Pluto is covered by numerous deposits of methane, either diluted in nitrogen or as methane-rich ice. Within the dark equatorial region of Cthulhu, bright frost containing methane is observed coating crater rims and walls as well as mountain tops, providing spectacular resemblance to terrestrial snow-capped mountain chains. However, the origin of these deposits remained enigmatic. Here we report that they are composed of methane-rich ice. We use high-resolution numerical simulations of Pluto’s climate to show that the processes forming them are likely to be completely different to those forming high-altitude snowpack on Earth. The methane deposits may not result from adiabatic cooling in upwardly moving air like on our planet, but from a circulation-induced enrichment of gaseous methane a few kilometres above Pluto’s plains that favours methane condensation at mountain summits. This process could have shaped other methane reservoirs on Pluto and help explain the appearance of the bladed terrain of Tartarus Dorsa. Pluto is covered by numerous deposits of methane. Here, the authors show that the formation of methane frost on mountain tops and crater rims in Pluto’s equatorial regions completely differ from those forming snow-capped mountains on Earth. In particular, west of Sputnik Planitia, the region of Cthulhu is characterized by a spectacular dark mantling that has been interpreted as an accumulation of haze particles that have settled from the atmosphere7, or in some locations a cryovolcanic deposit8.An important observation of Pluto made by the New Horizons spacecraft in July 2015 was the great geomorphological diversity and albedo contrast of terrains in its equatorial regions3, the Long-Range Reconnaissance Imager (LORRI) instrument onboard New Horizons revealed the presence of patchy bright deposits at specific locations6 and the Multispectral Visible Imaging Camera (MVIC) showed that they contain methane (CH4)2. Figure 9. They are capped with bright frosts  above 1.5 km altitude with a striking resemblance to terrestrial alpine landscapes 11.Although most of Cthulhu’s surface appears volatile-freepes Fig. . MVIC sh4 ice form on top of these mountains? It has been suggested that the sublimation and condensation of volatile ices could drive the ices out of thermodynamic equilibrium and result in altitude segregation with N2-rich ice dominating at low-elevations and CH4-rich ice dominating at high elevations13. Here we explore an alternative scenario that involves an atmospheric process. On Earth, atmospheric temperatures decrease with altitude, mostly because of adiabatic cooling and warming in upward and downward air motions, respectively. As a consequence, surface temperatures also decrease with altitude because the surface is cooled by the dense atmosphere through sensible heat fluxes. In such conditions, as moist wind approaches a mountain, it rises upslope and cools adiabatically, leading to condensation and formation of snow on top of mountains. Note that here frost is defined as ice crystals that form directly on a below-freezing surface via a phase change from gas in the atmosphere, whereas snow is defined as individual ice crystals that grow while suspended in the atmosphere and subsequently fall as precipitation onto the surface.Why does CH15 because of the heating resulting from the absorption of solar radiation by CH4 gas . The atmosphere is too thin to affect the surface temperature itself and in the absence of N2 ice, the surface remains in local radiative balance everywhere, independent of the altitude and colder than the atmosphere above. One consequence of this is that the near-surface air is cooled and tends to flow downslope because it is denser than the air away from the slope at the same level. Climate simulations confirm this trend and indicate that these katabatic downslope near-surface winds dominate everywhere and at all times of day on Pluto14. Under such conditions, it is impossible to explain the condensation of CH4 by upward air motion as on the Earth, and a different mechanism must be identified.Could this process apply to Pluto too? Both observations and modelling show that, unlike on Earth, there is a strong increase in atmospheric temperatures with altitude in the first kilometres above the surface4-rich ice. We then use a numerical climate model of Pluto to investigate the origin of their formation. Our simulations reproduce the accumulation high-altitude CH4 ice where the frost-capped mountains are observed, in particular on the ridges and crests of the Pigafetta and Elcano Montes in eastern Cthulhu. They show that CH4 condensation is favoured by sublimation-induced circulation cells that seasonally enrich the atmosphere with gaseous methane at those higher altitudes.Here we demonstrate that the bright frosts observed in Cthulhu are mostly made of CH4 and nitrogen (N2) band depth and CH4-phase index maps3 derived from the highest spatial resolution spectral-image of the Linear Etalon Imaging Spectral Array (LEISA) instrument, presented alongside the latest digital elevation model9. These maps indicate that the bright frosts on the top of the mountain chain are mostly composed of CH4-rich ice. Only a few very small N2-rich ice patches occur in this area, mostly located at the bases of these mountains and in the valleys separating them.Figure 4 ice was detected was on north-facing walls and rims of many craters in Cthulhu, including Edgeworth Crater and many smaller examples surrounding Pigafetta Montes. The composition and phase maps also show that these deposits are CH4-rich ice3. We can straightforwardly explain this behaviour by calculating that these slopes received less insolation than the south-facing slopes during the previous northern fall and winter . It includes a full description of the volatile exchange between surface ice and atmospheric gas , transport and turbulent mixing of gaseous CH4 in the atmosphere and an implicit scheme for the formation of CH4 clouds. The radiative effect of CH4 and CO is also included14. The model provides an evolution of surface pressure and of the CH4 and CO mixing ratio in good agreement with New Horizons and terrestrial observations17. In the model, we rely on Raoult’s law as a substitute for the ternary equation of state. We consider that CH4-rich ice behaves like pure CH4 and that N2-rich ice contains 0.5% of CH4 . We note that this approximation can lead to some uncertainties in CH4 solid-phase stability but that it remains a reasonable approach for the focus of this paper (CH4-rich deposits) and does not qualitatively change the results of this paper.Here, we use the Pluto Global Climate Model (GCM) of the Laboratoire de Météorologie Dynamique (LMD), designed to simulate the climate and CH16 but with the following improvements. Firstly, we used the latest topography data of the encounter hemisphere of Pluto9, which includes Sputnik Planitia, eastern Cthulhu, and Tartarus Dorsa . Secondly, perennial CH4 deposits were added on the sub-Charon side of Pluto (covered by low-resolution New Horizons imaging) wherever terrain with diagnostic characteristics of the bladed terrain was detected18 . Thirdly the initial state of the simulation was obtained for Earth year 1984 from a 30-million-year simulation performed with the Pluto volatile transport model20 , thus allowing a steady state for ice distribution, surface and soil temperatures to be reached. We used two horizontal resolutions: a baseline resolution of 7.5° in latitude, 11.25° in longitude (i.e. ~150 km) for the years 1984–2014 and a higher resolution of 2.5° in latitude and 3.75° in longitude (i.e. ~50 km) for the years 2014–2015. This allowed us to better represent the atmosphere-topographic interactions during the New Horizons encounter.To simulate Pluto as observed in 2015, we performed a simulation similar to the one presented in Bertrand et al.4 atmospheric mixing ratio reaches 0.4–0.7% in 2015, with slightly higher values in the northern hemisphere due to increasing CH4 ice sublimation from the mid-to-high northern latitude plains, as predicted by previous climate simulations14. A net CH4 ice deposition of ~20 μm over the year 2015 is obtained locally in Cthulhu, at the summits and on the flanks of the highest mountains of Pigafetta and Elcano Montes, in qualitative agreement with New Horizons observations  to 0.5 . However, on Pluto, it is likely that once a fresh initial CH4 frost grows to a few millimetres, the surface albedo increases to values even higher than 0.5, inducing a stronger cooling (reduced solar heating) of the surface and allowing more CH4 to condense there. This positive feedback should favour even stronger katabatic winds and the accumulation of more ice on top of Pluto’s mountains. To explore its effect, we performed a second simulation in which the CH4 ice–albedo was slowly increased up to 0.9 as soon as the ice thickness reached more than 1 µm . In this second simulation, the modelled CH4 condensation rates are stronger than in the first, in