The δ15N in BOM isolated from surficial sediment (δ15NBOM) was significantly correlated with δ15N in filter feeding unionid mussels  from lakes and with hard-shell clams  from estuaries. In lakes, δ13CBOM was significantly correlated with δ13CUN, but δ13CBOM was not significantly correlated with δ13CMM in estuaries. Values of δ15NBOM and δ15NUN were significantly and positively correlated with increasing amounts of impervious surface, urban land cover, and human populations in watersheds surrounding lakes. In estuaries, δ15NBOM was only significantly and positively correlated with greater percent impervious surface in the watersheds. Correlations of δ13CBOM in lakes and estuaries, δ13CUN, and δ13CMM with land use and human population were mostly non-significant or weak. Overall, these results show that δ15NBOM can serve as a proxy for δ15N of filter feeding bivalves in lakes and estuaries and is useful for assessing anthropogenic impacts on aquatic systems and resources. Our study area was limited in size, but our results support further studies to test the application of this sediment stable isotope-based technique for assessing and ranking aquatic resources across broad geographical areas.Excessive inputs of nitrogen from anthropogenic activities in watersheds can cause detrimental effects to aquatic ecosystems, but these effects can be difficult to determine based solely on nitrogen concentrations because of their temporal variability and the need to link human activities to ecological responses. Here, we (1) tested the use of stable isotopes of nitrogen (δ The e15N ratios change depending on the mix of nitrogen inputs from human wastewater, atmospheric deposition, and agricultural fertilizers entering aquatic sites [15N ratios of chironomids were found to be useful for understanding nitrogen inputs, processing, and transformations in streams and lakes as well, and their δ15N ratios were associated with landscape, water body, and biological factors [15N ratios in both soft tissues and shells of hard-shell clams  have been found to directly relate to the percentage of wastewater input to small estuaries [15N in these organisms as a time integrative measure of human impact; however, the use of chironomids, which are almost exclusively freshwater taxa, and the use of filter feeding bivalves as indicators can be limited by the availability of organisms and constrained by the need to compare among species with potentially differing feeding strategies and physiologies [To provide a time integrated metric of nitrogen pollution in aquatic sites, studies have identified relationships between stable isotopes of nitrogen in aquatic organisms and variables indicative of anthropogenic land development in the surrounding watersheds -19. Relaic sites -24. Rece factors . In saltstuaries ,27. Overiologies ,28. Thes15N in benthic organic material (BOM) (δ15NBOM) isolated from surficial sediments is one sample type potentially useful as a baseline; especially because of its nearly universal availability in lakes and estuaries. δ15NBOM can be significantly correlated with δ15N of organisms at different trophic positions despite BOM being composed of a mixture of detritus, benthic microbes, and deposited living and dead plankton from the water column. For example, significant relationships were found between δ15NBOM and δ15N of fish and mussels collected from lakes representing a wide range anthropogenic impact [15N in sedimented organic material from surface sediments of tidal flats and δ15N in manila clams, (Ruditapes philippinarum) among sites spanning a wide range of eutrophication levels [15N ratios in two species of bivalves from estuaries on Cape Cod, USA had significant relationships with δ15N in organic material from sediments [15N in sediment as an indicator is further supported by studies that found a correspondence of δ15N ratios between sediment and seston [13C) provide additional information that complements δ15N results by establishing a baseline value for food chains and for calculation of organismal trophic positions in aquatic systems [The δc impact ,28. Addin levels . In anotediments . The used seston  and findd seston . Carbon  systems ,28,33,3415NBOM and δ15N of other organisms, we surveyed 51 lakes and 28 sites in nine estuaries to document and compare these relationships among sites in these two distinct ecosystems and to examine their possible use as indicators of the effects of anthropogenic disturbance in their watersheds. Our goals were: (1) to determine if δ15NBOM and δ13CBOM sampled from estuaries and lakes represented corresponding isotopic ratios in filter feeding bivalves (primary consumers), and (2) to evaluate the use of δ15N and δ13C in BOM and filter feeding bivalves as indicators of human impact on aquatic systems resulting from anthropogenic land development in watersheds. Documenting these relationships is important to developing the use of δ15NBOM as an indicator of nitrogen effects, which could be particularly useful because of the widespread availability of BOM in lakes and estuaries. Results from this study can be useful to monitoring programs interested in identifying effects of nutrient pollution and for informing decisions and management strategies seeking to protect aquatic resources and to improve those experiencing negative effects of eutrophication from human sources. Results from the present study also may provide support for the use of stable isotopes in BOM for national assessments and rankings of environmental conditions in estuaries [With multiple studies showing the potential usefulness of relationships between δ2.Elliptio complanata) were collected from the littoral zones by hand or with a clam rake within ~5 m of where the sediment was sampled for BOM, but they were observed and sampled in only 26 of the 51 lakes. Triplicate samples of BOM from surficial sediments were collected from a total of 28 sampling stations along the shorelines of four Rhode Island and five Massachusetts estuaries, which had watersheds ranging from 250 ha in the smallest salt pond to 275,000 ha for Narragansett Bay. Hard shell clams  were collected with a clam rake within a ~10 m2 area around the location of the BOM sample.Lakes and estuaries were selected to represent a wide range of anthropogenic land development of surrounding watersheds. Single samples of BOM from surficial sediments were collected from littoral zones of 51 lakes in Rhode Island, USA. These lakes had small surface areas, ranging from 3.4 to 430 ha, but were within watersheds that ranged from 23 ha to 2250 ha for lakes that were part of larger river systems . Unionid15NUN and δ13C values where only one bivalve was found, were used in comparisons. We report the number and sizes of bivalve samples found at sample sites in Bivalve tissue samples were from a section of the foot of UN and MM. These individual samples were oven dried at 60° C for >2 days, ground and weighed for isotope ratio mass spectrometry (IRMS) analysis. Each BOM sample and bivalve sample was analyzed individually, and means, or individual δ® screen  and collected in 55 mL centrifuge tubes. Samples were shaken and centrifuged at 1000× g for five minutes. The supernatant was poured off and a spatula was used to take samples from the top ~1.5 cm of the material in the bottom of the tube. Samples were dried in an oven at 40 °C for at least three days, ground with mortar and pestle to a fine powder, weighed to 0.01 mg on a microbalance, and analyzed using an Isotope Ratio Mass Spectrometer (IRMS).Sediment samples in both lakes and estuaries were collected with a hand-held piston coring sampler, which obtained a 6.5 cm diameter core. In both lakes and estuaries, samples were taken by wading where water depths were <1 m. The corer was driven into the sediment, which varied from sandy to highly silty. The corer was withdrawn and, while maintained vertically, a core cap was pushed onto the bottom of the core tube, which was then detached from the sampler. Samples of benthic organic material (BOM) were obtained from the surface layer (~the top 1.5 to 2.0 cm) of the intact sediment core. The surface layer was re-suspended by stirring, and the suspended material was removed with a 30 mL pipette. Samples were placed in clean plastic bottles, capped, placed on ice, and transported to the laboratory. Samples were refrigerated (4 °C) until they were processed within 96 h of sampling. Samples were stirred, poured through a 0.5 mm Nytex15N ‰) from that of the reference material, which was N2 in air. Carbon isotopic composition is expressed as a part per thousand deviation (δ13C ‰) from the reference standard Pee Dee Belemnite. All samples were analyzed in duplicate with a typical difference of about 0.1‰ and reported as mean values. An examination of results from wheat flour taken through all steps of the sample preparation procedure with original unprocessed flour showed no appreciable change in δ15N or δ13C ratios demonstrating that no contamination of samples resulted from sample preparation.The nitrogen and carbon isotopic composition of the BOM samples and tissue samples were determined by IRMS using a Vario Micro Elemental Analyzer interfaced to an Elementar Isoprime 100 Mass Spectrometer . The nitrogen isotopic composition of the samples is expressed as a part per thousand deviation (δ13C of carbonate (CaCO3) in mollusk shells [13CBOM for comparisons with δ13C in bivalves, and for relating to land cover variables. In lakes, we conducted a pilot study to determine whether removal of