A significant share of the world’s undiscovered oil and natural gas resources are assumed to lie under the seabed of the Arctic Ocean. Up until now, the exploitation of the resources especially under the European Arctic has largely been prevented by the challenges posed by sea ice coverage, harsh weather conditions, darkness, remoteness of the fields, and lack of infrastructure. Gradual warming has, however, improved the accessibility of the Arctic Ocean. We show for the most resource-abundant European Arctic Seas whether and how a climate induced reduction in sea ice might impact future accessibility of offshore natural gas and crude oil resources. Based on this analysis we show for a number of illustrative but representative locations which technology options exist based on a cost-minimization assessment. We find that under current hydrocarbon prices, oil and gas from the European offshore Arctic is not competitive on world markets.The online version of this article (doi:10.1007/s13280-017-0957-z) contains supplementary material, which is available to authorized users. A significant share of the world’s undiscovered oil and natural gas resources is assumed to lie under the seabed of the Arctic Ocean. The US Geological Survey estimates that “about 30% of the world’s undiscovered gas and 13% of the world’s undiscovered oil” are north of the Arctic Circle, including onshore resources  with satellite-derived observations to select four CMIP5 models, which compute sea ice concentration best compared to these observations. The selected models are further analyzed to estimate the future sea ice distribution in our four target areas. We focus on results from the emission scenarios RCP 4.5 and RCP 8.5 , to project possible climate change magnitudes until 2100 . Alternatively and in case of water depth restriction, export of the products can also be realized by means of pipelines to backup treatment plants and further to the local network (continuous export). Such continuous pipeline connection could be used to produce LNG , CNG , or GTL (Gas to Liquids) in a specialized treatment plant, allowing further long distance exports to clients worldwide.Floating platforms can be designed to work in even harsher environments and especially in deep water conditions. An example is the FPSO  technology, where a floating terminal vessel (ship shaped or barge type) comprises capacity for production, processing, and storing of hydrocarbons. The product can be exported either via shuttle tanker (mostly if the step-out distance exceeds roughly 200 km) or via pipeline, depending on cost effectiveness. All floating production units are permanently moored to the seafloor. Most units can be temporarily disconnected from the moorings in emergency, e.g., when large icebergs or excessive thick drifting ice approaches, which cannot be managed by ice-breaking service vessels.The latest development is the subsea processing technology where no permanently floating or fixed platform is required for production. Instead the production facilities are directly installed on the seafloor so that the environment at the sea surface does not harm the operations. These facilities export the products via pipeline to shore  configuration) or via flowlines and risers to floating storage or production and storage facilities, such as FSOs (Floating Storage and Offloading facilities), FPSOs, or FLNG ). It should be mentioned that each production unit mainly dedicated to gas production (or to oil production) will also produce condensate (comparable to oil) or associated gas and vice versa, which also has to be handled, or even stored and exported at the floating facility.The choice of the actual production technology employed at a given location depends on a number of environmental constraints, most importantly step-out distance, water depth, wave conditions , ice condition , and wind conditions . Assuming iceberg management for all technologies, Fig. It is important to note that deepwater areas, which are permanently ice covered, are very complicated (or even today not possible) to develop. This is caused by the need to get  access to the production wells for planned and emergency workover drilling and maintenance works by means of dedicated vessels (ships or semi-submersibles) and remotely operated vehicles (ROV). Directional drilling from aside (from onshore) is not feasible when the distance to the well exceeds about 8 km . Table Further constraints for operations at remote locations are e.g., set by rescue and evacuation systems and helicopter flights for personnel transfer. An installation is considered remote if it is located more than 40 nautical miles from the nearest manned installation or airport/heliport. Consequently, consideration should be given to provide Jet-A1 refueling facilities, where the distance from shore to an installation or vessel  exceeds 50 nautical miles.S3 for more details). Note that the calculations have been carried out in the years 2011–2013. Meanwhile prices might have changed and technology further developed. Also, political constraints have changed (partly completely) in major Arctic areas since then. Therefore, we decided not to focus on this issue in greater detail.In the following, we present the main cost components of the various technologies, including all project development costs, equipment, procedures, and operations needed to meet the specific requirements. The cost estimates are based on realistic technical and economic assumptions and cost data derived from comprehensive literature and internet source reviews (see Table All cost estimates include (if applicable) iceberg management by ice-breaking OSVs, project development, shore base, supply and tug boats, development drilling and completion, subsea components and installation, flowlines to gathering point, service vessels, structure decommissioning and removal as well as production, service, maintenance, and insurance. Shipping and onshore processing (if applicable) is included, too.However, it is evident that the cost elements can only provide a rough estimate of the total costs. We do, for example, not include political costs, which may be required by the concessionaires of the deposits in the Arctic region. Political costs often include e.g., direct costs related to the permission award or to the provision of safety and security measures required by most of the involved communities or states. These political costs may drastically change the economic viability of an Arctic hydrocarbon export scheme up to conditions, where political costs have to be considered as a “show stopper.”Table We combine the information from the suitability of a technology and the sea ice projections with data on bathymetry and step-out distance. Figure Regarding ice conditions, we have to distinguish between summer and winter ice conditions. Fixed and subsea technologies can generally cope with harsh conditions and can be employed if ice conditions are favorable only in summer, where a time window with manageable ice conditions (1.5 m ice thickness or less) is needed for initial construction and maintenance. Floating production units as well as LNG carriers and tankers, however, need year-round sufficiently low ice thickness under 1.5 m. Figure While ice coverage in winter is for long time (still) different to summer conditions, we do not find that it is a decisive obstacle for energy production. Even though the Kara Sea (WSP2) is not ice free in the winter irrespective of the climate scenario, and also a significant part of the assessment unit off West Greenland is ice covered, in both cases the sea ice is thin enough to allow both for floating production units as well as LNG export. This is especially relevant as floating production units are the cheapest options for gas production in the Arctic Below is the link to the electronic supplementary material.


Ice-shelf channels are long curvilinear tracts of thin ice found on Antarctic ice shelves. Many of them originate near the grounding line, but their formation mechanisms remain poorly understood. Here we use ice-penetrating radar data from Roi Baudouin Ice Shelf, East Antarctica, to infer that the morphology of several ice-shelf channels is seeded upstream of the grounding line by large basal obstacles indenting the ice from below. We interpret each obstacle as an esker ridge formed from sediments deposited by subglacial water conduits, and calculate that the eskers' size grows towards the grounding line where deposition rates are maximum. Relict features on the shelf indicate that these linked systems of subglacial conduits and ice-shelf channels have been changing over the past few centuries. Because ice-shelf channels are loci where intense melting occurs to thin an ice shelf, these findings expose a novel link between subglacial drainage, sedimentation and ice-shelf stability. The formation mechanisms of ice-shelf channels remain poorly understood. Here, using ice-penetrating radar data, the authors propose that ice-shelf channel morphology in the Roi Baudouin Ice Shelf, East Antarctica, is seeded by esker ridges, indenting the ice from below. Water beneath the Antarctic Ice Sheet promotes the formation of ice streams that rapidly slide over wet sediments and a lubricated base. Ice streams discharge the majority of Antarctic ice into floating ice shelves, which surround about 74% of the Antarctic perimeter459In this study we use satellite data and ice-penetrating radar to show that ice-shelf channels on the Roi Baudouin Ice Shelf, East Antarctica, are seeded upstream of the grounding line by basal obstacles indenting the ice from below. These obstacles align with predicted hydrological outlets, and thus we interpret them as eskers (sediment ridge composed of gravel and sand) formed by the overlying subglacial water conduits. Our findings confirm a recognized linkage between ice-shelf channel formation and subglacial hydrology6911Here we survey three hydrologically predictedIn full details below, we argue that scenario 3 accords best with our observations, and we interpret each basal obstacle as an actively evolving ramp-shaped esker whose size increases towards the ocean due to subglacial conduit widening and decreasing water flow speed. Eskers, a glacial landform used in the reconstruction of palaeo ice sheets1416We estimate the grounding-line position using satellite-based interferometric synthetic aperture radar by picking the landward limit of the tidal flexure zone in interferograms from 1996, 2007 and 2016 . The lim20212) of reflector A upstream of the grounding line. We see no reflections delineating lateral walls, so either no walls exist or they are too steep to be imaged by our nadir-looking radar. The former interpretation here implies an essentially two-dimensional internal reflector, which is inconsistent with the seaward extension of these features into ice-shelf channels where ice is lacking compared to the neighbouring areas  bedforms is supported by more evidence on the ice shelf: surface ridges dating from earlier times can be found there, implying past changes in the coupled systems.The seeding of ice-shelf channels A–C at the grounding line means that their morphology can record grounding-line history as well as changes in sub-shelf melt plume dynamics as ice flow advects them towards the shelf front. At sites A and C, the surface ridges decay seawards to grade into the depressed surface of the ice-shelf channels downstream  although−1 as an upper limit, which is an order of magnitude higher than model estimates of sedimentation rates at melt-stream portalsIn this interpretation, the surface ridges at sites A and C are currently below their critical threshold so they do not intrude far into the ice-shelf channels. But the isolated hills suggest that these ridges may soon be advected into the ice shelf. An end-member scenario is that the ramp-shaped esker at site A was completely removed by flooding 175 a ago and has since re-grown to its present size. This requires a sedimentation rate of ∼1.4 m aAt site B, the basal disruption imaged by reflector B is offset from the surface ridge, and we lack ground-based radar data for examining its geometry along flow. In plan view, the corresponding