Cystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Mutations associated with CF cause loss-of-function in CFTR leading to salt imbalance in epithelial tissues. Kalydeco  was approved for CF treatment in 2012 but little is known regarding the compound’s interactions with CFTR including the site of binding or mechanisms of action. In this study we use hydrogen/deuterium exchange (HDX) coupled with mass spectrometry to assess the conformational dynamics of a thermostabilized form of CFTR in apo and ligand-bound states. We observe HDX protection at a known binding site for AMPPNP and significant protection for several regions of CFTR in the presence of Kalydeco. The ligand-induced changes of CFTR in the presence of Kalydeco suggest a potential binding site. CFTR is an essential part of salt homeostasis in epithelial tissues. In the lungs, loss-of-function CFTR mutations cause thicker mucus that is difficult to clear and increases susceptibility to bacterial infection2. Chronic infection leads to lung trauma and eventual organ failure. In addition to pulmonary effects, CF patients also suffer from pancreatic deficiency, poor nutrient absorption, and sterility in males2. There are 242 known CF-causing mutations in CFTR, though as many as 92% of patients have a deletion of phenylalanine 508 (ΔF508) on at least one allele (https://www.cftrscience.com/cftr-mutations). Common CF-causing mutations were used for a phenotypic screen that resulted in the development of two novel small molecule therapeutics, Kalydeco and Lumacaftor  that aid the majority of patients3. Although not curative, Kalydeco and Lumacaftor are a breakthrough in CF therapy as they demonstrate defects associated with CFTR can be targeted by molecular therapeutics.Loss-of-function mutations to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) cause Cystic Fibrosis (CF), a condition affecting 4. CFTR is the only known ABC transporter that is a chloride selective ion channel, coupling ATP-binding to the gating of chloride down its electrochemical gradient. CFTR has two transmembrane domains (TMDs), two nucleotide binding domains (NBDs) and a Regulatory Domain (RD) connecting NBD1 and TMD2. RD phosphorylation (by Protein Kinase A [PKA] and other kinases7) enables channel opening. CFTR activity is also controlled by the binding of ATP, which is thought to cause dimerization of the NBDs8. Mutations that affect CFTR gating are known to cause CF, including the two most common, ΔF508 and G551D. Although the mechanism remains unclear, evidence suggests the CF-therapeutic Kalydeco increases channel opening by an ATP-independent mechanism after RD phosphorylation10.CFTR is a member of the C-subgroup of ABC transporters, which typically couple ATP hydrolysis to the transport of ligands against their concentration gradient11; however, the site of binding, and how it affects the function and stability of CFTR are unknown. In a purified liposome ion flux assay system, Eckford and colleagues showed Kalydeco could enhance phosphorylated wild type and mutant CFTR-dependent ion flux. This effect was ATP-independent, but in the wild type and ΔF508 the responses were additive with ATP. Interestingly, ion flux in the G551D mutant was not enhanced by adding nucleotide. Furthermore, Kalydeco can potentiate channels that completely lack NBD213 as well as a number of other CF mutants with gating defects15. These have led to the hypothesis that Kalydeco may act by reducing the energy barrier to channel opening via binding somewhere in the TMDs9 or TMD/NBD interfaces16, thereby facilitating the conformational changes associated with channel opening in a mechanism independent of ATP-induced activation.Kalydeco potentiates CFTR21. Aleksandrov et al. showed AMPPNP bound preferentially at site 122 and Weinreich et al. observed slowed opening kinetics when ATP was applied with AMPPNP, implying that AMPPNP could bind to the closed state and in fact was inhibitory at site 123. Zerhusen et al. and Mathews et al. noted inhibition of AMPPNP in the presence of ATP when AMPPNP was added at millimolar concentrations, presumably because it begins to out-compete ATP for site 125. It is also interesting to note that in the crystal structure of the related bacterial ABC transporter TM287/288 the bound AMPPNP was only found at site 1, with the protein adopting a closed (inward-facing) conformation26. However, additional studies are needed to clarify the structural effect that AMPPNP has on CFTR.To date, there have been no reports to show the combined effect of AMPPNP and Kalydeco. In addition the CFTR literature has not yet clarified the effect that AMPPNP has on the channel, and some conflicting results exist, but in general they lead to the conclusion that this ATP analog may be inhibitory when bound to site 1 only. First, many labs have shown that AMPPNP fails to activate the channel without a small amount of ATP28. Integral membrane proteins present significant technical challenges for HDX, such as the production of suitable protein samples and the ability to achieve sufficient sequence coverage. Prior to this work, HDX experiments were performed on isolated NBD1 of CFTR to compare the stability of wild type versus ΔF508 constructs29 and investigate the effect of Lumacaftor binding30. Recently, HDX was successfully employed to compare the open and closed conformations of the bacterial ABC transporter BmrA31. Here we apply HDX to a full length human CFTR construct and find differential effects by two ligands on the phosphorylated form of the protein. The non-hydrolysable ATP analog AMPPNP imparts protection to exchange in the nucleotide’s binding site. We were able to detect significant protection to exchange by Kalydeco in close proximity to F508. AMPPNP diminishes conformational changes imparted by Kalydeco which would support AMPPNP acting as an inhibitor. This study represents the first application of HDX to characterize ligand binding interactions of a full length mammalian membrane ABC transporter. In addition this work provides a structural mechanism of action for Kalydeco’s ability to potentiate CFTR and suggests a potential binding site.Hydrogen/deuterium exchange (HDX) mass spectrometry is a well-established tool for characterizing protein conformational dynamics and protein-ligand binding interactions40 and enabled these HDX studies on active phosphorylated CFTR, rather than the non-phosphorylated form used for recent structural studies41. This is hereafter referred to as thermostabilized CFTR (hCFTRTS). With this construct we observe an approximate 5-fold increase in expression over wild type in transiently transfected HEK Expi293 cells . Protein purified from Expi293 cells is partially phosphorylated, but can be dephosphorylated with Lambda Phosphatase treatment and subsequently phosphorylated with PKA  that were confirmed by mass spectrometry , but was about 45-fold slower compared to activity measured in the presence of lipids or digitonin44  of hCFTRTS in the presence and absence of ligands. Since this dye detects denatured protein by associating with hydrophobic surfaces it cannot normally be used for determining the Tm of membrane proteins because of the necessary presence of detergent, which increases the SO background fluorescence. By decreasing the detergent concentration to the CMC , the background fluorescence was small enough to clearly observe the unfolding transition  dye assay was used to measure the melting temperature  compared to published results for wild type42, consistent with the observed increase in expression  in the presence of 1 mM AMPPNP  dephosphorylated hCFTRTS. HDX analysis covered 60% of the sequence  PKA phosphorylated full length hCFTRe Figure  at all ee Figure . Low seqTS was monitored over six time points ranging from 10 s to 1 h , which are predominantly formed by NBD1 and NBD2. Binding at two sites has also been shown for ADP23. Both sites have interactions contributed from multiple motifs including the phosphate-interacting Walker A motif 8. The two ATP-binding sites exhibit distinct binding and hydrolysis behavior; site 1 has higher affinities for ATP and AMPPNP than site 2 and is unable to hydrolyze ATP due to mutations in the NBD1 Walker B motif and NBD2 signature sequence47. AMPPNP caused protection to deuterium exchange for peptides at site 1, with the greatest protection for residues 456–468 (wild type hCFTR sequence numbering) of the Walker A motif  and the average protection of these peptides (from 18% to 12%)  Fig. . While A508 Figs  and 5. Tm measurements and HDX was achieved by combining previously identified mutations and deletions proven to enhance CFTR expression and stability. This stabilized construct enables future biophysical studies that require milligram quantities of protein.Efforts to isolate full length hCFTR for biophysical characterization have been hindered by low expression even in mammalian cell systems. Purification of hCFTR in quantities suitable for both TTS construct by Kalydeco  of the thermostabilizing mutations are in NBD1, which may artificially rigidify hCFTRTS NBD1 compared to native NBD1. The four intracellular coupling helices that connect the TM segments and are positioned in parallel to the plane of the membrane demonstrated rapid deuterium exchange rates  without ATP also present. These results therefore support previous studies that AMPPNP alone is not an activator at these concentrations though it does appear to bind at site 121. It is unclear if the lack of CFTR activity previously reported is due to the apparent lack of binding at site 2, or if AMPPNP binding at site 1 is actually inhibitory, or both. These data support a model for AMPPNP-bound CFTR where the channel remains closed and AMPPNP is only present at site 1 ([AMPPNP] ≤400 µM).The nucleotide binding experiments showed the greatest protection for peptides in ATP-binding site 1. These results suggest HDX is suitable for detecting direct ligand-binding interactions for full length CFTR. Interestingly, no protection was observed for site 2, suggesting AMPPNP may not be a strong binder at this site, which is consistent with prior reports for AMPPNP at site 262. This could indicate a compound binding site and this region has been previously proposed as a CFTR site for rational drug design62. Interestingly, F508 makes van der Waals interactions with residues from ICL4 and TM11 in this region. The ΔF508 deletion is the most common CF-causing mutation. Kalydeco is effective against this mutation and the HDX protection at ICL4 provides a structural molecular mechanism of action for potentiation of CFTR ΔF508. Given the proximity of ICL4 to F508 Kalydeco may cause protection to HDX in this region as well. Unfortunately an ion signal for the peptic peptide in this region was not reproducibly observed in these HDX experiments. ICL4 resides between ICL1 and F508 and it is interesting to note that ICL1 also shows protection to HDX in the presence of Kalydeco, albeit to a lower extent which could indicate allosteric stabilization in a region with close proximity to the potential binding site. ICL1 has also been reported to modulate CFTR channel activity via interactions with NBD163. A number of mutational studies show this ball and socket joint region is involved in interdomain communication between NBD1 and the TMDs65. Cysteine cross-linking experiments revealed interactions between F508 and several aromatic residues from ICL4, including F1068, Y1073 and F107465. Deletion of F508 would disrupt such interactions, therefore destabilizing the ICL4/NBD1 interface and potentially the whole channel. Stabilization of ICL4/NBD1 by HDX upon Kalydeco binding provides a mechanism of action to account for the drug’s ability to increase channel activity of ΔF508. The HDX data with Kalydeco is also suggestive of a potential binding site for the drug though it is important to note that regions protected in an HDX experiment can also indicate allosteric conformational changes induced by compound binding.The region with the largest decrease in deuterium uptake in the presence of Kalydeco is in ICL4 of the ball and socket joint978, a site known to cause ATP-independent, constitutive activity when mutated