We study theoretically the bio-sensing capabilities of metal nanowire surface plasmons. As a specific example, we couple the nanowire to specific sites (bacteriochlorophyll) of the Fenna-Matthews-Olson (FMO) photosynthetic pigment protein complex. In this hybrid system, we find that when certain sites of the FMO complex are subject to either the suppression of inter-site transitions or are entirely disconnected from the complex, the resulting variations in the excitation transfer rates through the complex can be monitored through the corresponding changes in the scattering spectra of the incident nanowire surface plasmons. We also find that these changes can be further enhanced by changing the ratio of plasmon-site couplings. The change of the Fano lineshape in the scattering spectra further reveals that “site 5” in the FMO complex plays a distinct role from other sites. Our results provide a feasible way, using single photons, to detect mutation-induced, or bleaching-induced, local defects or modifications of the FMO complex, and allows access to both the local and global properties of the excitation transfer in such systems. Photosynthesis, the transformation of light into chemical energy, is one of the most crucial bio-chemical processes for life on earth345673910111215Recently, hybrid quantum systems, which combine two or more physical systems, enable one to combine the strengths and advantages of individual systems. Work in this area has led to new phenomena and potentially new quantum technologies18192122232425262729312419in vivo FMO complex can experience both mutation-induced and bleaching-induced local defects or non-functional sites, leading to blocked excitation transferring pathways. This has been demonstrated in recent experiments3418Like all other species, the N = 7 sites , εn is the site energy of chromophore n, and n-th and n′-th sites. For simplicity, here the recently discoveredwhere the state |In the bacterial photosynthesis, the excitation from the light-harvesting antenna enters the FMO complex at sites 1 or 6 and then transfers from one site to another. When the excitation gets to the site 3, it hops irreversibly to the reaction center. In the regime that the excitonic coupling in vivo. A SP with energy vg and k are the group velocity and wave vector of the incident SP, respectively, and vg is set to be unity throughout this paper. The incident SP can then be scattered by the two sites, due to these strong SP-site couplings. Alternatively, it can be absorbed by these two sites and be dissipated due to loss in the FMO complex. The total Hamiltonian TH of this SP-FMO hybrid system can be written as refs HT = Hsp + Hsp-site + HFMO, withAs shown in Hsp stands for the energy of the SPs with k-mode SP, Hsp-site denotes the interaction between the SPs and the sites of the FMO complex. In Hsp-site, d is the separation between site 1 and 6, while g1(6) between the SPs and site 1 (6) is assumed to be independent of k under the Markovian approximation43g1 and g6 are consistent with a recent analysis of nanowire SPs coupled to J-aggregates1819where When the SP is propagating on the surface of the metal nanowire, it inevitably suffers from dissipation, such as Ohmic loss, that can be described by a Markovian channel. Thus, here we include the Markovian channels for irreversible excitation transfer from site 3 to the reaction center, as well as the Ohmic loss, by introducing non-Hermitian terms in the total Hamiltonian. Moreover, since we are interested in time-independent solutions of the photon state, the effect of quantum jumpsγn are mostly zero, except for the rates referring to the Markovian channels, γ1 = γ6 = γdp + γol and γ3 = γs. Here, the Ohmic loss rate γol is set to be γol = 20−1γsp2124d = 0. Similarly, since the separation is small compared with the distance (~30 nm) that SPs propagate during the dephasing time of scattering49γdp on sites 1 and 6, and neglect dephasing for other sites, because the broadening of these sites, which couple to the SPs, typically dominates the scattering spectrawhere Ek = vgk, can be written as24The energy eigenstate of the hybrid system with an energy matching the incident SP, g, 0sp〉 describes that the FMO complex is in the ground state with no SPs, and |n, 0sp〉 stands for that the excitation is in the site n, while ξn is the probability amplitude that the site n absorbs the excitation. We also assume that the SP field is incident from the left of the waveguide, the scattering amplitudes where |t and r are the transmission and reflection amplitudes, respectively, and θ(x) is the unit step function. The total Hamiltonian HT can be further transformedHere, cR(x) [cL(x)] is a bosonic operator annihilating a right-going (left-going) photon at x, and δ(x) is the Dirac delta function. This real-space total Hamiltonian can be applied to the energy eigenstate [T = |t|2 and the probability amplitudes ξn can then be obtained by solving the eigenvalue equation γdp, proportional to the temperature−1.where genstate . The traJ5,6, between sites 5 and 6 leads to the enhancement of the coherence between other the sites of the FMO complex, the Fano resonance is strongly altered [It has been demonstrated experimentally34 altered . This reg1 = g6 = 10 cm−1. In the following section, we will discuss the effects of asymmetric couplings.Similarly, mutation-induced defects or local environment modification34J1,2 [g1/g6 = 100 . The changes in the transmission spectra are enhanced compared to those in g1/g6 reduces the communication between discrete and continuous channels. As a result, the two channels become more disparate, leading to an enhancement in the transmission spectra.So far we have shown that local defects or missing sites can be detected via changes in the transmission spectra of the incident SP. However, if these changes in the transmission spectra can be enhanced, then the observations of such an effect in experimental realizations would become more feasible. We have observed that the transmission spectra contain Fano lineshapes, thus we can predict that a discrepancy between the discrete (the FMO sites) and continuous (the SPs) channels will strongly affect the behavior of such Fano resonances5657J1,2  and entiJ1,2  with asyJ5,6 [g1/g6 = 0.01 . Again we see that the changes in the transmission spectra are enhanced compared with those in J1,2 (−104.1 cm−1) is larger than J5,6 (89.7 cm−1), we would expect this effect to be obvious in Similar results can also be obtained as shown in J5,6  or complJ5,6 . CorrespSince the FMO protein complex is in reality a trimer, the validity of the results obtained by studying the monomer alone shall be addressed here. Typically studies have shown that the subunits of the trimer act as separate transport channelsIn summary, we have shown that by observing the scattering of an incident SP, coupled to sites 1 and 6 of the FMO complex, both the inhibition of the excitonic coupling (excitation-transferring pathway) and entirely missing sites can be detected. We also show that by making the discrete (sites) and the continuous (SPs) channels more disparate, it is possible to further enhance the changes in the transmission spectra of the incident SP. These results provide an alternative method to detect the mutation-induced local defects or the non-functional sites in the FMO complex. We expect that future work will reveal even more uses for such hybrid bio-sensors, including probing the role of quantum coherence and environmental effects in photosynthetic complexes.How to cite this article: Chen, G.-Y. et al. Plasmonic bio-sensing for the Fenna-Matthews-Olson complex. Sci. Rep.7, 39720; doi: 10.1038/srep39720 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We suggest the presence of a floating ice tongue since at least 300 AD lasting until 1900 AD followed by elevated 20th century glacier calving due to the loss of the tongue. We attribute this regime shift to 20th century unprecedented low sea-ice occurrence in the East Greenland Current and conclude that properties of this current are important for the stability of the present ice tongues in NE Greenland.The early 2000s accelerated ice-mass loss from large outlet glaciers in W and SE Greenland has been linked to warming of the subpolar North Atlantic. To investigate the uniqueness of this event, we extend the record of glacier and ocean changes back 1700 years by analyzing a sediment core from Sermilik Fjord near Helheim Glacier in SE Greenland. We show that multidecadal to centennial increases in alkenone-inferred Atlantic Water SSTs on the shelf occurred at times of reduced solar activity during the Little Ice Age, when the subpolar gyre weakened and shifted westward promoted by atmospheric blocking events. Helheim Glacier responded to many of these episodes with increased calving, but despite earlier multidecadal warming episodes matching the 20 A large part of this change has been attributed to increased iceberg calving from tidewater outlet glaciers3, highlighting the role of marine-terminating glaciers in regulating ice-sheet stability. Mass change of the Greenland Ice-Sheet has only been continuously tracked with satellite data since the early 1990s. To understand whether the mass loss of the recent decade represents an outstanding event, or whether it is part of recurring phenomena acting on annual, decadal, or centennial timescales, we lack a longer-term history of the Greenland Ice Sheet. The answer to this question has potentially strong implications for the kind of ice-sheet changes we can expect in the context of future warming climate.In the last two decades there has been a rapid increase in the loss of ice from the Greenland Ice Sheetst century4. Specifically Jakobshavn Isbræ, in West Greenland, and Kangerdlugssuaq and Helheim glaciers in SE Greenland  documents several episodes of increased iceberg calving of which the youngest marked episode occurred between 2000 and 2005. This study found that regional climatic modes  may have influenced glacier calving through modulation of warm subsurface waters on the SE Greenland shelf and inside the fjord and that, conversely, episodes of increased cold polar waters occurrence may have potentially stabilised the glacier termini. However, almost nothing is known about the behaviour of Helheim Glacier before the 20th century and thus how on-going climate changes will affect the dynamics of outlet glaciers.The link between climatic changes and outlet glacier variability during the past century has been demonstrated by a reconstruction of Helheim Glacier calving based on the combination of three sediment cores from Sermilik Fjordth century record of ocean and glacier variability back to 300 AD by analysing sediment core ER07 from Sermilik Fjord. Specifically, we use alkenone thermometry to reconstruct sea-surface temperature (SST). We assess the impacts of ocean conditions and sea-ice on the stability of Helheim Glacier using a reconstruction from the same core of iceberg sediment rafting while discussing the relationship between iceberg calving and sediment rafting. These new data provide the first-ever multi-centennial record of ocean and glacier changes that includes the 21st century changes in detail and thereby places the current changes into a longer temporal context embedding low frequency ocean variability.Here we extend the 202. The ocean waters that circulate on the SE Greenland shelf dominate the subsurface waters in Sermilik Fjord. The hydrological vertical structure of the fjord is characterised by a 10–20 m thick surface layer of glacial melt water, a 100–150 m thick intermediate layer of cold Polar water, and a lowermost layer of warm saline water derived from the IC15. The primary driver for SST variability in coastal SE Greenland is the varying influence of the IC and the East Greenland Current (EGC), the two major currents that dominate the local hydrology16.Sermilik Fjord is situated in central SE Greenland; it is 80 km long, up to 15 km wide, and 600–900 m deep , or Medieval Warm Period (MWP)20 reported in numerous paleoclimate records though with different duration. Most MCA records, however, document warmer climatic conditions in the Northern Hemisphere, including near our study area in west and summit Greenland22 and off North Iceland23 with concomitant low sea-ice cover24.Our record shows multidecadal to centennial shifts in the temperature of surface ocean waters off SE Greenland with progressive cooling until the