particular in the diurnal zone above ∼20°S with a net CH4 ice deposition over the year 2015 of ~40 μm and on top of Pigafetta Montes and a better agreement with New Horizons observations  over multi-annual timescales, during past climate epochs with larger amounts of gaseous CH4 available for condensation20. The lack of image resolution, the uncertainties on the starting season of the frosts and the model approximations for computing CH4 condensation and sublimation rates  prevent us for estimating the thickness of CH4-rich deposits with great confidence. Nevertheless, the 2015 simulation sheds light on the possible processes forming the CH4-rich deposits seen at mountain summits in eastern Cthulhu.In our simulation, the CHons Fig. . In thisons Fig. . By extr4 mixing ratio is greater than the CH4 mixing ratio at saturation . As mentioned above, on a N2-free surface such as in Cthulhu, there is no dependence of surface temperature on altitude , the equatorial regions in Cthulhu in 2014–2015 are cold enough during night-time (~40–42 K) to trigger CH4 condensation onto the surface and the formation of μm-thin CH4 frosts  than in the depressions , the CH4 condensation is also controlled by the near-surface winds, which mix gaseous CH4 near the surface (turbulent mixing). The model predicts that on Pluto, these winds tend to be stronger on the steepest slopes, such as on the flanks of Pigafetta Montes where slopes can range up to 45°9 . In such conditions, initially elevated portions of the CH4 deposits may represent sites of increased CH4 condensation relative to lower portions, and so their positive topographic relief may gradually be amplified. This would complement the ‘penitente model’21 in which the bladed texture is suggested to be mostly induced by sublimation processes.Overall, the formation of CH16 to simulate Pluto’s climate and the methane cycle in 2015. The model includes atmospheric dynamics and transport, turbulence, radiative transfer and molecular conduction as well as phase changes for N2, CH4 and CO. The GCM reproduces well the thermal structure measured by the New Horizons spacecraft in the lower atmosphere  and the threefold increase in surface pressure observed from stellar occultations between 1988 and 2015 with ~1.1 Pa in 201522. Recent improvements to the model include incorporation of perennial high-altitude CH4 deposits in the equatorial regions (bladed terrain), based on mapping of Pluto’s far side18, and the use of the latest topography from New Horizons data9. We use flat topography for the non-observed southern hemisphere. Adding topography in the southern hemisphere does not impact the results of this paper. The general circulation is strongly controlled by the north-to-south N2 flow and is not significantly impacted by the presence of N2 ice deposits outside Sputnik Planitia16.We used the LMD Pluto GCM20. These long-term simulations allow the surface ice distribution , surface temperatures and soil temperatures to reach a steady state for current-day Pluto. The initial state of the atmosphere in the 3D GCM is an isothermal profile. We run the GCM from 1984 to 2015 which is sufficient to reach a steady state for the atmosphere and a realistic circulation regime insensitive to the initial state14.The initial state for the GCM is derived from simulations performed with the 2D LMD Pluto volatile transport model over 30-million years, taking into account the orbital and obliquity changes of Pluto over timez1 = 5 m).The long-term volatile transport simulations and the low-resolution GCM simulations are performed with a horizontal grid of 32 × 24 points to cover the globe  and 27 vertical levels. Simulations for the years 2014 and 2015 are then performed with a higher spatial resolution by using a grid of 96 × 72 points  and 47 vertical levels  are set to 0.1 and 1, respectively, which corresponds to a terrain covered by dark materials such as Cthulhu10. Methane ice emissivity is fixed to 0.8 in all simulations. We use a CH4 ice–albedo of 0.5 for the equatorial deposits  and of 0.65 for the polar deposits, which is consistent with the available albedo maps of Pluto10.Our GCM simulations have been performed using an NId = 20 J s−0.5 m−2 K−1, to capture the short-period diurnal thermal waves and a larger thermal inertia below set to Is = 800 J s−0.5 m−2 K−1 to capture the much longer seasonal thermal waves which can penetrate deep into the high-TI substrate. The rest of the settings are similar to the previous simulations of Pluto with the LMD GCM16. We assume a density for CH4 ice of 500 kg m−3 20.The thermal conduction into the subsurface is performed with a low thermal inertia near the surface, set to 4 and CO are minor constituents of Pluto’s N2 atmosphere and their surface–atmosphere interactions depend on the turbulent fluxes given by:qsurf the saturation vapour pressure mass mixing ratio (in kg kg−1) at the considered surface temperature, q the atmospheric mass mixing ratio, ρ the air density, U the horizontal wind velocity and Cd the drag coefficient at 5 m above the local surface (Cd = 0.06). qsurf is computed using the thermodynamic Claudius–Clapeyron relation23 for CH4 with a latent heat for sublimation of 586.7 kJ kg−1.CH24. We note that sophisticated equations of state exist for the N2–CH4 and N2–CH4–CO systems under Pluto surface conditions (CRYOCHEM12). These ternary and binary systems, as currently understood12, are able to explain a great diversity of phases  that are seen on Pluto within the range of expected temperatures and relatively unvarying surface pressure and strongly N2-dominated vapour composition seen on Pluto.On Pluto’s surface, the volatile ices should form solid solutions whose phases follow ternary phase equilibria; they do not exhibit ideal behaviour4 deposits modelled in this paper , the ternary phase equilibria shown by Tan and Kargel12 predict that two phases coexist: a very nearly pure CH4 solid (≪1% impurities of N2 and CO in solid solution) and N2-rich vapour. This is consistent, to first order, with the observations by New Horizons and with what we report in this paper and in Fig. At the temperature of the CHHowever, as these equations of state have not been coded for use in a GCM or been applied to the specific distribution of ices and temperatures seen on Pluto or in a GCM, we have substituted the alternative of relying on Raoult’s law, as described next. It is possible that for conditions of rapid frost deposition, the solid condensates may be amorphous mixtures, which might tend to exhibit thermodynamics somewhat like those implied by Raoult’s law. Accordingly, we interpret Pluto to be a non-equilibrium dynamical environment with continuous exchange of materials , including on daily timescales where departures from equilibrium could be likely.Future work involving laboratory experiments, spectroscopic analyses, thermodynamic models and GCMs is strongly needed to improve the models, constrain the timescales for ice relaxation toward thermodynamics equilibrium and explore in detail the effect of the ternary phase equilibrium on Pluto (and on other Trans-Neptunian objects).17. We consider the mixtures N2:CH4 and N2:CO with 0.5% of CH4 and 0.3% of CO respectively, as retrieved from telescopic observations and from New Horizons observations25. However, we note that Raoult’s law was at first intended for vapour–liquid equilibria and ideal solutions only, which do not include the N2–CH4–CO system observed on Pluto. Despite the fact that this approximation gives good results and allows for reproducing the atmospheric mixing ratios observed by New Horizons observations17, it may still introduce errors on sublimation and condensation rates of the different types of ice. This approximation seems sufficient for our present needs and for the study of CH4-rich ice deposits on Pluto’s surface, which are the focus of this paper, but we acknowledge that it leads to some unevaluated uncertainties in CH4 solid-phase stability.Here, for simplicity in coding with a GCM, we have adopted Raoult’s law, as in previous GCM studies4 ice–albedo to change depending on the thickness of ice Q present on the surface. We assumed a minimal albedo of Amin = 0.1 for deposits thinner than Qlim = 1 μm, and a maximal albedo of Amax = 0.9 for the thickest deposits. In the model, this change is applied to CH4 ice in the equatorial regions only, outside the bladed terrain deposits (i.e. only recent frost). We use a simple hyperbolic tangent function for the transition between the extreme albedo values :We tested the impact of an amplifying surface ice–albedo feedback on the results. To do that, we allowed the surface CH4 condenses on the surface, as it lowers the surface temperature and therefore the near-surface CH4 mixing ratio at saturation, thus allowing for a further increase of the CH4 condensation rate. Note that the goal here is to boost the effect of albedo feedback with a simple representation within the time of the simulation, but these changes in surface albedo may actually occur