to cysteine, serine, or proline66. Both the human homology model and the phosphorylated zebra-fish structure of the open CFTR conformation shows these residues may make new contacts across the pore with ICL467  of AMPPNP at 25 °C can lead to weak channel opening, but no opening was observed at lower AMPPNP concentrations, including concentrations similar to what were used in these HDX experiments72. Therefore, when interpreted in the context of the literature, our HDX data suggest AMPPNP can act as a site 1 binder and an inhibitor of CFTR. Under this model, binding of both compounds would be expected to shift CFTR conformation back to the closed state compared to Kalydeco alone. Allosteric conformational changes would be diminished while protection at the binding sites would remain. In fact, protection to exchange is only observed for two regions in the presence of both compounds, the TMD2/NBD1 ball and socket region and ATP-binding site 1. This would further support the ball and socket region of TMD2/NBD1 as the Kalydeco binding site.To date, there have been no reports to show the combined effect of AMPPNP and Kalydeco. The HDX data with both AMPPNP and Kalydeco showed that AMPPNP-interacting peptides remained protected although most of the regions that were protected by Kalydeco alone showed reduced or no protection Figure . This re11. Although HDX cannot unambiguously identify binding sites, the regions identified in this study (e.g. the ICL4/NBD1 interface) are consistent with current theories16, suggesting potential binding site locations for Kalydeco. This work also presents an HDX platform for characterizing and comparing the molecular mechanisms of compound action for future CF drug discovery programs.The results discussed here illustrate the utility of HDX for determining how ligands bind to and affect the dynamic structure of CFTR in solution. HDX has been used to confirm the binding site of model CFTR ligands, AMPPNP and ADP, and identified conformational changes induced by the drug Kalydeco. This supports Kalydeco’s ability to directly bind CFTR and its effects are independent of activation by ATP6-FLAG tagged fusion. Quikchange (Agilent) mutagenesis was used to modify the sequence to include selected thermostabilizing mutations .Wild type human full length CFTR  DNA was codon optimized for mammalian cell expression and synthesized by Genewiz into a pUC57 vector. CFTR was then subcloned into a modified pCDNA3.1 vector, yielding an N-terminally SUMO* tagged, C-terminally EGFP-HisTS) was expressed in the Expi293 suspension cell line (Invitrogen) using the manufacturer’s instructions with some modifications. Cells were transfected at 2.5 × 106 viable cells per milliliter with Expifectamine and 10 µg of DNA per 30 mL transfection  volume and grown 16–18 hours on an orbital shaker in an incubator set to 37 °C and 5% CO2. Enhancers 1 and 2 were subsequently added to the transfection and expression was continued for an additional 48 hours under the same conditions. Cells were harvested by centrifugation at 1850 g for 20 minutes at 4 °C, washed with ice cold PBS, and spun again. Cell pellets were then flash frozen in liquid nitrogen and stored at −80 °C until purification.Thermostablized, full length human CFTR  40 times on ice. Broken cells were then centrifuged at 40,000 rpm in a 70 Ti rotor in a Beckman Ultracentrifuge for 25 minutes at 4 °C. Pelleted material was dounce homogenized in solubilization buffer  40 times on ice and then stirred for 1.5 hours at 4 °C. Solubilized membranes were then spun again for 45 minutes. Soluble material was passed over a packed 10 mL anti-FLAG M2 resin (Sigma) column connected to a Bio-rad FPLC system and washed with FLAG Wash Buffer  until the UV280 absorbance dropped and stabilized. Bound protein was eluted with 20 mL FLAG Elution buffer . Purified hCFTRTS was treated with Lambda Phosphatase (NEB) for 30 minutes at room temperature before addition of 2 mL of Talon resin (Clontech) and incubated for an additional 30 minutes. The slurry was then poured into a 30 mL gravity column (Bio-rad) where unbound material was allowed to flow through. Talon resin with bound protein was washed with 5 column volumes of Talon Wash Buffer  and either eluted with 3 column volumes of Talon Elution Buffer  or treated with PKA (NEB) for 1 hour at room temperature with mixing. PKA treated, resin-bound protein was washed again with 5 column volumes of wash buffer and subsequently eluted. TEV and ULP1 proteases were then added to the eluted protein overnight at 4 °C. Cleaved protein was concentrated  and injected into a Superose 6 10/300 gel filtration column equilibrated in SEC Buffer . Peak fractions were combined and concentrated, followed by determination of concentration via 280 nm absorbance  before aliquoting. Aliquots were flash frozen in liquid nitrogen and stored at −80 °C until use. Protein purity was verified using Bio-Rad Mini-PROTEAN TGX Stain-Free SDS-PAGE gel analysis. Presence or absence of phosphorylation was assayed in the same gel with Pro-Q Diamond Phosphoprotein Gel Stain (Molecular Probes).To purify hCFTRTS was measured using the Sypro Orange fluorescent dye (Molecular Probes). Recombinant hCFTRTS (15 µg/ml) was mixed with dye (5×) and indicated ligands in assay buffer . Reactions were incubated at room temperature for 10 minutes in 200 µl quartz cuvettes (Hellma) prior to fluorescence measurement. The Sypro Orange fluorescence signal was monitored every 0.5 °C at 300 nm excitation and 600 nm emission using a temperature controlled Cary Eclipse Fluorescence Spectrophotometer between 30–90 °C . Curves were differentiated and fitted to a Lorenzian distribution to determine the melting temperature with each measurement performed in triplicate.Thermal melting temperature for hCFTRTS was measured using the UNit (Unchained Labs). Recombinant hCFTRTS (100 µg/ml) and indicated ligand (or vehicle) was diluted in assay buffer . Reactions were incubated at room temperature for 30 minutes prior to addition of 8.9 µL (with three replicates per condition) to the UNi quartz capillary strip. The full fluorescence spectrum (250–700 nm) and SLS signals were monitored continuously using lasers at 266 and 473 nm as the temperature was increased at a rate of 1 °C per minute between 20–85 °C. SLS signal versus temperature for each sample are plotted together in Supplementary Figure Thermal aggregation for hCFTR2, 0.03% DDM). Recombinant hCFTRTS at 0.5 µM was combined with 20/30 U PK/LDH, 250 µM NADH, and 2 mM PEP. The plate was incubated in the reaction mixture for 10 minutes at room temperature, after which the reaction was started by the addition of ATP (5 µM). Plates were assayed at 25 °C in a Tecan microplate reader (Infinite M1000), in which the absorbance at 340 nm was monitored every 30 seconds for 60 minutes. ADP concentration was determined using NADH absorption and initial velocities were calculated.ATPase activity measurements were performed using the PK/LDH Assay following the manufacturer’s instructions in a black 384-well, low-volume, clear-bottom plate (Corning type 3544) in a total volume of 20 µl in assay buffer . The buffer for HDX on-exchange experiments had the same composition except H2O was replaced with D2O (99.9%) (referred to as D2O buffer hereafter). A total of 5 µL of 5 µM hCFTRTS protein solution was manually mixed with 20 µL of the H2O buffer and 25 µL of the quench solution containing 100 mM Na2HPO4, 0.02% DDM and 1% (v/v) TFA. The above 50 µL protein sample was then digested online by passing through an immobilized pepsin-coupled column  and the digest was subjected to high performance liquid chromatography (HPLC) analysis using a trap column , an analytical column  and 1/16” o.d. × 75 µm i.d. PEEKsilTM tubing . Sample loading, digestion, and desalting were driven by a capillary-scale HPLC pump , while gradient elution was performed with a separate HPLC pump of the same make and model. For HPLC, buffer A was H2O containing 0.1% (v/v) formic acid, buffer B was acetonitrile containing 0.1% (v/v) formic acid. The flow rate was 50 µL/min with a gradient from 0.5% to 40% B over 60 min. The mobile phase for the immobilized pepsin-coupled column was buffer A and the flow rate was 50 µL/min. MS and collision-induced dissociation (CID) MS/MS spectra were acquired in positive ion mode on an Orbitrap Velos Pro hybrid ion trap-Orbitrap mass spectrometer  equipped with an ESI source operated at capillary temperature of 250 °C and spray voltage of 4 kV. Mass spectra were acquired for m/z 300–2000 using a maximum injection time of 10 ms and automatic gain control (AGC) target value of 1 × 106. MS/MS spectra were acquired using data-dependent acquisition with dynamic exclusion where the top 10 most abundant ions in each scan were selected and subjected to CID. MS/MS spectra were acquired at an activation Q value of 0.25, activation time of 30 ms, normalized collision energy of 35, isolation width of 2 m/z, AGC target value of 1 × 104 and maximum injection time of 100 ms. Each MS scan consisted of 1 microscan under normal scan mode and each MS/MS scan was the average of 4 microscans under normal scan mode.For the tandem mass spectrometry (MS/MS) sequence coverage experiment, the purified protein (25.7 µM) was diluted to 5 µM using a buffer composed of 50 mM Tris-HCl, 150 mM NaCl, 2% (v/v) glycerol, 0.03% (m/v) DDM, 2.5 mM MgClTS sequence using MASCOT  with a peptide mass tolerance of ± 10 ppm and a fragment mass tolerance of ± 0.8 Da. Peptides with an ion score of greater than 20 and mass error of less than 3 ppm were included in the peptide set used for HDX.The resulting spectra were searched against the hCFTRTS stock into 84.4 µL H2O buffer, then adding 2.1 µL 0.1 M Tris-HCl or 2.1 uL 20 mM AMPPNP or ADP followed by 2.1 µL DMSO or 5 mM Kalydeco. The final Kalydeco concentration was 100 µM, a 20-fold excess over hCFTRTS, to promote saturating conditions. The final concentrations of AMPPNP or ADP were 400 µM. The protein samples were allowed to equilibrate at 4 °C for 1 h before HDX analysis. For HDX MS experiments, 5 µL of 5 µM hCFTRTS solution was mixed with 20 µL of the D2O buffer , or H2O buffer for controls, and incubated for various lengths of time at 4 °C, before being added to 25 µL quench solution. Deuterated protein was then digested online using the same procedure as described above. The pepsin column, trap column and analytical column utilized in the HDX MS experiments were the same as in the MS/MS sequence coverage experiment. HPLC was performed with a flow rate of 50 µL/min, and a gradient proceeded by a 0.5% to 10% B over 0.2 min ramp followed by a 10% to 30% B gradient over 5.3 min. MS spectra were acquired in the range of m/z 300–1700 in positive ion mode on the Orbitrap Velos Pro mass spectrometer  equipped with an ESI source operated at capillary temperature of 250 °C and spray voltage of 4 kV. Protein and quench solutions, trap and analytical columns were kept at 4 °C inside of the chromatography refrigerator . The online pepsin column was kept at 15 °C using a temperature controller . HDX incubation was performed at 4 °C for 6 different time points: 10, 30, 60, 300, 900, and 3600 s. The experiments were performed at random order and the data for each on-exchange time point were obtained in 3 replicates. All HDX data were normalized to 100% D2O content, corrected for an estimated average deuterium recovery of 70%, and processed using the HDX WorkBench .Before each HDX experiment, 5 µM protein samples were made by diluting 16.4 µL 32 µM hCFTRThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