inorganic carbon in CaCO3 was necessary prior to analysis of BOM by IRMS. We selected a total of 67 BOM samples from 42 lakes and removed inorganic carbon by acid fumigation [13CBOM after acid fumigation. The mean difference of δ13CBOM untreated − δ13CBOM fumigated was = −0.05‰, range (−0.23 to + 0.07‰), n = 67); due to this small difference in lakes, we used untreated δ13CBOM samples. In estuaries, δ13CBOM from three stations showed large decreases in δ13CBOM after acid fumigation. Therefore, in estuaries we used results from acid fumigated δ13CBOM samples for comparisons.We were concerned that collection of BOM samples might also include shell fragments, and if so, that the elevated δk shells  and in smigation . Results15NUN and δ13CUN or individual values (where only one organism was found) were used for the comparisons with δ15NBOM and δ13CBOM and with land cover variables and population. In estuaries the mean values of the triplicate δ15NBOM and δ13CBOM ratios were compared with the corresponding mean δ15NMM and δ13CMM ratios, or with individual values where only one bivalve was found, at a total of 28 stations from nine estuaries. Only one station was sampled in Narragansett Bay, but the nitrogen and carbon isotope ratios in this station, δ15NBOM = 7.8‰ ± 0.5‰ SD; δ13CBOM = −18.3‰ ± 0.3‰ SD and δ15NMM = 13.0‰ ± 0.2‰ SD; δ13CMM = −17.4‰ ± 0.2‰ SD, agreed with summary values for Narragansett Bay calculated from other sources. These sources measured nitrogen and carbon isotopes in the surface sediment (top 2 cm) of core samples from the axis of the Bay and found δ15N = 7.9‰ ± 0.35‰ SD, and δ13CBOM = −19.9‰ ± 0.84‰ SD [M. mercenaria samples were from stations taken throughout the Bay and had means of δ15NMM =13.2‰ ± 0.54‰ SD, and δ13CMM −16.76 ± 0.61‰ SD [15NBOM and δ15NMM from all stations within each of the nine estuaries were used for comparison with land cover and population variables (n = 9).In lakes, the means of δ0.84‰ SD . The M. 0.61‰ SD . The gra15N ratios in filter feeding bivalves with those in BOM or surface sediment, we developed two relationships: (1) δ15N in mussels (δ15NUN) as a function of δ15NBOM in the 26 lakes, and (2) δ15N in hard-shell clams (δ15NMM) as a function of δ15NBOM from a total of 28 stations from nine estuaries. These regressions were compared using analysis of covariance (ANCOVA). Similar relationships with δ13CUN as a function of δ13CBOM, and δ13CMM as a function of δ13CBOM were developed and analyzed.For comparisons of δLand cover data within the watersheds of lakes and estuaries were obtained from RIGIS (2003–2004) and Mass15NBOM, δ13CBOM in 51 lakes and in the nine estuaries. Similar comparisons were also made using only data for δ15NBOM, δ13CBOM and with δ15NUN, δ13CUN in the 26 lakes where mussels were found. Comparisons were also made between δ15NBOM and δ15NMM and between δ13CBOM and δ13CMM in the nine estuaries. Correlations used individual δ15NBOM and δ13CBOM values and site mean values of δ15NUN and δ13CUN for lakes and grand means of δ15NBOM, δ13CBOM, δ15NMM, and δ13CMM from all stations within each estuary. We compared the results from both correlation methods to determine the extent to which the presence of non-normality of some variables in the Pearson correlations impacted results.We compared Spearman and Pearson correlations of land cover variables and population in watersheds with δ15NBOM from the lakes and estuaries.We compared relationships of land cover variables and population with each other and selected log % impervious surface as the most appropriate variable for representing anthropogenic land development. We developed regression relationships with log % impervious surface as a function of δt-tests, F-tests for equality of variance, and regression analyses were done using Microsoft Excel. We reported results of Pearson correlation coefficients with significance indicated for pairwise comparisons to show relationships within the data. The level of significance for statistical tests was p ≤ 0.05.Correlations, least squares regressions, ANCOVA, and regression model selection were done using SAS software version 9.4 . Student’s 3.3.1.15N in mussels (δ15NUN) from lakes and increased δ15N in hard shell clams (δ15NMM) from estuaries were associated with greater δ15NBOM , but their intercepts were significantly different (p < 0.001). Increased δ13CUN were associated with greater δ13CBOM in lakes , but δ13CMM was not significantly correlated with δ13CBOM in estuaries  of regression relationships between δ15NUN as a function of δ15NBOM in lakes and δ15NMM as a function of δ15NBOM in estuaries. This level of agreement indicates that δ15NBOM from sediment closely duplicates δ15N found in bivalves, which are often considered primary consumers and have been used in environmental assessments [15N of mussels taken from the same sites have been reported [15N in hard shell clams and in soft shell clams (Mya Arenaria) collected from the same small estuaries on Cape Cod, USA have been found [For environmental assessment of how anthropogenic activities in watersheds affect lakes and estuaries, our results suggest that a sediment-based method presents a useful alternative to mussels and other organisms, which commonly can be absent from sites. In the present study, we found a statistical similarity of slopes and Ressments -24,43. Wessments ,45, and reported . Additioen found .13CUN as a function of δ13CBOM was significant; however, the relationship of δ13CMM from estuaries as a function of δ13CBOM was not significant. The diet of M. Mercenaria is planktonic microalgae [13CMM relative to δ13CUN Our findings indicate that although δ15NBOM could be used as a surrogate for bivalves in assessments of nitrogen concentrations and impacts in lakes and estuaries, δ13CBOM appears to have utility as a surrogate for δ13C only for bivalves in lakes.In lakes of the present study, the relationship of δcroalgae , whereascroalgae ,46. Thes15NBOM with variables indicative of anthropogenic land development of watersheds surrounding lakes showed significant positive correlations with log population, log % impervious surface, % urban residential, and a negative correlation with % forest. These results support the use of δ15NBOM for assessments in watersheds surrounding lakes. Due to the dynamic nature of estuaries, it is often difficult to assess their environmental condition.Our comparisons of correlations of δ15NBOM may be useful for these assessments.Environmental assessments in estuaries have used a variety of methods to develop and refine benthic organism-based indices to evaluate ecological status of sites -50. Howe15NBOM with land use and population variables in estuarine watersheds were not as strong as in lakes, and δ15NBOM was only significantly correlated with log % impervious surface. These results likely reflect the highly dynamic nature of the estuarine environment and the influence of freshwater and seawater inputs, tides, complex flows, and variable marine algal (food) sources on the composition of the organic material in sediments and therefore on δ15NBOM in estuarine sediments, but still show that δ15NBOM in estuaries is associated with watershed development. One of our estuaries, Narragansett Bay, has a watershed which is ~38 times larger than the next largest estuarine watershed, and has been found to be highly influenced by nutrients from wastewater [15NBOM to indicate effects of anthropogenic land development in watersheds of estuaries.The correlations of δ15N in bivalves or δ15NBOM. Our results showed that variables indicating anthropogenic land development  were principal explanatory variables and likely indicated the consequent input of wastewater to sites. One study suggested that drivers of δ15N through nitrogen inputs in lakes may show regional differences [In other studies, the sources of nitrogen inputs to aquatic sites have been found to consist of human or livestock wastes, inputs of human wastewater, or from application of nitrogen in fertilizers in surrounding watersheds. In the present study, the percentage of agricultural land use in watersheds surrounding lakes and estuaries was low and did not represent a significant variable for predicting δferences . Our stuferences ,52.15NBOM. However, in larger waterbodies comparisons of δ15NBOM with anthropogenic activities in watersheds may be influenced by a large range of activities and inputs which are difficult to quantify. Therefore, in large lakes and estuaries, studies to determine the appropriate sampling strategies for using stable isotopes in sediment will be needed for development of adequate assessments.The lakes and estuaries of the present study, except for Narragansett Bay, are relatively small; therefore, land use in the surrounding watersheds was more likely to have been reflected in the δ15N in bivalves and BOM, which indicated that δ15NBOM may be used as a surrogate for δ15N in bivalve mollusks. Our findings suggest that δ15NBOM provides an effective summary metric for assessing and ranking the environmental conditions of lakes and estuaries resulting from anthropogenic land development of watersheds.Overall, the present study showed highly significant positive relationships between δSupplement1Table S1: Location data for study lakes and watershed size (ha) for lakes and estuaries; Table S2: Number, size data, δ15N and δC13 for Mussels (UN) from 26 Lakes; Table S3: Number, size data, δ15N and δC13 and sample site locations for Mercenaria mercenaria from a total of Estuaries 28 stations from nine Estuaries.