onset of the 20ury Fig. . The timth century marks the transition to a new temperature regime from 1250 AD to 1900 AD. This interval coincides with the Little Ice Age (LIA), a period of general atmospheric cooling and glacier advance in most sites of the Northern Hemisphere and expansion of sea-ice in the North Atlantic Ocean25. The reconstructed SSTs during this interval are high  and reveal a subtle cooling trend and greater variability than during the DACP. Three notable warm periods are salient around 1270–1300 AD, 1430–1460 AD, and 1620–1670 AD.The abrupt SST increase at the end of the 12th century to present-day, average SSTs increased slightly  to values that are comparable to the mean conditions during earlier warm episodes of both the DACP and the LIA. The higher variability of SSTs during the 20th century could in part be due to the higher temporal resolution of the record in this interval   when compared to randomly generated red-noise data, and a positive correlation for the period 300–900 AD , although only marginally significant, which is probably due to the low data-resolution in core MD99–2322 . These results strengthen our confidence in the age model for core ER07.Due to the scarcity of dateable material in glacioproximal sedimentsyer Fig. . In simiThe comparison of our new SST record with a reconstruction of past changes in total solar irradiance (TSI) Fig.  indicate29. The physical mechanisms at play remain debated30, but considering the small amplitude of the variations in TSI, amplifying mechanisms are likely to explain the magnitude of climate variability in paleo-records. Climate modelling has demonstrated that atmospheric circulation responds to low solar activity by resembling the negative phase of the winter NAO32. Other modeling studies have shown that solar minima of the 20th century and the Maunder Minimum favor persistent North Atlantic anticyclonic blocking33. This weather regime has been related to the negative phase of the East Atlantic pattern (EA-) and shown to be responsible for the most severe winters on record in Europe34. This mode of variability has a similar North South dipole structure as the NAO but its centers of action are displaced southeastward. Both NAO and EA are important drivers of the North Atlantic Ocean circulation. The response of the ocean to weaker westerly winds (negative NAO) as well as persistent blocking events across the Atlantic is a wind-driven reduction and shrinking of the subpolar gyre36. Indeed, oceanic warming in West Greenland at the turn of the 21st century has been paralleled to a decrease from high positive NAO index values in the early 1990s to negative state in 1996 and the strong decline of the subpolar gyre circulation9. In a recent study, the role of negative EA and blocked events on the subpolar gyre circulation has been evidenced35. Therefore, negative NAO and persistent blocking events favored by reduced solar activity are likely to explain the warm oscillations of the Atlantic waters seen off SE Greenland (Sermilik and Kangerdlugssuaq29) and the generally higher temperatures during the LIA than MCA off Sermilik Fjord. In contrast to SE Greenland, in the RAPID17–5P core south of Iceland, solar activity positively correlates with SSTs28 . We suggest that those of the pre-20th century multidecadal ice rafting episodes that are concurrent with increased SSTs relate to climatically forced increased iceberg calving. This indicates that some ice rafting events may be related to intrinsic glacier behavior and that some of the IC warming episodes did not result in glaciological changes.The 1700-year long record from Sermilik Fjord reveals a decrease in iceberg rafting from 300 AD to 1300 AD Fig. . This isth century is unexpected since average SSTs during the 20th century were similar to those during previous multidecadal warming episodes. We suggest that the unprecedented high iceberg rafting from Helheim Glacier after 1900 AD links with unprecedented reductions in sea-ice in the EGC after 1900 AD  is 0.06 cm yr−1 and 0.13–0.5 cm yr−1 (average 0.25 cm yr−1), respectively. Material for grain size analysis was continuously sampled at intervals of 0.5 cm down to 26 cm, at 1 cm intervals between 26 and 31 cm, and at 2 cm intervals below 31 cm. The grain size distribution (volume %) was estimated by a Malvern Mastersizer 2000 laser particle sizer. Samples were dispersed with natrium pyrophosphate (0.01 M Na4P2O7 × 10 H2O) and treated in an ultrasound bath for two minutes before measuring the 0.3–1000 µm fraction. The flux of sand (>63 µm) was calculated as a function of the wt % of sand, sedimentation rate and dry bulk density.Sediment core ER07 was obtained using a Rumohr corer, using 80 mm core liner, from 525 m water depth . X-ray imagery37 to C39 ketones with two or three double bonds that are produced primarily by the ubiquitous marine coccolithophorides Emilinia huxleyi. SSTs were obtained from the unsaturation ratio of C37 alkenones (37:2/(C37:2 + C37:3) and the global calibration .Supplementary Information


However, the molecular mechanism of Ih inhibition by this drug has been limitedly explored. Here, we show that inhibition of Ih by lidocaine, recorded from Chinese hamster ovary (CHO) cells expressing the HCN1 channel, reached a steady state within one minute and was reversible. Lidocaine inhibition of Ih was greater at less negative voltages and smaller current amplitudes whereas the voltage-dependence of Ih activation was unchanged. Lidocaine inhibition of Ih measured at −130 mV (a voltage at which Ih is fully activated) was reduced, and Ih amplitude was increased, when the concentration of extracellular potassium was raised to 60 mM from 5.4 mM. By contrast, neither Ih inhibition by the drug nor Ih amplitude at +30 mV (following a test voltage-pulse to −130 mV) were affected by this rise in extracellular potassium. Together, these data indicate that lidocaine inhibition of Ih involves a mechanism which is antagonized by hyperpolarizing voltages and current flow.Lidocaine is known to inhibit the hyperpolarization-activated mixed cation current (I In addition to its well-known action on voltage-gated sodium channels5, lidocaine may produce its therapeutic actions, as well its known adverse effects, by acting on other target proteins, including Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. For example, lidocaine has been shown to inhibit native HCN channels in cardiac Purkinje fibres7, sinoatrial node pacemaker cells8, dorsal root ganglion neurons9, and thalamocortical neurons10, with an IC50 ranging from 38–99 µM and maximum inhibition varying from 70 to 100%. Two studies have examined the inhibition of heterologously expressed cloned HCN channels by lidocaine, with IC50 and maximum inhibition values of 67–276 µM and 42–44%, respectively12. The molecular mechanism underlying the action of lidocaine on HCN channels and the basis for the variation in lidocaine’s effect among studies of native and cloned HCN channels remain unknown.Lidocaine is a drug that depresses both sinus node activity and cardiac conduction, acts as a local anesthetic and topical analgesic, and, when applied systemically, produces central analgesia and sedationh, by lidocaine. Four HCN genes have been identified in mammals (HCN1-HCN4) and they are similar in primary sequence to the Shaker-related voltage-gated potassium (Kv) channels15. However, HCN channels have recently been shown to have a structure that is similar but not identical to those channels17. Like Kv channels, HCN channels are tetrameric and each subunit is multi-membrane spanning and possesses an intracellular N and C-terminus16. However, the recently solved human HCN1 channel structure16 shows that the selectivity filter (the portion of the pore closest to the outside of the cell) of the HCN channel contains fewer binding sites for cations, which may help to explain the permeability to both sodium and potassium ions18. The fourth S4 transmembrane segment, the voltage sensor, is long and appears to hold the pore (transmembrane S6 helices) in a compact and potentially closed conformation. Such a structural arrangement suggests that, unlike Kv channels19, the pore is naturally open at hyperpolarized potentials and that the voltage sensors apply pressure to close the pore during depolarization; this interpretation made from the HCN1 structure is consistent with that made previously by a combined functional and mutagenesis study of the HCN2 channel20, which is the isoform that is phylogenetically the closest to the HCN1 isoform21.In this study, we examine the mechanistic basis of the inhibition of the HCN channel and associated hyperpolarization-activated current, I22 and has been shown in HCN1-knockout mice to contribute to the electrical and cellular activity of different types of cells, including sinoatrial node pacemaker myocytes23, somatosensory neurons24, cortical neurons25, cerebellar Purkinje neurons26, and hippocampal neurons27. We show that lidocaine inhibition of HCN1-mediated Ih is near complete within one minute of exposure, is reversible, and involves a mechanism which is antagonized by hyperpolarizing voltages and current flow.Here, we focus on lidocaine inhibition of the HCN1 isoform, which has been found throughout the brain and in the conduction system of the heart2. Transfection of a mammalian expression vector encoding mouse HCN1  (2 µg per dish) was performed using the FuGene6 transfection reagent  according to the manufacturer’s protocol. The DNA plasmid encoding Green Fluorescence Protein (GFP) was co-transfected for visualization of transfected cells.Chinese hamster ovary (CHO) cells were maintained in Ham’s F-12 medium  supplemented with 50 µg/ml penicillin/streptomycin (Life Technologies) and 10% fetal bovine serum (Life Technologies). Cells were grown on glass coverslips (22 × 22 mm) in 35 mm dishes at 37 °C with 5% CO2, 0.5; CaCl2, 1.8; HEPES, 5, and titrated to pH 7.4 with NaOH. It should be noted that “low” refers only to the other solutions used in this study (see below) whereas a concentration of 5.4 mM potassium in the extracellular solution is usually considered to be a normal concentration in vivo. Cells expressing GFP were selected for patch clamp recording. After attaining the whole-cell configuration, cells were perfused by using a Fast Step SF77B perfusion system , with either the same, low potassium extracellular solution or solutions containing different proportions of KCl and NaCl. The 30 and 60 mM potassium extracellular solutions contained 30 mM KCl and 110 mM NaCl and 60 mM KCl and 80 mM NaCl, respectively, and were titrated to pH 7.4 with either NaOH or KOH. The calculated osmolarities for the internal and external solutions used were ~290 mOsm. Patch pipettes were pulled from borosilicate glass  using a P-100 electrode puller  and filled with an intracellular solution containing (mM): KCl, 130; NaCl, 10; MgCl2, 0.5; EGTA, 1; HEPES, 5, adjusted to pH 7.4 with KOH, and had a resistance of 4–5 MΩ. Whole cell patch-clamp recordings were performed with an Axopatch 200B amplifier  and a Digidata 1320A data acquisition system  using pCLAMP software . The membrane currents were low-pass filtered at a frequency of 2 kHz and digitized at 5 kHz. Data were collected more than 5–10 min after whole cell access to allow the internal pipette solution to equilibrate with the cell. Experiments were carried out at room temperature (21–23 °C).Approximately 24 to 48 hours post-transfection, a small piece of coverslip was paced into a recording chamber (900 µl) on the stage of an inverted microscope. Cells were perfused (0.5–1 ml/min) with a low potassium extracellular solution containing (mM): KCl, 5.4; NaCl, 135; MgCl29. By Western blotting and patch clamp electrophysiology, we showed that untransfected CHO cells do not express HCN channel protein or HCN-mediated currents. Upon transfection, robust Ih currents were elicited from GFP-expressing cells and HCN1 channel protein is abundant in Western blots. As we found previously, robust hyperpolarization-activated currents were observed in the current study when recording from cells that expressed GFP.In previous studies, we characterized aspects of HCN1 channel expression in CHO cellsLidocaine HCl was purchased from Sigma–Aldrich Canada Ltd. , diluted in corresponding extracellular solution and applied by using the Fast Step SF77B perfusion system. Although the transfection efficiency was quite good, we recorded from only one