ice-shelf channel is less developed compared to ice-shelf channels A and C and deviates from the ice-flow direction towards ice-shelf channel C. These observations suggest that the esker/conduit B began developing more recently and has been migrating eastward to reach its current position.Numerous subglacial processes in scenario 3 require further investigation. Our mechanism for the formation of ramp-shaped eskers involves a reduction of water flow speed towards the grounding line, which has not been directly measured. Also, without access to the bed, we lack precise information about the current subglacial conduit arrangement and sediment transport regime on/near the eskers, which determine how their form continues to evolve. Since all three sites lack sediment sources on the surface, a negligible sediment delivery to the bed is expected from supraglacial melt-water streamsin situ in their formation environment, and confined by ice, which prevents sediment-flank slumping. If the grounding line retreats, rapid degradation by slumping and erosion would occur, especially as their sediments are probably weakly consolidated in the subaqueous environment; given enough time, a drastic height reduction is hence not inconceivable. These ideas seem to us consistent with the fragmented nature of esker networks from the Wisconsinan glaciation , which typically consist of ridge segments with major gaps in between. Indeed, many of them may be the meagre remnant or core of originally much higher eskers.It is noteworthy that our eskers are an order of magnitude larger than most eskers in deglaciated areas, which usually do not exceed 50 m in heightFinally, the inferred ramp shape of our eskers has counterparts in the deglaciated landform record, notably eskers of type I–III described by BrennandOur findings have several implications. First, the understanding of ice-shelf channel formation is now improved and more complicated than previously assumed. Three mechanisms have been suggested in other studies: first, ice-shelf channels can develop through meltwater plume flow combined with transverse variability in ice thickness1112691188Second, evidence for channelized, subglacial water outlets supports the hypothesis that the meandering of ice-shelf channels seen in many ice shelves is an archive for the history of the subglacial hydrology in the respective drainage area6Third, large ramp-shaped eskers upstream of the grounding line require stability of the subglacial hydrological system for their development. The Roi Baudouin Ice Shelf is a good candidate for this, because many ice-shelf channels extend from the grounding line to the ice-shelf front along flowlines, indicating temporal stability of the respective source at the grounding line over several hundreds of years. Analysis of an ice rise in that ice shelf suggests an even longer period of stability of the large-scale flow regime spanning several thousands of yearsFourth, the surface ridges and the relict ridge in the ice-shelf channel are key to our interpretation of the ramp-shaped esker formation. Therefore, our data provide field evidence for the evolution of a subglacial landform which is extensively investigated in other areas where ice-sheets have retreatedFifth, the ramp-shaped esker/subglacial conduit system is clearly visible in our ground-based and airborne profiles about two ice thicknesses upstream of the grounding line. Their locations can be inferred from remote-sensing data, by aligning calculated water outlets with ice-shelf channelsIn summary, we have discovered an actively evolving system of ramp-shaped eskers and subglacial conduits upstream of the grounding line of an Antarctic ice shelf. Seawards, ice-shelf channels are likely further incised by a buoyant melt-water plume forced by the meltwater exiting the conduit. The ice-shelf channel amplitudes, however, can already largely be determined by the height of the ramp-shaped esker. The latter develop through a generic process of conduit widening at the grounding line where creep closure is small. Diminished water outflow speed increases potential for sedimentation. Some ramp-shaped eskers are large enough to form corresponding ridges at the surface of the overriding ice. Depending on the height-to-width ratio, these ridges are preserved in the adjacent ice shelf and testify to an evolving subglacial hydrological system including rerouting of conduits and potentially flushing/eroding of the ramp-shaped eskers. Our findings improve the understanding of ice-shelf channel formation by exposing a novel link between subglacial hydrology and ramp-shaped esker formation impacting ice-shelf stability. Moreover, our data set is the first evidence for ramp-shaped esker formation beneath a contemporary ice sheet and sheds light on a subglacial landform that has been intensively studied in geomorphology for decades, but for which the formation mechanisms are only poorly constrained. Further research should be directed to more rigorously infer the existence and the type of sediments deposited by the subglacial conduit , which will facilitate quantitative modelling of sediment transport at the basal boundary of ice sheets.−1 and traces were horizontally stacked to common postings (∼12 m) during the post-processing. After bandpass filtering (frequency range between 3 and 9 MHz), the data were migrated to account for off-angle reflections from sloped interfaces  using Kirchoff-Depth migration implemented in the open-source software Seismic Unix. The required radio-wave velocity model varies with depth using a depth–density parameterization8 m s−1) everywhere. We, therefore, chose the parameters (surface density and densification length) of the depth-density function so that the equivalent firn-air content is below 1 m. Picking of internal reflections and 3D-visualization is done using seismic interpretation software (OpenDtect).Ground-based and airborne radars image the thickness and internal stratigraphy of ice by measuring the traveltimes of reflections from actively emitted electromagnetic waves in the MHz range. Reflections originate from changes in ice density, ice acidity or a preferred orientation of the ice crystalsBandpass filtering/migration causes ringing near the strong reflections originating from the bed and the upper surface of the subglacial conduit. These artefacts obstruct analysis of the wavelet's phase structure, which is therefore done using the raw data only. We find no phase reversal for both reflections types, indicating a transition from an optically less dense (with faster radio-wave velocity) to an optically denser (with a slower radio-wave velocity) background medium. This is consistent with a water and or sediment-filled conduit which is overlain by (less-dense) ice.The kinematic GNSS data were collected with a dual-frequency GNSS receiver attached to the radar's receiver. We estimated the position of the moving station at 1 Hz sampling rate using the GAMIT/GLOBK v10.5 softwareThe satellite-derived surface elevations stem from TanDEM-X, a radar satellite pair imaging the ice-sheet surface using the X-band with small signal penetration into the surface. The elevation model is mosaicked out of 40 TanDEM-X single look complex scenes acquired in austral winter 2013. Scenes were processed individually using the SARscape software. The processing includes coregistration with the CryoSat-2 surface elevationAll data are available from the authors on request.How to cite this article: Drews, R. et al. Actively evolving subglacial conduits and eskers initiate ice shelf channels at an Antarctic grounding line. Nat. Commun.8, 15228 doi: 10.1038/ncomms15228 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Notes, Supplementary Table, Supplementary Figures and Supplementary References


CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors.Recently we reported the discovery of a potent and selective CK2α inhibitor  Although initial hits usually have lower potency than those derived from High-Throughput Screening campaigns (HTS), FBDD is considered to be more efficient in the optimization phases of drug discovery.1.2CK2 is a hetero-tetrameric enzyme composed of two catalytic subunits (α and/or α′) and two regulatory subunits (β and/or β′) which confer stability, control selectivity and enhance enzyme activity.CK2 is involved in multiple intracellular pathways including the regulation of cell proliferation and cell growth. It is also believed to be an apoptosis-suppressor in both healthy and cancer cells.9Among other features, cancer cells show dysregulated proliferation and apoptotic activity leading to uncontrolled cell growth.The most common strategy to inhibit CK2 is through small molecules that target the ATP-binding catalytic site.15Therefore, as with other kinases, increased interest has arisen in the development of inhibitors that do not target the conserved ATP site and demonstrate better selectivity. CK2 inhibition exploiting allosteric binding sites has been achieved using a variety of approaches which target CK2 substrates,21CAM4066). CAM4066 has nanomolar affinity for CK2α and clearly increased selectivity relatively to other known CK2 inhibitors.Recently, we reported the discovery of a new binding pocket, the αD site, within the catalytic CK2α subunit and described the development of a novel class of inhibitors of CK2α utilising this pocket , the partially open conformation (5CVH) where Tyr125 partially fills the pocket and an inactive conformation where the αD pocket is filled by Leu124 (PDB: 5CVG) which distorts the hinge region and prevents ATP binding. To allow the binding of 1 the residue that fills the αD site in the different apo forms  is displaced and Met225 rotates opening the bottom of the αD site. The amine of the fragment forms hydrogen bonds with the backbone carbonyl of Pro159, two waters at the top of the pocket and forms possible π-cation interactions with Phe121. The dichlorophenyl part of the fragment sits deep in the pocket interacting with hydrophobic core of the pocket, exposed when Met225 moves out of the pocket.As reported previously, inst CK2 .22 Three3.21, given that it bound weakly to multiple sites and therefore was of no use to probe the function of one site. The initial plan was to pursue a fragment linking strategy in order to quickly develop inhibitors that would bind at the αD site but inhibit by linking to fragments in the ATP site. This strategy would have 4 phases;1.Fragment optimisation: The aim was to develop fragments with a higher affinity and selectivity for the αD site than 1, as 1 was found binding to a number of different sites and hence thought to be less than ideal as a starting point for the development of specific inhibitor.2.ATP site fragment identification: Warhead fragments bound in the ATP site would be identified using X-ray fragment screening. Ideally, such fragment would interact with the ATP site only weakly so that the affinity for CK2α would be dominated by the affinity for the non-conserved αD site and thus drive selectivity and ATP site warhead would provide only steric hindrance to displace the nucleotide from the kinase. The chosen fragment would also contain a suitable chemical functionality in a correct position for linker attachment.3.Linker optimisation: Fragment linking is seen as the more challenging route of fragment optimisation as introducing a linker that does not interfere with the binding mode of the original fragments is difficult. Therefore, the optimised αD site fragment would be iteratively grown towards the ATP site in an attempt to generate an efficient linker.4.Fragment linking: Finally, the optimised αD site fragment would be linked to the weakly binding ATP site fragment using the knowledge gained from the linker optimisation to give the final chemical tool which will be used to probe the potential of the αD site to develop selective CK2α inhibitors.Prior to conducting any studies to assess the potential of the αD site, a chemical tool to probe the αD site had to be developed from 3.2.11 as it was observed to bind to the ATP site, the interface and the αD site. Therefore, the initial aim was to identify more selective αD binding fragment, at the expense of the ATP and α-β interface