The NEGIS was ~20–70 km behind its present ice-extent ~41–26 ka and ~7.8–1.2 ka; both periods of high orbital precession index and/or summer temperatures within the projected warming for the end of this century. We show that the NEGIS was smaller than present for approximately half of the last ~45 kyr and is susceptible to subtle changes in climate, which has implications for future stability of this ice stream.The sensitivity of the Northeast Greenland Ice Stream (NEGIS) to prolonged warm periods is largely unknown and geological records documenting such long-term changes are needed to place current observations in perspective. Here we use cosmogenic surface exposure and radiocarbon ages to determine the magnitude of NEGIS margin fluctuations over the last 45 kyr (thousand years). We find that the NEGIS experienced slow early Holocene ice-margin retreat of 30–40 m a The outlet glaciers that comprise the Northeast Greenland Ice Stream (NEGIS) have experienced accelerated retreat in recent years, yet their longterm stability remains unclear. Here, via cosmogenic surface exposure and radiocarbon ages, the authors investigate the stability of the NEGIS for the past 45 kyr. A prominent feature of the GrIS is the Northeast Greenland Ice Stream (NEGIS)3, which constitutes an ~600-km-long ice stream that drains ~12% of the interior GrIS via three fast-flowing marine-terminating outlet glaciers: Nioghalvfjerdsfjord Gletscher (NG), Zachariae Isstrøm (ZI), and Storstrømmen Gletscher (SG)  scenarios7. To assess these modern observations and modeling scenarios, as well as the possibilities of future NEGIS collapse, a long-term perspective is urgently needed to understand the (in)stability of the NEGIS.Greenland Ice Sheet (GrIS) mass loss has doubled since the beginning of the 20th century8. High-resolution multibeam swath bathymetry and shallow seismic data from the shelf offshore of the NEGIS show a number of glacial landforms including mega-scale glacial lineations suggesting that the ice sheet margin extended to the shelf edge in Northeast Greenland12. The age of the landforms is, however, poorly constrained due to a lack of marine sediment cores from the area. It is assumed that they were formed during the LGM as they appear fresh and are not draped with significant sediment cover. Farther north at 81°N, the reduction in ice-rafted debris and lower sedimentation rates in marine sediment cores suggest that deglaciation from the shelf began ~20 ka13, whereas the shelf areas at Kejser Franz Josef Fjord (72°N) and Scoresby Sund (70°N) ~700–1000 km farther south experienced regional deglaciation ~17–19 ka17. Radiocarbon dates of postglacial marine shells from the coastline outside NEGIS indicate deglaciation around 9.7–9.1 ka; the later deglaciation of this area compared to adjacent areas to the north and south has been used to suggest more extensive glaciation reaching the shelf edge18. However, these 14C dates are only minimum-limiting ages, which are often significantly younger than 10Be ages of deglaciation in Greenland19. These limitations highlight the need for better age constraints to resolve the long-term ice-margin fluctuations of NEGIS.During the global Last Glacial Maximum , the GrIS reached the continental shelf in Northeast Greenland, but it has been contentious whether it reached the shelf edge ~250–350 km from the present ice margin or remained on the inner shelf Fig. 8. High-r10Be) on glacial boulders with radiocarbon dates (14C) from reworked marine shells in moraines to reconstruct the last 45 kyr of NEGIS-margin fluctuations (see Methods). In addition, we investigate the climate forcings that may drive long-term ice-margin variability on these time scales. We find that the NEGIS experienced slow early Holocene ice-margin retreat of 30–40 m a−1 from the outer coast to the present ice margin, likely as a result of the buttressing effect of sea or shelf ice. We furthermore show that the NEGIS was smaller than present ~41–26 ka and ~7.8–1.2 ka, or approximately half of the last ~45 kyr as a result of air and ocean temperature forcings similar to or slightly higher than present.Here we combine cosmogenic surface exposure ages  in Northeast Greenland (77.4–79.4°N) during the last deglaciation  ka (uncertainty in parentheses includes the production rate uncertainty). Adjacent to the present ice margins at Blåsø, Lambert Land, ZI, Søndre Mellemland, and Bloch Nunatakker we dated eighteen samples. With one outlier excluded, we obtained a mean deglaciation age of 9.3 ± 0.2 (0.4) ka. Our 10Be ages demonstrate that the deglaciation of the outer coast to present ice margin, a distance of 60–100 km, was completed within ~2.4 kyr at an average retreat rate of 30–40 m a−1.Cosmogenic exposure dating is a widely used method to constrain the timing of deglaciation in Greenlandion Fig. . By assu29. We have combined new and existing 14C dates from NEGIS’s three marine terminating outlet glaciers in the study area  and the Holocene  in moraines is a method often used to infer times when the ice margins in Greenland were further inland than at presentrea Fig. . Overall10Be ages demonstrate that deglaciation of the outer coast to the present ice margin occurred between 11.7 ± 0.6 and 9.3 ± 0.4 ka. This is ~2 kyr older than the oldest recorded 14C dates of ~9.7 ka from the outer coast18. The timing of early Holocene NEGIS retreat from the coast to its current extent generally coincides with both increased surface air and subsurface ocean temperatures  were 4–5 times slower than during the Holocene36 . Approximately 41–26 ka estimated summer temperatures were ~6–8 °C warmer than the LGM primarily because of higher boreal summer insolation, but ~8–12 °C colder than the preindustrial period , when the 10Be ages indicate that the areas in front of the present ice margin were again deglaciated. The timing of maximum LGM extent of NEGIS agrees with a Northeast Greenland marine record that places the ice margin on the continental shelf ~26–20 ka13.The e36 Fig. . Howeveriod Fig. . The comiod Fig.  seems to14C dates of marine shells and whale bones from the left-lateral margin of NG show that the floating ice margin was smaller than present extent and reached a minimum of at least 70 km behind its present extent ~7.8–4.6 ka27. At SG, reworked shells in Little Ice Age moraines suggest that it was smaller than present ~5.4–1.2 ka28. The timing of the retracted ice margin of the NEGIS outlet glaciers ~7.8–1.2 ka generally agrees with a smaller than present GrIS extent during the mid-Holocene thermal maximum seen in southern Greenland38. The timing of retracted NEGIS also coincides with local chironomid-based temperature maxima ~8–5 ka from Store Koldeway in Northeast Greenland39 and warm subsurface temperatures in Fram Strait32  for the end of this century Fig. . In cont42. Topographic relief ranges between 0 and 500 m but locally mountains are up to ~1000 m high. The landscape is characterized by selective linear erosion; signs of glacial erosion are significant, particularly at lower elevations. During the LGM, the ice sheet advanced on to the continental shelf in Northeast Greenland45, but it has been contentious whether it reached the shelf edge ~250–350 km from the present ice margin or remained on the inner shelf . We used the CRONUS-Earth online calculator49, the Arctic production rate50, and time invariant scaling of Lal/Stone52 to calculate sample ages , 11.7 = mean 10Be age (in ka); 0.037 = the uncertainty associated with the Arctic production rate50 and “St” scaling52.σ standard error of the mean (in kyr); 9.3 = mean 10Be age (in ka), 0.037 = the uncertainty associated with the Arctic production rate50 and “St” scaling52.In 2015 and 2016 we conducted fieldwork using helicopter and twin otter plane. We selected field sites using aerial and satellite imagery. Most samples were collected from boulders perched on ice scoured bedrock, except for three samples from Kap Amelié collected from boulders on drift and on Lambert Land where we collected two boulder samples on a moraine outside the Little Ice Age moraine Fig. . We aime10Be inherited from a previous period of exposure47.We excluded two outliers based on the most general knowledge of the regions glacial history that are older than the LGM  and most likely contain 14C analysis; all contained enough carbon for AMS radiocarbon measurement. All radiocarbon ages have been calibrated into calendar years using IntCal1355 and a reservoir age of 550 years (∆R = 150)56 in Oxcal 4.357. The 14C ages are reported with 2-sigma uncertainty  temperatures are more relevant than mean-annual temperatures when considering past margin positions. For the last 22 kyr Greenland ice core δ15N-based temperature reconstructions59 were merged with climate model simulations62 to generate Greenland-wide, seasonally resolving temperature reconstructions31. Supplementary Fig. oN, 20oW). Coupled ocean atmosphere GCM simulations are not available through MIS 3; therefore the same approach cannot be used to investigate summer temperatures during that time period. Instead, we rely on a multiple regression approach in which it is assumed that three key forcings dominate the Greenland temperature evolution: AMOC strength, greenhouse gas radiative forcing, and local orbital forcing. Summer temperature at the site (TJJA) is then given by:2 dry mixing ratio in ppm, a1 through a3 are linear scaling coefficients. The CO2 mixing ratio is converted to radiative forcing using the approach by ref. 63, with a pre-industrial reference concentration of 280 ppm. All forcings are shown in Supplementary Figs. TJJA, we use a multi-ice core 64, and insolation values calculated following ref. 65. The AMOC strength is the most uncertain of the three forcings and is reconstructed as follows. We start from the Greenland Summit67 δ18O record (average of GRIP and GISP2 δ18O records) corrected for mean ocean δ18O68, and convert it to site  temperature using an effective isotope sensitivity of α = 0.29 ‰ K−159. Using the logic underlying Eq. 2 forcing (b2 = 3.05 Km2 W−1) and insolation  from the GISP2 temperatures, where the stated  sensitivities were obtained from the single-forcing deglacial GCM simulations of ref. 61, in which greenhouse gas and orbital forcings were applied separately. It is then assumed that remaining temperature variability is due solely to AMOC variability, which we estimate using a sensitivity  of b1 = 1.07 ± 0.25 K Sv−1, which optimizes the correlation to the reconstruction by ref. 31, as shown in Supplementary Fig. a1 = 0.238 ± 0.1 K Sv−1, a2 = 3.76 ± 1.5 Km2 W−1, and a3 = 0.137 ± 0.03 Km2 W−1; these coefficients are found using multiple regression analysis on the 0-22ka NEGIS JJA reconstruction; the coefficients are in good agreement with those found from the single-forcing coupled GCM experiments of ref. 61. The MIS 3 JJA reconstruction is shown in Supplementary Fig. Greenland ice cores provide detailed records on the timing and magnitude of past mean-annual temperature change. However, GrIS mass loss occurs during the summer monthsThe data that support the findings of this study are available in the supplementary information or it can be acquired from the corresponding author on request.Supplementary Information