cell per dish to avoid carrying out voltage protocols on cells that had already been exposed to the drug. New dishes were also employed if measurements were unfinished because of issues such as cells becoming unresponsive to voltage or large and irreversible alterations in access resistance or leak current. Lidocaine was perfused for 1–2 minutes in order to obtain steady state inhibition.50 and Hill coefficient, concentration-response curves were normalized and fitted using the Hill equation as follows:I represents the current measured in the presence of a given drug concentration; Imax is the control current measured in the absence of the drug; [C] is the drug concentration; IC50 is the half-maximal inhibitory concentration; and h is the Hill coefficient.Data were analyzed using ORIGIN 8 . To determine the ICG is the conductance estimated after the current activation at a hyperpolarizing step [V]; Gmax is the maximum conductance obtained after the most hyperpolarizing step; V50 is the half-maximal activation potential; and k is the slope factor.The normalized conductance-voltage relationship for steady-state activation of HCN1 current was fitted by the Boltzmann equation:h, the relationship between concentration and current obtained from individual cells was fitted to the Michaelis-Menten model:I represents the current measured at a given potassium concentration, Imax is the maximum current, [K+]o is the extracellular potassium concentration, and Kapp is the apparent dissociation constant.To model the effect of extracellular potassium on inward steady-state IReversal potentials in different concentrations of extracellular potassium were determined from leak-subtracted steady state current obtained from a hyperpolarizing pulse to −90 mV and current obtained from a subsequent pulse to −60 mV. For each cell, values for the steady state at −90 mV and for the subsequent tail current at −60 mV was plotted versus voltage and joined by a straight line, which was extrapolated to cross the x-axis to yield an estimate of reversal potential.t test; Statistical tests were two-tailed and results were considered significant at α = 0.05.Data are presented as mean ± SEM. We used one-way ANOVA to test for comparisons of more than two groups. Comparisons between two groups were conducted with the use of a paired or unpaired Student h at different voltages and of Gh, which was estimated as the magnitude of the tail Ih at a constant potential (−65 mV) in 30 mM extracellular potassium . The fraction of steady state Ih inhibited by lidocaine increased from ~44% at −135 mV to ~62% at −90 mV  of steady-state Iium Fig. . This co mV Fig. . Inhibitges Fig. . The lach. In order to do this, we raised the proportion of extracellular potassium in order to increase current flow, which is one of two approaches used previously to examine how current flow impacts ivabradine inhibition of Ih; inhibition of Ih by that drug is less at more negative potentials and, for Ih in the sinoatrial node (If) and Ih associated with the HCN4 isoform, inhibition is reduced by an increase in current flow31.We next examined the effect of increasing current flow on lidocaine inhibition of Ih conductance and the voltage-dependence of Ih activation when measured in CHO cells expressing the HCN1 isoform. In addition to a depolarizing shift in reversal potential, raising extracellular potassium is known to increase maximum Ih conductance in both native tissue37 as well as in cells that express cloned HCN channels40. We previously examined this unusual potassium-induced increase in conductance of Ih in CHO cells expressing the HCN1 isoform by substituting potassium for sodium in the extracellular solution and thereby leaving unchanged the concentration of both the total and permeant cations28. We used the same approach here to first characterize the effect of extracellular cations on Ih conductance and voltage dependence of Ih activation, and then to examine the effects of extracellular cations on inhibition of Ih by lidocaine.We first determined how raising extracellular potassium impacts Ih activated by a hyperpolarization to −130 mV and outward Ih recorded as the tail current at +30 mV (deactivation) from prior hyperpolarization to −130 mV. Increasing extracellular potassium from 5.4 mM to 60 mM produced a reversible increase in the magnitude of the inward Ih, which reached a maximum in ~1 minute  and slope factor (k), as described in the Methods. Raising extracellular potassium did not significantly modify the range of Ih activation, although the slope factor was slightly increased  to activate Ih, followed by a repolarizing pulse to +30 mV to deactivate the current (0.5 s), was applied every 15 seconds. Bath application of 100 µM lidocaine at 5.4 mM extracellular potassium reversibly inhibited inward Ih (at −130 mV) by ~48%  inhibited by 100 µM lidocaine was less at 30 and 60 mM extracellular potassium  , the degree of inhibition is greater at lower levels of potassium  Fig. . When IhSee Fig. . Howeverh in the presence of lidocaine was normalized to that measured under control conditions and fitting the obtained kinetics with a double-exponential function . Indeed, plotting the total normalized degree of inhibition of tail current area versus lidocaine concentration and fitting with the Hill plot produced three curves with similar values for maximum inhibition. Fitting also produced Hill coefficients of ~1 and values for IC50 of ~56, 87, and 56 μM. Values for maximum inhibition and IC50 are most similar to those values determined by lidocaine inhibition of Ih measured at −130 mV in 5.4 mM extracellular potassium.Lastly, we determined the degree of lidocaine inhibition of outward Ih in CHO cells that express the HCN1 channel depends upon the extracellular concentration of potassium and voltage. We show that inhibition of Ih is increased at less negative potentials whereas the dependence of Ih activation over the same range of voltages is not affected. We also show that the inhibition of Ih at −130 mV is reduced, and Ih conductance are increased, by raising potassium from 5.4 mM to 60 mM, whereas inhibition at +30 mV, and Ih conductance, are unaffected by the same changes in potassium. Thus, the data show that lidocaine inhibition of Ih and Ih conductance are inversely correlated and, together with concentration response curves, suggest that inhibition of Ih by lidocaine is antagonized by current flow and that the increase in current is antagonized by the drug. Finally, we show that lidocaine has no effect on the Ih activation curve.In this study, we demonstrate that lidocaine inhibition of Ia of approximately 7.9, exists in an uncharged basic form and a charged cationic form. As has been proposed for other channels41, the charged form may inhibit the HCN1 channel from the extracellular side or the uncharged form may diffuse through the plasma membrane, become re-protonated in the cytoplasm and inhibit the channel from the inside. Alternatively, the uncharged form of the drug may interact with the channel by traversing a lipophilic pathway in the plasma membrane towards a site on the channel which is embedded in the plasma membrane. Studies have shown that the application of QX-314, a quaternary derivative of lidocaine which is protonated and unable to cross the lipid bilayer, has no effect on Ih in neurons when applied on the external side whereas it inhibits Ih when applied from the intracellular side43. Thus, we currently favor the view that uncharged lidocaine diffuses through the plasma membrane, becomes re-protonated inside the cell and inhibits the HCN1 channel from the inside.Lidocaine, a weak base with a pKh by other drugs. For example, inhibition of Ih by the drugs ZD7288, UL-FS49, DK-AH3, and ivabradine is reduced at more negative voltages and these molecules have only small or negligible effects on the voltage-dependence of Ih activation48. Like the lidocaine analog, QX-314, the aforementioned drugs are thought inhibit Ih in their cationic form by acting on a site which is accessible from the inside of the cell49. Like lidocaine inhibition of Ih in our study, ivabradine inhibition of Ih in rabbit sino-atrial node cells and Ih in HEK cells heterologously expressing the HCN4 isoform is also increased when current is reduced; in those studies, current was reduced by reducing the proportion of extracellular potassium (as we did in our study) or by adding millimolar levels of extracellular cesium31. In these studies, it was proposed that the dependence of ivabradine inhibition on current was due to its interaction with permeant ions on the inner aspect of the pore. Thus, together with the intracellular action of QX-314 on Ih in other systems, it seems possible that, in our study, the reduced inhibition of Ih by lidocaine at larger current amplitudes is due to its greater interaction with permeant ions on the inner side of the HCN1 pore. Further studies will be required to understand the interaction between current amplitude and lidocaine, and why drug potency is reduced mainly in 30 mM potassium whereas both potency and maximum inhibition of Ih by the drug are impacted by the drug in 60 mM potassium.Our data on lidocaine are similar to previous data on the inhibition of Iin vivo. The greatest inhibition of Ih by lidocaine in our study occurred at the lowest concentration of extracellular potassium utilized, 5.4 mM, and the highest concentration of sodium (130 mM); these are close to the usual physiological concentrations for these cations. Our data in Fig. h by lidocaine increases progressively as the membrane potential becomes less negative suggesting that the effect of this drug might vary with membrane potential in vivo e.g. during an action potential. The concentration of potassium and sodium are also known to vary in vivo. In the heart, the concentration of extracellular potassium may range between 3 and 10 mM, and has been proposed to contribute to bradycardia that is mediated by HCN channels52. In the central nervous system, a range of potassium concentrations between 3 and 12 mM has been measured, while extracellular sodium may decrease by up to 7 mM during repetitive activity55. This variation in cation concentration encountered in vivo could influence the inhibition of Ih by lidocaine.The present findings are important for understanding the myriad of effects of lidocaine f sodium 30 mM; thin vivo by acting on the HCN1 isoform. For example, knockout of the HCN1 isoform from somatosensory neurons in the mouse has been shown to reduce cold allodynia and to contribute to action potential generation . Thus, inhibition of HCN1 by lidocaine likely contributes to its analgesic effects, and it will be important to determine the extent to which lidocaine inhibition of HCN1 contributes to this drug’s in vivo actions and how inhibition is impacted by voltage and current. However, lidocaine inhibits three of the four mammalian HCN isoforms (HCN1 as well as HCN2 and HCN4), all of which are found in neurons and cardiomyocytes11. While it remains unknown if lidocaine inhibition of HCN2 and HCN4 is as sensitive to current and voltage as HCN1, there are noted differences in the inhibition of HCN1 and HCN4 isoforms by ivabradine, with only the latter being prominently current-dependent31. In brain slice whole-cell recordings of rat thalamocortical neurons, we previously found that lidocaine inhibition of Ih (100 μM) was not dependent on voltage and was comparatively potent (IC50 = 72 μM) and near complete; on the basis of the present findings, these differences could be explained by the relatively low extracellular potassium concentrations utilized (2.5 mM)10. Differences in lidocaine inhibition of Ih in thalamocortical neurons versus Ih in HCN1-expressing CHO cells could be due to the different HCN isoforms that are expressed in the former, thought to be mainly HCN2 and HCN460, or to other factors such as other differences in recording conditions and cellular context.The results from our study and others support the probability that lidocaine exerts some its effects h in CHO cells expressing the HCN1 isoform is reduced at hyperpolarizing voltages and inversely correlated with increases in Ih amplitude. Our data, together with data on lidocaine inhibition of Ih in other systems, suggest that lidocaine may diffuse through the plasma membrane, becomes re-protonated inside the cell and inhibits the channel by binding to the inner side of the HCN1 pore, where it interacts with permeant ions. Future studies will be required test this idea and to determine if inhibition occurs in the same way in other members of the HCN channel family and other members of the lidocaine family of drugs.In summary, the results of the present study show that lidocaine inhibition of I