sites.Promiscuity was a particularly acute problem with the original fragment 1, identified from the Zinc database,in silico against the αD site. From this screen, a number of commercially available compounds were purchased and co-crystal structures were obtained to determine the binding mode. These compounds explored a range of structures around the initial fragment and included variations in the distance from the hydrophobic core to the amine group as well as changes in the substitution pattern at the 3 and 4 positions . All 5 tructure f. In theK2α_FP10 f, blue, d by ITC  , Lys69 binders  and fragments that interact with both the hinge region and Lys69 . The hinge region is very conserved amongst kinases9) that gave the highest shift was then further investigated in a phosphorylation inhibition assay and its Kd determined via ITC. This showed Compound 9 has a Kd of 58 μM , was screened against CK2α. The screening was performed by soaking the fragments into crystals as cocktails of four fragments. Of these, 23 fragments showed weak electron density in the ATP site. These compounds could be divided into 3 groups, based on their binding mode: hinge binders  and it contained a suitable synthetic handle for linking into αD binding compounds , amine (14), amides (15–17) and sulfonyl amide (18) moieties. The X-ray structure of compound 13 showed the trajectory of the linker was still away from the target region of the ATP site (14–17), the side chain of Met163 changed its conformation, turning into the ATP site 8 and 9 were both co-soaked into CK2α with compound 19. Unfortunately, the crystals containing 8 did not give data with high enough resolution to be able to fit the ligand. On the other hand, crystal structures of 9 with 19 showed that the benzoic acid core fragment at the opposite end of the ATP site did not prevent the movement of Met163 and thus the opening of the channel. The ester of the linker in 19 was slightly displaced compared to the structure without the fragment 9 as the end of the linker bound in close proximity to the fragment . Both compounds contained a seven atom linker as this was the required length established through the optimisation and design process, however, the position of the amide groups within the linkers was varied. In order for compounds 19 and 8 to be linked together to provide the optimal inhibitor, the linker would have to bend by 90° as 8 and 19 are in different planes 1JWH)4DGL)M by ITC a and thi of CK2α b. The IC to CK2α c and d.  to CK2α c. Likewi4CAM4066 has validated the concept of using the newly discovered αD site to achieve selectivity. Furthermore, it showed a case of successfully applying a fragment linking strategy which is known to be particularly challenging. In-silico molecular modelling was a remarkably beneficial tool as, together with X-ray, it provided a means to development a suitable linker to attach the high-affinity molecule lying in the αD pocket to the low-affinity fragment binding in the ATP site. Moreover, this work provided knowledge regarding the new cryptic pocket and the linker channel, showing potential for the development of a new class of CK2 inhibitors.In the present work we give a detailed description of fragment-based inhibitor development that utilized the unforeseen discovery of a novel binding pocket on CK2α. The αD site was discovered by the serendipitous binding of fragments in this new site. These fragments were first optimised to give a selective anchor in αD site that was then linked to a weakly binding fragment in the ATP site which acted as the warhead to provide the inhibition. In order to achieve the super additivity possible from linking fragments an extensive linker optimisation program was pursued to identify the length and composition of the linker. The resulting compound vindicated this strategy of carefully optimising the linker as the affinity of the final compound (320nM) was greater than could be expected from the combination of two fragments with 250 µM and >500 µM affinities. Although further work is required to improve this chemical tool into a lead candidate, the discovery of


This inference was supported by the positive correlation  between the net production of FDOM and the modeled degradation rates of particulate organic carbon sulfate reduction. The production of FDOM was more pronounced in a shallow shelf site S1 with a total net production ranging from 17.9 to 62.3 RU for different FDOM components above the SMTZ depth of ca. 4.1 mbsf, which presumably underwent more accumulation of particulate organic matter than the other three deeper sites. The sediments were generally found to be the sources of CDOM and FDOM to the overlying water column, unearthing a channel of generally bio-refractory and pre-aged DOM to the oceans.Little is known about the production of fluorescent dissolved organic matter (FDOM) in the anoxic oceanic sediments. In this study, sediment pore waters were sampled from four different sites in the Chukchi-East Siberian Seas area to examine the bulk dissolved organic carbon (DOC) and their optical properties. The production of FDOM, coupled with the increase of nutrients, was observed above the sulfate-methane-transition-zone (SMTZ). The presence of FDOM was concurrent with sulfate reduction and increased alkalinity (R A large stock of organic carbon 1100–1500 Pg C), including permafrost, is contained in its drainage basin area, which is sensitive to climate change00 Pg C, ca. 80%) of the organic matter sinking to seafloor is channeled into benthic respiration1718Although the origins of sediment organic matter are diverse including riverine runoff, coastal erosion, sea ice, aeolian input, and autochthonous primary productivity in the Arctic910111314Although there have been a number of DOM studies on Arctic Ocean seawaters and arctic rivers12202122232425910112829Optically active DOM is widely used to trace the dynamics of DOM in aquatic environments. These DOM pools  from terrestrial-derived humic substances in surface waters are generally observed to be bio-refractory while some fresh biomass-derived fractions could be readily utilized1314333435ca. 10.5 ka39The objectives of this study were (1) to characterize the Arctic sediment PW-DOM with different depths (up to 10.5 meters below seafloor (mbsf)) in the Chukchi-East Siberia Sea area along a shelf-slope-basin gradient, (2) to infer the potential origins, diagenetic transformations, and the paleoenvironments associated with the DOM variations, and (3) to explore the potential production of FDOM in the sediments and benthic flux of DOM in the water-sediment interface. The study area is of interest for this topic in that (1) there is a seasonal sea ice cover in the region so it is very sensitive to climate change, (2) it is currently affected by nutrient-rich Pacific water inflow through the Bering Strait so it is easily affected by high primary productivity, which is known to be an important source of organic matter in this region, and (3) the marginal seas are characterized by high sedimentation rates. Sediment cores roughly reflect the geological time spanning up to mid- to late Quaternary deposit based on the age-depth models at sites in close proximity37ca. 33.7 to 36.8 psu among the four sites (2O + SO42− → H2S + 2HCO3−) and anaerobic oxidation of methane (AOM: CH4 + SO42− → HS− + HCO3− + H2O) reactions in the sediments. The rapid changes of both alkalinity and sulfate signify the fast POCSR and AOM reactions which might be due to large amount of decomposable organic matter inputs to the shallow shelf site S1. The sulfate downcore profiles revealed that the sulfate-methane-transition-zone (SMTZ) is about 4.1 mbsf at site S1, while the other sites had not reached SMTZ yet up to the studied depths a(254) and a(350), displayed more obvious trends, especially for site S1, with a general increase followed by a gradual decrease with the core depth. Meanwhile, an apparent inflection point was observed at the shelf site S1, which coincides with the SMTZ depth (ca. 4.1 mbsf) inferred from the sulfate profile.The bulk DOC data for pore waters at sites S1, S2, S3, and S4 exhibited the average concentrations of 6.6 ± 2.4, 4.4 ± 1.9, 2.5 ± 0.8, and 8.4 ± 3.6 mM, respectively. These DOC values were 1–2 orders of magnitude higher than those reported for the surficial sediments (0–10 cm) from the Amundsen and Nansen Basins located in the Eurasian Basin (0.04–0.35 mM)ca. 4.1 mbsf) followed by a gradual decrease to the deeper sediments  were identified from the EEM-PARAFAC modeling . From th1314ediments .in situ marine phytoplankton productivity. This explanation is supported by the low aromaticity proxy *) at the site. The possible causes for the high phytoplankton productivity in this area will be discussed in the following section. The East Siberia Continental Shelf site S3 demonstrated a relatively lower DOC level  among the four studied sites, implying that other factors such as autochthonous production, sea ice cover, and ocean currents rather than riverine runoff could be dominantly operative for the carbon preservation. It is notable that even the labile phytoplankton-derived DOM could be preserved in sediments in the case of a fast sedimentation , in which the fresh organic materials could escape the respiration and preserved in the sediments4p < 0.0001) of a(254) (211 ± 78 m−1) and a(350) (53 ± 18 m−1), as compared to the other sites that displayed an order of magnitude lower values of a(254) (12–23 m−1) and a(350) (3–9 m−1). These results are interesting in that this site is relatively remote from a large river discharge. Similar to the CDOM patterns, the absolute abundances of the fluorescent components showed the highest level at site S1 (p < 0.0001).The average bulk DOC values were 6.6 ± 2.4 mM at the Chukchi Shelf site S1, which may be explained that the shallower shelf area usually traps more terrestrial organic matter, resulting in a higher organic carbon burial42 > 0.95, p < 0.0001) were observed for either CDOM or FDOM with nutrients *, and slope ratio (SR) were utilized here to trace the origins and characteristics of the optically active DOM. Detailed definitions and descriptions of these proxies are found elsewhere46474849org/Norg were reported to be generally <10 in the Chukchi Sea regiona(254)* was relatively low (0.6 ± 0.5 L(mgC-m)−1) and the protein-like C4 a(254)* (0.1 ± 0.1 L(mgC-m)−1) and the high protein-like C4% (72 ± 9%) at site S4. Although the potential linkage of the protein-like component with phenolic compounds was previously reporteda(254)*. Isotopic and biomarker analyses indicated a significant fraction of terrigenous organic matter in the Arctic Ocean910org/Norg) of <10 at nearby sites also supports the dominant source of algal-derived organic matter in the Chukchi Shelf 29The optical indices of humification index (HIX), biological index (BIX), fluorescence index (FI), specific absorption coefficient ca. 4.1 mbsf). For the studied depths at the sites S2, S3, and S4 (3.5 to 10.5 mbsf), the net increases ranged from 0.2 to 4.5 RU for all EEM-PARAFAC components. The FDOM increases with depth should not be due to the diffusive loss to the water column giving the huge differences of depth profiles between the shelf and basin sites. The quantity of the FDOM production was two orders of magnitude higher at site S1 than at the other sites. The shallower shelf site has a much shallower water depth of 100 m in comparison with the other slope and basin sites with much deeper water depths , and they are involved in the two chemical reactions of POCSR and AOM. The POCSR potentially produces DOM. POCSR was reported to dominate organic matter degradation above SMTZ in sediments without seismic vents or faults4+ and PO43−), concurrent with increased alkalinity and reduced sulfate  as well as significant test value of p < 0.0001 for all four EEM-PARAFAC components C1–C4. The correlation remains strong  for most of the components (C1 and C3) at the relatively clustered sites S2, S3, and S4. Our observation is also in line with a previous report on the assimilation of particulate organic carbon by archaea above SMTZ in sedimentsIn this study, the production of FDOM was accompanied by the increases of