over longer timescales, and be efficient for deposits thicker than ~1 mm.In our model, this ice–albedo scheme leads to a strong positive feedback when CH3 is an index allowing discrimination between CH4 diluted in N2 ice and CH4-rich ice phases based on the position of a set of CH4 near-infrared bands. It is thus based on the measure of the shift of these bands upon dilution in N2 ice as measured in the laboratory27.The phase index, as defined in Schmitt et al.2-rich and CH4-rich ice at scale below a few kilometres, with an intimate mixture of the crystals of both phases or a vertical stratification . However, taking into account the noise and detection level, the maps show that nitrogen may be only present in some very localized area at low altitude. The phase index maps clearly point to a CH4-rich dominant composition.At the spatial resolution of the LEISA measurements (2.7 km in the high-resolution strip used for Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Potyviruses encode a large polyprotein that undergoes proteolytic processing, producing 10 mature proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb-RdRp, and CP. While P1/HC-Pro and HC-Pro/P3 junctions are cleaved by P1 and HC-Pro, respectively, the remaining seven are processed by NIa-Pro. In this study, we analyzed 135 polyprotein sequences from approved potyvirus species and deduced the consensus amino acid residues at five positions  in each of nine cleavage sites. In general, the newly deduced consensus sequences were consistent with the previous ones. However, seven NIa-Pro cleavage sites showed distinct amino acid preferences despite being processed by the same protease. At position −2, histidine was the dominant amino acid residue in most cleavage sites , except for the NIa-Pro/NIb-RdRp junction where it was absent. At position −1, glutamine was highly dominant in most sites (88.2–97.8%), except for the VPg/NIa-Pro junction where glutamic acid was found in all the analyzed proteins (100%). At position +1, serine was the most abundant residue (47.4–86.7%) in five out of seven sites, while alanine (52.6%) and glycine (82.2%) were the most abundant in the P3/6K1 and 6K2/VPg junctions, respectively. These findings suggest that each NIa-Pro cleavage site is finely tuned for differential characteristics of proteolytic reactions. The newly deduced consensus sequences may be useful resources for the development of models and methods to accurately predict potyvirus polyprotein processing sites. To propagate in host cells, most RNA viruses generate multiple proteins from relatively small-sized genomes , 2. RNA To overcome this obstacle, various strategies have been adopted by viruses to generate multiple proteins from single RNA genomes , 3. For Potyvirus, which is named after the type species potato virus Y, has 183 approved species and is the largest genus in the family Potyviridae  -[ethivg]-[Hq]-[Yf]/S .The previously known P1 consensus sequence was [ILMV]-X-[HQ]-[FY]/S . The preS2 site or HC-Pro/P3 junction is self-cleaved by HC-Pro protease . AnalysiAs per the previously mentioned criteria, we deduced the S2 site consensus sequence for the HC-Pro protease self-cleavage site as Y-[rl]-V-G/G . The precis or in trans -[Hf]-[Qe]/[sga] . The preThere are seven NIa-Pro cleavage sites (from S3 to S9) in a potyvirus polyprotein. Since these sites are recognized by the same protease, all the seven sites are usually combined into a single data set to deduce the consensus sequence as described earlier in this report. However, it is possible that some sites may have different amino acid sequence preferences to accommodate site-specific structure or cleavage reaction characteristics.To investigate whether there are any site-specific sequence preferences, we individually analyzed the seven NIa-Pro cleavage sites . ConsensWhen we examined the amino acid frequencies and consensus sequences, we found similarities and differences between the consensus sequence derived from all the NIa-Pro sites and those from individual sites as well as among the seven consensus sequences. Some sites exhibited unique amino acid preferences distinct from other sites, which was noticeable in the bubble plots, sequence logos, and consensus sequences. The most notable site was the VPg/NIa-Pro junction (S7): at the position −1 of the S7 site, glutamic acid was exclusively observed  while glutamine was highly dominant in the other six sites with frequencies ranging from 88.2% to 97.8%.To more precisely compare the frequencies of amino acids at the same positions among the seven NIa-Pro sites, we rearranged the frequency data according to positions . At posiThe position −3 had no dominant (≥50%) residue in the consensus sequence or in any of seven sites. When the seven sites were individually examined, the highest frequency of a residue was observed in the S7 site, where glutamic acid was present in 48 out of 135 proteins (35.6%). The next highest frequency was 31.1% (42 proteins), which was observed for arginine in the S4 site. Ten amino acids  were assigned as a consensus residue in at least one of the seven sites as these satisfied the minimum threshold frequency condition (≥10%): arginine and tyrosine were assigned as consensus residues in four sites in different combinations; glutamic acid and serine in three sites; histidine and lysine in two sites; and alanine, glutamine, threonine, and valine in one site.At position −2, histidine was the dominant residue in six sites  with frequencies ranging from 57.8% to 60.7%. At the S8 site (NIa-Pro/NIb-RdRp junction), however, no histidine was observed whereas glutamic acid showed the highest frequency with 37.0% (50) of the proteins containing it. Six different amino acids were designated as consensus residues in at least one of the seven sites: histidine and phenylalanine in six sites; leucine and threonine in two different sites; and glutamic acid and valine in one site.As mentioned above, glutamine was highly dominant (88.2–97.8%) at position −1 in six sites . However, in the S7 site (VPg/NIa-Pro junction), all 135 proteins had glutamic acid. In the other six sites, glutamic acid was the second most frequent residue, although its frequency was rather low (0.7–11.9%).When all the 945 NIa-Pro recognition sites were analyzed, 837 (88.6%) proteins had one of the following three amino acids with very small side chains at position +1: serine, glycine, and alanine. However, the abundance of these amino acids varied from site to site when the seven NIa-Pro cleavage sites were analyzed individually. Serine was the most abundant residue in five sites  with frequencies of 86.7%, 66.7%, 47.4%, 47.4%, and 48.2%, respectively. Serine was the second most common residue in the S3 site, albeit with low frequency (11.9%), and was scarce in the S6 site (0.7%). Glycine was highly dominant (82.2%) in the S6 site and was the second most abundant residue in sites S5 (10.4%), S7 (28.9%), and S8 (25.2%). Alanine was the most common residue (52.6%) in the S3 site, the second most abundant residue in sites S6 (17.0%) and S9 (36.3%), and the third most abundant residue in sites S7 (20.2%) and S8 (17.0%). Therefore, although the three amino acids with very small side chains were favored at position +1 in the NIa-Pro cleavage sites, each site exhibited different preferences for them. Interestingly, lysine was the third most abundant residue in site S3 (10.4%).Analysis of individual NIa-Pro cleavages sites revealed that only position −4 had the same consensus residue  in all the seven sites. Other positions (from −3 to +1) showed varied preferences for consensus residues among the seven sites, although their physicochemical properties were similar. Site-specific sequence preferences for NIa-Pro, observed in this study, may be associated with local substrate environments or cleavage reaction parameters in the different sites.Potyvirus is the largest genus of known plant viruses, containing 183 currently approved species -V-G/G (new). The HC-Pro recognition sequences are extremely conserved among potyviruses, especially at positions −4, −2, −1, and +1 , 26. TheThe previous consensus sequence of NIa-Pro protease cleavage sites was V-X-[FHL]-[EQ]/[AGS]. The new consensus sequence, deduced from 945 NIa-Pro recognition sites by combining all seven sites in each of the 135 representative polyproteins, was Y-[eyr]-[Hf]-[Qe]/[sga]. At each of the five core positions, one of the consensus residues was observed in 88.6%, 36.5%, 64.0%, 99.3%, and 89.5% of the 135 representative polyproteins, respectively. If more residues were added at positions −3 and −2 to achieve the 70% capture level, the extended consensus would be Y-[eyrshqtv]-[Hfl]-[Qe]/[sga], which could capture 73.5% and 73.5%, respectively, at positions −3 and −2.The most interesting finding in this study was that NIa-Pro showed different preferences for recognition sequences among the seven cleavage sites (S3 to S9 sites). The most prominent distinction was observed at position −1 in the S7 site (or VPg/NIa-Pro