This is roughly twice the estimate of a previous analysis that did not include glaciers and ice caps outside of Greenland and which extended only to 2010. From 2010 onward, the total freshwater flux is about 1,300 km3/yr, equivalent to 0.04 Sv, which is roughly 40% of the estimated total runoff to the Arctic for the same time period. Not all of this flux will reach areas of deep convection or Arctic and Sub‐Arctic seas. We note, however, that the largest freshwater flux anomalies, grouped by ocean basin, are located in Baffin Bay and Davis Strait. The land ice freshwater flux displays a strong seasonal cycle with summer time values typically around five times larger than the annual mean. This will be important for understanding the impact of these fluxes on fjord circulation, stratification, and the biogeochemistry of, and nutrient delivery to, coastal waters.The freshwater budget of the Arctic and sub‐polar North Atlantic Oceans has been changing due, primarily, to increased river runoff, declining sea ice and enhanced melting of Arctic land ice. Since the mid‐1990s this latter component has experienced a pronounced increase. We use a combination of satellite observations of glacier flow speed and regional climate modeling to reconstruct the land ice freshwater flux from the Greenland ice sheet and Arctic glaciers and ice caps for the period 1958–2016. The cumulative freshwater flux anomaly exceeded 6,300 ± 316 km Freshwater has been accumulating in Baffin Bay for 20 years3The freshwater anomaly has reached 6,300 kmImportant source of labile nutrients to coastal water We partition the FWF between solid and liquid phases and resolve the sources at the individual catchment and glacier scale at monthly time steps. Such information is not only important for the large scale hydrography of the surrounding oceans but also for modeling and understanding fjord circulation, ice/ocean interactions  and a solid component (D), here defined as the flux of ice crossing the grounding line of marine‐terminating glaciers around the margins of the GrIS. In sections 2.13 difference in their annual estimates of D for the same epoch  is the product of surface velocity and ice thickness along an outlet glacier flux gate, typically located near and upstream of the grounding line: the junction between the grounded ice sheet and the ocean. Thickness data are generally obtained from ice penetrating radar data measurements, while velocities are derived from synthetic aperture radar interferometry . This provided a monthly time series for the whole ice sheet for 1958, 1964, and 1992–2016. For those years with no observed discharge value we followed the approach in  added to the uncertainty on an individual measurement as the error estimate, which equates to about 11% of the mean D value for the interpolated years .Errors in discharge, where measured ice thickness data are available, are about 2% and 6% where thickness is interpolated  in conjunction with a digital elevation model , which has been statistically downscaled to 1 km from the native resolution of the model of 11 km . This absolute difference in FWF is comparable to, for example, the five‐year variability in P‐E over sub‐polar basins , while less, remain significantly above the mean for the 59 year period studied (972 km3/yr). As noted in the introduction, a reconstruction of the 20th Century surface mass balance for the GrIS, indicates that recent runoff fluxes (since the mid‐2000s) are unprecedented for the last 115 years and their acceleration since 1995 is also unprecedented since at least 1900  that is of more relevance to the hydrography of the Arctic and SNA oceans rather than the absolute change in FWF. To examine the cumulative anomalies, we partitioned the FWF into ocean basins as defined by the International Hydrographic Organization (IHO) . The domain and basins are shown in Figure CAFWF  IHO,  using thCAFWF by ocean basin, alongside the total for all basins, is shown in Figure 3/yr), which we do not discuss further. Not surprisingly, anomalies begin to increase during the mid‐1990s when runoff over Greenland also shows a marked acceleration .The time series of 3/yr although the accelaration since the 1990s is similar. Second, the regional climate model output has been downscaled to 1 km, which provides improved resolution of, in particular, peripheral glaciers and ice caps (PGIC) around the margins of Greenland that are not directly connected to the ice sheet , which contribute a mean FWF of 109 km3/yr over the 59 years of our data but, more importantly, show a marked and steady increase, predominantly into Baffin Bay, from the early 2000s. Fourth, and most importantly for the cumulative anomaly, we have extended the time series from 2010 to the end of 2016. This increases CAFWF by about 2,000 km3  higher than during the early 1990s and still contributing to a positive FWF anomaly to all ocean basins.Four factors are responsible for the differences in the magnitude and characteristics of our new time series when compared to the previous analysis  suggests that the anomaly will exceed 7,000 km3 by 2018 and 10,000 km3 by 2028.Several studies, using ocean GCMs, have explored the potential impact of GrIS melting on the hydrography of the SNA and the strength of the AMOC (Boning et al., n Figure  (Boning Other studies, taking a different approach, have also suggested that the enhanced melting from Arctic land ice is already influencing overturning strength and convection (Rahmstorf et al., To date, however, no freshwater forcing experiments have been undertaken with our new times series and, as a consequence, the potential oceanographic impacts remain somewhat speculative. We aim to address this in a companion paper, in preparation, where the FWF anomalies presented here will be used to force an ocean GCM with one objective being to update the study of Boning et al. , and sim3/month is due to D and increases gradually from the early 1990s. Evidence from ice mélange in two Greenland fjords suggests that the solid ice discharge provides a continuous and dominant FWF to the fjord, except during high summer when runoff can exceed submarine melt rates (Enderlin et al., it is important to note that the FWF is not delivered uniformly in space or time. Significant surface runoff only occurs during summer months, predominantly, from June to August: a fact that has been incorporated in some recent freshwater forcing experiments (e.g., Dukhovskoy et al., Not only is the FWF distributed unevenly geographically but also vertically within the water column. In the case of marine terminating glaciers some proportion of the surface runoff reaches the bed and is released at the base of the glacier (Sole et al., In this paper we derive a new estimate of freshwater fluxes from terrestrial land ice that contributes to the Arctic and North Atlantic Oceans. Compared to a previous assessment for the Greenland Ice Sheet alone, the cumulative freshwater anomaly is about double. This is partly due to the addition of glaciers and ice caps outside of Greenland but also to the additional years considered here, which extends the time series to the end of 2016. Due to limitations in data coverage and model domain, we did not include FWF estimates for the Russian Arctic and discharge changes in Svalbard. We estimate that these omissions represent about 5% of the FWF anomaly we obtained, which is below the uncertainties in the time series. The total cumulative FWF anomaly is close to reaching a value where it will be comparable to the interannual variability in FWF driven by other processes in the climate system (Boning et al.,


The study of autoimmunity mediated by immunoglobulin E (IgE) autoantibodies, which may be termed autoallergy, is in its infancy. It is now recognized that systemic lupus erythematosus, bullous pemphigoid (BP), and chronic urticaria, both spontaneous and inducible, are most likely to be mediated, at least in part, by IgE autoantibodies. The situation in other conditions, such as autoimmune uveitis, rheumatoid arthritis, hyperthyroid Graves’ disease, autoimmune pancreatitis, and even asthma, is far less clear but evidence for autoallergy is accumulating. To be certain of an autoallergic mechanism, it is necessary to identify both IgE autoantibodies and their targets as has been done with the transmembrane protein BP180 and the intracellular protein BP230 in BP and IL-24 in chronic spontaneous urticaria. Also, IgE-targeted therapies, such as anti-IgE, must have been shown to be of benefit to patients as has been done with both of these conditions. This comprehensive review of the literature on IgE-mediated autoallergy focuses on three related questions. What do we know about the prevalence of IgE autoantibodies and their targets in different diseases? What do we know about the relevance of IgE autoantibodies in different diseases? What do we know about the cellular and molecular effects of IgE autoantibodies? In addition to providing answers to these questions, based on a broad review of the literature, we outline the current gaps of knowledge in our understanding of IgE autoantibodies and describe approaches to address them. Friend or Foe? This is the major question that the immune system must address in every individual from the time of conception, through the development of the fetus and the child, right into adulthood. But sometimes it gets this wrong and raises an inappropriate immunological response against self. Although there are several types of autoimmunity this review will address solely immunoglobulin E (IgE)-mediated autoimmunity. Because its mechanisms have much in common with classical allergy involving exogenous allergens, such as grass pollen or house dust mites, IgE-mediated autoimmunity is generally termed “autoallergy” and the target molecules of the response are called “autoallergens.”Although the presence of an allergic antibody has been postulated since the innovative serum transfer experiment of Prausnitz and Kustner in 1921 . IgE, whAlthough IgE has a well-documented role in classical allergy to exogenous allergens, this review will address only IgE-mediated autoimmunity. It will focus on three related questions: what do we know about the prevalence of IgE autoantibodies and their targets in different diseases? What do we know about the relevance of IgE autoantibodies in different diseases? What do we know about the cellular and molecular effects of IgE autoantibodies? In addition to providing answers to these three questions, based on a comprehensive review of the literature, we outline the current gaps of knowledge in our understanding of IgE autoantibodies and describe approaches to address them.Within a decade of its discovery, reports began to incriminate IgE as a possible contributor to the pathogenesis of several chronic inflammatory disorders, such as rheumatoid arthritis (RA) –7, bulloDecades before the discovery of IgE, it was reported that human dander extract can elicit immediate-type skin reactions in patients with severe atopy and that this skin sensitivity could be passively transferred with serum –35. ManyInterestingly, this phenomenon has already been seen in very young infants of less than 1 year where 15% of the children with atopic eczema mounted IgE autoreactivity and raised in the age group of 2–13 years with moderate to severe AE and total IgE serum levels higher 1,000 kU/L up to 80% .In SLE, several IgE autoantigens have been described , 47, andN-methylpurine-DNA glycosylase and CAP-Gly domain-containing linker protein family member 4 in some SLE patients but not healthy controls. These autoantigens were unique in that they seemed specific targets of IgE autoantibodies but not autoantibodies of the IgG class  , 57 althSeveral studies were able to detect IgE autoantibodies against the intracellular BP230 autoantigen. Engineer et al. reported IgE-anti-BP230 without detection of BP180-specific IgE autoantibodies in 100% of six BP patients . In contPemphigus vulgaris (PV) is another severe autoimmune bullous skin disease and is primarily associated with IgG against desmoglein 3 (dsg3), a desmosomal adhesion protein. However, raised IgE in these patients has