nutrients of ammonium and phosphate o) showed that most of the sites served as the sources of CDOM and FDOM (primarily for shelf site S1) to the overlying water column  and Bering Shelf Water with a low level of nutrients (NO3− ≤ 1 μM)Sampling sites are located in the shallow Chukchi Shelf (site ARA06C-JPC-1a), Northwind Basin (site ARA06C-JPC-2), East Siberia Continental Slope (site ARA06C-JPC-3), and Chukchi Basin (site ARA06C-JPC-4) of the western Arctic Ocean . For simA stable stratification is generally maintained in Arctic seawater due to a large riverine freshwater discharge and sea ice melting8® high density polyethylene). The pore and bottom water samples for DOM analyses were both immediately stored in a freezer. The samples for the onboard analyses and ion measurements were stored in a refrigerator (about 4 °C).Sediment cores were collected at the sites using both Jumbo Piston Corer (JPC) and Box Corer from August to September 2015, during the RV ARAON Arctic Expedition (ARA06C). The surficial sediment porosity  was calc43− and NH4+ were measured using colorimetric methods using a spectrophotometer (Shimadzu UV-2450) at 885 nm and 640 nm, respectivelySalinity was determined by a Reflectometer, and alkalinity was analyzed by titration using 0.1 N HCl onboard. The reproducibility of alkalinity was <2% based on the repeated analyses of the IAPSO  standard seawater. The POHow to cite this article: Chen, M. et al. Production of fluorescent dissolved organic matter in Arctic Ocean sediments. Sci. Rep.6, 39213; doi: 10.1038/srep39213 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The vast majority of birds were observed at one site only, and the probability of the few detected movements between sites was negatively correlated with the distance among each pair of sites. Hardly any movements were recorded over more than 15 km, suggesting small home ranges. SFI values indicated that juveniles were less site-faithful than adults which may reflect the accumulated knowledge and/or dominance of older animals. Among adults, females were significantly less site faithful than males. A sexual difference in winter site fidelity is unusual in shorebirds. SFI values show site-faithfulness is lower when multiple winters were considered, and most birds seem to chose a wintering site early in the season and use that site throughout the winter. Sanderlings show a very limited tendency to explore alternative wintering options, which might have implications for their survival when facing habitat change or loss .Many migratory bird species show high levels of site fidelity to their wintering sites, which confers advantages due to prior knowledge, but may also limit the ability of the individual to move away from degrading sites or to detect alternative foraging opportunities. Winter site fidelity often varies among age groups, but sexual differences have seldom been recorded in birds. We studied a population of individually colour-marked sanderlings wintering in and around the Tejo estuary, a large estuarine wetland on the western coast of Portugal. For 160 individuals, sighted a total of 1,249 times between November 2009 and March 2013, we calculated the probability that they moved among five distinct wintering sites and how this probability is affected by distance between them. To compare site fidelity among age classes and sexes, as well as within the same winter and over multiple winters, we used a Site Fidelity Index (SFI). Birds were sexed using a discriminant function based on biometrics of a large set of molecularly sexed sanderlings ( Although migratory birds are extremely mobile, they are often remarkably site-faithful to their breeding e.g., , winteriHowever, when confronted with a rapidly changing environment, the regular use of the same set of sites over the years may expose individuals to increasingly poorer conditions and habitats , when a Calidris mauri showed larger home ranges and weaker homing behaviour than adults in winter , and two are located on the oceanic coast near the mouth of the Tejo river , we only sexed adult birds through this method as wing is a key biometric sexing parameter  ≤ 0.75; n = 24) were excluded from the sex-related fidelity analysis. This resulted in 102 sexed birds, 64 males and 38 females.A total of 374 sanderlings were captured and ringed with individual colour-ring combinations in all three sites within the Tejo estuary in five consecutive non-breeding seasons between 2008/9 and 2012/13. Some birds were caught during late August and thus could still have been passage migrants, but since the re-sighting data refers only to the wintering period, this will not affect our winter site fidelity analysis. At capture, all birds were aged based on plumage characteristics, being classified as either first winter (hereafter referred as juveniles) or adults. Biometric data  were also collected for most individuals to determine their sex. Sex of measured birds was determined based on a function derived from a generalised linear model (GLM) of biometric data from 990 molecularly sexed sanderlings captured in Mauritania and Ghana in a concurrent study see , which ieter see . The GLMeter see , but to From 2009/10 to 2012/13, during the sanderling wintering period (November–March), the study sites were visited frequently by us and many volunteer observers and the presence and identity of colour-marked birds was recorded. A total of 317 marked individuals were sighted, including 302 birds ringed locally and another 15 marked elsewhere . The full dataset included 2,358 sightings. Since the dataset includes many sightings obtained during haphazard visits of volunteers , we do not know whether the searching effort was similar for all sites, because volunteer observers will not have contacted us when they did not find a colour-ringed sanderling during their visits. We also suspect that the sites outside the estuary are likely to have been visited less often than the sites located within the estuary. For the same reason, sightings are not evenly spread over each winter and over different winters .In order to minimize the risk of having incorrect readings affecting our analyses, we limited our analysis to include birds that had been sighted a minimum of three times in a given area/year which resulted in 160 individuals (36 juveniles and 124 adults) for which we had a total of 1249 sightings . For 101ni is the number of sites used by individual i, n is the total number of sites surveyed, pi is the observed number of changes between sites performed by individual i and oi is the total number of sightings of individual i. SFI ranges from zero (no site fidelity) to one (complete site fidelity). For each individual, SFI values were calculated within each winter  and for all winters combined .In order to test differences in site fidelity between sexes and age classes, as well as differences in the site fidelity within a single winter and over multiple winters we calculated a site fidelity index (SFI) described by To investigate sex differences in site fidelity, we compared both intra-annual and inter-annual SFI values for males and females. For birds with data for multiple winters we used the average intra-annual SFI, and in both cases we used Mann–Whitney tests to compare SFI scores. Two approaches were used to compare site fidelity among age classes. One compared the intra-annual SFI values for juvenile and adult birds for the winter when each individual was ringed/aged, through a Mann–Whitney test; the other used only birds ringed as juveniles and sighted over multiple winters, in which case we made a pair-wise comparison of intra-annual SFI values calculated for each juvenile in their first winter and in the subsequent winter (when adult) with the most sightings of that particular individual, using a Wilcoxon matched pairs test.Finally, in order to compare site fidelity within the same winter and over multiple winters, we made a pair-wise comparison of intra and inter-annual SFI values for all adult birds sighted over multiple years using a Wilcoxon matched pairs test. There could be some biases when comparing inter-annual SFI of individuals with different numbers of sightings in each winter, but this problem would only affect comparisons among individuals and not pair-wise comparisons for each individual. Note that birds ringed as juveniles were excluded from this analysis because the tests described above showed that sanderlings are less site faithful in their first winter (see below). Data are presented as average±SE.n = 4 winters with an average 73 ± 25 individuals/winter) were always sighted at the same site within a given winter (n = 65) for 2 winters, 67.9% (n = 28) for 3 winters and 62.5% (n = 8) for 4 winters. Bird movements were more likely to occur between sites in close proximity, namely between Alcochete and Samouco, between Samouco and Seixal and between Seixal and Caparica  and over multiple winters . In fact, there was only a single case of dispersal between sites located at a distance of over 20 km. No marked birds were ever detected at Oeiras even though the site was frequently used by non-marked sanderlings.In fact, the GLMMs indicated that the probability of movement was negatively affected by distance, with lower probabilities of dispersal between sites further away both within one winter , and which disperses over a huge latitudinal range during the winter , while Caparica which is just 14.5 km away from the same site was used by 6 marked individuals. This suggests that 15–20 km is indeed the maximum distance sanderlings are likely to move among wintering sites under stable conditions. As is the case for most studies focusing on animal dispersal , our stuStill, even when considering these biases and limitations, there is a clear pattern of site fidelity and decreasing chance of movements over increasing distances. The few birds ever detected outside the estuary originated from the sites nearer the mouth of the estuary , and never from the site located the furthest (Alcochete) even though this was the site with the largest number of individual birds ringed and sighted. The few sightings elsewhere along the Portuguese coast of sanderlings that were colour-marked in other countries than Portugal, suggest similar site fidelity: from 37 sightings of 12 individuals , each observed in a single winter, only one was observed at two locations which were separated by less than 4 km (Póvoa de Varzim 41°23′N/8°46′W and Vila do Conde 41°21′N/8°45W).Altogether, our data suggest that sanderlings in and around the Tejo estuary rarely move among wintering sites located at distances of more than 15 km. Such a level of site fidelity during winter is likely to confer advantages related to previous knowledge of foraging locations, potential predation risks, and social interactions with other individuals using the same areas e.g., , but mayEnsis americanus on the shore , suggesting that sanderlings are able to react to the availability of new foraging opportunities. Also, in the Solent estuary, UK, marked sanderlings seem to move regularly among feeding sites located 15–20 km apart . There is no evidence that any of the three areas we studied within the Tejo estuary suffered significant changes during the course of this study, so we cannot predict whether the birds would move if there was any decline in habitat quality, but these anecdotal observation from The Netherlands and UK suggest that they are able to move to a larger extent than we observed in the Tejo. However, at Caparica, the beaches are known to be suffering from severe coastal erosion and are estimated to be retreating inland at an average rate of 7 m year−1 as a result of river damming and dredging, inadequate coastal management and urban pressure, and sea-level rise (In the Netherlands, individually recognisable sanderlings have been observed to move over tens of kilometres in response to a sudden increase in food availability generated by a storm washing up large amounts of American Jack knife clam vel rise . These bvel rise  which cavel rise . In factvel rise . Despitevel rise .10.7717/peerj.2517/supp-1Data S1Click here for additional data file.10.7717/peerj.2517/supp-2Appendix S1Click here for additional data file.