Microstructured composites with hierarchically arranged fillers fabricated by three-dimensional (3D) printing show enhanced properties along the fillers’ alignment direction. However, it is still challenging to achieve good control of the filler arrangement and high filler concentration simultaneously, which limits the printed material’s properties. In this study, we develop a magnetically assisted drop-on-demand 3D printing technique (MDOD) to print aligned microplatelet reinforced composites. By performing drop-on-demand printing using aqueous slurry inks while applying an external magnetic field, MDOD can print composites with microplatelet fillers aligned at set angles with high filler concentrations up to 50 vol%. Moreover, MDOD allows multimaterial printing with voxelated control. We showcase the capabilities of MDOD by printing multimaterial piezoresistive sensors with tunable performances based on the local microstructure and composition. MDOD thus creates a large design space to enhance the mechanical and functional properties of 3D printed electronic or sensing devices using a wide range of materials. 3D printed composites with hierarchically arranged fillers have been challenging to fabricate. Here, the authors make use of magnetically assisted droplet-based printing to 3D print voxelated structures with high filler content, localized control of filler material, and orientation. More recently, 3D printing has been extended to fabricate microstructured composites consisting of orderly arranged 1D fibrous or 2D plate-shaped anisotropic reinforcement fillers. Microstructured composites are interesting as they give rise to superior properties. For example, bioinspired Bouligand and nacre-like structures are formed by stacked layers of aligned stiff fibres and platelets respectively, within a soft matrix. These structures have high stiffness owing to the high filler content, while their layered architecture toughens the structure5. Aside from mechanical properties, microstructured composites containing functional fillers such as aligned graphene or hexagonal boron nitride (hBN) microplatelets also display enhanced thermal and electrical properties7. Although exciting progress has been made in the field of 3D printing, it is still challenging to fabricate these microstructured composites due to the complex anisotropic and multimaterial layered structure required.Three-dimensional (3D) printing is a manufacturing technology that generates freeform 3D structures using layer-by-layer deposition. Traditionally, 3D printing is used for small batch prototyping with limited material compatibilities. However, recent advancements have allowed more classes of materials to be 3D printed for use in multidisciplinary fields such as aerospace, robotics, biomedical and electronic applications10. However, such techniques are usually limited to printing polymers with varied mechanical properties and cannot fully realise the same properties as composites containing actual stiff fillers. Current methods that utilise inks containing stiff fillers generally make use of shear-induced or field-assisted alignment of the fillers12. 3D printing of microstructured composites reinforced by 1D fillers is easier to achieve as the fillers only need to be aligned along one direction. Conversely, the fabrication of microstructured 2D platelet-based composites is more demanding as the fillers have an additional axis to align. Despite so, the 3D printing of platelet-based composites has been accomplished in various research groups. Yang et al. 3D printed nacre-inspired graphene nanoplatelet structures within a photocurable resin using an electric field-assisted stereolithography (SLA) method13. Magnetic fields have also been used in similar ways to biaxially align alumina microplatelets in 3D printed photocurable polymers15. While these methods are effective in printing microstructured materials, the use of photocurable polymer inks limits the filler loadings as high filler loadings lead to high ink viscosities which impede the rotation of the microplatelets during alignment. As a result, the final composites often have low solid loadings of <15 vol% which limits their properties. To fabricate composites with higher solid loadings, Feilden et al. used direct ink writing (DIW) to print ceramic composites using an alumina-based hydrogel ink. The shear forces developed during printing aligned the microplatelets along the circular circumference of the dispensing nozzle16. Although samples with high solid contents of ~50 vol% were printed, the filler alignment cannot be freely controlled. Hence, a printing technique that allows the control of filler orientation while maintaining high solid loadings in the printed structures would be highly desirable.One strategy to print microstructured composites is to use multimaterial methods such as polyjet printing or fused deposition modelling to print combinations of soft and stiff materials17. This strategy has been previously used in magnetically assisted slip casting to fabricate high solid content microstructured composites with controllable microplatelet alignment18. Using similar aqueous slurries in a drop-on-demand 3D printing technique augmented with magnetic fields for alignment, additional voxelated control in the printed structures could be achieved.To achieve this goal, the use of solvent-based slurry ink is a promising option. These types of inks generally have lower viscosities than resin-based inks, which allow the fillers to be easily aligned. At the same time, densification occurs as the solvent evaporates, leading to structures with high solid contentsIn this work, we apply this principle and develop a 3D printing technique, which we call magnetically assisted drop-on-demand printing (MDOD). MDOD can print microplatelet-based microstructured composites with high filler loadings up to ~50 vol%, and with locally varying filler orientation and composition. To achieve this, aqueous inks with magnetically responsive anisotropic microplatelets are deposited in droplets onto a substrate while a magnetic field is applied to induce filler alignment. We also show that MDOD can be easily applied to different types of microplatelets, leading to the possibility of multimaterial printing. The material and filler alignment in each individual droplet can be tuned to give voxelated control of the overall printed structure. To demonstrate the advantage of MDOD, multifunctional devices such as piezoresistive sensors are fabricated and their performances and limitations are assessed. By controlling their local microstructure and composition, the mechanical and functional properties are enhanced. The ability to vary the material composition and microstructure creates a large design space to tune the device performance based on the needs of the target applications. The work presented here can be leveraged to fabricate novel microstructured materials and also offer an alternative approach to improve the performances of 3D printed functional devices.19. During the printing process, a droplet-on-demand printer deposits ink droplets with microplatelet concentrations ϕi onto a substrate One key factor for attaining good control of microplatelet alignment and densification is the optimisation of the microplatelet concentration in the ink. We first used titania-coated alumina  microplatelets to study the ink densification and alignment as they have a reflective platelet surface which allows easy identification of alignment during the printing process.ϕmax = 10.5 vol%  than what is currently reported in other methods. In addition, the anisotropic nature of these functional platelets leads to anisotropic properties that are tunable in the 3D printed structures  but had a much lower stiffness of 2.17 MPa. By building alternating 0–90° aligned layers, the multi-alignment sample can achieve a compromise between stiffness (3.6 MPa) and energy dissipation (176 kJ m−3). While the vertically aligned layers contribute to stiffness, the horizontal layers help prevent catastrophic failure by creating energy dissipating layers through mechanisms such as crack deflection  was in the range of 0.1 nF , polyvinylpyrrolidone MW 360,000 (Sigma-Aldrich), superparamagnetic iron oxide nanoparticles ferrofluid (Ferrotec EMG-605), hBN microplatelets , graphite , PDMS (SYLGARD™ 184) and epoxy resin (Weicon MS1000) were purchased and used without modification.The different microplatelets were first made magnetically responsive by adsorbing SPIONs on their surfaces. In a typical procedure, 2 g of dry microplatelets were first dispersed in 200 ml of deionized (DI) water using magnetic stirring. Ferrofluid EMG-605 was then added to the suspension such that the SPIONs made up 5 vol% to the relative mass of the platelets. The mixture was stirred overnight and filtered using vacuum filtration to recover the magnetised microplatelets. The microplatelets were then dried in a drying oven overnight. To make the ink, the dried magnetised platelets were mixed with 1 wt% PVP aqueous solution at the desired microplatelet content. The resulting mixture was then sonicated and vortexed until a homogenous ink was produced.