junction), where all 135 representative proteins contained glutamic acid. In the other six sites, glutamine was highly dominant (88.2–97.8%). At position −2, histidine was dominant (57.8–60.7%) in all but the S8 site (or NIa-Pro/NIb-RdRp junction), where histidine was absent and glutamic acid was the most common residue instead, with a frequency of 37.0%.The most variable sequence preference of NIa-Pro was observed at position +1, where one of the following three residues with very small side chains were highly preferred: alanine, glycine, and serine; however, preference for these three residues varied from site to site and the consensus residues at this position in the seven sites (from S3 to S9) were [Ask], S, [Sg], [Ga], [sga], [sga], and [sa], respectively, which could capture 74.8%, 86.7%, 77.0%, 99.3%, 96.3%, 89.6%, and 84.4% of the 135 representative proteins, respectively. Serine was the most frequent residue in five sites , whereas alanine and glycine were most abundant in the S3 and S6 sites, respectively.The observed consensus sequence variation among NIa-Pro recognition sites may be associated with site-specific fine-tuning of cleavage reaction. For example, the cleavage reaction at the VPg/NIa-Pro junction S7 site) was slower compared to other junctions [ site wasA comprehensive previous study showed that several parts of potyvirus genomes were under positive selection including the NIb-RdRp/CP junction (S9 site) in many potyvirus species , 29. It The findings of this study reveal distinct sequence preferences in seven NIa-Pro processing sites, suggesting that each site is finely tuned for differential characteristics of proteolytic cleavage reactions. To the best of our knowledge, this is the first study reporting consensus sequences for the seven NIa-Pro recognition sites. The newly deduced consensus sequences for nine cleavage sites and the representative polyprotein sequences obtained may be useful resources for the development of computational models and bioinformatics tools to accurately predict the proteolytic processing sites.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Escherichia coli, ppGpp regulates transcription of as many as 750 genes within 5 min of induction by binding directly to RNA polymerase (RNAP) at two sites ~60 Å apart. One proposal for the presence of two sites is that they have different affinities for ppGpp, expanding the dynamic range over which ppGpp acts. We show here, primarily using the Differential Radial Capillary Action of Ligand Assay (DRaCALA), that ppGpp has a similar affinity for each site, contradicting the proposal. Because the ppGpp binding sites are formed by interactions of the β’ subunit of RNAP with two small protein factors, the ω subunit of RNAP which contributes to Site 1 and the transcription factor DksA which contributes to Site 2, variation in the concentrations of ω or DksA potentially could differentially regulate ppGpp occupancy of the two sites. It was shown previously that DksA varies little at different growth rates or growth phases, but little is known about variation of the ω concentration. Therefore, we raised an anti-ω antibody and performed Western blots at different times in growth and during a stringent response. We show here that ω, like DksA, changes little with growth conditions. Together, our data suggest that the two ppGpp binding sites fill in parallel, and occupancy with changing nutritional conditions is determined by variation in the ppGpp concentration, not by variation in ω or DksA.During nutrient deprivation, the bacterial cell undergoes a stress response known as the stringent response. This response is characterized by induction of the nucleotide derivative guanosine tetraphosphate (ppGpp) that dramatically modulates the cell’s transcriptome. In  Escherichia coli, the basal level of ppGpp (from ~1 to 10 μM) only moderately affects gene expression, but induction of RelA in response to the accumulation of deacylated tRNA(s) increases the ppGpp concentration 100–1,000-fold, dramatically changing gene expression  and guanosine pentaphosphate , respectively, collectively referred to here as ppGpp  displays a modest lag in recovering from a downshift from a rich to a minimal medium  on transcription in vitro, crosslinking of 6-thio-ppGpp and binding of ppGpp to the RNAP-DksA complex suggested that Site 2 is at the interface of DksA and the β’ subunit rim helices at the entrance to the RNAP secondary channel (The identification of a second ppGpp-binding pocket (Site 2) in  channel . These r channel , and the channel . A recen channel .rrnB P1 promoter at saturating ω concentrations (~3-fold), and does not activate transcription from amino acid biosynthesis promoters. ppGpp binding to Site 2 alone  has a larger inhibitory effect on rrnB P1 (~6-fold) and is sufficient for full activation of amino acid biosynthesis promoters . These results are consistent with the growth properties of strains lacking ppGpp  has a modest inhibitory effect on transcription from the romoters . When bong ppGpp , ω  of rpoZ was inserted into pET23a-His10-SUMO  at the BamH1 and HindIII sites using HiFi DNA Assembly (NEB) to create RLG15371.Strains and plasmids are listed in Purification of RNAP -ppGpp to RNAP, RNAP/DksA, RNAP/TraR, or RNAP/GreB complexes was measured by the DRaCALA, adapted from Binding of -ppGpp (~5 nM). The observed fraction of [32P]-ppGpp bound at each RNAP concentration from each of seven independent experiments was fit to a one site saturation binding curve . The Kd,app’s for each independent binding curve were then averaged, resulting in a Kd,app of 6.1 ± 1.3 μM for ppGpp binding to Site 1 . DksA was not included in the binding reactions with wild-type RNAP and ppGpp, eliminating binding to Site 2 . Increaso Site 1 ,B.32P]-ppGpp bound  obtained by purification of RNAP from a strain deleted for rpoZ (ΔrpoZ). We showed previously that [32P]-ppGpp does not bind to DksA in the absence of RNAP  to which no DksA was added (and thus lacked Sites 1 and 2) was >100 μM (rpoZ) overexpressing ωΔ2–5 (RNAPΔω + ωΔ2–5) also had a Kd,app >100 μM . The data were fit for each of seven separate experiments, and the Kd,app values were averaged . If the nominal concentration of RNAP were used on the X-axis as in d,app would be even tighter, 1.7 ± 1.2 μM. Thus, when plotted either way, there was a significant difference in the Kd,app of the enzyme for ppGpp with both sites vs. only one site.Even though Sites 1 and 2 are located ~60 Å apart on RNAP  and haver Site 2  with [32averaged . For thed,app. The Hill coefficient was ~1, and the curve was not sigmoidal when the data were plotted on a linear scale  compared to the affinity for RNAP with only one site . In the presence of DksA-K98A, ppGpp bound to Site 1 with a Kd,app of ~1.3 ± 0.1 μM  with a K± 1.3 μM . ppGpp b± 0.5 μM , ~3-fold± 0.1 μM , althougd,app for ppGpp binding to Site 1 in the wild-type RNAP-TraR complex was at least 4.2 ± 0.6 μM  was ~6.4 ± 0.9 μM , but it declined ~2-fold when cells transitioned to stationary phase  to the two sites in RNAP. Available data indicate that, in of ppGpp . Structuof ppGpp , suggestin vitro  must result primarily from changes in ppGpp, not ω or DksA concentrations.Depending on which step during transcription is affected, ppGpp function requires the presence of ω or DksA. However, since the concentrations of ω and DksA change little during exponential growth in d,app = 3.3 ± 1.2 μM average affinity for the two binding sites on wild-type RNAP versus 6.1 ± 1.3 μM or 7.9 ± 1.3 μM, respectively, for RNAPs containing only Site 1 or 2). In addition, the affinity of ppGpp for the RNAPs in complex with the “separation of function” DksA variants is 1.3 ± 0.1–1.8 ± 0.5 μM. These results suggest that the increased affinity of Site 1 for ppGpp in the presence of DksA accounts for the reduced average affinity of wild-type RNAP for ppGpp relative to the affinity for either Site 1 or Site 2 alone. Thus, DksA allosterically enhances binding of ppGpp to Site 1, even though DksA and Site 1 are separated by ~30 Å at their position of closest approach (Interestingly, our data indicate that DksA increases the affinity of the wild-type RNAP for ppGpp (Kapproach . Althougapproach . The phyE. coli bind ppGpp, only a subset of which bind ppGpp with an affinity as high as RNAP (Finally, the concentrations of ppGpp observed during the stringent response appear to be much higher than needed for full occupancy of the two sites on RNAP. Although the high concentrations undoubtedly evolved in part to increase the kinetics of ppGpp occupancy of RNAP, some 70 proteins in  as RNAP . We suggAll datasets presented in this study are included in the article/AM designed, performed, and analyzed experiments and wrote the paper. WR analyzed experiments and wrote the paper. DT performed and analyzed experiments. RG designed and analyzed experiments and wrote the paper. All authors contributed to the article and approved the submitted version.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.


