  was determined after oven-drying of the whole aboveground parts (at 70 °C until weight stabilization). After one month, the five remaining replicates of each species were harvested and pooled. Leaves and stems were separated. The total leaf area at one month (LA1) was measured with WinRhizo Pro and the leaves and stems biomasses were determined after oven-drying (at 70 °C until weight stabilization). The aboveground biomass at one month  was calculated as the sum of the leaf and stem biomasses.Seed mass (SM) was determined after seed oven-drying at 70 °C until mass stabilization. When the first leaf was fully developed, five replicates of each species were harvested and pooled [see plant-scale leaf specific area (SLAps) was calculated as the ratio of the total leaf area and the total leaf mass. The aboveground leaf mass fraction (LMFa) was calculated as the ratio between the leaf mass and the total aboveground mass. The aboveground leaf area ratio (LARa) was calculated as the product of SLAps and LMFa. The aboveground absolute growth rate was calculated both on a biomass basis and on a leaf area basis , using the equations:Eight functional traits associated to leaf area development were then calculated . The plaaboveground relative growth rate was calculated both on a biomass basis and on a leaf area basis , using the equations:The net assimilation rate (NARa) was calculated as the ratio between RGRa and LARa.The ps were measured according to the standardized protocols of trait measurements [All traits, except SLAurements .Gaëlle Damour: Formal analysis, Data curation, Writing, Funding acquisition. Chloé Guérin: Methodology, Data acquisition, Formal analysis. Marc Dorel: Methodology, Supervision, Funding acquisition.