−1 to 500 s−1. Each measurement was repeated three times. The ink contact angle was characterised by taking side view optical images of ink droplets ~6 µL using a USB microscope (Dino-lite AM7915MZTL). The contact angles of five ink droplets were measured for each type of ink. Sedimentation rates were measured by taking a time-lapse of ink droplets ~6 µL until they totally dry.The ink rheology was characterised using a shear rheometer (Bohlin Gemini HR Nano). Viscometry measurements were made using a 15 mm diameter serrated plate system with 200 µm gap size and shear rates from 0.1 s3D printing was performed using an automated fluid dispensing system (Nordson 3-Axis PROPlus). The ink was filled into 5 ml syringes (Nordson) before loading into the printer. A 0.33 mm inner diameter stainless steel flat tip needle (Nordson) was then installed onto the printhead. The input air pressure was set at ~0.5 bar. The copper substrates were prepared by attaching copper foil (Sunhayato) onto glass slides (VWR) to keep them flat. Glass coverslips (VWR) were used as glass substrates. The gold-sputtered substrates were prepared by sputtering (Joel JFC-1600) the glass coverslips with ~10 nm of gold. The substrates were cleaned with ethanol prior to printing to ensure consistent printing as impurities on the substrates may lead to a variation in ink contact angle and could cause defects in the print.The printing steps of the desired pattern were programmed onto the printer’s software interface. A rotating magnet consisting of a permanent Neodymium magnet attached to a DC motor (RS Components) was set up at the side of the printer. During each printing step, several droplets were deposited on the substrate and the stage was programmed to move the droplets to the rotating magnet set up for alignment  and ImageJ analysis to identify the alignment of the printed microplatelet composites. The angles of at least 50 microplatelets were taken on each sample.θ = 0°, 20°, 45°, 70°, 90° and diameter of ~4 mm and heights of ~0.2 mm were printed on glass substrates. Three samples of each alignment were printed. 4 thin strips of carbon tape ~1 mm × 3 mm were attached to opposite edges of each droplet along the x and y directions as defined in Fig. Graphite droplets with varying alignment of θ = 0° and 90° and diameters of ~4 mm were deposited onto the middle of a 1.8 × 1.8 cm2 silicon wafer (Merck). After drying, the samples were placed in a convection oven (IKA 125-control) and preheated at 80 °C for 10 mins. The samples were then removed and placed onto an insulating cardboard under a thermal camera (FLIR ETS320). A movie of the sample cooling down was recorded. The cooling rate was then obtained from the slope of the temperature vs time profile of each sample. The measurements were performed at least three times for each sample.hBN droplets with platelet alignments of −1 to a maximum load of 100 mN and dwell time of 10 s. 20 indents were made on each sample. For the Vicker’s hardness test, a load of 1 kg was applied for 10 s. Nine indents were made on each sample.Four by four xirallic droplet structures with varying alignments were printed with an overall length and breadth of 8 mm and height of 1.5 mm. Once the droplets were dried, they were sintered in a high-temperature furnace (Nabertherm LHT 08/18) first at 500 °C for 1 hour for binder removal and then at 1600 °C for 2 hours for sintering. Once the samples were cooled back to room temperature, they were cold mounted in an epoxy resin for subsequent preparation for further characterisation. The mounted samples were first grinded using sandpapers with increasing grits of 400, 800, 1200 and 2400. This was followed by polishing using OPS solution (Struers). The mechanical properties of the polished samples were characterised using nanoindentation  and Vicker’s hardness tester (Future Tech FM-300E). The nanoindentation tests were performed using a Berkovich tip with a loading rate of 1 mN s−1. Three samples of each orientation were tested for repeatability.Xirallic-PDMS structures were also fabricated for toughness measurements using compression tests. Xirallic micropillars with diameters of 4 mm and height of 3 mm were printed and infiltrated with PDMS. The samples were then subjected to compression testing (Instron 3366) using a 500 N load cell and a loading rate of 0.2 mm min−1, up to a maximum of 40 N was applied to the sensor. To check the repeatability and stability of the sensor signals, the measurements were performed after every 10 compression cycles up to 30 cycles.Three by three arrays of graphite droplets were printed into micropillars with diameters of ~2.0 mm and heights of ~1.5 mm on a copper foil substrate to form a sensor area of ~1 cm by 1 cm. Rings of hBN were then deposited around each micropillar and the structure was infiltrated with PDMS to form the final device. The stress–strain curve and electromechanical properties of the resultant sensors were tested using a compression tester (Instron 3366). The two pieces of copper foils sandwiching the printed graphite micropillars were connected a two-probe multimeter. The electrical resistance was monitored while a compressive force with a ramp rate of 5 N minThe thermal properties of the sensors were characterised using a thermal camera (FLIR ETS320). A lever system using a tweezer and a support was used to apply a force onto the sensor area. This was done to ensure that the sensor area would be unobstructed for the camera to record its temperature. Weights were placed onto the middle of the tweezer to apply pressures equivalent to 1 kPa and 25 kPa onto the sensor area. For each loading and unloading step, the temperature was recorded until a saturation temperature was reached.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


Exploring sociodemographic and disease characteristics that contribute to patient-reported outcomes can inform targeting of strategies to support recovery and adaptation to stroke. This study aimed to examine among a sample of stroke survivors at three months post-hospital discharge: (1) self-reported physical and mental health quality of life scores; (2) self-reported depressive and anxiety symptoms; and (3) sociodemographic and clinical predictors of these outcomes.This cross-sectional survey study recruited stroke survivors from eight hospitals in one Australian state. Adult survivors recently discharged from hospital stroke wards (within 3 months) were mailed a study information package and invited to complete a pen-and paper survey. Survey items assessed: quality of life (SF12v2), depression (PHQ-9), anxiety (GAD-7) and sociodemographic and clinical characteristics. Predictors were examined using multiple linear regression analysis.Of the 1161 eligible patients who were posted a recruitment pack, 401 (35%) returned a completed survey. Participants reported a mean SF-12v2 Physical Composite Score (PCS) quality of life score of 44.09 (SD = 9.57); and a mean SF-12v2 Mental Composite Score (MCS) quality of life score of 46.84 (SD = 10.0). Approximately one third of participants  were classified as depressed (PHQ-9 ≥ 10); and 27% (n = 104) were classified as anxious (GAD-7 ≥ 8). Lower PCS was associated with being female, not employed and having a comorbid diagnosis of diabetes and atrial fibrillation. Lower MCS was associated with a history of transient ischemic attack (TIA). Males and those with higher levels of education, had greater odds of having lower depression severity; those with a history of TIA or diabetes had lower odds of having lower depression severity. Males had greater odds of having lower anxiety severity; those with a history of TIA had lower odds of having lower anxiety severity.Sub-groups of stroke survivors may be at-risk of poorer quality of life and psychological morbidity in the early post-discharge phase. These findings support the role of early identification and prioritisation of at-risk survivors at discharge, as they may require modifications to standard hospital discharge processes tailored to their level of risk. Globally, the lifetime risk of having a stroke is 25% . An estiThere has been a move toward the administration of Patient-Reported Outcome Measures (PROMS) to obtain stroke survivors’ own perspective of their health, in addition to traditional clinical outcomes , 7. ThisThere is also emerging evidence of a relationship between early phase and longer-term outcomes for stroke survivors. One Australian study reported that stroke survivors’ quality of life in the early period following stroke was linked to the development of longer-term unmet needs at a median of 2 years post-stroke . SymptomExploring sociodemographic and disease characteristics that contribute to patient-reported HRQoL, depression and anxiety symptoms can inform targeting of strategies that support recovery and adaptation to stroke . SeveritA cross-sectional survey was undertaken with stroke survivors recruited from eight hospitals located in New South Wales.Eligible stroke survivors were: admitted for stroke who were conscious