GBA1), implicated in the rare, autosomal recessive lysosomal disorder Gaucher disease, are the most common known genetic risk factor for Parkinson disease. Insights into the inverse relationship between glucocerebrosidase and alpha-synuclein have led to new therapeutic approaches for the treatment of Gaucher disease and GBA1-associated Parkinson disease. Unlike the current drugs used to treat Gaucher disease, which are highly expensive and do not cross the blood-brain-barrier, new small molecules therapies, including competitive and non-competitive chaperones that enhance glucocerebrosidase levels are being developed to overcome these limitations. Some of these include iminosugars, ambroxol, other competitive glucocerebrosidase inhibitors, and non-inhibitory chaperones or activators that do not compete for the active site. These drugs, which have been shown in different disease models to increase glucocerebrosidase activity, could have potential as a therapy for Gaucher disease and GBA1- associated Parkinson disease. Some have been demonstrated to reduce α-synuclein levels in pre-clinical studies using cell-based or animal models of GBA1-associated Parkinson disease, and may also have utility for idiopathic Parkinson disease.Parkinson disease, the second most common movement disorder, is a complex neurodegenerative disorder hallmarked by the accumulation of alpha-synuclein, a neural-specific small protein associated with neuronal synapses. Mutations in the glucocerebrosidase gene ( GBA1, encoding the lysosomal enzyme glucocerebrosidase (GCase) (Gaucher disease (GD), first described in 1882 by Dr. Phillipe Gaucher, is an inborn error of metabolism due to mutations in the gene  (GCase) . GCase,  (GCase) . In pati (GCase) . Clinica (GCase) . Acute n (GCase) . Chronic (GCase) . There iGBA1 are one of the most common genetic risk factors for PD, as well as dementia with Lewy bodies (DLB). Mutations in GBA1 were found in 5–20% of sporadic PD cases with an estimated odds ratio of 5.4. In DLB the odds ratio was 8.3 (GBA1-associated PD (GBA1-PD) resemble those with sporadic PD but exhibit an earlier disease onset. The lysosome, the central player uniting GD and PD, is the main organelle responsible for α-Syn degradation   which alGBA1-PD are missense mutations leading to misfolded enzyme, pharmacological chaperones which cross the blood-brain-barrier provide an attractive therapeutic strategy. Here we review studies of chaperones as a treatment for GD and GBA1-PD, focusing on preclinical developments.Pharmacological chaperones, small molecules designed to bind to a specific target protein and assist in the folding of the protein, are also being developed as an alternative treatment approach for GD or GD-associated PD. They further function by stabilizing the enzyme to prevent misfolding and to enhance accurate translocation of the protein from the ER to the Golgi and lysosome . Since mN-(n-nonyl) deoxynojirimycin (NN-DNJ). The first chaperone considered for GCase was an iminosugar, ∗ (V394L/V394L + saposin C−/−) showed that IFG administration extended lifespan and increased GCase activity and protein levels in the brain and visceral tissue, with attenuation of proinflammatory responses , was reported to bind to the GCase active site, increase GCase activity in cell lysates and restore lysosomal trafficking of GCase in N370S cells . Incubatesponses . Furtherextracts . Whetherextracts . Anotherd miRNAs . Treatin therapy .De La Mata et al. reported that a distinct iminosugar, NAdBT-AIJ restored mitochondrial dysfunction and GCase activity in L444P mice in combination with coenzyme Q10 . Anotherin vitro, demonstrating low cytotoxicity and significantly increased GCase activity in GD and GD+PD fibroblasts with different GD mutations, without any serious adverse effects , a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was found to be a pH-dependent, mixed-type inhibitor of GCase, and thus a potential therapy for GD. With its inhibitory activity maximal at neutral pH, ABX is found in the ER, and is undetectable at the acidic pH of lysosomes . GCase a effects . Anothern levels . Testingn levels . In nonhn levels . In Drosife span . Severalife span . One stuife span . Anotherife span . Howeverife span , and reaife span .GBA1-PD. Since fibroblasts do not store GlcCer, monocyte-derived macrophages from 20 patients with GD and GD iPSC-differentiated macrophages were examined. The non-inhibitory chaperone, NCGC758, enhanced GCase activity, reduced glycolipid storage, and normalized chemotaxis and the production of reactive oxygen species in the macrophages. NCGC758 also reversed inflammatory defects in GD macrophages by inducing autophagy and reducing IL-1b secretion  assay using an extract of spleen from a patient with GD as the source of mutant enzyme . In thisecretion . Anotherecretion . NCGC607ecretion . NCGC758ecretion .Histone deacetylase inhibitors (HDACi) have also been explored for the treatment of GD by modulating a GCase-associated ubiquitin–proteasome pathway . It was GBA1 mutation c.84dupG and mutations in other PD genes  has been evaluated as an inhibitory chaperone with fewer side effects and better efficacy, because its inhibitory effect does not occur at the low pH present in lysosomes. A recent comparative molecular docking analysis showed that ABX has more broad binding affinity toward GCase than NN-DNJ and other SRT drugs  and SRT  drugs to treat GD. However, ERT does not have utility for icacious . Small micacious  by stronicacious . Howeverutations  and alsoutations . IsofagoRT drugs . A phaseRT drugs .GBA1-PD because they avoid the primary problem associated with inhibitory chaperones, the inhibitory competition with substrates of GCase in the lysosome. A few non-inhibitory chaperones identified by HTS were shown to have disease-reversing effects in cellular models of GD or GBA1-PD (Non-inhibitory chaperones are highly attractive candidate drugs for the treatment of GD or  GBA1-PD , 2016a.  GBA1-PD .in vitro and in vivo disease models which mimic key features of the disease are cessary. DA neurons differentiated from patient iPSCs accumulate α-Syn and oxidized dopamine, and thus can be used to test the efficacy of small molecules for PD (GBA1-PD animal models need to be developed. Three dimensional in-vitro cell culture systems can be also considered for drug screening and preclinical validation of candidate drugs, because they more closely resembles the in-vivo cell environment than routine in-vitro cell culture (To increase the likelihood of success in clinical trials, a properly designed pre-clinical drug testing strategy is essential. Thus, appropriate s for PD . Howevers for PD . Howevers for PD . While hs for PD  and enhas for PD , it is n culture .T-UH contributed to the conceptual idea, reviewed, analyzed the literature, and wrote the manuscript and table. RS assisted in compiling background material and in writing the manuscript. ES provided the conceptual idea, reviewed, and edited 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.