TLC) produces cholestasis by inhibiting biliary solute secretion in part by retrieving MRP2 from the plasma membrane (PM). Tauroursodeoxycholate (TUDC) and cAMP reverse TLC‐induced cholestasis by inhibiting TLC‐induced retrieval of MRP2. However, cellular mechanisms for this reversal are incompletely understood. Recently, we reported that TLC decreases PM‐MRP2 by activating PKCε followed by phosphorylation of myristoylated alanine‐rich C kinase substrate (MARCKS). Thus, cAMP and TUDC may reverse TLC‐induced cholestasis by inhibiting the TLC/PKCε/MARCKS phosphorylation pathway. We tested this hypothesis by determining whether TUDC and/or cAMP inhibit TLC‐induced activation of PKCε and phosphorylation of MARCKS. Studies were conducted in HuH‐NTCP cell line and rat hepatocytes. Activation of PKCε was determined from the translocation of PKCε to PM using a biotinylation method. Phosphorylation of MARCKS was determined by immunoblotting with a phospho‐MARCKS antibody. TLC, but not cAMP and TUDC, activated PKCε and increased MARCKS phosphorylation in HuH‐NTCP as well in rat hepatocytes. Treatment with TUDC or cAMP inhibited TLC‐induced activation of PKCε and increases in MARCKS phosphorylation in both cell types. Based on these results, we conclude that the reversal of TLC‐induced cholestasis by cAMP and TUDC involves, at least in part, inhibition of TLC‐mediated activation of the PKCε/MARCKS phosphorylation pathway.Taurolithocholate ( Results of our studies in both cell types are consistent with the proposed hypothesis.ε), Calbiochem (actin), and BD Transduction Laboratories (E‐Cadherin). These antibodies react with both human and rat proteins. Sulfosuccinimidyl‐6‐(biotin‐amido)‐hexanoate (Sulfo‐NHS‐LC‐Biotin) was purchased from Pierce . Streptavidin beads were purchased from Novagen . HuH‐NTCP cells (HuH7 cells stably transfected with human NTCP) were generously provided by Gores ‐cAMP (CPT‐cAMP), TUDC, and TLC were purchased from Sigma‐Aldrich . Commercial sources of other antibodies were Cell Signaling Technology  and cultured as described previously , and the relative band densities were quantitated using ImageJ® from NIH. All values were expressed as mean ± SE. Analysis of variance followed by Fisher's least significant difference (LSD) test was used to statistically analyze the data, with P < 0.05 considered significant.Phosphorylation of MARCKS was determined using phospho‐MARCKS (Ser152/156) antibody as described previously (Schonhoff et al. ε in HuH‐NTCP and rat hepatocytes. We determined the effect of cAMP and TUDC on PM‐PKCε for two reasons. First, translocation to membranes is a readout for activation of conventional and novel PKCs (Reyland ε (Beuers et al. ε to PM has not been reported. Thus, TUDC was also included in this study.TUDC and cAMP have been shown to prevent TLC‐induced decreases in PM‐MRP2 (Beuers et al.  Reyland . Second, Reyland . Althougε. This result is consistent with previous studies in rat hepatocytes (Beuers et al. ε. Similar effects were observed in HuH‐NTCP cells (Fig. ε significantly compared to the control. Neither cAMP nor TUDC affected the basal level of PM‐PKCε. However, TLC‐induced increases in PM‐PKCε were inhibited by cAMP as well as TUDC (Fig. ε.Our studies in rat hepatocytes showed that, TLC, but not cAMP or TUDC Fig. , increaslls Fig. . TLC eleUDC Fig. . These rε followed by phosphorylation of MARCKS (Schonhoff et al. We next determined whether cAMP and TUDC also inhibit TLC‐induced increases in MARCKs phosphorylation in HuH‐NTCP cells and rat hepatocytes, since TLC decreases PM‐MRP2 by activating PKCε. Previous studies showed that cAMP and TUDC reversed TLC‐induced retrieval of PM‐MRP2 (Beuers et al. ε followed by MARCKS phosphorylation (Schonhoff et al. ε and MARCKS phosphorylation in both hepatocellular carcinoma cell line and primary hepatocytes. These results are consistent with the hypothesis that cAMP and TUDC can reverse TLC‐induced PM‐MRP2 retrieval by inhibiting TLC/PKCε/MARCKS phosphorylation pathway.The aim of this study was to test the hypothesis that cAMP and TUDC reverse TLC‐induced retrieval of PM‐MRP2 by inhibiting TLC‐mediated phosphorylation of MARCKS via PKCα (Beuers et al. α as well as inhibiting PKCε/MARCKS phosphorylation. It may be noted that PKCα has also been shown to induce cholestasis (Kubitz et al. ε/MARCKS phosphorylation may raise the possibility that TUDC may act via cAMP. This, however, seems unlikely since TUDC does not increase the intracellular level of cAMP (Wimmer et al. ε/MARCKS phosphorylation. Mechanisms by which TLC activate PKCε, and cAMP and TUDC inhibit this activation remains to be determined.The present study provides further insights into the mechanism of reversal of TLC‐induced cholestasis. A previous study suggested that the anticholestatic effect of TUDC against TLC‐induced cholestasis may involve activation of PKCReversal of cholestasis may also involve other mechanisms. It is believed that PM localization of a transporter at any given time is the result of a net balance between the rate of exocytic insertion and endocytic retrieval (Marinelli et al. ε and MARCKS phosphorylation in human‐derived hepatic cell line as well as in rat hepatocyte. It is concluded that cAMP and TUDC reverse TLC‐induced cholestasis by inhibiting the TLC/PKCε/MARCKS phosphorylation/PM‐MRP2 retrieval pathway.In summary, the present study shows for the first time that cAMP and TUDC inhibit TLC‐induced activation of PKCThis study does not include discussion of off‐label/investigational use or application of a product or device.


The delivery of five stimuli to each cranial site is recommended during transcranial magnetic stimulation (TMS) mapping. However, this time-consuming practice restricts the use of TMS mapping beyond the research environment. While reducing the number of stimuli administered to each cranial site may improve efficiency and decrease physiological demand, doing so may also compromise the procedure's validity. Therefore, the aim of this study was to determine the minimum number of stimuli per cranial site required to obtain valid outcomes during TMS mapping. Map volume and centre of gravity (CoG) recordings obtained using five stimuli per cranial site were retrospectively compared to those obtained using one, two, three, and four stimuli per cranial site. For CoG longitude, one stimulus per cranial site produced valid recordings . However, this outcome is rarely explored in isolation. As two stimuli per cranial site were required to obtain valid CoG latitude  and map volume  recordings, it is recommended that a minimum of two stimuli be delivered to each cranial site during TMS mapping in order to obtain valid outcomes. Transcranial magnetic stimulation (TMS) has been used extensively as a noninvasive tool to explore corticomotor physiology , 2. DuriDuring nonnavigated TMS mapping, five stimuli are commonly delivered to each site on a 10 × 10 cm grid positioned over the participant's scalp . When usThere exists a small amount of literature investigating the validity of TMS mapping protocols with varying numbers of stimuli per cranial site. For instance, it has been demonstrated that five stimuli per cranial site are sufficient to obtain centre of gravity recordings within 2 mm of those obtained when using 20 stimuli per cranial site . While tWhile reducing the number of stimuli administered to each cranial site during TMS mapping has the potential to improve efficiency and decrease physiological demand, such changes may also compromise the procedure's validity. Despite these considerations, the optimal number of stimuli for use during TMS mapping remains unclear. Therefore, the aim of this study was to determine the minimum number of stimuli per cranial site required to achieve valid outcomes during TMS mapping.This study utilised a retrospective analysis to assess the validity of TMS mapping protocols involving varying numbers of stimuli per cranial site. Participant-level data were retrieved from two previously conducted studies , 14. MapMethods were reported in accordance with the TMS-specific checklist developed by Chipchase et al.  Centre of gravity latitude (CoG lat) coordinates (cm away from vertex)Centre of gravity longitude (CoG long) coordinates (cm away from vertex)The following indices of corticospinal plasticity, measured by TMS, were analysed:Both previous studies employed protocols involving the delivery of five stimuli per cranial site. The same person performed TMS mapping on all participants. All data were obtained from single-pulse, monophasic stimuli delivered using a Magstim 200 stimulator  and a figure-of-eight coil. Neuronavigation was not used in any of the TMS mapping sessions. A five-second interstimulus interval was utilised. The magnetic coil was oriented at a 45° angle in all trials to preferentially induce current in a posterior-to-anterior direction in the cortex .μV) within the target muscle [All assessments were performed under resting conditions. The optimal cortical site (“hotspot”) was determined in both studies by identifying the coil position that evoked a maximal peak-to-peak motor evoked potential (MEP). Resting motor threshold (rMT) was defined as the minimum stimulator intensity at which fifty percent of stimuli, applied at the optimal scalp site, evoked a response of at least 0.05 mV , that site was considered “active” [zixi/Σzi; Σziyi/Σzi  [Peak-to-peak MEP amplitudes were initially analysed in the blocks of five stimuli administered to each cranial site during TMS mapping. This was performed using MATLAB 7  . If the “active” , 20. Act“active” . The cenplitude) . This prThis study used TMS mapping involving the delivery of five stimuli per cranial site as the reference standard. Outcomes obtained using five stimuli per cranial site were therefore considered to be the “true” values. For a mapping protocol involving fewer than five stimuli per cranial site to be considered valid, both analyses of variance  and intraclass correlation coefficients had to reveal no significant differences between outcomes obtained using that protocol and those obtained using the true value. This strict definition of validity was employed so that recommendations would not be influenced by subjective interpretations of “acceptable” levels of variability. Similar definitions have also been utilised in previous TMS studies , 22, 23.Assumptions of normality and sphericity  for parametric analyses were assessed using the Shapiro-Wilk test and Mauchly's test of sphericity, respectively . The Grep < 0.05, but a Bonferroni correction was applied during all post hoc tests to compensate for multiple comparisons, resulting in a significance level of <0.0125 for these tests.As map volume data failed normality testing, nonparametric analyses, in the form of Friedman's tests and post hoc Sign tests, were employed to compare map volume recordings obtained using five stimuli per cranial site and those obtained using one, two, three, and four stimuli per cranial site . As CoG Absolute intraclass correlation coefficients (ICCs) between recordings obtained using five