and not requiring a high level of medical care; aged ≥ 18 years; ≤ 3 months post first ischemic or haemorrhagic stroke, or transient ischemic attack; being discharged home, or to private rehabilitation; and able to provide informed consent, as indicated by return of baseline survey. Survivors were ineligible if they  had: severe neurological impairment not associated with stroke; severe language or cognitive impairment; and/or insufficient English to complete patient-reported measures.Eligible stroke survivors discharged from participating hospitals were posted a study information and recruitment pack by clinic staff on behalf of the research team. Consenting survivors were asked to complete and return the survey to the researchers via a provided reply-paid envelope. A reminder pack was sent to all eligible survivors approximately two weeks after the initial information pack was mailed. Recruitment and data collection took place from April 2018 to December 2019.Quality of life was assessed using the Medical Outcomes Study Short Form version 2 (SF-12v2) [SF-12v2) , 25. It SF-12v2) . ScoringDepression was assessed using the Patient Health Questionnaire 9  (9-items) [9-items) . This is9-items) .Anxiety was assessed using The Generalized Anxiety Disorder Scale-7  is a 7-item, self-rated scale [ed scale . Particied scale . Based oed scale .Sociodemographic and clinical characteristics: age, level of education attained, employment status, and type of stroke were obtained via standard self-report items. Treatment/s received was obtained ”; “Carotid endarterectomy (surgery to remove plaque from the main artery in your neck)”; “Carotid angioplasty (surgery to place a stent in the main artery in your neck)”; “Endovascular clot retrieval (surgery to remove a clot from your brain)”; “Unsure”; and “Other”. Comorbid medical conditions were obtained by the item “Which of these health conditions apply to you?”, with response options: “History of Transient Ischaemic Attack (TIA)”; “Irregular Pulse ”; “Diabetes”; “Fibromuscular Dysplasia (FMD)”; “High blood pressure”; “High cholesterol”; “Smoker”; “Obese (Body Mass Index above 30)”; “Overweight (Body Mass Index over 25)”; “Drink six or more standard alcoholic drinks per day”.p < 0.05 (two-tailed) was used to indicate statistical significance.Statistical analyses were programmed using SAS v9.4 . Descriptive statistics for categorical data are presented as count (%), or mean (SD) and median  if continuous. For participants with no more than 2 missing PHQ-9 items, missing values were imputed by mean imputation of non-missing responses. Mean imputation was performed on the GAD-7 where there were no more than 1 missing GAD-7 item per participant. Participants with 3 or more missing PHQ-9 item responses did not receive a total score for PHQ-9. Similarly, participants with 2 or more missing GAD-7 item responses did not receive a total score for GAD-7. Mean imputation is consistently used in previous works to address missing responses for PHQ-9 and GAD-7 –33. AnalA total of 1328 patients were assessed for study eligibility of which 1161 were identified as being eligible: adults with no or mild to moderate cognitive impairment, able to communicate in English, and discharged home or to rehabilitation from hospital. Reasons for ineligibility included: > 3 months post stroke (n = 3) and not discharged home (n = 165). Of the 1161 eligible patients who were posted a recruitment pack, 401 (35%) consented to participate and returned a completed survey. Table p = 0.001) on the PCS scale than females. Those who responded ‘Working’ scored on average 3.35 points higher (p = 0.030) on the PCS compared to those that responded’Not working’. Those that responded ‘Yes’ to having diabetes scored on average 4.48 points (p < 0.001) lower on the PCS scale than those that responded’No’ to having diabetes. Those that responded ‘Yes’ to having atrial fibrillation (AF) scored on average 2.71 points (p = 0.014) lower on the PCS scale than those that responded’No’.Participants reported a mean SF-12v2 Physical Composite Score (PCS) quality of life score of 44.09 (SD = 9.57). Table p = 0.030) points lower than those who did not have a history of TIA.Participants reported a mean SF-12v2 Mental Composite Score (MCS) quality of life score of 46.84 (SD = 10.06). Table p < 0.001) of being in a lower depression severity category compared to females. Participants with tertiary level education had approximately 2.52-fold higher odds  of having a lower depression severity compared to those with secondary school or below level of education. Participants with a history of TIA had approximately 54% lower odds  of being in a lower depression severity category compared to those with no history of TIA. Those with diabetes had approximately 45% lower odds  of being in a lower depression severity category than those without diabetes. Those who have 6 or more standard drinks of alcohol a day had approximately 62% lower odds  of being in a lower depression severity category than those who drink less than 6 standard drinks of alcohol a day.The mean depression score on the PHQ-9 was 7.64 . Approximately one third of participants  were classified as depressed using a cut-off score of ≥ 10. Overall, 41% of survivors were classified as having minimal depression ; 26% were classified as having mild depression ; 18% were classified as having moderate depression ; 9% were classified as having moderately severe depression  and 6% were classified as having severe depression . Table p = 0.022) of being in a lower anxiety severity category compared to females. Participants with a history of TIA had approximately 58% lower odds  of having a lower anxiety severity compared to those with no history of TIA.The mean anxiety score was 4.82 , 27% of participants (n = 104) were classified as anxious using a cut-off score of ≥ 8; and 22% of participants (n = 80) were classified as “anxious” using a cut-off score of ≥ 10. Overall, sixty percent of survivors were classified as having minimal anxiety ; 19% were classified as having mild anxiety ; 13% were classified as having moderate anxiety ; and 7% were classified as having severe anxiety . Table This study examined the self-reported quality of life, depressive and anxiety symptoms among a sample of stroke survivors three months post-hospital discharge; and the sociodemographic and clinical predictors of these outcomes.Our findings suggest that physical and mental quality of life scores of stroke survivors in our sample were lower than those reported in studies of the Australian general population . SurvivoDiabetes mellitus is an established risk factor for stroke and has been associated with poorer outcomes after stroke , includiLower MCS was associated with a history of TIA. Recurrence of stroke after TIA or minor stroke has previously been associated with poor health related quality of life (HRQoL) and depression . NationaOur findings are consistent with previous literature which demonstrates that one third of stroke survivors are likely to experience post-stroke depression , with otBeing male was associated with higher odds of having lower severity depression and anxiety, and having higher education was associated with higher odds of having lower severity depression, among our sample. This finding is consistent with a meta-analysis of longitudinal studies which showed that female gender is predictive of depression . Having There may be benefits in offering additional support to those stroke survivors with clinical and/ or sociodemographic risk factors for poor psychosocial outcomes following stroke. For instance, our findings suggest that female stroke survivors may represent an important sub-group who may be in greater need of support in the early discharge phase, given their poorer quality of life and greater severity depression and anxiety. This is consistent with previous studies which have found that female survivors have poorer outcomes than males even up to 5 years post-stroke . FurtherThe recruitment of participants from multiple centres is a strength of this study. However, there are a number of limitations to the study. Only 35% of invited survivors completed a survey. As we did not have clinical and sociodemographic information about those who did not participate, it was not possible to assess responder bias. Further, we excluded those with severe cognitive impairment or aphasia limiting their ability to provide informed consent, or those survivors discharged to residential care. Therefore, the results of the study may not be generalizable to all stroke survivors.There are sub-groups of survivors who may be at-risk of poorer quality of life, psychological morbidity and a greater number of unmet needs in the early post-discharge phase. These findings represent an opportunity to improve the delivery of patient-centred care for stroke survivors, by supporting the identification and prioritisation of survivors who are most at-risk of poor outcomes in the early post-discharge phase and may therefore require modifications to standard hospital discharge processes, tailored to their level of risk.