Relief learning is the association of environmental cues with the cessation of aversive events. While there is increasing knowledge about the neural circuitry mediating relief learning, the respective molecular pathways are not known. Therefore, the aim of the present study was to examine different putative molecular pathways underlying relief learning. To this purpose, male rats were subjected either to relief conditioning or to a pseudo conditioning procedure. Forty-five minutes or 6 h after conditioning, samples of five different brain regions, namely the prefrontal cortex, nucleus accumbens (NAC), dorsal striatum, dorsal hippocampus, and amygdala, were collected. Using quantitative Western blots, the expression level of CREB, pCREB, ERK1/2, pERK1/2, CaMKIIα, MAP2K, PKA, pPKA, Akt, pAkt, DARPP-32, pDARPP-32, 14-3-3, and neuroligin2 were studied. Our analyses revealed that relief conditioned rats had higher CREB phosphorylation in NAC 6 h after conditioning than pseudo conditioned rats. The data further revealed that this CREB phosphorylation was mainly induced by dopamine D1 receptor-mediated activation of PKA, however, other kinases, downstream of the NMDA receptor, may also contribute. Taken together, the present study suggests that CREB phosphorylation, induced by a combination of different molecular pathways downstream of dopamine D1 and NMDA receptors, is essential for the acquisition and consolidation of relief learning. Relief learning is the association of a cue with the relief from an aversive event and can be observed across species, e.g., in humans, rodents, or flies . Since tWhile there is some basic knowledge on the neuroanatomical and neuropharmacological basis of relief learning in mammals ,7,10 theA key protein in synaptic plasticity and memory is the cAMP response element-binding protein (CREB) which functions as a transcriptional activator after its phosphorylation . PreviouBased on these findings, protein expression and phosphorylation patterns after relief conditioning in male rats were analyzed at two time points, 45 min, and 6 h, in the present study. First, CREB expression and phosphorylation levels were examined in the NAC and four other brain regions that at least partly may play a role in relief learning . Second, to identify the molecular pathways related to CREB phosphorylation in the nucleus accumbens during relief conditioning, the expression of selected kinases in different signaling pathways underlying NMDA and dopamine D1 receptor activation was investigated. Sprague-Dawley rats, 7 to 9 weeks old at the time of conditioning were used. They were bred in our animal facility  and kept in temperature-controlled rooms  under a light/dark cycle of 12/12 h (lights on at 6:00 a.m.) with food and water ad libitum. Experimental subjects were housed in groups of 4 to 6 in standard cages filled with wood chip bedding . Behavioral tests and tissue isolation were performed during the light phase from 9:00 A.M. to 4:00 P.M. All studies were conducted under the European regulations for animal experiments (2010/63/EU) and approved by the local authorities .Male Behavioral experiments were performed in a startle system consisting of wooden chambers  equipped with animal cylindric enclosures (9 cm inner diameter and 16 cm in length) made of acrylic glass. Motion-sensitive transducers mounted underneath these enclosures detected the movements of the animals. For data acquisition, the output signal of the transducers was digitized at a sampling rate of 1 kHz and stored on a computer. As startle magnitudes, the mean transducer output in the time window 10 to 30 ms after the startle probe onset was taken [As a conditioned stimulus (CS), a light cue  was presented to the animals with a 10 W white bulb mounted on the back of the test chambers. As an unconditioned aversive stimulus, scrambled foot shocks  were administered by a floor grid of six parallel bars (0.5 cm in diameter and 1 cm apart) inserted into the enclosures during conditioning. A white background noise of 50 dB sound pressure level (SPL) and the acoustic startle probe  were generated by a loudspeaker mounted at the ceiling of the chambers.On the first day, a baseline startle test was performed. The rats were introduced into the enclosures of the startle boxes and after an acclimation time of 5 min, 10 startle probes stimuli with an inter-trial interval (ITI) of 30 s were presented. On day 2, animals were conditioned. For relief conditioning, the US, followed by the CS (US offset to CS onset: 2.5 s), were presented 15 times with a mean ITI of 150 s. In the control-conditioning group, the US and the CS presentations were uncorrelated in time . No startle probes were presented during the conditioning session. Forty-five minutes or 6 h after the conditioning procedure, most of the rats were sacrificed for tissue collection (see below). However, a small sample of animals was subjected to a retention test one day later. The retention test consisted of 5 min of accommodation time, followed by 10 startle probes to habituate the startle response. Then, 20 further startle stimuli were presented, half of them in the absence and half of them in the presence of the light CS .The baseline startle test was used to group the rats into the groups “Relief” and “Control” with similar mean baseline startle magnitudes. One day later, as shown in Rats were sacrificed 45 min or 6 h after the conditioning session. They were capitated under anesthesia with isoflurane  and the brains were quickly extracted and dissected in coronal slices (1 mm) using rat brain slicer matrix . The brains were dissected on ice into 5 different regions, namely the prefrontal cortex (PFC), nucleus accumbens (NAC), dorsal striatum (STR), dorsal hippocampus (HIP), and amygdala (AMY) . Samples®Nuclease  using K-Control™ TLC homogenizer. For protein extraction the homogenized samples were solubilized in 4xSDS sample buffer containing 1% (w/v) SDS, 40% (v/v) glycerol, 20% (v/v) β-mercaptoethanol, 250 mM Tris pH 6.8, 0.004% (w/v) Bromophenol blue and, boiled at 95 °C for 5 min. Protein quantification was performed by amido black assay [The brain samples were homogenized on ice in 1xPBS (pH 7.4), containing proteinase inhibitor , phosphatase inhibitor , and 250 U Benzonaseck assay . For Wesp ≤ 0.05 was used for all tests. Outliers (Q = 5%) were removed from the analysis.Data were expressed as means ±SEMs. Behavioral data were analyzed with analysis of variance (ANOVA). Planned comparisons of protein expression and phosphorylation in control- and relief conditioned rats were performed with non-parametric t-tests . A significance level of F1,20 = 7.31, p = 0.01) and a significant effect of trial type  but not of group . Furthermore, post-hoc Sidak’s tests showed significant startle attenuation in the Relief group  but not in the Control group . In the retention test on conditioned relief, the light stimulus attenuated the startle magnitude in the relief-conditioned rats but not in the control-conditioned rats . This isThe first part of the protein analysis was focused on CREB expression and phosphorylation, 45 min and 6 h after conditioning. p = 0.04). After 6 h, pCREB (p = 0.007), as well as phosphorylation ratio of CREB (p = 0.04), were significantly higher in relief-conditioned animals compared to the control group.In the NAC a, total . After 6 h, no significant differences between the two conditioned groups were seen, but a clear tendency towards higher phosphorylation could be observed in the fraction of relief learners. Interestingly, in the PFC (p = 0.01) and the phosphorylation ratio (p = 0.002) after 6 h, while no differences were seen after 45 min. In STR and AMY, the expression and activation level of CREB in both conditioned groups, as well as both time points, were similar.In the HIP b, the ex the PFC c, relief2+-calmodulin-dependent kinase (CaMK) that is activated by the calcium influx [We have previously shown, that accumbal NMDA receptor activation plays a key role in the acquisition of conditioned relief . NMDA rem influx . To deteF1,20 = 6.20, p = 0.02,). This suggests that relief conditioning activates both pathways together and that CREB phosphorylation could be mediated by their joint activation. There were no effects of relief conditioning on these kinases in the four other brain regions at both time points  compared with the control-conditioned . Akt phosphorylation can occur at Thr308 or Ser473. In this study, the Akt expression and phosphorylation on Thr308 were analyzed. After 45 min, no Western blot signal of pAkt was detectable in the NAC, and after 6 h, no effects of relief conditioning on Akt expression and phosphorylation were found b,e. 2+ signaling pathways, dopamine D1 receptor stimulation can modulate the expression and phosphorylation of DARPP-32 (Dopamine and cAMP-regulated phosphoprotein), which is another important regulator for CREB phosphorylation. Phosphorylation of DARPP-32 can happen at Thr34 or Thr75. These different phosphorylation sites lead to either induction or reduction of Ser133 CREB phosphorylation [Via PKA and Carylation ,20. Neitp = 0.02; Motivated by the data of a comprehensive collaborative study of our group on the proteome after instrumental learning , we alsoThe aim of this study was to reveal molecular underpinnings of relief learning in male rats. Based on previous findings, the coincidence activation of NMDA and dopamine D1 receptors in NAC is crucial for relief memory formation ,8. ThereOur molecular analyses were focused on brain regions that have been shown to be involved in relief learning or