stimuli per cranial site and those obtained using one, two, three, and four stimuli per cranial site were calculated for each outcome. Intraclass correlation coefficients, including their confidence intervals, were interpreted using the following values: less than 0.50 = poor; 0.50 to 0.65 = moderate; 0.65 to 0.80 = good; and greater than 0.80 = excellent . Thus, fχ2(4) = 27.88, p < 0.001). Post hoc testing  revealed that map volume was smaller when five stimuli were delivered than when one stimulus was delivered . There was no difference in map volume between five stimuli per cranial site and two , three , or four  stimuli per site, suggesting that a minimum of two stimuli per cranial site was required to achieve valid map volume recordings (significance level set at p < 0.0125). This was supported by ICC analyses, which revealed that a minimum of two stimuli per cranial site was required for the entire 95% confidence interval surrounding the ICC to be greater than 0.80 (see t(33) = −10.149, p < 0.001). Median (IQR) map volume recordings for one, two, three, four, and five stimuli per cranial site are shown in 0.80 see . RecordiF = 5.15, p = 0.02). Post hoc testing  revealed that CoG latitude was smaller when five stimuli were delivered than when one stimulus was delivered  = 8.23, p = 0.01). There was no difference in CoG latitude between five stimuli per cranial site and two  = 0.71, p = 0.41), three  = 1.90, p = 0.18), or four  = 1.66, p = 0.21) stimuli per site, suggesting that a minimum of two stimuli per cranial site was required to achieve valid CoG latitude recordings (significance level set at p < 0.0125). Intraclass correlation coefficient analyses showed that all of the varying numbers of stimuli produced valid CoG latitude recordings . Therefore, two stimuli per cranial site was the minimum number required to satisfy both of the requirements for valid CoG latitude recordings .Mean (SD) CoG latitude recordings obtained using one, two, three, four, and five stimuli per cranial site are shown in F = 1.92, p = 0.15), suggesting that one, two, three, and four stimuli per cranial site all produced valid CoG longitude recordings. This finding was supported by the ICC analyses presented in There were no differences in CoG longitude across the varying numbers of stimuli per cranial site  recordings during TMS mapping. For CoG longitude, one stimulus per cranial site was sufficient to produce valid recordings. However, a minimum of two stimuli per cranial site was required to achieve valid recordings for both CoG latitude and map volume. As CoG longitude is rarely explored in isolation and is typically measured in conjunction with CoG latitude, the results of this study indicate that a minimum of two stimuli can be delivered to each cranial site during TMS mapping in order to achieve valid outcomes when measuring map volume and organisation.Transcranial magnetic stimulation mapping is a common technique used to explore corticomotor adaptation , 2. HoweThe results of the present study support anecdotal observations that the delivery of five stimuli per cranial site during traditional TMS mapping is inefficient and unnecessary . By implIn this study, map volume recordings obtained using one stimulus per cranial site were larger than those obtained using five stimuli per cranial site. Such findings may be indicative of habituation to repeated stimulation. This decrease in the responsiveness of corticospinal neurons with repeated stimulation is thought to be due to depression of excitatory synaptic activity or an increase in the inhibition of excitatory interneurons . HoweverDespite a rigorous approach towards data collection and synthesis, this study is not without limitations. The exclusion of clinical populations means that the results of this study are only applicable to healthy participants. Likewise, as this study was limited to upper limb musculature, the results may not be generalised to muscles of the lower limb or spine, which have deeper representations within the motor cortex . As neurThe findings of this study are also only applicable to “traditional,” systematic TMS approaches. Alternate mapping techniques, involving unevenly spaced stimulation grids and “pseudorandom,” rather than systematic, site stimulation have been shown to be reliable when only one stimulus is delivered to each target site , 40. HowAlthough this study highlights the potential for improving the efficiency of TMS mapping procedures, further research is required. Exploration of lower limb and spinal musculature, including studies involving active rather than resting conditions, is required to determine the optimal number of stimuli per cranial site during TMS mapping involving these regions. A greater emphasis should also be placed upon clinical populations in order to determine if the results of this study are generalizable beyond healthy individuals. Future research should also seek to validate the present study's findings using alternate mapping protocols and grid configurations. For example, while a denser stimulation grid (less than one cm spacing) would increase the number of target sites over a particular area, it could reduce the need for repeated stimulation at those sites and potentially improve the overall accuracy of the motor maps. Similarly, alternate analysis techniques may also influence the optimal number of stimuli per cranial site. Various interpolation and smoothing techniques have been employed during TMS mapping in order to account for within-subject fluctuations in MEP amplitudes and reduce the need for evenly spaced stimulation grids . Future The results of this study indicate that valid map volume and CoG recordings can be obtained using a minimum of two stimuli per cranial site during TMS mapping of the upper limb. Such findings have the potential to greatly reduce data acquisition times and participant discomfort. Future research should seek to determine if these findings can be applied to clinical populations and lower limb or spinal musculature.


Leucine rich repeat kinase 2 is a complex enzyme with both kinase and GTPase activities, closely linked to the pathogenesis of several human disorders including Parkinson’s disease, Crohn’s disease, leprosy and cancer. LRRK2 has been implicated in numerous cellular processes; however its physiological function remains unclear. Recent reports suggest that LRRK2 can act to regulate the cellular catabolic process of macroautophagy, although the precise mechanism whereby this occurs has not been identified. To investigate the signalling events through which LRRK2 acts to influence macroautophagy, the mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) and Beclin-1/phosphatidylinositol 3-kinase (PI3K) pathways were evaluated in astrocytic cell models in the presence and absence of LRRK2 kinase inhibitors. Chemical inhibition of LRRK2 kinase activity resulted in the stimulation of macroautophagy in a non-canonical fashion, independent of mTOR and ULK1, but dependent upon the activation of Beclin 1-containing class III PI3-kinase. Substantia nigra pars compacta. Coding mutations in the LRRK2 gene are the most frequent genetic cause of familial PD, with polymorphisms in LRRK2 associated with an increased risk of idiopathic PD123LRRK2 locus as being involved in the risk for PD7LRRK2 region was identified as being subject to frequent carcinogenic alterationsLRRK2 gene is therefore related to the etiopathogenesis of at least four human diseases, making it the focus of increasing attention as a putative drug target.Leucine rich repeat kinase 2 is one of the key genetic factors contributing to the risk of developing Parkinson’s disease (PD), an irreversible, progressive neurodegenerative movement disorder primarily associated with neuronal cell loss in the The physiological function of LRRK2 is as yet unclear. It is a complex enzyme, with active kinase and GTPase domains that are thought to reciprocally regulate one another’s activity11151718202527The data presented herein demonstrate that the kinase activity of LRRK2 acts as a negative regulator of macroautophagy in astrocyte cell models. Our results suggest that LRRK2 may act to control a non-canonical pathway alternative and parallel to that regulated by the mammalian target of rapamycin (mTOR) and Unc-51 Like Kinase 1 (ULK1), but dependent on the presence of an active Beclin-1 complex. These data have important implications for the study of the physiological and pathological functions of LRRK2, in particular for any pharmacological intervention based upon LRRK2 inhibition.LRRK2 is expressed at high levels in astrocytes within the brain3018We have previously suggested that the increased levels of LC3-II after LRRK2 kinase inhibition were consequence of an increase in autophagosome production rather than a decrease in degradation. We here corroborate this data by improving the experimental setting including Torin-1 control, evaluating two distinct time-points of treatment, assessing the drug Chloroquine alongside with Bafilomycin (BafA1) and performing co-localization analysis of LAMP1 and p62. As first, H4 cells were co-treated with LRRK2 kinase inhibitor and BafA1 or Chloroquine. Results showed an additive effect of LRRK2in1 over BafA1 (or Chloroquine) treatment alone  that app34In our previous workIn order to assess whether ULK1 is required for LRRK2-induced macroautophagy, ULK1 protein levels were stably reduced by 70% in H4 cells transfected with ULK1 shRNA. 