also been reported . InterceIn mucus membrane pemphigoid, one of four patients showed IgE reactivity to the gamma2 subunit of laminin-332 . In endeIn summary, reports in pemphigus are limited, but IgE reactivity seems to be more prevalent in the endemic form of pemphigus foliaceus than in PV.An important role of IgE and FcεRI reactions was postulated in both CSU and CindU for several decades  before iPerhaps the first evidence of autoreacitivitiy in CSU came with the clinical observation that CSU patients have high rates of thyroid diseases . DetaileBeside thyroid diseases, the prevalence of individual autoimmune diseases in chronic urticaria is increased in general . In a review, the rates of comorbidity were ≥1% for insulin-dependent diabetes mellitus, RA, psoriasis, and celiac disease, ≥2% for Graves’ disease, ≥3% for vitiligo, and ≥5% for pernicious anemia and Hashimoto’s thyroiditis. Interestingly, >15% of CSU patients have a positive family history for autoimmune disease .A recent review investigated also the correlation of chronic urticaria with other autoimmune diseases such as SLE. The prevalence of CSU and CSU-like rash in SLE was investigated from 42 independent studies. Comorbidity in adult patients reportedly ranged from 0 to 22% and 0.4 to 28%, respectively . In children with SLE, CSU was reported in 0–1.2% and CSU-like rash in 4.5–12%  . SpecifiIn vitro studies showed functional relevance, and clinically IgE-anti–IL-24 levels showed an association with disease activity. Detailed analysis of the remaining identified IgE targets had not been provided yet.Beside these IgE antibodies that target antigens known in autoimmune diseases, patients with CSU can also develop IgE autoantibodies against probably disease-specific antigens. Schmetzer et al. found more than 200 IgE autoantigens in patients with CSU that were not found in healthy controls . Of the In many diseases, single or few reports have indicated a presence or potential role of autoreactive IgE.Beside IgG autoantibodies, in autoimmune uveitis specific IgE to retinal S antigen was positive in 69% of 32 patients. In contrast, patients suffering from bacterial uveitis, as well as the healthy controls were negative for autoantibodies to retinal S antigen . FurtherA role of IgE antibodies in RA was first suggested several decades ago , 100. AnA role of IgE antibodies in the disease was suggested, despite the fact that there was no association between MS and allergies . In a stThe presence of IgE autoantibodies in Grave’s disease was first proposed 40 years ago . ElevatiElevated total IgE levels are frequent in patients with autoimmune pancreatitis , 112, anAutoallergic mechanisms have been proposed in murine models of asthma , 114. InAspergillus fumigatus P2 protein . In many investigations, a close correlation between levels of auto-IgG and auto-IgE in the same patients has been shown, making it difficult to estimate the contribution of auto-IgE to the pathology of SLE , 130. HoIn a recent publication, Pan and colleagues have shown that SLE patients not only have high levels of IgE directed against various autoantigens but also strongly decreased numbers of circulating basophils, suggesting auto-IgE-dependent basophil activation in the blood . These aThe largest body of evidence for a pathological role of autoreactive IgE in the literature can be found for BP. In those patients presenting with specific IgE directed against the autoantigen BP180, a correlation of auto-IgE and disease severity has been reported , 60, 63.In a number of case reports and case series, excellent efficacy of treatment with omalizumab has been shown in BP patients –140. WhiThe evidence for a pathogenetic role of auto-IgE in chronic urticaria has been extensively discussed in a recent review . This paex vivo, IL-24 was able to activate mast cells after pre-incubation with serum from IgE-anti-IL-24 positive patients, indicating a biological relevance for IgE-anti-IL-24 in CSU patients  and galectin-3 are capable of binding IgE and inducing cellular responses. Thus, mast cell and basophil activation may be only the tip of the iceberg when considering the biology of IgE autoantibodies and their contribution to diseases. In particular, the function of CD23 in IgE-dependent sensitization of the host to autoallergens, which in turn may further strengthen the production of autoreactive IgE might be of great importance in the development of autoallergies.When looking on the effects of IgE autoantibodies, mast cells and basophils are usually the focus of attention as they express high levels of the high-affinity receptor for IgE (FcεRI). Antigen-dependent activation of mast cells and basophils In general, all IgE, whether it is autoreactive or whether it recognizes exogenous antigens, can trigger the same cellular responses as there is no evidence yet that autoreactive IgE have special molecular characteristics that drive certain functions/responses such as, for instance, cytokinergic activity. However, certain activities are not necessarily triggered by autoreactive IgE such as the CD23-mediated transepithelial transport of IgE. Here, we will focus on IgE-binding receptors with regards to their contribution in autoallergy.10 M−1), but does not contain intracellular signaling motives, one transmembrane-spanning β chain, which crosses the plasma membrane four times and contains an immunoreceptor tyrosine-based activation motive (ITAM), and two identical γ chains each containing an ITAM (de novo synthesis of lipid mediators such as prostaglandins and leukotrienes as well as the production of cytokines and chemokines and the expression of various surface markers. These include chemokine receptors such as CCR7 on plasmacytoid dendritic cells (pDCs) (The high-affinity receptor for IgE (FcεRI) consists of one IgE-binding α chain, which binds IgE with a very high affinity   or the ss (pDCs)  that facs (pDCs) , 130. Fcs (pDCs) . Thus, ain vivo relevance of this “cytokinergic” activity has not been demonstrated yet, it may have an impact in autoallergy. Cytokinergic IgE molecules associate with themselves and show an enhanced propensity to be polyreactive to various autoantigens  that is comparable to that of FcεRI . In FAP, the antigen-loaded IgE-CD23 gets internalized and loaded onto MHC-II molecules for presentation on the B-cells surface . By this, CD23-FAP can lead to epitope spreading, a process where the response to one epitope encountered by the BCR induces epitope-specific immunoglobulin production against the allergen internalized by CD23 (PATH) also binds to the autoantigen  or from B cells that were previously allergen or pathogen reactive?•Does the production of auto-IgE precede the onset of autoallergic signs and symptoms and when and why does it stop?Many questions on IgE autoantibodies remain unanswered.These and other questions are currently addressed by ongoing research. The answers that will come from these and future studies will help to better understand the biology and relevance of IgE autoantibodies in disease and to develop better approaches for the prevention and treatment of autoallergies.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The collagen-binding receptor tyrosine kinase DDR1 (discoidin domain receptor 1) is a drug target for a wide range of human diseases, but the molecular mechanism of DDR1 activation is poorly defined. Here we co-expressed different types of signalling-incompetent DDR1 mutants (‘receiver’) with functional DDR1 (‘donor’) and demonstrate phosphorylation of receiver DDR1 by donor DDR1 in response to collagen. Making use of enforced covalent DDR1 dimerisation, which does not affect receptor function, we show that receiver dimers are phosphorylated in trans by the donor; this process requires the kinase activity of the donor but not that of the receiver. The receiver ectodomain is not required, but phosphorylation in trans is abolished by mutation of the transmembrane domain. Finally, we show that mutant DDR1 that cannot bind collagen is recruited into DDR1 signalling clusters. Our results support an activation mechanism whereby collagen induces lateral association of DDR1 dimers and phosphorylation between dimers.DOI:http://dx.doi.org/10.7554/eLife.25716.001 The membrane surrounding each living cell contains a variety of proteins that carry out different roles. For example, proteins called receptor tyrosine kinases help a cell to receive signals from its external environment. Receptor tyrosine kinases span the membrane so that one part of the protein known as the ectodomain sticks out from the surface of the cell, while another part  sits inside the cell.When a signalling molecule binds to the ectodomain, the kinase domain becomes active and starts to add chemical groups called phosphates to other proteins. This process, known as phosphorylation, changes the protein’s activity, which in turn influences the cell’s behaviour. In most cases, the signalling molecule causes two receptor tyrosine kinase proteins to bind to each other and form a “dimer” in which the kinase domains are able to phosphorylate, and thus activate, each other.Female mammals need a receptor tyrosine kinase called DDR1 to develop mammary glands (the glands that produce milk). DDR1 is activated when a signalling molecule called collagen binds to its ectodomain. Unlike many other receptor tyrosine kinases, DDR1 exists as a dimer even before it binds to collagen, so it is not clear how collagen activates DDR1. One possibility is that collagen causes several DDR1 dimers to form clusters on the membrane so that kinases on neighbouring dimers can phosphorylate each other. Juskaite et al. explored this idea by pairing up normal DDR1 proteins with mutant versions that are unable to bind to collagen.The experiments show that when collagen binds to the normal DDR1 molecules, DDR1 dimers do indeed form clusters. This enables the normal protein molecules in neighbouring dimers to phosphorylate each other as well as the mutant proteins. In this way, the clustered DDR1 dimers can become active even if the clusters contain one or more mutant versions that are unable to detect collagen. Further experiments show that specific contacts need to form between neighbouring dimers for this phosphorylation to occur.Abnormal DDR1 activity is associated with several diseases including cancer, inflammation and fibrosis. The findings of Juskaite et al. suggest that developing new drugs that can prevent DDR1 from forming clusters may help to treat people with these conditions. Further work is also needed to analyse the size and structure of DDR1 clusters and investigate if other proteins also associate with the clusters.DOI:http://dx.doi.org/10.7554/eLife.25716.002 The discoidin domain receptor (DDR) subfamily of receptor tyrosine kinases (RTKs) comprises two members, DDR1 and DDR2. The DDRs regulate cell adhesion, cell migration and differentiation in a number of mammalian tissues . Both DDUniquely among RTKs, the DDRs bind to key structural proteins found in all types of extracellular matrices, namely different types of collagen. Fibrillar collagens are ligands for both DDR1 and DDR2, while non-fibrillar collagens have different DDR preferences, with collagen IV exclusively binding to DDR1 and collagen X preferring DDR2 over DDR1 . The intLike all RTKs, the DDRs are composed of a ligand-binding extracellular region, a transmembrane domain and a cytoplasmic region that contains the catalytic kinase domain. DDR1 and DDR2 share a high degree of homology, in particular in their globular domains. The extracellular DDR region contains two globular domains: an N-terminal, ligand-binding discoidin domain that is tightly linked to a discoidin-like domain . These gA crystal structure of the inactive DDR1 kinase revealed a typical bilobal architecture, with kinase autoinhibition through interactions of the activation loop with the active site . The DDRThe DDRs differ from typical RTKs by several unusual features, the mechanistic bases of which are poorly understood. One of their puzzling characteristics is the slow kinetics of collagen-induced autophosphorylation, which in some cell types can take hours to reach a maximum and then remains detectable for more than a day . AnotherBecause the DDRs are constitutively dimerised in the absence of ligand, the paradigm of ligand-induced RTK dimerisation  cannot aHere we show evidence for a mechanism that is compatible with ligand-induced clustering of DDR1 