Since then, structure–activity relationships of indoles as CB1R modulators have been reported. Targeting the allosteric site on CB1R, new families structurally based on urea and on 3-phenyltropane analogs of cocaine have been discovered as CB1R-negative allosteric modulators (NAMs), respectively, by Prosidion and by the Research Triangle Park. Endogenous allosteric ligands of different nature have been identified more recently. Thus, the therapeutic neuroprotection application of lipoxin A4, an arachidonic acid derivative, as an allosteric enhancer of CB1R activity has been confirmed in vivo. It was also the case of the steroid hormone, pregnenolone, whose negative allosteric effects on Δ9-tetrahydrocannabinol (Δ9-THC) were reproduced in vivo in a behavioral tetrad model and in food intake and memory impairment assays. Curiously, the peroxisome proliferator-activated receptor-γ agonist fenofibrate or polypeptides such as pepcan-12 have been shown to act on the endocannabinoid system through CB1R allosteric modulation. The mechanistic bases of the effects of the phytocannabinoid cannabidiol (CBD) are still not fully explained. However, there is evidence that CBD behaves as an NAM of Δ9-THC- and 2-AG. Allosteric modulation at CB1R offers new opportunities for therapeutic applications. Therefore, further understanding of the chemical features required for allosteric modulation as well as their orthosteric probe dependence may broaden novel approaches for fine-tuning the signaling pathways of the CB1R.In 2005, the first evidence of an allosteric binding site at the CB One traditional way of designing new drugs is the so-called one target–one disease approach. Indeed, this is the case of many commercially available drugs, which interact with enzymes, receptors, or ionic channels among others. One of the most important targets are the G-protein-coupled receptors (GPCRs) for which agonists and antagonist/inverse agonists have been developed.2 and structural knowledge on how these interactions take place has advanced on the basis that some cocrystals of a ligand bound to an allosteric site have been obtained.3These traditional ligands bind to the same site as the endogenous ligand, the so-called orthosteric site. However, recently, research has shifted to compounds that can interact with a different region of the receptor, termed the allosteric site, since this approach may result in pharmacological advantages such as higher specificity and thus reduced side effects. The best known mechanisms of action of these allosteric ligands are activation or inhibition of the receptor signaling, referred to as positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs). There are also neutral or silent ligands, silent allosteric modulators, which do bind to the allosteric site without affecting the response of the endogenous agonist. In addition, bitopic ligands that bind to both the orthosteric and allosteric sites have been described. In the last years, there has been considerable research in allosteric modulation of GPCRs both in academia and industry,4 and also pain for which PAMs of the μ-opioid receptor have been proposed.5In addition to two allosteric modulators marketed for HIV and hyperparathyroidism, recent potential therapeutic applications of allosteric modulators of GPCRs include central nervous system (CNS) disorders such as neurodegenerative diseases or schizophrenia1 and CB2 receptors (CB1R and CB2R), for which thousands of traditional ligands, belonging to many different chemical structures, have been synthesized and evaluated. However, this has had only limited success in bringing them to the clinic. In fact, only three natural derived medicines  are available largely due to their unwanted side effects.A particular kind of GPCRs are the cannabinoid CB6–9 According to structural features, CB1R allosteric modulators reported to date can be classified as follows: indole derivatives; urea derivatives; and structures acting as endogenous ligands and miscellaneous structures.One possibility to overcome the limitations of these traditional cannabinoids interacting with the orthosteric sites would be to use allosteric cannabinoid ligands. Despite the considerable research undertaken on traditional cannabinoid agonists and antagonists, there have been comparatively few reports on allosteric cannabinoid ligands. In this review, we will give an overview of the chemical structures that have been tested and proven to be allosteric cannabinoid ligands, but will not deeply deal with the pharmacological aspects of cannabinoid allosterism, which has been covered elsewhere.1R was revealed in 2005 by pharmacological studies realized on three indoles from the company Organon Research, Org27569, Org29647, and Org27759 3–6–7 region. Org27569 acts by modulating receptor activity through conformational changes in the receptor. The authors described how Org27569 sterically blocked movements of the extracellular loops and TMH6 that are involved in receptor activation. Recently, site-directed fluorescence labeling studies reported by Fay et al.15 showed that effectively TMH6 movements are associated with G-protein activation, whereas its attached helix eight could be involved in the binding of arrestin triggering the biased signaling pathway.The Org27569-binding site has been determined by Shore et al.1R. However, it was only in 2012 that Piscitelli et al.16 reported that the first indole-2-carboxamides structurally correlated with Org27569. The very close derivative of Org27759, the indole 1  studies were reported by various research groups confirming that indole-2-carboxamide was a good scaffold for developing allosteric modulators for CBindole 1 , was fou17–19 Among them, it is worthy to mention compound 2, the 3-pentyl-1H-indole-2-carboxamide derivative  that were isolated using supercritical fluid chromatography. Difference in pharmacology potencies of both enantiomers could be appreciated.After a first generation of Org27569 derivatives, new series of 2-phenyl-122 reported the pharmacological properties of 6-methyl-3-[2-nitro-1-(thiophen-2-yl)ethyl]-2-phenyl-1H-indole -stimulated signaling in [35S]-GTPγS binding, β-arrestin recruitment, and extracellular signal-regulated kinase (ERK) phosphorylation assays. A very interesting finding is that PAM effects of ZCZ011 could be confirmed by in vivo assays. The indole ZCZ011 exhibited antinociceptive effects in neuropathic and inflammatory pain models with no associated cannabimimetic effects.Ignatowska-Jankowska et al. ZCZ011; . ZCZ011 23 from the University of Aberdeen applied the bioisosteric replacement of carboxamide by sulfonamide leading to numerous N-arylalkyl-1H-indole-2-sulfonic amides claimed in a patent. Inventors reported that ABD1027 . Thus, this effect could be attributable to a potential probe dependence of lipoxin A4. Adding to the complexity, recent studies realized by Khajehali et al.13 on lipoxin A4 could not corroborate the PAM modulatory effects on either AEA- or CP-55,940-mediated cAMP inhibition in CHO-CB1R cells.One of these molecules is lipoxin A4 , an oxyg1R is the steroid hormone pregnenolone -induced ERK1/2 phosphorylation in CHO-CB1R cells. Furthermore, these NAM effects on Δ9-THC were reproduced in vivo in a behavioral tetrad model, in food intake and memory impairment assays. In contrast, in the neuronal model reported by Staiker et al.,11 pregnenolone failed to modulate 2-AG of synaptic transmission. Likewise, Khajehali et al.13 reported lack of modulatory effect of pregnenolone on either Δ9-THC- or WIN55,212–2-induced activation of ERK1/2 phosphorylation. These divergent data suggest probe- or pathway-dependent allosteric effects.Another putative endogenous allosteric modulator at the CBnenolone , a hydro30 described a 12-amino acid sequence, structurally corresponding to an N-terminal extended form of hemopressin, as a potent CB1R NAM of orthosteric agonist-induced cAMP accumulation, [35S]-GTPγS binding, and receptor internalization. This polypeptide, named pepcan-12, has been recently localized in discrete cells of the CNS and adrenal gland.31 It is worth mentioning that this endocannabinoid peptide was previously reported to interact with CB1R as an agonist.32 Furthermore, the NAM profile of pepcan-12 has been confirmed in the DSE study of 2-AG synaptic transmission performed by Staiker et al.11Until recently, synthetic or endogenous compounds acting on the endocannabinoid system (ECS) were of lipid nature. In 2012, Bauer et al.1R allosteric modulation.The interest gained in this field led to the identification of an assorted range of other small molecules that also display CB1R.33 These molecules had been previously characterized as dopamine transporter (DAT) inhibitors.34 Among this series, the tropanes, RTI-371 and RTI-370, and the benztropine JHW007 (1R activity in a cell-based calcium mobilization assay. RTI-371 was found to be of special interest because it is a potent and selective DAT inhibitor that lacks cocaine-like behavioral effects and abuse liability. According to the authors, these atypical cocaine-related effects could be due to positive allosteric modulation of the CB1R.33 This study highlights the potential of the 3-phenyltropane scaffold for the development of novel CB1R PAMs.In 2009, researchers from the Research Triangle Park screened several 3-phenyltropane analogs of cocaine for their activity at the CBe JHW007  stand ou1R binding site; however, it has been shown to display antagonism of CB1R agonists in vitro.35–37 More recently, Laprairie et al.38 provided evidence that CBD behaved as an NAM of Δ9-THC- and 2-AG-dependent CB1R internalization, β-arrestin recruitment, and phospholipase C β3- and ERK1/2-phosphorylation. These results may explain some of the in vivo effects of this promising nonpsychoactive compound providing novel insights in the intriguing pharmacology of CBD.The nonpsychoactive phytocannabinoid cannabidiol CBD;  has show39 (40 reported the pharmacological profile of fenofibrate at CBRs. [35S]-GTPγS binding experiments revealed partial agonism of fenofibrate at CB1R and full agonism at CB2R. Moreover, at higher concentrations, this PPAR-α ligand was also able to significantly decrease [35S]-GTPγS binding of CP-55,940 acting as an NAM of CB1R. Thus, the authors suggested two possible interpretations. Fenofibrate could be considered a bitopic ligand at CB1R since it binds both orthosteric and allosteric sites or fenofibrate could be an allosteric CB1R modulator that could be able to produce by itself a functional response by interacting with a nonorthosteric site at CB1R.The close relationship between the ECS and the PPARs led to explore the activity of the PPAR-α agonist, fenofibrate39 , in the 1R in the brain have limited the use of orthosteric CB1R ligands as drugs. However, this receptor plays an important role in diverse processes such as pain, cognition, and metabolism. Alternative targets within the ECS have been proposed, including CB2R, the fatty acid amide hydrolase, or the monoacylglycerol lipase. Allosteric modulation at CB1R offers new opportunities for therapeutic applications. Even though the first CB1R allosteric ligand, Org27569, was identified in 2005, significant interest by the research community only started few years ago. Then, there have been rapidly increasing research efforts directed into CB1R allosterism during these last 3 years. This review reflects the structural diversity and nature of the CB1R allosteric modulators identified so far. In particular, there has been an increasing identification of endogenous allosteric modulators that offer new insights in the intriguing pharmacology of CB1R. In 1R allosteric modulators is displayed. It is worth mentioning that in several of the studies discussed above, there is no correlation between the different cell-based pharmacological data. Moreover, the in vitro data do not always translate to in vivo effects. These divergent functional data may account for signaling-specific allosteric modulation as well as orthosteric probe dependence. Subsequently, there is a clear need for developing more allosteric pharmacological tools for understanding this complex pharmacology.The psychoactive side effects generated by activation of CB1Rs provides the basis for future design and synthesis of optimized allosteric modulators. Efforts need to be made to expand these scaffolds and obtain structural information about the ligand–allosteric site interactions. Further understanding of the chemical features required for allosteric modulation as well as their orthosteric probe dependence may broaden novel approaches for fine-tuning the signaling pathways of the CB1R.The characterization of small molecules as allosteric modulators of CB