similar associative learning processes, namely the NAC, PFC, AMY, or HIP while the STR is most probably not involved in such learning processes and, hence, served as an arbitrary control brain region. First, we analyzed the expression and phosphorylation level of CREB. CREB plays a key role in memory acquisition and consolidation  and CREBTo demonstrate the involvement of CREB, the expression of CREB and phosphorylated CREB-Ser-133 were studied in all candidate regions of the brain at both time points. Western blot data revealed a biphasic pattern of CREB phosphorylation in the NAC. First, 45 min after conditioning, there was a decrease of CREB phosphorylation, and later, 6 h after conditioning, the CREB phosphorylation level in relief-conditioned animals was significantly higher than in the control group. Both processes may be caused by the activation of NMDA receptors during relief conditioning. NMDA receptors are linked to protein phosphatases and kinases, and activation of NMDA receptors in extrasynaptic or synaptic regions of neurons lead to pCREB dephosphorylation or CREB-phosphorylation, respectively. Calcium-dependent signals per se can also act as a switch between intracellular signaling pathways that can modulate neuronal plasticity through CREB phosphorylation and/or dephosphorylation in different types of learning ,30. Our Interestingly, the decreased CREB phosphorylation in the NAC at 45 min was accompanied by decreased CREB phosphorylation in the hippocampus in relief-conditioned rats a,b. Howe2+-calmodulin-dependent kinases (CaMK), protein kinase B (PKB or Akt) extracellular-Signal-regulated kinase (ERK) or mitogen-activated protein kinase (MAPK) protein kinase C (PKC), p90 ribosomal S6 kinase (p90RSK), casein kinase I, and casein kinase II [2+-calmodulin-dependent kinase (CaMKII) [After analyzing the modulation level of CREB, we further asked which signaling pathways are involved in the observed CREB phosphorylation in the nucleus accumbens during relief learning. Several studies demonstrated that CREB phosphorylation at Ser-133 may occur through any of its upstream protein kinases including protein kinase A (PKA), Cainase II ,13,32. Binase II  and is ainase II ,18,33. Oinase II . Activat2+ signaling pathways through dopamine D1 stimulation mediate the expression and phosphorylation of DARPP-32. The activation of DARPP-32 is dependent on its phosphorylation state and modulated by different kinases and phosphatases. DARPP-32 has four sites for phosphorylation: Thr-34, Ser-75, Ser-97, and Ser-130. It is reported that, following the induction of dopamine D1 receptor, PKA can phosphorylate DARPP-32 at Thr-34 and simultaneously, dephosphorylate Thr-75 and Ser-97 by PP2A/B56δ, resulting in amplification of the dopamine D1 signaling and inhibition of protein phosphatase1 (PP1) [In addition to NMDA receptors, stimulation of dopamine D1 receptors is also required for relief learning . PKA is e1 (PP1) . Phosphoe1 (PP1) ,20. Moree1 (PP1) ,37. Since1 (PP1) . In addition to the signaling pathways underlying dopamine D1 and NMDA receptors, other proteins may also be involved in relief memory formation. A recent comprehensive analysis on protein rearrangements in mouse brain after auditory learning revealed several protein candidates, including 14-3-3 and neuroligin2, to be overexpressed upon learning . 14-3-3 There are several limitations of our study. For example, we only tested male rats and sex might affect the results. However, we believe that sex is not a critical factor in relief learning since we did not detect sex differences in relief learning in previous experiments . Another limitation is that CREB can also be phosphorylated by cGMP-PKG . HoweverPrevious studies of our group, in rodents revealed a critical role of coincident activation of dopamine D1 and NMDA receptors within the NAC in relief learning. The aim of the present study was to decipher the molecular pathways involved in relief learning, by focusing on the signaling pathways underlying NMDA and dopamine D1 receptors. We found robust CREB phosphorylation in the NAC, 6 h upon relief learning . Lu et aTaken together, our study strongly supports a critical role of the NAC in relief learning and is the first one elucidating the involved signaling pathways.


Mauritia flexuosa palm swamp, the prevailing Peruvian Amazon peatland ecosystem, is extensively threatened by degradation. The unsustainable practice of cutting whole palms for fruit extraction modifies forest's structure and composition and eventually alters peat‐derived greenhouse gas (GHG) emissions. We evaluated the spatiotemporal variability of soil N2O and CH4 fluxes and environmental controls along a palm swamp degradation gradient formed by one undegraded site (Intact), one moderately degraded site (mDeg) and one heavily degraded site (hDeg). Microscale variability differentiated hummocks supporting live or cut palms from surrounding hollows. Macroscale analysis considered structural changes in vegetation and soil microtopography as impacted by degradation. Variables were monitored monthly over 3 years to evaluate intra‐ and inter‐annual variability. Degradation induced microscale changes in N2O and CH4 emission trends and controls. Site‐scale average annual CH4 emissions were similar along the degradation gradient . Site‐scale average annual N2O emissions (kg N ha−1 year−1) were lower at the mDeg site (0.5 ± 0.1) than at the Intact (1.3 ± 0.6) and hDeg sites (1.1 ± 0.4), but the difference seemed linked to heterogeneous fluctuations in soil water‐filled pore space (WFPS) along the forest complex rather than to degradation. Monthly and annual emissions were mainly controlled by variations in WFPS, water table level (WT) and net nitrification for N2O; WT, air temperature and net nitrification for CH4. Site‐scale N2O emissions remained steady over years, whereas CH4 emissions rose exponentially with increased precipitation. While the minor impact of degradation on palm swamp peatland N2O and CH4 fluxes should be tested elsewhere, the evidenced large and variable CH4 emissions and significant N2O emissions call for improved modeling of GHG dynamics in tropical peatlands to test their response to climate changes. Mauritia flexuosa palm swamp peatlands showed significant N2O emissions and large CH4 emissions, the later exhibiting an exponential rise with increased precipitation. Their degradation, which results from cutting whole palms for fruit extraction, induced microscale  changes in N2O and CH4 emission trends and controls but no obvious changes at the macroscale. Monthly and annual emissions were mainly controlled by variations in: WFPS, water table level (WT), and net nitrification for N2O; WT, air temperature and net nitrification for CH4.Over 3 years of monitoring, Peruvian Amazon  This degradation translates into changes in vegetation cover and composition, reductions in biomass C stocks and litter inputs, and could potentially result in destabilization of peat deposits , with emission rates in the tropics found to be maximum around a WFPS of 60% and to remain high at 80% WFPS , and several studies have reported a decline in net CH4 flux accompanying a lowering of the WT  How do N2O and CH4 fluxes vary spatially at the microscale and macroscale under undegraded and degraded conditions? (b) How do the fluxes vary intra‐annually and inter‐annually? (c) How do environmental variables control the spatial and temporal variation of the fluxes? Spatial variability at the microscale was assessed by differentiating hummocks supporting live or cut palms from surrounding hollows. The macroscale analysis considered structural changes in vegetation and soil microtopography as impacted by degradation.This study investigated soil fluxes of N22.1The study was carried out in the province of Loreto, southwest of Iquitos, in the Northern Peruvian Amazon. Based on 1948–1994 weather data, the climate of the region is warm and humid with mean annual temperature of 27°C and mean annual rainfall of 3,087 mm Marengo, . Most moM. flexuosa palms, located near the Itaya River, one of the tributaries of the Amazon River , while the hDeg site was dominated by Cecropia membranacea, a pioneer tree species , a site moderately degraded , and a heavily degraded site . The Intact site was located within the protected Quistococha Regional Reserve. The mDeg and hDeg sites were adjacent to the reserve and used by local communities for extraction of 2.22 measurement sections, separated by ~150 m  and its adjacent hollow (live‐hollow). At the degraded sites, a plot consisted of two subplots: One with a live M. flexuosa palm similarly to the Intact site, the other with the stump of a cut M. flexuosa palm on a hummock (cut‐hummock) and its adjacent hollow (cut‐hollow). M. flexuosa palms are typically cut at 1 m height with the stump left on‐site while the remaining cut trunk is used to build walking paths inside the forest.The experiment was installed in April 2014 at the Intact site, in July 2014 at the hDeg site and in February 2015 at the mDeg site. At each site, we established three 250 m4 and N2O fluxes were monthly measured by the static closed chamber method in opaque PVC chambers  permanently inserted 3–5 cm into the ground. The measurements were systematically performed between 08:30 and 15:30 hr to avoid potential diel variation in gas fluxes. Dial fluctuations of CH4 fluxes were found to be negligible at the Intact site  flow of 30 ml/min. Soil respiration was measured concomitantly with fluxes of N2O and CH4 using the dynamic closed chamber method  amounted to 40 months. CH2O and