dimers. Co-expression studies using a range of signalling-incompetent DDR1 mutants with different forms of signalling-competent DDR1 and DDR2 showed that collagen-induced DDR1 activation involves phosphorylation in trans between neighbouring DDR1 dimers. Phosphorylation between DDR1 dimers occurs both on the juxtamembrane region and the kinase activation loop and can be elicited by different types of ligand. Moreover, phosphorylation in trans requires specific contacts between the transmembrane domains, but not between the ectodomains. These findings define a unique mechanism of transmembrane signalling by DDR1.In this study we explored the mechanism of ligand-induced phosphorylation of DDR1 by co-expression of signalling-defective DDR1 mutants with signalling-competent DDR1. Receptor phosphorylation was visualised by Western blotting with a phospho-specific antibody (Ab) against a key tyrosine phosphorylation site in the intracellular juxtamembrane region, tyrosine-513 . DDR1 haThe signalling-defective DDR1 mutants had mutations in the ectodomains but intact cytosolic juxtamembrane regions and kinase domains. As detailed below, co-expression with signalling-competent DDR1 resulted in ligand-induced phosphorylation of the signalling-defective mutants, which on their own could not be phosphorylated in response to collagen. Hence, in our co-expression experiments, we use the terms ‘donor kinase’ for the signalling-competent DDR1 and ‘receiver kinase’ for the signalling-defective mutants, respectively. In order to distinguish the cytoplasmic domains of receiver and donor DDR1 we replaced tyrosine-513 in the donor receptor with a phenylalanine, thereby creating DDR1b-Y513F, which cannot be phosphorylated at this residue . This alWe first confirmed expression and collagen-induced activation of DDR1b-Y513F in transiently transfected HEK293 cells. Collagen-induced autophosphorylation of the activation loop (anti-pY792) was detected at similar levels as in wild-type DDR1b, whereas, as expected, no signal was detectable for DDR1b-Y513F with the Ab against phosphorylated tyrosine-513  did not impair the ability of DDR1b-Y513F to promote collagen-induced phosphorylation of the ectodomain deletion mutant  resulted in efficient collagen-induced tyrosine-513 phosphorylation of the signalling-incompetent receiver dimers . A more So far we monitored activation of DDR1 mutant receiver dimers by tyrosine-513 phosphorylation. Blotting with phospho-specific Abs against the activation loop tyrosines, tyrosine-792 or tyrosine-796, showed that DDR1 receiver dimers were also phosphorylated on the activation loop , indicatThe requirement for kinase activity of the donor kinase was addressed by co-expression of the ectodomain deletion dimer, DDR1b-ΔECD-Cys, as the receiver kinase, with the kinase-dead form of DDR1a, DDR1a-KD, as the donor kinase . NeitherIn addition to stimulation by authentic collagen ligands, DDR1 phosphorylation can also be induced by an activating Ab, mAb-513 . This moFibrillar collagens are triple-helical proteins that assemble into fibrillar structures in tissues. In our co-expression experiments we incubated cells with soluble collagen I, but we cannot rule out that small fibrils form during the 90 min incubation times. These fibrils could provide multiple binding sites for DDR1, thereby clustering the receptor. We previously showed that DDR1 activation could be induced by collagen-mimetic synthetic triple-helical peptides encompassing a DDR1 binding motif . BecausePhosphorylation between DDR1 dimers requires close apposition of DDR1 molecules, which is likely a result of ligand-induced receptor clustering. We used immunofluorescence to study collagen-induced DDR1 redistribution on the cell surface. Eph receptors are RTKs that require clustering by ligand for receptor activation . It was DDR1 lacking its ectodomain cannot bind ligand. Expression of DDR1b-ΔECD in Cos-7 cells did not result in DDR1b-ΔECD recruitment to collagen-coated beads, as expected . HoweverHere we demonstrate an activation mechanism for DDR1 that is distinct from that of all other RTK families. Previous work showed that DDR1 forms constitutive dimers (or higher oligomers) in the absence of ligand  ruling oOur experiments with disulphide-linked constructs clearly demonstrate that inactive DDR1 mutants can be phosphorylated in a dimeric state. Phosphorylation of a key juxtamembrane tyrosine in the receiver construct (Y513) requires kinase activity of the donor but not of the receiver kinase. These data indicate a mechanism where the juxtamembrane regions of DDR1 dimers act as substrates for the kinase activity of neighbouring DDR1 molecules, rather than being phosphorylated exclusively through kinase activity within the same dimer. In addition to juxtamembrane phosphorylation, the activation loop of the receiver kinase was phosphorylated in our co-expression studies, again independently of receiver kinase activity, indicating that the activation loop can also act as a substrate for neighbouring DDR1 dimers. Activation loop phosphorylation is a major regulatory mechanism that releases cis-autoinhibition of kinases, including the majority of RTKs . The DDRCo-clustering with, and phosphorylation by, catalytically active DDR1 was independent of ectodomain contacts but required specific transmembrane domain interactions. These data reinforce a key role for the DDR1 transmembrane domain in signalling. DDR1 transmembrane helices have the highest propensity for self-association amongst all RTKs , and DDRDDR1 activation in our co-expression experiments was achieved with fibrillar and non-fibrillar collagens, a collagen-mimetic peptide, as well as by clustering with a multimeric anti-DDR1 mAb. The fact that a short collagen-mimetic peptide efficiently induced signalling between DDR1 dimers suggests that DDR1 clustering is receptor-mediated, rather than achieved through cross-linking via the ligand. While most RTKs are thought to signal as dimers, ligand-induced clustering is a key step in signalling of the Eph family of RTKs . DDR1 acInterestingly, signalling between DDR1 dimers did not require identical cytoplasmic regions and was also observed with DDR2 as the donor kinase. These findings have functional implications in that catalytically incompetent DDR1 isoforms  could coRecent studies have shown that oligomerisation beyond dimerisation also plays a role in signalling of RTKs that have traditionally been assumed to signal exclusively as dimers. Thus, EGF binding to the EGF receptor can result in formation of tetramers and higher-order oligomers, which may boost autophosphorylation of C-terminal tail tyrosines . However2, 100 units/ml penicillin, 100 µg/ml streptomycin, 10% foetal calf serum, 5 mM L-glutamine, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 0.1 nM cholera toxin, and 10 ng/ml epidermal growth factor. All cells were grown at 37°C, 5% CO2. Cells were tested for mycoplasma contamination every four months and were confirmed negative.Human embryonic kidney (HEK) 293 cells, monkey Cos-7 fibroblast-like kidney cells, and human breast cancer MCF-7 cells were from ATCC, Manassas, VA, and were cultured in Dulbecco’s modified Eagle’s medium/F12 nutrient mixture  supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin and 10% foetal bovine serum. Human breast cancer T47D cells were obtained from the Cell Production Laboratory, Imperial Cancer Research Fund, UK, and were cultured in Roswell Park Memorial Institute medium (Invitrogen) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin and 10% foetal bovine serum. MCF-7 cells  and T47D cells were only used as example cell lines with endogenous DDR1 expression, for comparison of their DDR1 surface levels to those in our HEK-293 cell expression system; their authenticity was not confirmed. Neonatal normal human epidermal keratinocytes  were cultured on a mitomycin C-treated feeder layer of 3T3 fibroblasts in DMEM/F12 medium supplemented with 1.8 mM CaClN-(9-fluorenyl)methoxycarbonyl) chemistry as a C-terminal amide on TentaGel R RAM resin in an Applied Biosystems Pioneer automated synthesizer and purified as described  was obtained from Fisher Scientific . Collagen I  and collagen IV  were purchased from Sigma . The collagen-mimetic DDR-selective peptide, (GPP)5-GPRGQOGVNleGFO-(GPP)5GPC-NH2, was obtained from Prof. Richard Farndale, University of Cambridge, UK. The peptide was synthesized by Fmoc ; goat anti-DDR1 extracellular domain Ab ; goat anti-DDR2 (AF2538 from R&D Systems); rabbit anti-phospho-DDR1 (Tyr513) and rabbit anti-phospho-DDR1 (Tyr792) from Cell Signalling ; rabbit anti-phospho-DDR2 (Tyr740) from R&D Systems; mouse anti-Flag IgG1 clone M2 (Sigma); rabbit anti-Flag (Sigma); and mouse anti-tubulin (Sigma). Anti-phospho-DDR1 (Tyr513), here referred to as anti-pY513, is a phospho-specific Ab that recognises phosphorylated tyrosine-513 in DDR1b. Anti-phospho-DDR1 (Tyr792), here referred to as anti-pY792, recognises phosphorylated tyrosine-792 in the activation loop of DDR1a and DDR1b. Anti-phospho-DDR2 (Tyr740), here referred to as anti-pY796, recognises phosphorylated Tyr740 in the activation loop of DDR2 and cross-reacts with the equivalent residue in DDR1, phosphorylated tyrosine-796. Mouse anti-DDR1 mAbs, 7A9 and IF10, were generated in our lab . The mul23DYKDDDKSGSAEGSPTA417ILI . An N-terminally Flag-tagged DDR1b cDNA, cloned into pGEM-3Z vector, was used as a template. DDR1b-ΔECD-Cys was generated by introducing the T416C mutation using the QuickChange method. DDR1b-ΔECD-TM1 and DDR1b-ΔECD-Cys-TM1 were generated by introducing the L430G/L431P mutation  for transient transfection in cells. Restriction and modification enzymes were purchased from New England Biolabs  or Promega . All PCR-derived DNA constructs were verified by DNA sequencing. PCR primers used for generating the mutant constructs can be obtained on request.HEK293 cells were grown in 24-well (for DDR1 autophosphorylation assays) or 6-well (for flow cytometry and comparison of protein expression levels) tissue culture plates and transfected with relevant DDR1 expression plasmids by calcium phosphate precipitation. Cos-7 cells were grown on coverslips in 24-well tissue culture plates and transfected with Fugene HD  according to the manufacturer’s instructions.The assay was performed as described before . BrieflyHEK293, T47D and MCF7 cells were grown in 6-well tissue culture plates and primary keratinocytes were grown in 10 cm dishes. HEK293 cells were transfected with varying amounts of DDR1b expression plasmid. Cells were serum-starved for 16 hr and lysed as above. Equal amounts of protein were analysed by reducing SDS-PAGE on 7.5% polyacrylamide gels. This was followed by blotting onto nitrocellulose membranes. The blots were probed with anti-DDR1 Ab followed by horseradish peroxidase conjugated goat-anti-rabbit secondary Ab, or anti-tubulin Ab followed by horseradish peroxidase conjugated sheep-anti-mouse secondary Ab. Signal detection was performed as above.Cos-7 cells transiently expressing the ectodomain deletion constructs, DDR1b-ΔECD, DDR1b-ΔECD-TM1, DDR1b-ΔECD-Cys or DDR1b-ΔECD-Cys-TM1, which contain N-terminal Flag tags, were washed with PBS, and incubated with mouse anti-Flag IgG1 for 1 hr on ice to label cell surface DDR1 constructs. This was followed by washing, fixing with 4% paraformaldehyde (Sigma) in PBS for 15 min at room temperature, blocking in 8% BSA in PBS for 1 hr, and incubation with anti-mouse IgG1 Alexa-Fluor-488 secondary Ab. Cos-7 cells transiently expressing DDR1b wildtype were stimulated with 10 µg/ml collagen I or a mixture of 10 µg/ml collagen and 10 µg/ml mouse anti-DDR1 7A9 IgG1 mAb, for 10 min at 37°C. The cells were washed with PBS, and incubated with mAb 7A9 for 1 hr on ice to label cell surface DDR1. This was followed by fixation and incubation with secondary Ab as above. The coverslips were mounted on slides with Prolong Gold Antifade Reagent (Invitrogen). The cells were examined using an Olympus BX-51 widefield microscope.For detection of cell surface expression of DDR1 ectodomain deletion mutants, HEK293 cells in 6-well tissue culture plates were transfected with N-terminally Flag-tagged DDR1 ectodomain deletion expression plasmids  and grown for 48 hr before being dissociated and resuspended in PBS containing 1% BSA. The cells were incubated for 30 min on ice with rabbit anti-Flag Ab in 100 μl of PBS/BSA. Cells were then washed three times with PBS/BSA and incubated with goat anti-rabbit