Sorghum bicolor (L.) Moench] in comparison with sub-humid climate and cropland (SHC) in northern China. Results showed that environment significantly (p < 0.05) influenced plant growth, biomass yield and components, and subsequently the ethanol potential of energy sorghum. Biomass and theoretical ethanol yield of the crop grown at SHS  and SAW  were both statistically (p < 0.001) lower than values at the SHC site . Higher desirable contents of soluble sugar, cellulose, and hemicellulose were observed at SHS and SHC sites, while sorghum grown at SAW possessed higher lignin and ash contents. Biomass type sorghum was superior to sweet type as non-food ethanol feedstock. In particular, biomass type hybrid GN-10 achieved the highest biomass (17.4 t ha−1) and theoretical ethanol yields  after averaging data for all environmental sites. The most productive hybrid, biomass type GN-4, exhibited biomass and theoretical ethanol yields >42.1 t ha−1 and 14,913 L ha−1, respectively, at the cropland SHC site. In conclusion, energy sorghum grown on marginal lands showed a very lower ethanol potential, indicating a considerable lower possibility for being used as commercial feedstock supply when compared with that grown on regular croplands. Moreover, screening suitable varieties may improve energy sorghum growth and chemical properties for ethanol production on marginal lands.Field experiments were conducted in marginal lands, i.e., sub-humid climate and saline-land (SHS) and semi-arid climate and wasteland (SAW), to evaluate ethanol potential based on the biomass yield and chemical composition of biomass type  and sweet type (var. GT-3 and GT-7) hybrids of energy sorghum [ Due to China's fairly limited cultivatable land resources, national policy has implemented land-use planning. As part of the overall plan, biofuel feedstock production will be limited to marginal lands to avoid land-use competition with food crops to maintain greater food security  to compare the variation in calculated ethanol potential based on biomass yield and chemical composition of energy sorghum grown on marginal and arable cropland under different climatic conditions; (2) to clarify the difference in biomass yield and chemical composition between biomass and sweet sorghum; and (3) to screen for suitable energy sorghum hybrids which could achieve high biomass yield and quality under marginal and arable land conditions for maximal ethanol production in northern China. The expected findings of this work could be helpful to evaluate the possibility of growing energy sorghum on marginal lands for commercial ethanol production in northern China.Field experiments were conducted in northern China at three different sites with distinct environmental characteristics, i.e., sub-humid climate and saline-land (SHS), semi-arid climate and wasteland (SAW), and sub-humid climate and cropland (SHC) Table . These l2 in size and divided into a sampling area (12 m2) and a harvest area (24 m2) for all replicates. Because soil and meteorological conditions were different each year at each experimental site, thus each year-location combination was considered an “environment” with its own specific characteristics.Five energy sorghum hybrids including biomass type  and sweet type (var. GT-3 and GT-7) were arranged in a randomized complete block design with four replicates at the SHS site in 2013, at the SWA site in 2013 and 2014, and at the SHC site in 2014. The selected hybrids were developed by the National Energy R&D Center for Non-food Biomass, China Agricultural University. Each plot was 36 mTwo to three seeds were sown at 0.6 × 0.2 m intervals oriented in a north–south direction using a manual hill-drop method. At the three-leaf growth stage, seedlings were manually thinned to leave one vigorous plant per hole and concurrently weeds were manually removed. All trials were carried out in accordance with good agricultural practices. However, due to concerns about extreme soil and arid conditions at the SAW site, irrigation and a higher fertilization dose were applied to the crop grown there, but not at the SHS and SHC sites. Sprinkler irrigation of approximately 30 mm of water was applied per month. Main agronomic practices and growth periods are presented in Table On the harvest dates, tiller number was recorded for 10 hills in each plot and afterwards all aboveground plants in the harvest area of each plot were cut and weighed to estimate the fresh yield. Concurrently, 10 aboveground sorghum plants chosen randomly were harvested at the soil surface in the sampling area of each plot and were used to measure plant size (plant height and stem diameter). Next, each individual sample plant was divided into stems, leaves, and panicles, and their fresh weights were separately measured. For sampled stems, every other internode was taken from the base of each individual plant. All leaves, panicles, and sampled internodes were cut into pieces 2-to-3 cm in length and subsampled using a point-centered quarter method. Each subsample was weighed and oven-dried at 75°C until constant weight was achieved for gravimetric determination of moisture content and calculation of plant dry biomass yield.2SO4 method and assayed using a UV–VIS spectrometer  according to Li et al. , cellulose, hemicellulose, and lignin were extracted using a two-step sulphuric acid hydrolysis process  were ground using a Wiley mill and passed through a 0.5-mm mesh for total soluble sugar determination and through a 1-mm mesh for cellulose, hemicellulose, lignin, and ash determinations. Soluble sugar was determined in the supernatants using the anthrone-Hi et al. . AccordiTheoretical ethanol yield (TEY) values from soluble sugar, cellulose and hemicellulose were individually calculated using the following formulas:TEYsugar represents the TEY from soluble sugar; TEYcellu represents the TEY from cellulose and hemicellulose; F1 represents the coefficient of conversion factor of ethanol from sugar (0.51); F2 represents the process efficiency of ethanol from sugar (0.85); F3 represents the coefficient of 1.11 for the conversion factor of sugar from cellulose and hemicellulose; F4 represents the process efficiency of sugar from cellulose and hemicellulose (0.85); ρ represents the specific gravity of ethanol, 0.79 g mL−1.Where, F-protected least significant difference (LSD) test at 5% level of significance for each evaluated parameter. The coefficients of variation (CV) were calculated from all original determinations and defined as the ratio of the standard deviation to the mean value.Means and standard errors were calculated for the four replicates for each parameter. Two-way ANOVA was performed using the SPSS 19.0 analytical software package  to assess the effects of genotype, environment, and their interaction. A mean separation test was performed by using the Soil and weather variables differed considerably during the energy sorghum growing period among the three sites Table . Cumulatp < 0.05), with the exception of non-significant effects of genotype on tiller number and ash yield and non-significant effects of environment and genotype interaction on tiller number, stem diameter, plant moisture, lignin content, and ash yield (Table p < 0.001) and that differences among various years were ultimately attributed to variations in environmental conditions.Effects of variables of environment, genotype, and their interaction on all measured parameters of plant growth were significant  among the experimental sites and the energy sorghum hybrids  with biomass yield (r = 0.663 for plant height and r = 0.471 for stem diameter). In addition, the longer growth period at the SHC site also contributed to higher biomass yield, as did lower tiller number, as observed previously  or SHS site (12.2 t ha−1) were statistically (p < 0.01) lower compared to average yield for hybrids grown at the SHC site (32.6 t ha−1)  higher biomass yield (41.9%) than at the SAW site. In general, salt stress at the SHS site or infertile soil coupled with higher evaporation probably leading to soil water stress at the SAW site decrease biomass yield relative to the regular cropland conditions at the SHC site. The dramatic differences in biomass yield at different sites in this study could be attributed to considerable diversity in environmental factors, such as climate  and soil type and fertility. Tang et al.  due to its superior adaptability to abandoned marginal land. In another study conducted in northern Greece, significantly lower sweet sorghum biomass yield (13.7 t ha−1) was observed in soil with high salinity  higher biomass yield (17.3 t ha−1) than sweet type (14.7 t ha−1), with a particularly greater difference in biomass type vs. sweet type yields at the SHC site   and is particularly well-adapted to adverse environmental conditions such as water deficits, salinity, and alkalinity. Considering only biomass yield performance as the major priority, biomass type hybrid GN-4 demonstrated a very high biomass yield of 42.1 t ha−1 after growth on cropland  under sub-humid climate conditions at the SHC site. Other research groups have also achieved successful growth of energy sorghum in sub-humid climate conditions, including Gnansounou et al.  the lowest proportion of total biomass, ranging between 4.6 and 9.7% at SHC site  of total dry biomass at the SHC site to the values at the SHS site (50.4–66.1%) and SAW site (39.5–60.2%). Panicle biomass was found to be significantly  was observed for soluble sugar 34.5%), lignin (26.1%), and ash (33.7%), whereas cellulose and hemicellulose content exhibited relatively lower variability, with CV values of 13.4 and 10.4%, respectively  higher at the SHC site  higher (by 27.0–34.8%) yields of cellulose, hemicellulose, and lignin than the sweet type. However, reverse trends were observed for yields of soluble sugars and ash, which were lower  for biomass type sorghum when averaged across all hybrids and sites (Table −1) of the first three aforementioned components across all sites. Between the two hybrids of sweet type sorghum analyzed, GT-7 produced higher yields of all chemical components except for the yield of soluble sugar.As an additional consideration, biomass type sorghum is predominantly composed of structural carbohydrates (cellulose and hemicellulose) Figure . It exhigher by 2.0–34.8% p < 0.05) higher (3.7 times) than in leaf when averaged across sites and hybrids  higher ethanol yield than that observed at the SHS site  and 5.7 times greater yield than at the SAW site   positive correlations with TEY; however, the content of ash, lignin, and hemicellulose and plant moisture were negatively correlated with TEY  averaged across all sites. In particular, hybrid GN-10 biomass type sorghum produced the highest TEY  highest ethanol yield at the SHC site relative to the other hybrids, exhibiting the highest value of 14,913 L ha−1.In this study, the biomass type sorghum exhibited a higher TEY magnitude compared with sweet type sorghum  and TEY  on cropland in a sub-humid climate. Ultimately, lower ethanol potential of energy sorghum grown on marginal land reflected a lower possibility for commercial feedstock supply than that grown on regular cropland. As well, screening suitable varieties could improve energy sorghum growth and chemical components for ethanol production.This study revealed environmental stress affecting biomass yield to guide future development of promising sorghum hybrids adapted to growth on marginal lands. As part of a larger sustainable agro-industrial framework, biomass type sorghum feedstock should be encouraged for industrial scale ethanol production due to its high productivity, adaptation to marginal growth conditions, and desirable qualities that facilitate efficient conversion of its biomass to ethanol. In particular, hybrid biomass type GN-10 possesses all of these attributes, while being especially well-adapted to growth in adverse environmental conditions such as water deficits, salinity, and alkalinity. However, from an output point of view, biomass type hybrid GN-4 achieved the highest values of biomass yield (42.1 t haCT: Analyzed the data, made tables and figures, and drafted the manuscript. SL and ML: Performed the laboratory experiments and collected the data. GX: Designed and supervised the field and laboratory work and finalized the manuscript.