150 minute treatment of LRRK2in1 led to a comparable increase of LC3-II production in stable ULK1 knock-down as compared to scrambled control cells. These data indicate that ULK1 is dispensable for macroautophagy induced by LRRK2 kinase activity inhibition .Co-treatment with LRRK2in1 and Torin-1 resulted in a stronger increase of LC3-II levels, as compared to single treatments with either LRRK2in1 or Torin-1 . This daIn addition to the ULK-kinase complex, canonical macroautophagy induction requires the class III PI3-kinase complex containing Beclin-1Finally, in order to assess whether Beclin-1 is required for LRRK2-induced macroautophagy, Beclin-1 protein levels were stably reduced in H4 cells transfected with Beclin-1 shRNA(s). Knock-down of Beclin-1 increased the basal level of LC3-II and this was not further increased after 150 minutes treatment with LRRK2-in 1, in contrast to scrambled shRNA controls which showed an enhancement of LC3-II compared to basal levels . This daDespite its key role in the etiology of a number of human diseases, there is as yet no consensus regarding the physiological function of LRRK2; leading to the suggestion that LRRK2 may have contrasting roles depending on the cell type and condition under investigationThe complex events regulating macroautophagy are yet to be fully characterised414344In the current study, pharmacological inhibition of LRRK2 kinase activity resulted in an increase of LC3 processing consistent with induction of macroautophagy. Inhibition of mTOR using Torin-1 causes a complete loss of ULK1 phosphorylation on Ser758 and this loss of phosphorylation on ULK1 is required for translocation of the ULK1/2 complex to the pre-autophagosomal structure535456The LRRK2in1 compound and the VPS34in1 were purchased from the Division of Signal Transduction Therapy, School of Life Sciences, and University of Dundee, UK. The GSK2578215A compound was purchased from Tocris. Bafilomycin A1 (B1793-2UG), Chloroquine (C6628) and Wortmannin (W3144-250UL) were purchased from Sigma-Aldrich. Torin-1 (CAY10997) was purchased from Cayman Chemicals.Antibodies used were as follows: LC3 antibody ; LRRK2 antibodies ; total S6 antibody ; phospho Ser235/236 S6 antibody ; total P70S6K antibody ; phospho Thr389 P70S6K ; total ULK1 antibody ; phospho Ser757 ULK1 antibody ; Beclin-1 antibody ; β-actin antibody ; β-tubulin .H4 cells (ATCC number HTB-148) were grown in DMEM containing 10% FCS. After 24 hours from plating, when at 80% confluence, H4 cells were treated with LRRK2 inhibitors LRRK2in1 or GSK2578215A, with BafA1 or Torin-1 or wortmannin at the concentrations and for the time reported in each experiment. For each experiment, DMSO vehicle controls were added. After washing in Dulbecco’s phosphate buffered saline (DPBS) cells were collected in a lysis buffer containing: 0.5% Triton X-100, 2 mM ethylen di-ammonium tetra acetic acid (EDTA), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), protease inhibitors  and phosphatase inhibitors  in 50 mM TRIS-HCl pH 7.5.et al.6 cells/well), and at 80% confluency the cells were treated with LRRK2in1 or DMSO (control) as reported in figures. Following treatment, the cells were washed in DPBS, collected by scraping and cell pellets were frozen until Western blot analysis.Primary astrocyte cultures were prepared from P3-4 mouse forebrain following a protocol adapted from Schildge http://rsbweb.nih.gov/ij/).Cell lysates were frozen immediately upon collection or kept at 4 °C for 30 minutes; following thawing, they were clarified by centrifugation at 13000 rpm for 5 minutes at 4 °C, protein concentration was assessed by BCA assay  and 10–15 μg aliquots were added of NuPAGE sample buffer containing 2-mercaptoethanol (Invitrogen), denatured for 10 minutes at 70 °C and analysed using NuPAGE, Novex precasted Bis-Tris 4–12% (Invitrogen), in MES running buffer (Invitrogen). After electrophoresis, gels were blotted onto 0.45 μm cut-off, PVDF through conventional blotting. Membranes were blocked and processed with peroxidase-conjugated antibodies for enhanced chemiluminescence (ECL) detection. Films were acquired as images in jpg format using an EPSON Perfection 4870 photo scanner and processed by the ImageJ software ( All the results have been repeated in at least 3 independent experiments (details are given in each figure legend). In the case of Western blot analysis for phospho-proteins, samples were divided in half and run in two parallel gels; one was processed for the phospho-antibody and one for the total-antibody (to avoid membrane stripping and re-probing). The total and the phospho bands were normalized against the respective β-actin loading control; then, the ratio of phospho over total protein was calculated. Statistical analyses were performed by the use of the Prism software (GraphPad) as described in each figure legend.H4 cells were transfected with 2 to 10 μg LRRK2 shRNA, ULK1 shRNA, Beclin-1 shRNA or scramble Open Biosystems GIPZ shRNAmir  using PEI (Sigma-Aldrich) transfection reagent according to the manufacturer’s instructions. ShRNA vectors contain a puromycin resistance gene. Cells were treated with 2 μg/ml puromycin supplemented DMEM 48 hrs after transfection and kept under selection for expansion. Puromycin selection was removed 24 hours before the experiment to avoid interference of the antibiotic with the treatment.How to cite this article: Manzoni, C. et al. mTOR independent regulation of macroautophagy by Leucine Rich Repeat Kinase 2 via Beclin-1. Sci. Rep.6, 35106; doi: 10.1038/srep35106 (2016).


Activation was achieved by rapidly increasing the temperature (temperature‐jump of 0.5–20ºC) of permeabilized trabeculae over a physiological range of sarcomere lengths (1.85–1.94 μm). The trabeculae were subjected to shortening ramps over a range of velocities and the extent of RLC phosphorylation was varied. The latter was achieved using an RLC‐exchange technique, which avoids changes in the phosphorylation level of other proteins. The results show that increasing RLC phosphorylation by 50% accelerates ktr by ∼50%, irrespective of the sarcomere length, whereas decreasing phosphorylation by 30% slows ktr by ∼50%, relative to the ktr obtained for in vivo phosphorylation. Clearly, phosphorylation affects the magnitude of ktr following step shortening or ramp shortening. Using a two‐state model, we explore the effect of RLC phosphorylation on the kinetics of force development, which proposes that phosphorylation affects the kinetics of both attachment and detachment of cross‐bridges. In summary, RLC phosphorylation affects the rate and extent of force redevelopment. These findings were obtained in maximally activated muscle at saturating [Ca2+] and are not explained by changes in the Ca2+‐sensitivity of acto‐myosin interactions. The length‐dependence of the rate of force redevelopment, together with the modulation by the state of RLC phosphorylation, suggests that these effects play a role in the Frank–Starling law of the heart.Force and power in cardiac muscle have a known dependence on phosphorylation of the myosin‐associated regulatory light chain (RLC). We explore the effect of RLC phosphorylation on the ability of cardiac preparations to redevelop force ( ktr) at submaximal [Ca2+].Regulatory light chain (RLC) phosphorylation has been shown to alter the ability of muscle to produce force and power during shortening and to alter the rate of force redevelopment (in vivo level in maximally [Ca2+]‐activated cardiac trabecula accelerates ktr.Increasing RLC phosphorylation ∼50% from the in vivo control level slows ktr and reduces force generation.Decreasing RLC phosphorylation to ∼70% of the ktr is dependent on sarcomere length in the physiological range 1.85–1.94 μm and RLC phosphorylation modulates this response.2+] activation and therefore does not necessarily require length‐dependent change in [Ca2+]‐sensitivity of thin filament activation.We demonstrate that Frank–Starling is evident at maximal [CaThe stretch response is modulated by changes in RLC phosphorylation, pinpointing RLC phosphorylation as a modulator of the Frank–Starling law in the heart.These data provide an explanation for slowed systolic function in the intact heart in response to RLC phosphorylation reduction. CFconversion factorDRduty ratiof−1)rate constant of cross‐bridge attachment F1.85–2)force of a maximally activated trabecula at a sarcomere length of 1.85 μm (kN mF1.90–2)force of a maximally activated trabecula at a sarcomere length of 1.90 μm (kN mF1.94–2)force of a maximally activated trabecula at a sarcomere length of 1.94 μm (kN mFiso–2)isometric force (kN m–1FL s–1)trabecula shortening velocity (trabecula lengths sFramp–2)force sustained during a shortening ramp at a steady velocity (kN mFrec–2)force recovered in isometric conditions after the end of protocol (kN mg−1)rate constant of cross‐bridge detachment (sk1.85ktr), at a sarcomere length of 1.85 μm (s−1)rate of force redevelopment (k1.90ktr), at a sarcomere length of 1.90 μm (s−1)rate of force redevelopment (k1.94ktr), at a sarcomere length of 1.94 μm (s−1)rate of force redevelopment (ktr–1)rate of force redevelopment (sNnumber available cross‐bridges for active cyclingRLCregulatory light chainSLsarcomere length (μm)2+ to bind the thin filament regulatory complex (troponin complex). This ‘on/off’ mechanism in striated muscle determines the availability of myosin binding sites on actin (thin filament activation)  (not created by stretch activation)  in cardiac tissue. Force redevelopment was described in 1957 by Huxley using a simple two‐state model, which described ktr as the sum of the forward (f), and backward (g) rate constants for the transition into (and out of) the load bearing acto‐myosin states, respectively  is given by:F is the force per cross‐bridge, N is the total number of cross‐bridges in the filament overlap region and f/(f + g) is the proportion of cross‐bridges in force generating states.Contraction in striated muscle is driven by interactions between actin and myosin, and is controlled by the availability of free Caf and g are complex terms, dependent on transitions between multiple biochemical intermediates   (2.2–2.3 μm), in the presence of either limiting or maximal [Ca2+], with conflicting results   Act 1986.ad libitum) weighing 250–350 g were anaesthetized using isofluorane inhalation, until unconscious. Rapid cervical dislocation was followed by removal of the heart by abdominal and subsequent thoracic resection. Hearts were excised with intact aortic arches for cannulation and retrograde perfusion with oxygenated ice‐cold Krebs–Henseleit solution . Left ventricular trabeculae (1–2 mm in length and 100–300 μm in width) were excised from the resected left ventricle. Trabeculae ends were then crimped in T‐clips and pinned at resting length on Sylgard for chemical permeabilization using relaxing solution  for 30 min at 5°C, incorporating 2% Triton X‐100. Trabeculae were stored post‐permeabilization at –20°C in 50% relaxing solution with 50% glycerol and protease inhibitors . Trabeculae were stored for no longer that 3 days prior to experimentation.Female Sprague–Dawley rats  were exchanged for native RLC in permeabilized trabeculae using an exchange buffer . Exchange was performed with 0.6 mg ml–1 RLC on the experimental set‐up at a sarcomere length of 2.1 μm at 0.5°C for 45 min. The extent of RLC phosphorylation in the trabeculae was calculated from the measured time course of RLC exchange  compared to native where the extent of phosphorylation is 0.44 mol Pi mol–1 RLC, reduced phosphorylation of 0.7 ± 0.05 (∼0.31 mol Pi mol–1 RLC) and control exchange mimicking native RLC phosphorylation levels of 1.1 ± 0.1  and low (0.5°C) temperatures to activate and relax muscle contraction, respectively  reduction in trabecula length, applied during the plateau of isometric force development at 20°C, starting at a sarcomere length of 2.1 μm. The speed and amplitude of the applied release is such that the trabecula becomes momentarily slack. Following the release, force re‐develops. Force re‐development is characterized in terms of the amplitude of force regained and the rate constant describing the speed of force redevelopment.et al. The second protocol, ‘release‐ramp’, consists of a small and fast step release, followed by a slower decrease in trabecula length at a steady shortening velocity at 20°C. The step release unloads the instantaneous elasticity of the preparations and the ramp shortening allows the trabecula to reach a steady tension, which is characteristic for the ramp velocity, thus defining the force–velocity relationship. The effects of RLC phosphorylation on the force–velocity relationship have been reported previously , –10% (1.90 μm) and –12% (1.85 μm) of the initial sarcomere length (2.1 μm) during activation. Isometric force redeveloped at each nominal sarcomere length, with an amplitude denoted as Frec is the steady‐state force recovered post protocol, F is the force at time t and ktr is the rate of force redevelopment post protocol.where t = 0, which was the time at which force was discernibly above zero (F = 0). This time point was when the slack had been taken up by active shortening of the trabeculae to the new lower sarcomere length . Figure D shows the exponential fit to three representative traces for force re‐development by the same trabecula at three sarcomere lengths. The fitted rate constants were k1.94 = 10 s–1, k1.90 = 9 s–1 and k1.85 = 8 s–1. Figure E shows the scatter of residuals for the fit to plots in Fig. D. This protocol was applied to each trabecula at each RLC phosphorylation level.The exponential was applied from gth Fig. C. FigurA). Depending on the ramp velocity, the amplitude of the initial rapid release length was altered to minimize series elasticity during ramp, which resulted in a relatively stable force signal during ramp release . This protocol provides a reliable method for determining the experimental relationship between force and velocity , with fibre length being the length of the trabecula between the T‐clips when held at a sarcomere length of 2.1 μm. The parameters describing the length change protocols are given in Table The release‐ramp protocols were designed to determine the force‐velocity relationship in trabeculae for a range of RLC phosphorylation levels. A byproduct of these protocols is the time‐course of force recovery following the period of shortening, which we find to be sensitive to RLC phosphorylation levels, providing information about the kinetics of actomyosin interactions. Isometric force was allowed to develop at a sarcomere length of 2.1 μm. At the plateau of isometric force, the length transducer applied a rapid shortening to the trabecula, followed by a slower shortening  and during ramp (Framp). The fit to the force recovery records after ramp shortening gave estimates for the rate constant describing the time‐course of force redevelopment. The rate constants were obtained for force recovery following ramps at three different velocities (see above) and are therefore denoted k1, k0.5 and k0.3 for the three ramp velocities of 1, 0.5 and 0.3 FL s–1. These rate constants were calculated for each of the three phosphorylation states.Each protocol reduced sarcomere length by ∼8% to 1.94 μm. Figure R is the relaxed state and A is the active, force‐generating state. The rate constants f and g determine transition into the force‐generating state and into the relaxed state, respectively.The model considers:t) is given by a single exponential equation as:In such a model, the amount of force at any time . According to this model, the rate constant describing force generation is (f + g). The rate of force development observed in the experiments described in the present study thus give estimates for (f + g) under various conditions of RLC phosphorylation and sarcomere length. The observed time course of force generation is well described by a single exponential process , which is given by:Calculating DR for each RLC phosphorylation level and SL allowed the approximation of force per cross‐bridge (FXB (in pN) and the total number of heads were derived by assuming a constant concentration of 200 μm of myosin heads  that SL affected ktr and (ii) that RLC phosphorylation affected ktr. Appropriate post hoc pairwise multiple comparisons were made using Bonferroni adjusted t tests, with a statistical significance cut‐off of P < 0.05. SigmaPlot  was used to perform statistical analysis. N is the number of trabeculae sampled for each protocol. Each treatment group comprised of five cardiac preparations.We hypothesized that: (i) RLC phosphorylation and (ii) sarcomere length affected et al. A), the extent of force recovery was higher at higher levels of RLC phosphorylation, and reduced by decreasing RLC phosphorylation. Trabeculae with control levels of RLC phosphorylation produced an isometric force at a SL of 1.94 μm (F1.94) of 63 ± 5 kN m–2 . This was not significantly different from trabeculae with increased RLC phosphorylation (78 ± 7 kN m–2) but was higher than in trabeculae with reduced phosphorylation . This was unsurprising because we have shown this phenomenon previously , was also affected by RLC phosphorylation. Unlike force, ktr is affected in a dose‐dependent fashion by RLC phosphorylation where control levels of RLC phosphorylation show an intermediate rate of k1.94 11 ± 1.7 s–1, to increased and reduced phosphorylation k1.94 24 ± 1.8 and 7.2 ± 0.9 s–1 respectively, with both being significantly different from control (P < 0.05) . At the shorter sarcomere lengths of 1.90 and 1.85 μm, differences in isometric force between control trabeculae and phosphorylated trabeculae were not observed . However, in both instances, trabeculae with reduced RLC phosphorylation exhibited a significantly lower force when compared to control and phosphorylated samples at a SL of 1.94 μM (P < 0.05). Phosphorylation level similarly affected the rate of force redevelopment at shorter sarcomere lengths in a manner the same as that for the longer starting sarcomere length of 1.94 μm (P < 0.05)  generated only a small amount of force (10–15%) compared to the values at control and 150% phosphorylation. (P < 0.05). The reason for this effect is unclear. However, reducing RLC phosphorylation reduces the ability of muscle to generate force at full thin filament activation, indicating that either force per cross‐bridge or cross‐bridge formation is significantly hindered.Measuring force redevelopment from slack allows the assessment of force production from a state where many of the cross‐bridges will be synchronized in the unbound and weakly bound states (diastole). Therefore, inferences can be made about the attachment and detachment kinetics of myosin and how these parameters can be altered by SL, as well as RLC phosphorylation. Force redevelopment from slack at sarcomere lengths of 1.94 , 1.90 and 1.85 μm was measured for three different RLC phosphorylation levels Fig. . These Sktr by allowing a direct comparison of the time‐course of force redevelopment at each phosphorylation level . This highlights the role of RLC phosphorylation on force redevelopment at the three physiological sarcomere lengths tested .Normalizing to the isometric force at each sarcomere length highlights the effect of RLC phosphorylation on ted Fig. A.ktr is slower at shorter sarcomere lengths . The gradients of linear regressions applied to the data in Fig. B were 79 ± 14, 8 ± 0.2 and 26 ± 3 s−1 μm−1 for phosphorylated, control phosphorylation and reduced phosphorylation, respectively (n = 5). Statistical significance was observed between each slope (P < 0.05).Additionally, for each RLC phosphorylation level, ths Fig. B. The g°C to a final sarcomere length of 1.94 μm . Release‐ramp shortening simulates active cross‐bridge cycling at different muscle loads, which would be encountered during systole. At the end of these manoeuvres, force redevelopment occurs in mixed populations of cross‐bridge states, unlike the release protocol, which synchronizes all myosin heads into weakly (or unbound) states. This assay allows us to assess whether changes in ktr can be attributed to enhanced thin filament activation by already bound cross‐bridges. The shortening velocities were 1, 0.5 and 0.3 FL s–1 , where 0.3 redevelops force from the highest muscle load with the most attached cross‐bridges, by proportion. For each RLC phosphorylation level, Fiso was constant across all ramp velocities . This was expected because Fiso was attained at a SL length of 1.94 μm after each manoeuvre velocity. Trabecular preparations with reduced RLC phosphorylation displayed a reduced Fiso post release (independently of release velocity) of 19 ± 2 kN m–2 (P < 0.05) versus control of 49 ± 4 kN m–2 and phosphorylated of 55 ± 3 kN m–2 .Trabeculae with one of three different RLC phosphorylation levels underwent release‐ramp shortening protocols during activation at 20ies Fig. C. This m–2 Fig. C.B, D and F). Trabeculae with control RLC phosphorylation displayed rate constants for force production of 12 ± 1.2, 11 ± 1.1 and 10 ± 0.5 s–1 at velocities of 1, 0.5 and 0.3 FL s–1 respectively  at 21 ± 1.6, 19 ± 1.7 and 18 ± 1.6 s–1 for velocities of 1, 0.5 and 0.3 FL s–1, respectively. Trabeculae with reduced RLC phosphorylation had slower rates of force production compared to control (P < 0.05) with rates of 10 ± 3.2, 7.6 ± 0.8 and 6.7 ± 1.1 s–1 at velocities of 1, 0.5 and 0.3 FL s–1, respectively.Normalizing force development at each RLC phosphorylation level for each ramp velocity illustrates the effect of RLC phosphorylation on the rate of force rise . Importantly, our measurements at saturating calcium allow us to consider processes that are independent of the Ca2+‐dependent contributions to the Frank–Starling law, and also show that RLC phosphorylation and the thick filament contribution both play a role in the Frank–Starling law.We have previously demonstrated the ability of RLC phosphorylation to influence myocardial force production, power and shortening under‐load  is close to the ATPase rate in trabeculae from rats under identical experimental conditions (∼8 s−1)  during maximal calcium activation, in contrast to the findings of another study at a higher SL (∼2.3 μm), which observed no change to ktr at this higher SL  , which does not fit the modelling of our experimental data and contradicts the existing literature  than the reduction in ktr (∼30%). This may be accounted for by a shift towards the super relaxed state of the myosin heads  of f and the myosin ATPase rate must be altered by reduced RLC phosphorylation to account for this reduction of force. Our modelling estimates a marked ∼5‐fold reduction in f in the reduced phosphorylation cohort, which, again, is probably explained by myosin proximity to actin . Our results complement these findings, highlighting RLC phosphorylation as being a thick filament level regulator of the Frank–Starling law of the heart, which is independent of the thin filament regulatory complex. We suggest that RLC phosphorylation modulates the Frank–Starling law of the heart in both health and disease. It does so by adapting myocardial contractile kinetics, allowing for the timely systolic expulsion of ventricular pre‐load.Under conditions where myosin heads are largely synchronized (post rapid release protocol) or unsynchronized (post release ramp protocol), RLC phosphorylation alters The authors declare that they have no competing interests.All data were collected at Imperial College London in the laboratory of Professor Michael A Ferenczi. CNT, TGW and MAF conceived and designed the experiments. CNT and MAF collected and analysed data. CNT, TGW and MAF interpretated data and produced the manuscript.The present study was supported by Wellcome Trust Grant 091460/Z/10/Z.