Alexa-Fluor-488 Ab for 30 min on ice. After three washes as above, the cells were resuspended in 2% formaldehyde in PBS. For comparison of surface DDR1 expression, HEK293, T47D and MCF7 cells were grown in 6-well tissue culture plates and primary keratinocytes were grown in 10 cm dishes. HEK293 cells were transfected with varying amounts of DDR1b expression plasmid. Cells dissociation, Ab staining and fixation were done as above, except that the primary Ab was anti-mouse DDR1 (mAb 7A9) and the secondary Ab was FITC-conjugated goat-anti-mouse IgG. Data were collected on a BD LSRFortessa cell analyser using BD FACSDiva software 6.0 (BD Biosciences) and further analysed on FlowJo software 10.2 .3 µm diameter latex beads (Sigma) were washed in distilled water, followed by washes in coating buffer  and incubation with 20 µg/ml collagen I in coating buffer overnight at 4°C on a rotating wheel. Control beads were coated with 20 μg/ml BSA. The beads were then washed in PBS, blocked in 1% BSA in PBS for 1 hr, and re-suspended in serum-free medium for use on cells.Cos-7 cells transiently expressing wild-type DDR1b were grown on coverslips and incubated with serum-free medium for 16 hr. Cells were then stimulated with collagen-coated beads or control BSA-coated beads for the indicated time at 37°C. After stimulation, cells were washed with PBS, and incubated with mouse anti-DDR1 7A9 IgG1 mAb for 1 hr on ice to label surface DDR1. This was followed by washing in PBS, fixing with 4% paraformaldehyde in PBS for 15 min at room temperature, washing in PBS, permeabilisation with 0.1% Triton X-100 (Sigma) in PBS for 5 min, blocking in 8% BSA in PBS for 1 hr, and incubation with rabbit anti-pY513 Ab. After washing, the cells were then incubated with anti-mouse IgG Alexa-Fluor-488 and anti-rabbit Alexa-Fluor-555 secondary Abs. Cos-7 cells transiently expressing the ectodomain deletion construct DDR1b-ΔECD , DDR1b-Y513F or both were stimulated with collagen-coated beads for 2 hr at 37°C. After stimulation, cells were washed with PBS, and incubated with mouse anti-Flag IgG1 and mouse anti-DDR1 ectodomain IF10 IgG2b Abs for 1 hr on ice to label surface DDR1b-ΔECD and DDR1b-Y513F, respectively. This was followed by washing, fixing, permeabilisation, blocking, and incubation with rabbit anti-pY513 Ab as described above. The cells were then incubated with anti-mouse IgG1 Alexa-Fluor-488, anti-mouse IgG2b Alexa-Fluor-555 and anti-rabbit Alexa-Fluor-647 secondary Abs. The coverslips were mounted on slides with Prolong Gold Antifade Reagent (Invitrogen). The cells were examined using an Olympus BX-51 widefield microscope.All experiments were performed at least three times with similar results. Blots show representative examples. Intensities of protein bands were quantitated using ImageJ software. All data are expressed as mean ± standard error of the mean (SEM) from at least three independent experiments. Statistical significance was tested with a Kruskal-Wallis multiple comparison test, followed by Mann-Whitney tests using GraphPad Prism 7 . Statistical significance was set at a p value < 0.05. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Fiona Watt (Senior Editor) and three reviewers, one of whom, Reinhard Fässler (Reviewer #1), is a member of our Board of Reviewing Editors.Thank you for submitting your article "Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The findings of your paper show that (1) ligand binding-induced clustering leads to the activation of DDR dimers, (2) active DDR dimers phosphorylate neighbouring DDR dimers in the juxtamembrane and activation loop, (3) the ectodomain is not required for these phosphorylation events in the receiving neighbouring DDR dimers, and (4) the TM domain interaction is essential for donor as well as neighbouring receiver DDR dimers. Despite some clear strengths of the submitted manuscript, the research has some important shortcomings and limitations, which the authors should address in a revised submission:Essential revisions:1) It is necessary to exclude that over-expressed DDR does not lead to artificial aggregates, How do the overexpressed surface levels compare to wild type level? Key experiments could be repeated at low expression levels if overexpressed levels are indeed much higher than endogenous levels.2) It is necessary to show functional relevance (e.g. adhesion) of DDR clustering.3) The dimer size is unclear. Confirm the dimer size in SDS-PAGE with appropriate molecular size markers. Indicate molecular weights in gels and blots.4) The authors use cysteine mutants to further separate phosphorylation within dimers and across different dimers. It is shown that for inter-dimer phosphorylation the receiver requires specific transmembrane sequences and the donor requires catalytic activity. Although activation loop phosphorylation can also occur across dimers, it appears that intra-dimer phosphorylation of the activation loop is more efficient . This ineLife. Your revised article has been favorably evaluated by Fiona Watt (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for resubmitting your work entitled "Collagen induces activation of DDR1 through lateral dimer association and phosphorylation between dimers" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance.Your figure provided to the reviewers demonstrates that DDR function prevents formation of larger aggregates. This figure should be included into the manuscript to underline the functional relevance of your findings.We also believe that activation loop phosphorylation across dimers occurs. However, your data suggest that the more efficient mechanism occurs within dimers. The weak dimer phosphorylation exclusively occurs across dimers (inter-dimer), whereas the strong monomer phosphorylation is the sum of intra and inter dimer phosphorylation, suggesting that this sum is dominated by intra-dimer phosphorylation. There is no evidence that inter-dimer phosphorylation of the activation loop is the major event or relevant. As long as phosphorylation within dimers occurs at least as efficient as across dimers, lateral association and activation loop phosphorylation is not important.Since the paper focuses on Y513 phosphorylation, it is not necessary to show that lateral association is responsible for activation loop phosphorylation. However, due to the two points above  to be activating nor (2) relevant for activation loop phosphorylation.) the statement 'Our data suggest that DDRs are activated by a mechanism in which collagen binding promotes lateral association of DDR1 dimers, which induces activation loop phosphorylation and thereby releases cis-autoinhibition of the kinase domain.' should be toned down and re-phrased appropriately. Essential revisions:1) It is necessary to exclude that over-expressed DDR does not lead to artificial aggregates, How do the overexpressed surface levels compare to wild type level? Key experiments could be repeated at low expression levels if overexpressed levels are indeed much higher than endogenous levels.We agree with the reviewers’ concern and have repeated key experiments at low DDR1 expression levels that clearly demonstrate dimer phosphorylation of receiver kinases, similar to the results of our original experiments. The new data rule out that the phosphorylation between neighbouring DDR1 dimers is due to aggregation of DDR1 at artificially high expression levels.DDR1 is expressed in a wide range of tissues but no systematic study has been carried out to compare expression levels of endogenous DDR1 in different cell types. Immortalised cancer cell lines have varying levels of surface DDR1. We used flow cytometry to compare surface levels of DDR1 in our HEK293 cell expression system with the levels on primary human keratinocytes and two commonly used breast cancer cell lines. The new data  show thaBecause our experimental approach relies on co-expression of mutant DDR1 constructs, our experiments can only be done under transfection conditions. Collagen-induced DDR1 phosphorylation is a characteristically slow process, which would be expected to occur with faster kinetics if over-expression lead to a different form, or enhanced, levels of DDR1 aggregation. However, this is not the case, further arguing against an activation mechanism that is dependent on very high levels of DDR1 expression.2) It is necessary to show functional relevance (e.g. adhesion) of DDR clustering.In a previous publication  we identified a number of function-blocking anti-DDR1 antibodies we believe are interfering with DDR1 clustering. Blocking DDR1 clustering by other means is likely to result in a similar complete inhibition of ligand-induced DDR1 phosphorylation, with functional consequences for any cellular process that relies on DDR1 kinase activity.In unpublished experiments that go beyond the scope of the current manuscript, we have shown that collagen binding to DDR1 leads to re-distribution of DDR1 on the cell surface from a punctate into a more compact structure see . This ph3) The dimer size is unclear. Confirm the dimer size in SDS-PAGE with appropriate molecular size markers. Indicate molecular weights in gels and blots.In a previous publication on disulphide-linked DDR1 constructs (including DDR1-T416C used in the present study), we showed blots of non-reducing gels with molecular mass markers up to 460 kDa . From these blots, it can be seen that full-length DDR1-Cys dimers are ~240 kDa, compared with ~120 kDa for DDR1 monomers on the same blot. The data shown in the new Figures  are show4) The authors use cysteine mutants to further separate phosphorylation within dimers and across different dimers. It is shown that for inter-dimer phosphorylation the receiver requires specific transmembrane sequences and the donor requires catalytic activity. Although activation loop phosphorylation can also occur across dimers, it appears that intra-dimer phosphorylation of the activation loop is more efficient . This inThere is indeed strong activation loop phosphorylation at the position of donor DDR1a or DDR1-Y513F , but we With regards to the activation loop phosphorylation signals shown in The reviewers would like us to clarify whether activation loop phosphorylation is due to inter-dimer phosphorylation or phosphorylation within a dimer. The experiments that we present in Our experimental design does not allow us to rule out that collagen-induced intra-dimer phosphorylation occurs. In fact, one might speculate that for closely apposed DDR1 dimers in signalling clusters, there is no functional difference between kinase domains that are linked via the transmembrane domain and those that are not. Due to the unusually large and unstructured intracellular juxtamembrane region, it is conceivable that within a signalling cluster all DDR1 kinase domains are at equal distance from one another.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance.Your figure provided to the reviewers demonstrates that DDR function prevents formation of larger aggregates. This figure should be included into the manuscript to underline the functional relevance of your findings.The revised manuscript now contains the requested figure as new We also believe that activation loop phosphorylation across dimers occurs. However, your data suggest that the more efficient mechanism occurs within dimers. The weak dimer phosphorylation exclusively occurs across dimers (inter-dimer), whereas the strong monomer phosphorylation is the sum of intra and inter dimer phosphorylation, suggesting that this sum is dominated by intra-dimer phosphorylation. There is no evidence that inter-dimer phosphorylation of the activation loop is the major event or relevant. As long as phosphorylation within dimers occurs at least as efficient as across dimers, lateral association and activation loop phosphorylation is not important.Since the paper focuses on Y513 phosphorylation, it is not necessary to show that lateral association is responsible for activation loop phosphorylation. However, due to the two points above  to be activating nor (2) relevant for activation loop phosphorylation.) the statement 'Our data suggest that DDRs are activated by a mechanism in which collagen binding promotes lateral association of DDR1 dimers, which induces activation loop phosphorylation and thereby releases cis-autoinhibition of the kinase domain.' should be toned down and re-phrased appropriately.As requested, the sentence has been re-phrased in the amended manuscript.