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 BET family of bromodomain‐containing proteins constitute epigenetic readers that bind to acetylated lysine residues of core histones, thereby translating epigenetic histone marks to effects on gene expression. BET inhibitors are currently emerging as promising therapeutic agents for treatment of various pathological conditions. Here, we explored the potential of using BET inhibition to modulate IgE‐mediated responses in mast cells.We assessed the effects of BET inhibitors PFI‐1, I‐BET151, and I‐BET762 on responses downstream of mast cell activation through IgE receptor cross‐linking.BET inhibitors were neither toxic for mast cells (at doses up to 20 μM), nor did they prevent IgE‐mediated mast cell degranulation. However, we found that BET inhibition, in particular by I‐BET151, suppressed IL‐6 gene expression and IL‐6 protein release in response to IgE‐mediated mast cell activation. This was observed in both bone marrow‐derived mast cells (BMMCs) and in mature peritoneal‐cell derived mast cells. Further analysis showed that BET inhibition also suppressed the expression of a number of additional genes of those that were upregulated by IgE receptor cross‐linking, including IL‐3, IL‐7R, CCR1, and ADAMTS9. However, BET inhibition was selective, i.e., several genes that were upregulated by IgE receptor cross‐linking were not affected by BET inhibitors.These findings suggest that BET inhibition can interfere with the upregulated expression of selected genes in mast cells activated by IgE receptor cross‐linking. Further, our findings introduce the concept of utilizing epigenetic mechanisms for modulating mast cell function in the context of IgE‐driven disease. Mast cells are well known for their detrimental impact on allergic disease, but are also important players in a range of additional pathologies Based on the powerful impact of mast cells on allergic and other pathological conditions, there is a great demand for therapeutic strategies that can counteract harmful mast cell activities. Such strategies include the use of various mast cell stabilizers and anti‐IgE therapy to prevent mast cell degranulation, as well as antagonists to the individual mediators that are released following mast cell degranulation Histone modification by various mechanisms, including lysine acetylation, methylation, phosphorylation, and sumoylation is now established as a major epigenetic mechanism with wide consequences for gene expression Here, we investigated the effect of BET inhibition on mast cell function. Our findings identify BET inhibition as a novel means of modulating the expression of selected genes in response to IgE‐mediated mast cell activation. Hence, the use of BET inhibitors could represent a new strategy to directly interfere with gene expression patterns in pathological settings where mast cells are involved.Penicillin‐streptomycin, L‐glutamine, DMEM plus GlutaMAX, and minimum essential medium (MEM) non‐essential amino acids were from Invitrogen , whereas Dulbecco Modified Essential Medium (DMEM), all salts for buffers, and the anti‐DNP IgE antibody were from Sigma . DNP‐HSA was from Biosearch Technologies . BET protein inhibitors PFI‐1 (PF‐6405761), I‐BET151 (GSK1210151A), and I‐BET762 (GSK525762) were from Sigma.Bone marrow‐derived mast cells (BMMCs) were obtained by culturing bone marrow cells from the femur and tibia of C57BL/6 mice as previously described 6 cells/ml) were sensitized overnight with IgE anti‐DNP at 0.1 μg/ml. The next day, cells were washed twice and suspended in fresh media or Tyrode's buffer  and stimulated for 1–24 h with DNP‐HSA (0.5 μg/ml). In samples where BET inhibitors were evaluated, mast cells were incubated for 1 h with BET inhibitor  at various concentrations, followed by activation with DNP‐HSA. For cytokine/chemokine release and Western blot analysis, activation with DNP‐HSA was performed for 4 or 24 h, while for mRNA expression, mast cells were activated for 1 h.Mast cells  were incubated with serial dilutions of BET inhibitors (ranging from 10 nM to 20 μM) for 1 h. Cell viability was measured using the Cell Titer‐Blue® cell viability assay  according to the recommendations of the manufacturer.Mast cells .Supernatants from activated mast cells were analyzed for their content of β‐hexosaminidase activity as described t relative to HPRT, where values of the IgE‐sensitized sample were set as 1. In samples, where no signal corresponding to the sensitized controls (IgE only) were detected, values for the IgE + DNP group were set as 1. Gene array analysis was performed using Affymetrix GeneChip® expression arrays (GeneChip® Mouse Gene 1.0 ST Array), as described previously Total RNA isolation and quantitative real time RT‐PCR (qPCR) was performed as described t‐test for two‐tailed distributions. Differences were considered significant if the P‐values were 0.05 or less.Data shown are means ± standard error of the mean (SEM). Statistical analyses were performed by using GraphPad Prism 7.0 (GraphPad Software) and paired Student's To evaluate the impact of BET inhibition on mast cell functionality, we first assessed whether BET inhibitors are cytotoxic to mast cells (BMMCs). At doses up to 20 μM, neither of three tested BET inhibitors  were cytotoxic to mast cells after 1 h of incubation . However, the possibility of using this type of inhibitors to intervene with mast cell functions has, to our knowledge, not been addressed before. Here, we show that gene expression in mast cells activated by IgE receptor cross‐linking can be modulated by inhibition of the BET proteins. One important implication of these findings is that responses downstream of IgE‐mediated mast cell activation can be influenced by epigenetic mechanisms. In line with our findings, it was recently shown that interference with BRD4, one of the BET family members can suppress growth and induce apoptosis in neoplastic mast cells It is apparent from the present study that BET inhibitors do not interfere with mast cell degranulation. It is therefore unlikely that BET inhibition has effects on the immediate response downstream of mast cell activation, that is events related to effects of the preformed mast cell mediators released from secretory granules. Instead, BET inhibition was shown to modulate gene expression patterns in response to IgE receptor cross‐linking, thus implicating BET inhibitors as possible agents to modulate events occurring in the late phase responses following mast cell activation. However, it was noted that BET inhibition also suppressed the baseline expression of several of the assessed genes. Hence, BET‐mediated regulation of gene expression in mast cells is not restricted to situations in which they are activated.One of the main findings was that BET inhibition potently suppressed the upregulation of IL‐6, both at the gene and protein level. This is thus in analogy with previous studies demonstrating that BET inhibitors can suppress IL‐6 expression in macrophages stimulated with TLR4 ligands Besides the IL‐6 gene, we here identified a number of additional genes whose expression in IgE‐activated mast cells was affected by BET inhibition. These included IL‐3, IL‐7R, and CCR1, all of which have established and important functions in inflammatory responses. Of note, IL‐3 is a potent growth factor for mast cells In summary, this study identifies BET inhibitors as novel agents with capacity to modulate mast cell gene expression. Importantly, the use of BET inhibition to modulate gene expression in mast cells constitutes a novel principle for intervening with mast cell function, in which gene expression is directly targeted rather than being modulated by targeting of signaling molecules that act upstream of the gene transcription apparatus.GGF planned and performed experiments, interpreted data, and wrote parts of the manuscript; ER planned and performed experiments, interpreted data, and contributed to the writing of the manuscript; MG planned and performed experiments, interpreted data; GP conceived of the study, planned experiments, interpreted data, and wrote the manuscript.The authors do not have any conflicts of interest in relation to this study.Additional supporting information may be found in the online version of this article at the publisher's web‐siteTable S1. Genes upregulated in peritoneal cell‐derived mast cells activated by IgE receptor crosslinking.Click here for additional data file.Table S2. Genes downregulated by BET inhibitor 151 in peritoneal cell‐derived mast cells activated by IgE receptor crosslinking.Click here for additional data file.


Bordetella pertussis subverts immune functions of host myeloid cells expressing the αMβ2 integrin . CyaA delivers into cytosol of cells an extremely catalytically active adenylyl cyclase enzyme, which disrupts the innate and adaptive immune functions of phagocytes through unregulated production of the key signaling molecule cAMP. We have used phosphoproteomics to analyze cAMP signaling of CyaA in murine bone marrow-derived dendritic cells. CyaA action resulted in alterations of phosphorylation state of a number of proteins that regulate actin cytoskeleton homeostasis, including Mena, Talin-1 and VASP. CyaA action repressed mTOR signaling through activation of mTORC1 inhibitors TSC2 and PRAS40 and altered phosphorylation of multiple chromatin remodelers, including the class II histone deacetylase HDAC5. CyaA toxin action further elicited inhibitory phosphorylation of SIK family kinases involved in modulation of immune response and provoked dephosphorylation of the transcriptional coactivator CRTC3, indicating that CyaA-promoted nuclear translocation of CRTC3 may account for CyaA-induced IL-10 production. These findings document the complexity of subversive physiological manipulation of myeloid phagocytes by the CyaA toxin, serving in immune evasion of the pertussis agent.The adenylate cyclase toxin (CyaA) of the whooping cough agent  Bordetella pertussis excels in sophistication of its immunomodulatory action. The bacterium causes the respiratory infectious disease called whooping cough, or pertussis, which can be lethal to unvaccinated infants1 and still accounts for an estimated 15 to 50 million cases and ~150,000–300,000 deaths annually world-wide2. Among the first cells of the immune system that respond to B. pertussis infection are the myeloid phagocytic cells that bear the complement receptor 3 . This includes macrophages, neutrophils and dendritic cells (DCs)3. B. pertussis employs several mechanisms to subvert their functions. A prominent role in paralysis of these sentinel cells is played by the CR3-binding adenylate cyclase (AC) toxin-hemolysin . CyaA is a member of the Repeat In ToXin (RTX) family of leukotoxins4 and consists of a cell-invasive adenylyl cyclase (AC) enzyme fused to a pore-forming RTX cytolysin (Hly) moiety5. Upon binding to CR3 on cell surface, the toxin translocates its AC domain directly across the plasma membrane into cytosol of phagocytes. There, the AC enzyme is activated by calmodulin and catalyzes unregulated production of a key signaling molecule, the 3′,5′-cyclic adenosine monophosphate (cAMP). Supraphysiological concentrations of cAMP then signal through protein kinase A (PKA) and Exchange Protein directly Activated by cAMP (Epac) pathways6 and rapidly annihilate the bactericidal capacities of phagocytes. Signaling of CyaA-produced cAMP provokes massive but unproductive cell ruffling, inhibits opsonophagocytic uptake of bacteria, blocks induction of nitric oxide (NO) production, inhibits NADPH assembly and oxidative burst and induces macrophage apoptosis9. The molecular details of how CyaA-triggered cAMP signaling interferes with phagocyte functions remain, however, poorly defined. The high specific activity of the CyaA-delivered adenylyl cyclase (AC) enzyme represents, hence, a unique tool for analysis of the impact of cAMP signaling on myeloid cell function in general.The Gram-negative coccobacillus 10 for quantitative shotgun phosphoproteomic analysis of cAMP signaling resulting from CyaA toxin action on primary mouse bone marrow derived dendritic cells (BMDC). The results reveal that CyaA action causes alteration of phosphorylation of a number of proteins involved in regulation of actin cytoskeleton homeostasis, phagocytosis, translation, chromatin remodeling, IL-10 secretion and tolerogenic DC shaping.We have used stable isotope labelling by amino acids in cell culture (SILAC)- toxoid were produced in Escherichia coli XL-1 Blue cells and purified as previously described11, including 60% isopropanol washes of the chromatography resin with bound CyaA, which reduced the endotoxin content of eluted CyaA below 300 IU/mg protein .CyaA toxin and its enzymatically inactive CyaA-ACThe handling of animals was approved by the ethical committees of the Faculty of Military Health Sciences of the University of Defence and of the Institute of Microbiology of the Czech Academy of Sciences. Handling of animals and all experiments were performed in accordance with