Large topologically associated domains (TADs) contain irregularly spaced nucleosome clutches, and interactions between such clutches are thought to aid the compaction of these domains. Here, we reconstituted TAD-sized chromatin fibers containing hundreds of nucleosomes on native source human and lambda-phage DNA and compared their mechanical properties at the single-molecule level with shorter ‘601’ arrays with various nucleosome repeat lengths. Fluorescent imaging showed increased compaction upon saturation of the DNA with histones and increasing magnesium concentration. Nucleosome clusters and their structural fluctuations were visualized in confined nanochannels. Force spectroscopy revealed not only similar mechanical properties of the TAD-sized fibers as shorter fibers but also large rupture events, consistent with breaking the interactions between distant clutches of nucleosomes. Though the arrays of native human DNA, lambda-phage and ‘601’ DNA featured minor differences in reconstitution yield and nucleosome stability, the fibers’ global structural and mechanical properties were similar, including the interactions between nucleosome clutches. These single-molecule experiments quantify the mechanical forces that stabilize large TAD-sized chromatin domains consisting of disordered, dynamically interacting nucleosome clutches and their effect on the condensation of large chromatin domains. Centromeric and telomeric chromatin display short linker DNA, and while certain nucleosomes are well mapped, particularly close to transcription start sites (TSS), other nucleosomes away from the TSS show more positioning freedom. Regions of DNA without nucleosomes, called nucleosome-free regions (NFR), of 100–300 bp length are prevalently found in vivo4. Overall, nucleosome positioning, histone–DNA stoichiometry, and the degree of chromatin compaction define the transcriptional level of the DNA5 and have a wide-ranging influence on cell and organism functioning, including, e.g., ageing6.Chromatin consists of DNA wrapped in two turns around histone octamers, consisting of two copies of H2A, H2B, H3, and H4 histones. The resulting nucleosomes are connected by linker DNA of variable length, generally below 100 bp. The positioning of nucleosomes is dynamic and varies from cell to cell. Nucleosomes can be repositioned depending on the transcription activity of the underlying genes, with silent chromatin having longer linker DNA8 and topologically associating domains (TADs)9. Three to ten neighboring nucleosomes on a DNA strand flanked by NFR form nucleosome clutches, and such clusters of nucleosomes may interact even if separated far apart11, with a large number of nucleosomes forming chromatin domains12. Genome-wide nucleosome positioning studies demonstrated that eukaryotic chromatin generally is heterogeneous without regular nucleosome positioning1, exhibiting such clutches formed by heterogeneous groups of nucleosomes13. A recent modeling study suggested that nucleosome-nucleosome interactions are essential for the formation of clutches, and the length of NFRs profoundly affects clutch size15. TADs arise from chromatin interactions within tens or hundreds of kilobases. While chromatin looping may occur within a TAD, active and inactive chromatin compartments may include several TADs17. TADs are suggested to represent local chromatin structures in interphase nuclei18.Two dynamic interaction types within megabase chromatin are nucleosome clutches (clusters)19. In vivo studies instead suggest that chromatin condenses as interdigitated 10-nm fibers22. Furthermore, recent data shows that regular nucleosome spacing and DNA-sequence dependent positioning are not observed in vivo, even for the artificial ‘601’ positioning sequences assembled into the genome using the CRISPR/Cas9 technique23. Hence, there is a need to establish and characterize in vitro models of condensed TAD-sized chromatin fibers.Therefore, chromatin in vivo can be imagined as an array of irregularly positioned clusters of nucleosomes that may not condense into the 30-nm fibers observed by reconstituted nucleosome arrays obtained from the Widom ‘601’ DNA high affinity nucleosome positioning constructs commonly used to investigate chromatin in vitro27 that T4 DNA (~ 166 kbp) with natural nucleotide sequence can be reconstituted to chromatin fibers with controlled nucleosome occupancy and used as in vitro model of long TAD-sized chromatin fibers, suitable for single-molecule studies. λ-phage DNA  chromatin displayed similar properties as T4 DNA chromatin26. The size of the resulting λ-DNA chromatin allows the overall compaction dynamics to be addressed using both single molecule fluorescent microscopy (FM), electron microscopy (EM), atomic force microscopy (AFM), and single molecule force spectroscopy. These methods enable the nucleosome occupancy of individual fibers as well as nucleosome-nucleosome interactions and the formation of nucleosome clutches and domains to be quantified.We have previously demonstrated29, λ-DNA has become a convenient object in single molecule force spectroscopy studies. The DNA elastic properties31, the DNA interaction with molecules causing DNA extension , bending33, loop formation31, or condensation36 were investigated using the λ-DNA template. A number of single-molecule studies addressed the properties of the chromatin formed on the λ-DNA by applying the chaperone-assisted reconstitution method40. These papers investigated events of DNA unpeeling associated with the complete release of the DNA from the histone cores or related to the forced unwinding of the last DNA turn. However, the earlier work did not study the low-force (below 10 pN) behavior of nucleosome arrays on long DNA (like λ-phage). This force regime is particularly relevant for the folding/unfolding of higher-order chromatin structures, including the formation of regular fibers and topologically associating domains.Since the pioneering observations of the double-helical DNA overstretching transition 42, as exploited in the ‘601’ template. Although λ-DNA features a wide sequence variety, it cannot be excluded that eukaryotic genomes have evolved sequence properties that direct nucleosomes to particular places, which has been speculated to mediate specific regulatory functions in vivo43. Therefore, it is imperative to validate features found on unnatural nucleosome DNA substrates by comparing these to native eukaryotic DNA. Although linker histones participate in the chromatin structure in higher eukaryotes, it has been shown that the viability of the cultured cells is resistant to significant H1 depletion44. In addition, complete linker histone knockout does not change the phenotype and transcription profile of the lower eukaryotes44. It has been found that the “native” folding of chromatin fibers can be reached without linker histone at millimolar Mg2+ concentrations46. These observations warrant the relevance of studying the nucleosome arrays without the linker histones.Though ‘601’ arrays and λ-DNA form suitable substrates for highly regular small nucleosome arrays and larger fibers, both are not natural substrates for nucleosome folding. It is an open question how this affects the stability of the formed nucleosomes, particularly their higher-order folding. Histone octamers have distinct sequence preferencesHere, we have characterized in vitro nucleosome fibers reconstituted on different long native natural DNA and artificial substrates at a single molecule level using FM, negative stain EM, AFM, and force spectroscopy. Most importantly, we demonstrate the spontaneous tendency of TAD-sized chromatin to form nucleosome clusters.ca. 2.5 μm to 1.0 μm reflecting the formation of nucleosome arrays and their compaction at 100 mM NaCl  with added 100 mM NaCl 50.The effect of Mg2+, and the long-axis length started to shrink at < 1 mM Mg2+. The long-axis length at 15 mM of Mg2+ was only slightly larger than the typical size of DNA in the globular state  and corresponding reconstituted chromatin fibers reported in our previous studies27. It was earlier demonstrated that a significant HO loading (HO:DNA > 0.7) is necessary for the condensation of short nucleosome arrays53. However, we observed that even unsaturated arrays of long chromatin molecules undergo notable compaction by Mg2+ , probably due to loops formed between nucleosomes that mediate attractive interactions. Note, that compaction due to the stacking of neighboring nucleosomes of the saturated short 12-mer nucleosomal arrays54, and also in long λ-arrays . This similarity suggests that, despite the difference in the compaction geometry of short54 and long25 arrays, their phase transition is primarily determined by the same overall charge neutralization of macromolecular chains.The essential difference between DNA and nucleosome arrays is the compaction at sub-millimolar and millimolar concentrations of divalent cations, in contrast to DNA that undergoes only modest contraction at [Mg27. The primary reorganization process is the association and dissociation of distant clutches of nucleosomes, mediated by the looping of the connecting DNA and chromatin. However, even in nanofluidic channels, FM does not have sufficient resolution to resolve these structures.Next, we directly imaged the clustering of nucleosomes in sub-saturated arrays using FM imaging in the nanofluidic channels. Figure Complementary to the FM, we used multiplexed magnetic tweezers (MMT) force spectroscopy to characterize the compaction and mechanical properties of the arrays reconstituted on the native human DNA, λ-DNA, and Widom ‘601’ nucleosome positioning sequence. This gives an independent assessment of individual fiber composition and compaction under similar physiological conditions as the FM at varying degrees of nucleosome occupancy. DNA templates for magnetic tweezers measurements (labeled by digoxigenin and biotin at the ends) were prepared and characterized as described in the “+, 10 mM Na+, 2 mM Mg2+) known to favor the formation of compact chromatin in the arrays reconstituted with the ‘601’ positioning sequence55. Our FM measurements clearly showed complete compaction of the λ-arrays under physiological conditions at HO:DNA = 1.0 with 2 mM Mg2+, while at HO:DNA = 0.5, the fibers appear not maximally but partially folded . This observed inhomogeneity should be attributed to the variation in nucleosome number and was determined for each individual fiber. Still, despite these variations, the successive steps of unfolding stretching curves of the nucleosome arrays display regular features that can be captured in a statistical mechanics model60 developed for short, well-defined ‘601’ arrays  model Fig. C, nucleoays Fig. B. After ays Fig. B, a forc62 and references cited therein) and allows reliable determination of the total number of nucleosomes in a given fiber . The number of nucleosomes obtained this way is generally larger than the corresponding level of condensation below the plateau region. Careful analysis of short, regular ‘601’-based fibers concluded that not all nucleosomes contribute to the folding of the fibers at low force and we, therefore, assign the number of folded nucleosomes (Nfolded) separately based on the expected DNA nucleosome repeat length (NRL) and the array extension at F < 3 pN59.At around 7 pN, the nucleosomes gradually yield more DNA until each nucleosome wraps 77–80 bp of DNA. Next, the stepwise release of this last turn of the DNA superhelix is observed as discrete 25–27 nm steps, corresponding to 77–80 bp, at forces > 10 pN. This characteristic feature has been studied in many single-molecule measurements of nucleosome array stretching . Ruptures shorter than 200 bp are not distinguishable from the step-wise unfolding of the fiber occurring at low forces  and ∆G1) in all types of fibers suggests that stacking interactions of such more remote nucleosomes have a relatively small effect.In the compacted inhomogeneous arrays, one would expect that the attractive forces between the inter-cluster contacts and nucleosome-nucleosome interactions inside the clusters have similar molecular features and strengths. Both folding of the 10-nm beads-on-a-string fiber and cluster–cluster interaction are facilitated by reducing the DNA-DNA repulsion due to charge screening by the positively charged histone tails, MgWe counted cluster–cluster ruptures and determined the length of the DNA release in each of these events see “” and theFrupture < 8 pN  and unfolding energy (∆G1) increase slightly with the HO:DNA ratio increase, though this change is not statistically significant. We found a considerable spread between individual molecules from the same batch, but the variance is similar for the longer fibers compared to the short 197-15 arrays and agrees with previous findings on the ‘601’ arrays58. Thus the difference between the levels of saturation is predominantly in the