Phoca vitulina richardii). The availability and spatial distribution of glacier ice in the fjords is likely a key environmental variable that influences the abundance and distribution of selected marine mammals; however, the amount of ice and the fine-scale characteristics of ice in fjords have not been systematically quantified. Given the predicted changes in glacier habitat, there is a need for the development of methods that could be broadly applied to quantify changes in available ice habitat in tidewater glacier fjords. We present a case study to describe a novel method that uses object-based image analysis (OBIA) to classify floating glacier ice in a tidewater glacier fjord from high-resolution aerial digital imagery. Our objectives were to (i) develop workflows and rule sets to classify high spatial resolution airborne imagery of floating glacier ice; (ii) quantify the amount and fine-scale characteristics of floating glacier ice; (iii) and develop processes for automating the object-based analysis of floating glacier ice for large number of images from a representative survey day during June 2007 in Johns Hopkins Inlet (JHI), a tidewater glacier fjord in Glacier Bay National Park, southeastern Alaska. On 18 June 2007, JHI was comprised of brash ice (2 (SD = 2.6 m2). We estimate the total area (± uncertainty) of iceberg habitat in the fjord to be 455,400 ± 123,000 m2. The method works well for classifying icebergs across scenes (classification accuracy of 75.6%); the largest classification errors occur in areas with densely-packed ice, low contrast between neighboring ice cover, or dark or sediment-covered ice, where icebergs may be misclassified as brash ice about 20% of the time. OBIA is a powerful image classification tool, and the method we present could be adapted and applied to other ice habitats, such as sea ice, to assess changes in ice characteristics and availability.Tidewater glaciers are glaciers that terminate in, and calve icebergs into, the ocean. In addition to the influence that tidewater glaciers have on physical and chemical oceanography, floating icebergs serve as habitat for marine animals such as harbor seals ( Tidewater glaciers, or glaciers that terminate in the ocean, are a prominent landscape feature in southeastern and south-central Alaska and play an important role in landscape and ecosystem processes. Changes in tidewater glaciers are cyclical in nature, with advance rates typically an order of magnitude slower and occurring over much longer time periods than retreats e.g., ,2). Thou. Thou2])Phoca vitulina richardii) to rest, whelp, nurse young, molt, and avoid predators )Herein, we present a case study with glacier ice habitat in an Alaskan tidewater glacier fjord to describe a novel method that uses OBIA to quantify the amount and fine-scale characteristics of glacier ice from high resolution aerial imagery. Our primary objective was to develop a semi-automated method to estimate ice habitat from high spatial resolution airborne imagery collected of floating glacier ice in a tidewater glacier fjord. Specifically, we (i) developed workflows and rule sets to classify airborne imagery of glacier ice using object-based image analysis; (ii) quantified the amount and fine-scale characteristics of floating glacier ice in the fjord; (iii) developed processes for automating the object-based analysis of ice for a large number of digital images; and (iv) present results describing ice characteristics and accuracy assessments of classification from a representative survey day during June 2007. The described method could be applied to ice habitats, including sea ice, in subpolar and polar regions, that are used by other pagophilic species.Aerial surveys were conducted under NOAA Fisheries Marine Mammal Protection Act permit #358-1787-01, and scientific permits issued by Glacier Bay National Park.2 of marine waters )Eumetopias jubatus) in Alaska, dugongs (Dugong dugon) in nearshore marine habitats in Australia, and penguins and pinnipeds in Antarctic [The OBIA technique that we have described could also be applied to imagery collected by unmanned Aerial System (UASs), which are relatively new low-cost platforms that can be used to quantify wildlife and the habitats that they use. For example, UASs have been successfully used to photograph several species and their habitats in remote regions including Steller sea lions (ntarctic –44.Ursus maritimus).This study demonstrates that OBIA is a method that can be used to assess the availability of glacier ice for seals and for quantifying the fine-scale characteristics of ice in tidewater glacier fjords. An understanding of the amount and fine-scale features of icebergs will be essential for assessing how future changes in glacier calving rates may influence ice habitat that is used by harbor seals in tidewater glacier fjords. The method described here can be applied in future studies to assess seasonal and inter-annual changes in the availability of icebergs, and also used to provide quantitative estimates of iceberg characteristics that can be used in habitat and population trend models for harbor seals or other pagophilic species. The approach we have presented could be broadly applied to quantify iceberg characteristics in other tidewater glacier fjords, as well as other ice habitats such as sea ice, in order to investigate the effects of changing ice cover on other pagophilic species such as penguins and polar bears (


Supplemental Digital Content is available in the text Left ventricular non-compaction cardiomyopathy (LVNC) is a rare heart disorder related to thrombosis. Anticoagulant therapy is suggested for the treatment of this disease. The success of the novel oral anticoagulant rivaroxaban as a treatment option for this disorder is unclear.A 43-year-old man who felt dizzy at rest was found to have an intraventricular thrombus.The thrombus was confirmed by echocardiography. And LVNC was diagnosed by cardiac magnetic resonance (CMR) and echocardiography.He was prescribed a low dose (10 mg daily) of rivaroxaban as treatment.After 3 months, the thrombus diminished, and the manifestation disappeared.Low dose of rivaroxaban may serve as a viable option for anticoagulation therapy in LVNC patients, with large clinical trials needed to determine the best course of treatment. The clinical diagnosis is based mainly on cardiac imaging including echocardiography, cardiac computed tomography (CT), as well as cardiac magnetic resonance (CMR), which are useful to identify and characterize the hyper-trabeculation of the heart.,7 Lacking a clear etiology, the therapy typically consists of a combination of heart failure, anti-arrhythmia, and anti-thrombosis treatment.Left ventricle non-compaction cardiomyopathy (LVNC) is characterized by “spongy” LV myocardium, abnormal trabeculation, and deep intratrabecular recesses resembling the endocardial surfaces of an early embryonic heart.,9 which necessitates the need for antiplatelet and/or anticoagulation therapy. However, the low prevalence of LVNC renders the efficacy of anti-thrombolytic therapy in the context of LVNC unclear. The majority of case reports have adopted a warfarin anticoagulant treatment strategy in LVNC. As the era of novel oral anticoagulations (NOACs) has arrived, NOACs may offer a safer and more convenient alternative therapeutic approach in LVNC patients. Specifically, rivaroxaban have been shown to provide effective thromboprophylaxis and a reduced risk of recurrent thromboembolism, without a significant increase in bleeding rates. Moreover, the benefits of rivaroxaban have been shown at low doses (10 mg) that are typically associated with prophylaxis. While rivaroxaban has proven effective in thrombus treatment postinfarction at doses >15 or 20 mg daily, there are no reports or clinical trials studying whether low-dose rivaroxaban may be effective at managing the thromboembolic risks associated with LVNC.The thromboembolic risks associated with LVNC are well-established,2A 43-year-old Asian male, with a 71-kg bodyweight and normal renal function, presented in the Cardiology Department at the China-Japan Union Hospital of Jilin University. He was found to have a mass in the left ventricle in May 2017 during his examination. The patient felt dizzy at rest without falling, spinning, or tinnitus. Dizziness persisted for 1 day and a routine echocardiographic examination discovered “suspected cardiac masses” in left ventricle, which led admission to our hospital. The patient suffered a brain injury 20 years prior, which caused an old trauma in a brain MRI examination without a novel ischemic lesion. The physical examination showed no abnormal signs on the heart.In our center, the echocardiography found a thrombosis-like mass in left ventricle, which was confirmed by cardiac acoustic contrast, and the patient presented with diminished systolic function characterized by an ejection fraction (EF) as low as 43%. The size of the mass was measured as 1.10 cm × 2.43 cm  such as rivaroxaban have shown therapeutic advantages to the commonly administered warfarin during systemic anticoagulation, including improved safety, reduced risk of bleeding, and convenience during therapy. However, more studies are warranted to prove rivaroxaban efficiency and safety in specific thrombosis-related conditions such heart failure or LVNC. Moreover, the ideal dose of rivaroxaban also warrants further consideration. Normally, the dose of rivaroxaban suggested for treating thrombosis, not prophylaxis, is >15 or 20 mg daily. In the present patient, only 10 mg of rivaroxaban was necessary for efficient thrombosis treatment, which suggests such a low dose might be enough for LVNC or early LVNC, in which the cardiac function as well as the blood flow within left ventricle are relatively preserved. Though our case is just one example, such an experience may inspire or imply a potential approach to treat thrombus in LVNC, a rare heart disease, before large clinical trials can be used to guide the therapy.As the thromboembolic risk in LVNC is well known, the choice of anti-thrombosis therapy in these patients is clear.4LVNC is a rare heart disease associated with obvious thromboembolic risk. In our case report, we have shown the success of low dose rivaroxaban in the anticoagulation therapy in LVNC patients. However, it is clear that the antithrombotic strategy for this disease requires more attention.