relevant guidelines and regulations, according to Guidelines for the Care and Use of Laboratory Animals, the Act of the Czech National Assembly, Collection of Laws No. 149/2004, inclusive of the amendments, on the Protection of Animals against Cruelty, and Public Notice of the Ministry of Agriculture of the Czech Republic, Collection of Laws No. 207/2004, on care and use of experimental animals.12 and outlined in detail in the Supplementary methods section. BMDCs were generated from bone marrow progenitors isolated from femurs and tibias of 6- to 8-week-old female C57BL/6 mice. GM-CSF induces catabolism of 13C/15N-labelled arginine, yielding heavy arginine-derived proline and 15N isotope incorporation, thus skewing estimation of SILAC ratios. Therefore an optimized SILAC-labeling medium was used to suppress these effects, as described earlier12.The generation and SILAC labelling of C57BL/6 murine BMDCs was carried out as previously described2+) for 2 hours at 37 °C. ‘Light’ isotope-labelled cells (12C6-arginine/12C6-lysine) were next treated for 10 or 30 minutes at 37 °C with 100 ng/ml of either CyaA toxin or CyaA-AC- toxoid dissolved in TUC buffer . Corresponding ‘heavy’ isotope-labelled cells (13C6-arginine/13C6-lysine) were treated by TUC buffer alone and served as controls for both CyaA and CyaA-AC--treated BMDCs  sodium deoxycholate (SDC)) containing a cocktail of phosphatase inhibitors . Lysis was accomplished by placing the cell suspension into a boiling water bath for 5 min13. After cooling of the lysed suspension to room temperature, the samples were subjected to benzonase treatment (Sigma) for 1 h and cell debris was removed by centrifugation . Protein concentrations in supernatants were measured using the Micro BCA kit (Thermo Pierce) and the corresponding ‘light’ and ‘heavy’ isotope-labelled lysates were mixed at a 1:1 ratio based on their protein content.SILAC-labelled BMDCs were first incubated in D-MEM (1.9 mM Ca14. The phosphopeptides were enriched on TiO2 resin (GL Sciences), separated by reversed-phase nano-scale LC and analyzed by mass spectrometry on a Q-Exactive MS instrument (Thermo Scientific) operating in data-dependent acquisition (DDA) mode. Data processing, identification and quantitation was performed with MaxQuant software16 and the MS data were deposited via the PRIDE partner repository17 in the ProteomeXchange Consortium database  under the dataset identifier PXD004733. Both WT CyaA and CyaA-AC–treated samples were first compared to buffer-treated controls in order to determine the changes in phosphorylation status of each phosphosite. The resulting ratios for individual phosphosites were derived from normalized ratios of the least modified phosphopeptides in a given replicate. Ratio values > 1 then represent dephosphorylation and values < 1 represent increase in phosphorylation. The statistical significance of differences in the phosphorylation status at individual sites was determined by the Global Mean Rank Test18. Significantly enriched phosphorylation motifs were assembled using the web-based motif-x algorithm19. Assignment to known kinase motifs was performed by Motif Matcher, a web-based online tool from the PHOSIDA posttranslational modification database21. Alternatively, Fisher’s exact test analysis of kinase motif enrichment was performed for significantly regulated phosphosites by using the Perseus software22 with built-in Human Protein Reference Database23 as the source of kinase motifs. Functional association between significantly regulated phosphoproteins was visualized using the online-based STRING database 24. Gene Ontology (GO)25 enrichment analysis was performed in Cytoscape26 using the plugin ClueGO27.Details of sample preparation and data processing are provided in the Supplementary methods. Briefly, proteins in SILAC-labelled BMDC lysates were digested with trypsin and the peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC)+) are a likely cellular target of the CyaA toxin in the course of B. pertussis infection of host airways. We thus assessed the impact of the subversive signaling of CyaA-produced cAMP in DCs by a quantitative phosphoproteomic analysis. Since human monocyte-derived dendritic cells exhibit high inter-donor variation, BMDCs from inbred C57BL/6 mice were used. The cells were labelled ex vivo with stable isotopes (SILAC)12 and advantage was taken of the use of a non-enzymatic CyaA-AC- toxoid as negative control. CyaA-AC- is unable to elevate cAMP in cells29 but retains the full capacity to bind the cell surface receptor CR3, to permeabilize cellular membrane and to elicit Ca2+ influx into cells and K+ efflux from cells, like intact CyaA. This control thus enabled exclusion of phosphorylation changes that were unrelated to cAMP signaling elicited by the CyaA toxin and were due to cell handling and/or cell permeabilization by the pore-forming activity of CyaA. To capture the early cAMP-triggered signaling events and to mimic the physiological concentrations of CyaA encountered by the phagocytes on B. pertussis-infected airway mucosa31, the SILAC-labelled BMDCs (106/ml) were exposed to 100 ng/ml of CyaA or of CyaA-AC- for 10 or 30 minutes.CR3-expressing intraepithelial DCs of myeloid origin , 1,700 threonine (11.81%) and 127 tyrosine (0.9%) phosphosites  were found to be the major kinases mediating cAMP signaling elicited by CyaA - toxoid for 10 minutes. Therefore, only samples from the 30 minutes time point for the toxoid were analyzed in more detail. When the significantly regulated phosphosites of the toxoid-treated sample were plotted against the matching phosphosites from the CyaA-treated condition, a prominent alteration of the phosphorylation state of the threonine residue 1465 (1462 in man) of tuberin (product of the Tsc2 gene) was observed ton Fig. . This waDCs Fig. . The netDCs Fig. , with thDCs Fig.  or Abl2 des Fig. . The int- . Deactivation of Akt kinase also yielded dephosphorylation of the threonine 247 residue of PRAS40, promoting mTOR inhibition47 , where hypophosphorylated 4E-BP1 strongly binds to eukaryotic translation initiation factor and prevents translation67. The proposed model of mTOR signaling regulation by CyaA in BMDCs is then shown in Fig. 69. Inhibition of mTOR also leads to the activation and nuclear translocation of NF-κB and CyaA-provoked inhibition of mTOR would go well with our previous observation that NF-κB is activated upon CyaA-produced elevation of cAMP in macrophages68. The CyaA-provoked inhibition of mTOR is likely to have rather pleiotropic effects and these would vary in different DC subtypes71. For example, inhibition of mTOR was reported to result in the inhibition of IL-4-dependent maturation of BMDCs and in lowering of their T cell stimulatory capacity73. This would go well with our observations that CyaA treatment skews TLR-induced maturation of dendritic cells, inhibits their capacity to present antigens to both CD4+ and CD8+ T cells and enables the toxin-treated DCs to expand regulatory T cells in vitro74.Pro-survival signaling of mTOR is negatively regulated by the protein tuberin, the activity of which is inhibited by phosphorylation of the threonine residue 1465 by AktBordetella infections, as hijacking of Crk appears to be implicated in bacterial pathogenesis through diverse mechanisms, including subversion of cellular ruffle formation . The Crk-associated proteins retained in the interaction network  by the cAMP-activated PKA was then shown to play an important role in membrane ruffle formation, causing a significant reduction of the retraction of membrane ruffles76. In line with that, increased phosphorylation of the known PKA/PKG target VASP on serine residue 235 was observed in WT CyaA-treated BMDCs samples 79. We observed that the action of CyaA provoked alterations of the phosphorylation state of several members of the GEF protein family, some of which were previously shown to directly promote RhoA activity80. For example, the protein known as Solo (or ARHGEF40) was shown to specifically induce RhoA activity in vascular endothelial cells81. Here, the treatment of BMDCs with CyaA resulted in dephosphorylation of the serine residue located in the region closest to the GEF domain of Solo. Another RhoA activator that is regulated by CyaA action is PDZRhoGEF . This protein was found to be phosphorylated on the serine residue  and GTP-bound (active) states and are regulated by the interplay of activating GEFs (activating guanine nucleotide exchange factor) and inhibitory GAPs (GTPase-activating proteins)Myo9b), whose depletion was shown to impair the migratory capacity of dendritic cells and alter their interaction with T cells83. The other GAPs found to be regulated upon CyaA action was the ARHGAP18 protein, which is required for cellular polarization involved in cell migration84 and the ARHGAP11A protein, a key regulator of RhoA during cell division 85.Among the GAPs known to be involved in RhoA regulation, three were found to be phosphorylated upon CyaA action on BMDCs (c.f. Supplementary Table S1), including the unconventional myosin-IXb  in the nuclear localization signal. This would go well with the report of Taniguchi et al.89 that cAMP-stimulated nuclear import of HDAC5 requires dephosphorylation of Ser 270 by protein phosphatase 2 A (PP2A). Furthermore, CyaA action on DC provoked alterations of phosphorylation state of additional proteins that are involved in chromatin remodeling and epigenetic regulation. Among them, the Cbx5 gene-encoded HP1α protein, involved in propagation and maintenance of pericentric heterochromatin90, was found to be phosphorylated on serine 93. Another Chromo domain-containing protein that was significantly phosphorylated (on serine 305) upon CyaA treatment of BMDC was the transcriptional repressor CBX8, also known as PC391. In addition, significant phosphorylation of the replication-dependent histone H1.4 (Hist1h1e) on threonine 35 and serine 36 was observed. This goes well with the observation that H1.4 can be phosphorylated by PKA during mitosis and when it dissociates from mitotic chromatin92. CyaA-triggered changes in chromatin state and histone modifications could then also result in alteration of pre-mRNA splicing93 and numerous other effects on gene expression and cell cycle progression.The prolonged course of the whooping cough disease, often lasting for up to 12 weeks or longer, raises the hypothesis that 60. Indeed, inhibition of SIKs by cAMP-activated and PKA-mediated phosphorylation was shown to suppress secretion of the pro-inflammatory cytokines IL-6, IL-12, and TNF-α by macrophages and appears to promote formation of regulatory (M2b) macrophages59. One of the SIK targets is the CREB-regulated transcription co-activator 3 (CRTC3) that can be phosphorylated at several serine residues. SIK-mediated phosphorylation was then shown to promote sequestering of CRTC3 into cytosolic complexes with the 14-3-3 protein, inhibiting CRTC3-assisted transcription of CREB-dependent genes, such as the anti-inflammatory cytokine IL-1059. In turn, phosphorylation of CRTC3 is inhibited by the cAMP-activated PKA and this enables translocation of the non-phosphorylated CRTC3 into the nucleus, where interaction of CRTC3 with CREB, activated by CyaA-produced cAMP94, may upregulate IL-10 gene transcription. In line with that we observed significant dephosphorylation of the serine 329 residue of CRTC3 and increased phosphorylation of the upstream regulatory kinases SIK1 (SIK), SIK2 (QIK) and SIK3 (QSK) in CyaA-treated BMDCs . These are known for their involvement in modulation of immune responsesSupplementary InfoSupplementary dataset