number of nucleosomes that have been reconstituted  and is significantly larger than that for NRL = 197 bp. This should be ascribed to the shorter linker length, which makes the compliance of chromatin fiber with unstacked nucleosomes less, or stacking the nucleosomes into a 2-start zig-zag fiber, which doubles the stiffness relative to a single stack of nucleosomes58. The parameters kfiber, ∆G1, and ∆G2 determined in this work agree with previously published tweezers results of ‘601’-DNA arrays65, albeit showing slightly lower values. These minor discrepancies might be caused by the difference in the sequences in the linker DNA58 or by unaccounted differences in the experimental protocols applied in the different laboratories.Since we did not observe differences as a function of HO:DNA ratio, we pooled the data for each type of array and compared averaged mechanical properties of the λ-, native-DNA to a range of ‘601’ arrays with different nucleosome repeat lengths  numbers for the λ-arrays  show similar mean values of ays Fig. C. This ikfiber) all feature a small broadening at higher values as compared to a normal distribution assemblies.The distributions of fiber stiffness  while static DNA attachment to the HO prevails at low salt. This difference in DNA dynamics results in the perturbation of the regular fiber structure without losing its compactness. Loss of energy in rupturing static contacts is compensated by increased entropy and the formation of numerous new weak and dynamic bonds. The observed absence of abrupt changes of the mechanical properties  in dependence of NRL, which is reported in this work and a recent detailed study58, supports the model of dynamic breathing chromatin structure.Recent cryo-EM studies reported that in vivo nucleosome arrays lack even a short-range uniformity in the linker DNA lengthsAn important finding of this work is that it is predominantly the reconstitution yield rather than the local structure of nucleosome clutches or mechanical properties of the fibers that defines the existence and frequency of the cluster–cluster contacts. We did not find that the difference in DNA sequence  makes a noticeable difference for cluster–cluster statistics.24, in which partially folded structures containing both unfolded and compacted globular regions of various sizes were observed. This was attributed to the heterogeneous distribution of nucleosomes on the DNA chain. The observed variance in the rupture forces and cluster–cluster distances is not the result of the low-resolution of the MMT measurements  but is intrinsic to the mechanics of the reconstituted fibers.The unfavorable contribution of bare DNA-DNA repulsion weakens the forces that hold the clusters together. At HO:DNA = 0.5, large stretches of bare DNA may not be included in the clusters of nucleosomes and contribute only to the bare DNA extension. A similar conclusion was earlier drawn based on stretching T4 chromatin fibers using a flow-stretching techniqueThe current methods for reconstitution, force spectroscopy, and data analysis may be used in further studies to test whether different chromatin fractions, such as heterochromatin or euchromatin, yield different reconstitution yields and/or folding properties. Our current results do not indicate that DNA sequence is essential in the higher-order folding of heterogeneous chromatin fibers.2 tetramer) followed by consecutive addition of the two H2A/H2B dimers. At the moderate monovalent salt concentration (150–400 mM), nucleosome-nucleosome interaction is attractive52, favoring nucleosome array folding. Hence, closely separated nucleosomes can form clusters since the favorable contribution of the attractive forces overcomes the entropy factor facilitating random and relatively uniform nucleosome distribution. This finding is consistent with a cooperative mode of nucleosome reconstitution69. Folding of the nucleosome arrays is a specific case of the general phenomenon of DNA condensation. DNA condensation is a cooperative process71 which means that under the conditions when polycationic condensing agents (like histones in the living cells) are present in an amount that is insufficient for compaction of all DNA, there will be a coexistence of regions of condensed and bare DNA.The question arises why the in vitro salt-dialysis reconstitution of the native DNA and the λ-arrays results in inhomogeneous clusters of the nucleosomes rather than in an entropy-advantageous random distribution of the nucleosomes along the entire DNA length? A plausible explanation is that the entropy gain associated with a random distribution of the nucleosomes along the entire DNA length is counteracted by the favorable attractive nucleosome–nucleosome interactions. Under the conditions of the nucleosome array reconstitution, the decrease of salt concentration leads first to the formation of the tetrasomes (complex of the DNA and (H3/H4)(A detailed account of DNA preparations is given in the MNase-digested human DNA for multiplexed magnetic tweezers (MMT) measurements was prepared by limited digestion of nuclei from Expi293 cells  followed by purification  to extraDNA templates with NRLs 197, 177, 172, 166, and 162 bp were prepared as detailed in the Supplementary Information –S3. In aThe quality of the DNA labeling was tested by the MMT measurements and fitting of the experimental data to the worm-like chain (WLC) model  ratio, and the assembled octamer was purified by gel filtration chromatography.Recombinant human histone proteins were individually expressed, purified, and refolded into human histone octamer, essentially as described19, excluding the addition of competitor DNA.The DNA templates and recombinant human histone octamers (HO) were reconstituted into nucleosome arrays by the salt dialysis method described previouslyDNA was mixed with HO at three ratios HO:DNA = 0.5, 0.8, and 1.0, calculated based on a ratio of one HO per 197 bp of DNA. The salt concentration was reduced from 2 M NaCl to 10 mM NaCl in continuous dialysis at a 0.65 ml/min flow rate overnight using Masterflex L/S digital Miniflex dual-channel peristaltic pump . The final buffer contains 10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 10 mM NaCl. The quality of the reconstituted arrays was accessed by electrophoretic mobility shift assay (EMSA); see Fig. For the arrays with NRLs shorter than 197 bp , the reconstitution protocol was similar to the one applied for the λ- and 197 bp NRL arrays (see above). The only difference was that the HO:DNA ratio was close to 1.0 (0.8–1.2). Each array was prepared in three batches of different HO:DNA ratios . Furthermore, two reconstitution runs were performed using DNA templates with long  and short (~ 100 bp) DNA handles. For NRL = 177 bp, the DNA template with long handles contained 36 repeats; the template with short handles had 38 repeats. Long handles of the 177-36 template created space for additional nucleosomes, so the abbreviation ‘177-38’ will be used for both 177-36 and 177-38 arrays.259.For all samples, the typical final concentration of the reconstituted arrays was 10–20 ng/µl measured as DNA concentration calculated from solution optical absorption at 259 nm, A2. Solutions were gently mixed after the addition of each new component. The final samples were incubated for 1 h at ambient temperature prior to observations. FM observations were performed using an Eclipse TE2000-U  microscope with a 100× oil-immersion lens. Fluorescent images were recorded and analyzed using an EM-CCD camera and an Argus 10 image processor . The long-axis length of the individual λ-DNA or nucleosome arrays was measured using ImageJ 1.52a software (NIH)74.λ-DNA  (New England Biolabs) was used without further purification in FM and negative stain EM experiments. Sample solutions for single molecule observations were prepared by successive mixing of TE buffer  containing 100 mM NaCl, a concentrated solution of λ-DNA or nucleosome array , fluorescent dye YOYO-1 (20 nM) and variable concentrations of MgCl27. The device consisted of an array of 90 μm long, rectangular channels with a cross-section of 50 × 70 nm2 (60-nm channel system) or 120 × 130 nm2 (125-nm system) and uncertainty in width and depth of ± 5 nm. Before fluorescence imaging, DNA was stained with YOYO-1  at a ratio of one dye molecule for each four base pairs. Then, the stained chromatin was driven into the nanochannels by electrophoresis. The fibers were visualized with a Nikon Eclipse Ti inverted fluorescence microscope equipped with a diode laser, 200 mW/488 nm , filter set, and a 100× oil immersion objective . Movie clips at a rate of 2.5 frames/s were recorded with an electron-multiplying charged coupled device camera (Andor iXon X3). The image pixel size of 0.16 × 0.16 μm2 was calibrated with the help of a metric ruler.Nanofluidic devices were fabricated in PDMS elastomer as previously describedA detailed account of experimental procedures and data analysis is given in the 60. First, a solution of anti-digoxigenin  was introduced into the flow cell and incubated for 2 h at room temperature, followed by flushing 1 ml of passivation solution (3.6% Bovine Serum Albumin (BSA) heat shock fraction, pH 7, ≥ 98% (Merck), 0.1% Tween 20 (Merck)) with subsequent storage at 4 °C. A detailed description of the MMT instrument, sample preparation, measurements, and data analysis is given in the “A homemade MMT setup and flow cells used in this work were assembled as described beforeNtotal, and the number of nucleosomes excluded from stacking and fiber folding Nunfold. Values of Ntotal and Nunfold were obtained by fitting the experimental force-extension curve in the range of moderate (8–15 pN) and low (0.5–2 pN) force worm-like chains that were reduced in contour length by 79 bp × Ntotal and NRL × Nfolded respectively. Next, force-extension curves were fitted with a statistical mechanics model for dynamic unfolding56 ; free energy (∆G1) associated with nucleosome unstacking, and the first step of DNA unwinding (from 146 to 92 bp); free energy related to the second stage of DNA unwrapping 62. The number of nucleosomes contributing to the fiber folding is defined as Nfolded = Ntotal − Nunfold. Values of kfiber, ∆G1, and ∆G2 were automatically fitted after their approximate manual setting. See The composition of the fibers varied and was quantified by the total number of nucleosomes in the fiber, Frupture < 15 pN range yielding an extension longer than 68 nm  were detected automatically using a Python script, after applying a 15-point median filter to reduce fluctuations and joining events that were separated less than 5 points. The XY-position (dR) change for each rupture was calculated to identify events corresponding to the bead's unsticking from the surface. A threshold of dR > 0.1 µm was used to discard such events.In this work, we have found that the low-force part of the force-extension curves of the long fibers exhibited abrupt extension jumps that are different from the gradual and reversible unfolding observed for the short arrays. We assign these events to ruptures of contacts between clusters of the folded nucleosomes, separated by stretches of nucleosome-free DNA or non-interacting nucleosomes. Ruptures in the 1 < λ-array was diluted to a DNA concentration of 5 µg/ml with TEN0.01 buffer and fixed with 0.02% glutaraldehyde on ice for 10 min. 4 µl of the sample was applied on the carbon-coated grid for 1 min and subsequently blotted from the edge of the grid using Whatman Grade 1 filter paper. Immediately, 4 µl of 2% uranyl acetate was applied on the grid and left to stain for another minute. The grids were visualized with a Tecnai T12 electron microscope operating at 120 kV, and micrographs were recorded on Eagle 4 K CCD Camera (FEI) at 30,000–49,000× magnification with a defocus value of − 1.2 μm. Micrographs were analyzed on Fiji and manually counted with the "multi-point" tool. Only arrays that were well defined as single molecules were counted. A total of 17 λ-arrays were counted for each HO:DNA ratio. Due to their large size, the λ-arrays were often entangled with each other, making it difficult to separate the arrays to get large datasets.A 5–10 µl droplet of the reconstituted λ-array at 30 ng/µl was spotted on a plasma oxidized silica surface. After 10 min to allow for sample adsorption onto the surface, the specimens were developed by flushing them with 1 ml of ultra-pure water followed by drying in a stream of nitrogen gas. Atomic force imaging was done with a Bruker Dimension Icon microscope  at room temperature in the air. Images were acquired in the standard tapping mode with silicon (Si) cantilevers (spring constant of 19–55 N/m) and operated just below their resonance frequency . The images were flattened, and the contrast and brightness were adjusted for optimal viewing conditions.Supplementary Information 1.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.