On polar coasts, seasonal sea-ice duration strongly influences shallow marine environments by affecting environmental conditions, such as light, sedimentation, and physical disturbance. Sea-ice dynamics are changing in response to climate, but there is limited understanding of how this might affect shallow marine environments and benthos. Here we present a unique set of physical and biological data from a single region of Antarctic coast, and use it to gain insights into factors shaping polar benthic communities. At sites encompassing a gradient of sea-ice duration, we measured temporal and spatial variation in light and sedimentation and hard-substrate communities at different depths and substrate orientations. Biological trends were highly correlated with sea-ice duration, and appear to be driven by opposing gradients in light and sedimentation. As sea-ice duration decreased, there was increased light and reduced sedimentation, and concurrent shifts in community structure from invertebrate to algal dominance. Trends were strongest on shallower, horizontal surfaces, which are most exposed to light and sedimentation. Depth and substrate orientation appear to mediate exposure of benthos to these factors, thereby tempering effects of sea-ice and increasing biological heterogeneity. However, while light and sedimentation both varied spatially with sea-ice, their dynamics differed temporally. Light was sensitive to the site-specific date of sea-ice breakout, whereas sedimentation fluctuated at a regional scale coincident with the summer phytoplankton bloom. Sea-ice duration is clearly the overarching force structuring these shallow Antarctic benthic communities, but direct effects are imposed via light and sedimentation, and mediated by habitat characteristics. The annual timing of sea-ice formation and departure is critical to the functioning of polar ecosystems, but in recent decades sea-ice dynamics have been changing. Higher temperatures and stronger winds have reduced the annual duration of Arctic sea-ice , 2, and Sea-ice dynamics directly impact biota living on or in the ice, such as mammals  and micrOf the ways in which sea-ice affects benthic communities, the most obvious is reduced solar irradiance , 15. DecSea-ice also mediates physical disturbance to the benthos by influencing both sedimentation and ice-scour. Sea-ice forms a barrier between the water column and the atmosphere, preventing wind-induced turbulence and water-column turnover to create relatively still, depositional environments, where flow is generally limited to tidal currents , 26. In Sea-ice attached to coast (‘fast-ice’) also prevents drifting ice-bergs from scouring the seabed and inflicting catastrophic physical disturbance . StudiesEvidence of links between sea-ice duration and shallow benthic community structure is growing, although patterns vary between study regions. A long-term study from the Arctic showed that macroalgae increased in abundance as sea-ice duration shortened , 29–a prLight, sedimentation, and disturbance to the benthos are likely to be mediated by habitat orientation and depth. Both light and sedimentation are vertically oriented processes, so their effects on benthic organisms should depend on the orientation of habitat relative to overhead inputs , 31. EffThis study was conducted around Casey Station , on the coast of Wilkes Land, East Antarctica . The sub™ cosine corrected photosynthetic irradiance sensors (400–700 nm) [™ 292 data loggers or Odyssey™ integrated data loggers. Light meters were mounted upright on stainless-steel tripods or star pickets, and were positioned and secured on the seabed by divers at depths of 7 to 10 m. They were calibrated against three Li-Cor LI-190SA sensors  for 1 w prior to deployment, and set to record ambient light levels every 20 min. Due to the deterioration in performance of light meters through time as a result of physical disturbance, shading or fouling, only the first 12 months of light data are presented. Raw data from light meters was converted into mol photons PAR m-2 yr-1.Light meters were deployed on the seabed at eight sites around Casey Station , Table 1–700 nm) , with daSediment flux was quantified by deploying sediment traps at six sites  for a ra2) from two depths, 6 and 12 m, 100–200 m apart. There was no significant difference in the size of boulders collected from different sites or depths . Surveys were conducted across two austral summers .Divers collected boulders from 11 sites across a gradient of seasonal sea-ice cover . At each2. Down-facing surfaces were not included because this surface was bare on boulders at silty sites. The placement of quadrats was constrained to be > 1 cm from any edge and at least 2 cm from another quadrat. Counts were made of serpulid and spirorbid polychaetes, and percent cover was estimated for all other taxa. All cheilostomate bryozoans were identified to species. Other taxa were categorised into morphospecies. Voucher specimens were collected and preserved. Sponges and calcified bryozoans were preserved in 80% ethanol with Milli-Q water, and other taxa were preserved in 7% formalin in seawater.Boulders were kept in aerated local seawater at -2°C, and communities were censused live under a dissecting microscope. Quadrats (2 x 2 cm) were randomly placed on a boulder, and all taxa occurring within a quadrat were recorded. On each boulder we sampled six quadrats on horizontal (up-facing) surfaces and six on vertical (side-facing) surfaces, giving a total of 48 cm-1, intermediate sites 7–11 mo yr-1, and open sites <7 mo yr-1.Sites were ranked according to their sea-ice duration , depth (6 and 12 m) and substrate orientation  were analysed with PERMANOVA  using PREffects of sea-ice cover (ice-rank), depth and substrate orientation on the abundance and diversity of individual taxa on boulders were analysed with generalized linear mixed models (GLMM) . Spatial-2 d-1). The response was modeled with a Gaussian distribution with log link function, and Site (bay) was modeled as a random intercept to account for autocorrelation within sites. Only data from traps deployed during summer were statistically analysed.Sediment trap data collected in summer were analysed with a GLMM, testing the relationship between deployment duration (number of days), position in a bay (inner or outer), and sediment flux , hydroids, sponges and ascidians . MultivaInversiula nutrix and spirorbid polychaetes. Algal cover was higher on horizontal surfaces within sites classified as having shorter sea-ice duration (I. nutrix and spirorbids) showed the opposite pattern, particularly on vertical surfaces at 12 m depth  were the second most abundant invertebrate taxa, and were considerably more abundant at open sites than sites with more sea-ice cover. Most sand tubes were usually inhabited by tanaids of the genus Nototanais, but some housed other mobile invertebrates and terrebellid polychaetes. Sponge cover was highly variable between sites, but showed an overall trend of greater cover with longer sea-ice duration. Encrusting algae were most abundant in the open zone, whereas arborescent algae peaked in cover at sites with intermediate sea-ice duration  found that glacial melting allowed macroalgae to colonize newly ice-free areas, but also report reduced light penetration due to increased terrigenous sediment input from land and more frequent ice scour . Other rInversiula nutrix in areas of longer sea-ice duration. Vertical surfaces were dominated by invertebrates regardless of sea-ice, but the identity of invertebrates changed along the sea-ice gradient. Communities on horizontal substrate showed stronger correlations to sea-ice duration than those on vertical substrate, consistent with expected response to greater exposure to light and sedimentation. In temperate and tropical systems, non-coral invertebrates tend to occupy shaded, vertical, or down-facing habitats [There was a strong biological gradient transitioning from invertebrate to algal dominance in the direction of decreasing sea-ice duration, but the nature of change depended on substrate orientation and depth. Horizontal surfaces were dominated by algae in areas of shorter sea-ice duration, and sand tubes and the bryozoan habitats , 31, 52,habitats  or outcohabitats , 54. Effhabitats , as sediThe relative effects of light and sedimentation can be inferred from changes in biota in relation to orientation, in areas of longer vs. shorter sea-ice duration. Assuming these physical factors affect horizontal more than vertical substrates, differences in biota between orientations at open sites  most likely reflect changes in light, while differences at closed sites  are more likely related to sedimentation. With this rationale, light in our study area drives a gradient between algae and invertebrates, whereas sedimentation facilitates fauna with sand tubes, and differentiates taxa based on morphology or other mechanisms of sediment tolerance. Light and sedimentation may also have interactive effects since negative consequences of reduced light on algae can be exacerbated under heavy sedimentation , and sucInversiula nutrix, appears related to both light and sedimentation. It is the only sessile invertebrate taxa able to reach substantial cover on horizontal substrate at closed sites, implying a degree of tolerance to sedimentation. In contrast, at open sites it dominates vertical substrate, and is rare on horizontal substrate. This switch in habitat use from horizontal to vertical substrate as sea-ice duration shortens is likely driven by tolerance to sedimentation giving way to out-competition by algae. It may also be more tolerant of UV light than other invertebrates, since few of the invertebrate taxa found in the closed ice-cover zone were present in the open ice-cover zone. Spirorbid polychaetes (mostly Spirorbis nordenskjoldi) were the other invertebrate taxa to show contrasting distributions to the majority of other invertebrates, being most abundant in the open zone. Interestingly, both of these taxa were by far the most abundant in the study region, and their ecological success may be related to an ability to exploit niches unavailable to other invertebrates.Habitat use by the most abundant species, Overall trends in algal cover are clearly driven by light, but sedimentation may contribute to more subtle differences in the distribution of algal functional groups. Both encrusting and arborescent algae were negatively correlated with sea-ice duration, though cover of arborescent algae peaked in areas with some sea-ice cover. This could reflect greater tolerance to sedimentation in arborescent vs. encrusting morphologies, since arborescent foliage are raised off the substratum and remain clear of sediment accumulation . ArboresThere was an intriguing peak in bare space in the intermediate ice-cover zone, which may reflect disturbance conditions or poor habitat quality. One hypothesis to explain the pattern is that iceberg scour in the intermediate ice-cover zone causes regular invertebrates mortality, but in contrast the open ice-cover zone, light is insufficient to support algal growth. Such conditions would create habitat unfavourable to both invertebrates and algae. The spatial mosaic of algae in the open zone suggests that iceberg scour is occurring and has ecological consequences , but ratIn summary, we found strong links between sea-ice duration and benthic community structure, and the nature of change was consistent with variation in light and sedimentation. Both light and sedimentation vary with sea-ice duration, and their effects on biota are mediated by the depth and orientation of habitat. Interactions between overhead sea-ice dynamics and benthic habitats undoubtedly contribute to the diversity of shallow Antarctic benthic communities, and understanding these interactions is critical to forecasting climate-related change. If climate change lengthens the period for which some areas are ice-free per year, species distributions are likely to shift accordingly. Taxa currently restricted to areas of prolonged ice cover are at risk of local extinction, which is ecologically significant because these areas contained a suite of invertebrates not found in areas of shorter sea-ice duration. Such change in benthic communities in response to sea-ice duration has recently been observed in both the Arctic  and AntaS1 TableThe number of deployments represents the number of sediment traps deployed at one site, with 2–5 sediment traps deployed at any one time. Traps were deployed for various time intervals between 08/12/2002 and 30/12/2006.(DOCX)Click here for additional data file.S2 TableAt each site we collected 16 boulders, 8 from 6 m depth and 8 from 12 m.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.