Late’iki  is a highly active volcano in the Tofua arc with at least four temporary island-building eruptions and one submarine eruption in the last 55 years. The most recent eruption, commencing in October 2019, resulted in lava effusion and subsequent phreatic explosions, the construction of a short-lived island that was quickly eroded by wave action and possibly further phreatic activity that continued into January 2020. The two-pyroxene dacite from the 2019 eruption is similar to the 1967/8 eruptions suggesting the magma is residual from earlier eruptions and has not undergone further differentiation in the last 50 years. New observations of the 2019 eruption site confirm the lava-dominant character of the volcano summit but a thin veneer of wave-reworked, finely fragmented lava material remains that is interpreted to have been produced by phreatic explosions from hot rock-water interactions during the effusive eruption. A notable absence of quench-fragmented hyaloclastite breccias suggests that non-explosive quench fragmentation processes were minimal at these shallow depths or that hyaloclastite debris has resedimented to greater depths beyond our summit survey area. Dacititic products were considered relatively uncommon and largely restricted to subaerial Fonulaei3; however, pumice rafts from submarine eruptions8 and dredged lavas from some submarine edifices are also dacitic10. Consequently, the volume of dacite magma erupted in the Tofua arc has been underrepresented due to the few available subaerial deposits. The ephemeral island-producing dacitic volcano of Late’iki , centrally located in the Tofua arc at 19.17° S and 174.85° W . It contains 20 known active volcanoes, including 13 that are submarine, which erupt basaltic andesite, andesite and dacite magmas both effusively and explosively12, each characterised by low explosivity and lava extrusion. Six temporary islands formed from the 1781–1995 eruptions; of these, the 1967–8 and 1979 eruptions also produced floating pumiceous material, and one eruption in 1990–1 is thought to have been entirely submarine and also produced floating pumice12. In contrast to subplinian to plinian pumice raft-producing eruptions from the nearby Home Reef and 0403-091  submarine volcanoes, where the main eruption phase lasts < 1 day14, eruptions at Late’iki last several weeks17. The earliest well-documented eruption (1967) began from a submarine vent with lava fountains and tephra ejections reaching 1.2 km altitude18, producing a small island 800 m long and ~ 15 m high that was removed by wave action within ~ 6 weeks15. Lavas were poorly—moderately vesicular19 dacites susceptible to breakage and wave erosion15. In May 1979 (possibly beginning earlier), Late’iki erupted again producing an island and explosions that ejected material ~ 150 m above the ocean with ash and steam reaching 1.5 km20. This eruption is presumed to be the source of an floating pumice raft observed NW of Late island in May 1979, which reached Fiji and the Solomon Islands21. A July 1979 survey by RV Balikula revealed a rapidly eroding island 300 × 120 m and ~ 15 m high, located approximately 1 km E of the former 1968 island22, with a tephra rampart marked by drainage gullies that was removed by early October 197923. In contrast, the next island-forming eruption (1995) produced an emergent lava dome. An early video recording by a citizen scientist (Allan Bowe) showed explosive activity continuing during lava extrusion with ash eruptions from two vents ejecting material to ~ 500 m above sea level and steam rising to 2 km24. The maximum island size was ~ 300 × 250 m and ~ 50 m high; it endured until its destruction in the 2019 eruption20.Late’iki is a shallow to emergent submarine volcano (summit currently < 10 m below sea level) and erupts regularly, with recorded eruptions in 1781, 1851, 1858, 1878, 1886, 1967–8, 1979, 1990–1, 1995 and 201916 for more details). A new island was formed 150 m WNW of the former 1995 Late’iki Island  until 19 November  until 14 December . Minor amounts of plagioclase microlites are present in the vesicular glass.Grab samples of the 2019 eruption deposits are clasts of grey lava that are finely vesicular, relatively crystal-rich with abundant glomerocrysts of plagioclase and pyroxene Fig. , and are89-76 (bytownite), similar to that of the 1967–8 lava , with two distinct compositions  and a rare Mg-rich bronzite (En83Fs13Wo4) that is occasionally rimmed by hypersthene and was interpreted by Ewart et al.2 as xenocrystic. Clinopyroxene typically occurs in glomerocrysts and exhibits two compositional populations: most is an unzoned calcic augite (En41Fs17Wo42) with the same composition as in the 1967–8 lava; we have also identified the minor occurrence of a phenocrystic Mg-rich augite (En52Fs11Wo37) that compositionally overlaps with augites in the basaltic andesite lavas2. Rare olivine is observed in the 2019 lava. Although not analysed in this study, rare, highly magnesian (Fo93) olivine phenocrysts with rims of hypersthene and Cr-spinel inclusions were reported in the 1967–8 lava15. We interpret the olivine, Mg-rich augite and bronzite to have to have been ultimately derived from basaltic magma and subsequently introduced into a very silicic melt.The mineralogy of most glomerocrysts is consistent with a plagioclase-poor gabbronorite bulk composition. Plagioclase compositions are highly calcic showing a limited range from Anava Fig. . Orthopy25), with only a few analyses for the 1967–8 eruption27.Geochemically, Late’iki remains poorly characterised compared to other volcanoes in the Tofua arc ; no molecular water (H2Om) was detected. H2O content and speciation in magma vary with pressure and temperature32. These data are consistent with the subaerial island-forming eruption and vesicular character of the lava where magma has degassed to atmospheric pressure prior to eruption and quench33. The lack of H2Om also confirms the sample was unaffected by post-eruption secondary hydration (e.g.35) from surrounding seawater or hydrothermal fluids, consistent with the slow diffusivity of H2O in glass at low temperatures (e.g.37) and the short timescale between eruption and sample collection (~ 4 months).FTIR analysis of the 2019 matrix glass reveals it has an H17. The only time series data that can be reliably derived from satellite observations prior to the eruption are the size and shape of the island, and the extent of hydrothermal venting from the summit , Volcano-F 2019 (VEI 2–3), Late 1854 (VEI 2), and Kao 1847 (VEI 2).Establishing Volcanic Explosivity Indices (VEIs)16, satellite images do not record the characteristic dense black ‘cock’s comb’ tephra jets associated with that style of emergent, typically basaltic, volcanism39. Instead, the 2019 eruption mostly generated only tephra-poor, steam plumes  time periods, and increases magma viscosities, which may stall a rising magma batch40. However, if not stalled, in a situation where exsolved gasses have not fully escaped the conduit system, higher viscosities can enhance explosivity42, and mineralisation and hydrothermal sealing of volcanic rocks43, particularly within lava domes45, may allow greater pressurisation of the system and more explosive eruptions. Thus, while the hydrothermal system at Late’iki may currently contribute to the lower explosivity of its eruptions compared to the similarly shallow dacitic Home Reef and 0403-091 volcanoes, the hydrothermal system here has the potential to enhance explosivity under other conditions.As evidenced by satellite imagery and bubbles observed in post-eruption seafloor photography Fig. , Late’ik47, the highly magnesian mineral assemblage is strongly out of equilibrium with the rhyolite glass and argues against simple melt extraction from a crystal mush as represented by the glomerocrysts.The overlap in phenocryst mineralogy and composition, glass and bulk-rock chemistry indicate the 2019 eruption has continued to evacuate magma initially injected into the volcanic system as recorded by the 1967–1968 eruption, with no significant fractionation occurring over the last 50 years. Since 1967, eruptions at Late’iki have been relatively closely spaced, occurring at intervals of 11, 11, 5 and 24 years. A similar cluster of eruptions occurred at Late’iki from 1851 to 1886, and the more substantial hiatus from 1886 to 1967 suggests the 1967 eruption recorded the eruption of a new magma batch. The presence of a minor ultramafic mineral assemblage, headlined by forsteritic olivine in a rhyolite glass, suggests mafic recharge may have been responsible for eruption initiation in 1967–68. The bulk rock chemical characteristics of the Late’iki lavas are highly unusual and apparently unique in the Tofua arc  may not be developed in sufficient quantities, and finely fragmented material from phreatic explosions is quickly remobilised and removed from the eruption site.The observed lithofacies at the summit of Late’iki contrast with current facies models for silicic volcanoes transiting from being submerged to subaerialion Fig. M, showed52) or explosive phreatic disruption of previously emplaced lavas. In any case, some ongoing phreatic or volcanic activity is required to explain these observations into 2020.On at least three dates after the main eruptive phase atypical activity is imaged at Late’iki by satellites. It is possible that other processes could explain several of these phenomena observed after the main eruptive phase. The concentric waves could be produced by refraction around a shallow region of the volcano, but because they are not present in other images and do not correlate with periods of anomalous wind or wave height this would require a shallowing and then deepening of the seafloor in this region on a short timescale (days to weeks) which would itself indicate active volcanic extrusion. Similarly, the white water could be produced by wave breaks on shallow seafloor but again these observations do not correlate with anomalous weather conditions and white water is not present in images taken within a few days of the inferred events. We therefore interpret the white water and concentric rings as evidence of intermittent, weak explosive activity continuing after October 2019, most likely resulting from steam explosions caused by seawater infiltration. The presence of pumiceous lava stringers in January may suggest a minor amount of newly fragmented material at this time but it is not definitive, since floating material could potentially be produced from the vesicular lava carapace at shallow water depths either by passive buoyant detachment from an active flow  with an accuracy of around 20 m. Additional sporadic, but very high resolution (as good as 30 cm per pixel) imagery was provided by the Maxar technologies DigitalGlobe platform collected using their WorldView-2 and WorldView-3 satellites53. Data were projected and compared using the open-source software QGIS . QGIS was also used to define island locations and boundaries and to measure the subaerial area of the 1995 island before the 2019 eruption and of New Late’iki for the period after the 2019 eruption until it because completely submerged.Data from three different satellite observation programs were used to reconstruct the timings and phases of the 2019 eruption. The European Space Agency’s Sentinel 2 Satellites’ Level 1C products (10 m per pixel) is pre-processed into 100 kmWe use the satellite imagery to qualitatively identify four different levels of hydrothermal activity. These are: No Activity—no discoloured water in the vicinity of the volcano  and the full videos can be found at https://figshare.com/s/a076ae26e952dafa51be.Sampling was conducted of the seafloor using a small grab sampler owned and operated by the Department of Geology and Palaeontology, National Museum of Nature and Science, Japan. Working conditions were challenging, with no accurate post-eruptive bathymetry available and changes in currents and tides causing the hydrothermal plume to move around the volcano during the day. As a result, sample sites were chosen opportunistically to the north west of the volcano, along the path of approach to allow for easy evacuation in the event of any heightened activity. Most sampling sites were NE of the former Late’iki and New Late’iki islands yet still within the area of eruptive activity imaged on 20 October Fig. . A 4 k r56. The EPMA (JEOL JXA-8230) analysis was conducted using an accelerating voltage of 15 kV, a probe current of 12 nA, and a spot diameter of 1 μm for mineral analysis. For glass analysis, accelerating voltage of 15 kV, a probe current of 8 nA, and a spot diameter of 10 μm was used to minimize Na loss. FTIR analysis was conducted on individual crushed shards as in Mitchell et al.57, using the mid-IR 3500 cm−1 H2Ot and 1630 cm−1 H2Om absorbance peaks with a species-dependent molar absorptivity coefficient for the 3500 cm−1 peak35.Geochemical data were collected on the one recovered summit sample large enough for comprehensive analysis, using XRF and EPMA (Appendix XSupplementary Information 1.Supplementary Information 2.