CH4 at ambient concentration and chamber deployment time. Mean and coefficient of variation of 35 N2O and CH4 ambient samples taken at random from monthly GC control procedure datasets amounted to, respectively, 330 ppb and 9% for N2O and 1,522 ppb and 1% for CH4. The detection limit was 6.8 g N ha−1 day−1 for N2O and 2 g C ha−1 day−1 for CH4. Following recommendations by Parkin et al. . The detection limit of the method was computed using the formula by Parkin et al.  based onn et al.  and Gilbn et al. , we repon et al. . The rel2.3In all, 24 samples per site (12 from hollows and 12 from hummocks) were collected from the upper 5 cm of the soil profile using a metal ring  equipped with a thermometer for hourly recording of air temperature. This climate station was situated between 1 and 3 km from the sites. Several environmental parameters were measured concurrently with soil GHG sampling. The WT relative to the soil surface was measured in a perforated PVC tube (5 cm in diameter and 1.5 m in length) installed permanently in the soil at 50 cm from each chamber. Soil temperature in the top 10 cm and air temperature were measured at the side of each chamber using a portable digital thermometer. Gravimetric soil moisture was measured from samples collected from the top 10 cm near each chamber. Samples were carefully transferred to sealed plastic bags taking care of not losing water, stored in a cool box and their wet mass weighted immediately upon return to the laboratory. Soil samples were oven‐dried at 60°C until reaching constant mass and soil moisture was determined on a dry mass basis. The soil WFPS was calculated based on the formula by Linn and Doran  as the p4+ content was analyzed using the formaldehyde method  concentration; net nitrification as the change in NO3− concentration over 10 days. Inorganic N stocks were calculated from the first extraction.Nitrogen availability was determined by measuring inorganic N pools and the rates of net mineralization and nitrification quarterly over 2 years starting in October 2016. Nine replicate soil samples per microtopography and site were collected around live palms to a 5 cm depth using a ring sampler. Samples were stored on ice, transported to the laboratory and kept at 4°C for a maximum of 3 days until incubation. Coarse roots and litter were manually removed from the samples before incubation. Net mineralization and net nitrification were determined following the procedure of Hart et al. . For eac2.52O and CH4 were examined using a mixed‐effects ANOVA model with four factors. Sampling month was treated as a random factor, whereas disturbance level, status and microtopography were treated as fixed effects. The design was asymmetric because not all status levels were present at all sites , and it was unbalanced because not all combinations of treatments had nine replicates per sampling month (due to missing data). Considering these limitations, adequate degrees of freedom, mean squares and F‐ratio were calculated using the PERMANOVA routine in PRIMER v7. Although this method was specially designed for multivariate data, it can be used for univariate data using Euclidean distance , status , microtopography (hummock and hollow) and temporal variability  on the fluxes of Nd Figure . Also, t2O and CH4 emissions to each variable. For the first significant interaction that included degradation level, status or microtopography, a paired t test with combined variances was applied after weighting by n and with probability correction for multiple comparisons using the Holm procedure . Relationships with annual averages were tested across sites considering palm status and microtopography. Detection of emission hotspots was performed using boxplots analysis. A chamber qualified as a hotspot when it displayed over the monitored period at least three values higher than three times the interquartile range from the upper edge of the 50% percentile . Soil macronutrients differed between sites only in hollows, with higher levels of Ca at the hDeg site than at the mDeg site and a higher concentration of Mn at the hDeg site than at the Intact site.Edaphic properties were homogeneous among sites and microtopographies Table . Bulk de4+ (>380 mg N/kg d.m.) while NO3− contents were quite low  was also much higher than net nitrification rate (<1 mg N kg−1 d.m. day−1). Soil NH4+ content was lower in hollows than in hummocks at the hDeg site (p < .0001). Likewise, net mineralization was lower in hollows than in hummocks at the Intact site (p < .0001), and in general (p = .05) corresponding to higher soil moisture levels (p < .0001). NH4+ content was lower at the hDeg site than at the other sites while net mineralization increased with degradation (p < .0001 for both). Soil mineral N content and dynamics did not follow a consistent temporal pattern but in April 2017 overall NH4+ contents, net mineralization and net nitrification were at their maximum, while NO3− contents were at their minimum  and increased when the water level went up in hollows at the hDeg site . Monthly net nitrification rate decreased when the soil WFPS rose in hollows at the mDeg site .Over the whole measurement period, inorganic N pools were dominated by NHMonthly and annual precipitation during the measurement period are presented in Figure F66,2,708 = 9.38, p < .001) between 28 and 32°C, 26 and 33°C and 27 and 34°C at the Intact site, mDeg site and hDeg site, respectively, with no apparent link to El Niño or La Niña events , with monthly means fluctuating between 19 and 30°C at the Intact site, 24–27°C at the mDeg site and 20–28°C at the hDeg site . In May and June 2015, the sites flooded, and the WT rose to 80–150 cm above the soil surface. These months were affected by the El Niño event and were preceded by 2 months of very high rainfall, between 450 and 530 mm , with high values in July 2015 following the May–June flooding.The soil WFPS was similar among microtopographies at the Intact site .Mean monthly N−1 day−1 detection limit band. Monthly N2O fluxes fluctuated over a wide range, from small uptakes (−4 g N ha−1 day−1) to high emissions , with alternating monthly increases and decreases.Measured flux values were 84% within the −6.8; 6.8] g N ha.8; 6.8 g2O fluxes were mainly driven by variations in mineral nitrogen content and dynamics at the Intact site .Monthly average NS Figure . Monthlye Figure . The pows Figure . This re2O emissions along the degradation gradient ranged from 0.5 ± 0.1 to 2.6 ± 0.7 kg N ha−1 year−1  to high emissions , but fluxes were consistently large across sites following the May–June 2015 flooding.Measured flux values were 4% within the  g C ha4 fluxes were linked to the WT at all sites  of site‐scale surface. At this microtopography, the lower WT level at the Intact site than at the degraded sites and the lower WFPS at the Intact and highly degraded sites than at the site of medium degradation were consistent over time. Hollows displayed a similar WT level among sites  and the presence of one hotspot at the sites could not be clearly associated with the environmental variables. Such high flux pulses have also been reported for other tropical peat forests  were within the detection limit band; as also reported by several studies conducted in undrained peatlands  or for annual site‐scale rates  reported for similar M. flexuosa palm swamp forests in the Peruvian Amazon  and are close to the average of 2.7 ± 1.9 kg N ha−1 year−1 reported for intact tropical peat swamp forests  for Amazonian floodplains and Smith and Lewis (−1 day−1) for temperate wetlands and in the low range of the 1.5–9 g C ha−1 day−1 limit evaluated by Christensen  and reported by Teh et al.  but lower than values recorded in forested peatlands of Panama  were moisture‐related suggesting that future changes in rainfall patterns in the region may substantially alter N2O and CH4 emissions. Our study also provided evidence of the significant magnitude of the emissions, particularly for CH4; highlighting the critical need for their inclusion in biogeochemical modeling of peatlands.Tropical peatlands are of global and regional importance but remain critically understudied especially outside of Southeast Asia. Very little is known about their state of conservation and their contribution to mitigate and/or exacerbate climate change. Our study showed that degradation of palm swamp peatlands in the Peruvian Amazon modifies soil–atmospheric exchanges of NSupplementary MaterialClick here for additional data file.


Out of the pre-specified biomarkers, seventeen candidate proteins were differentially altered between the two drugs (p < 0.05). These include IGFBP-2 and AGER associated with diabetes complications, and MMP-3 associated with extracellular remodeling. As a follow-up, IGFBP-2, MMP-3, and IGF-I were quantified with an ELISA using a larger sample size of DMD biosamples  over a longer treatment duration. MMP-3 and IGFBP-2 validated the SOMAscan® signal, however, IGF-I did not. This study identified GC-responsive biomarkers, some associated with safety, that highlight differential PD response between Dfz and Pred.Prednisone (Pred) and Deflazacort (Dfz) are commonly used glucocorticoids (GCs) for Duchenne muscular dystrophy (DMD) treatment and management. While GCs are known to delay the loss of ambulation and motor abilities, chronic use can result in onerous side effects, e.g., weight gain, growth stunting, loss of bone density, etc. Here, we use the CINRG Duchenne natural history study to gain insight into comparative safety of Pred versus Dfz treatment through GC-responsive pharmacodynamic (PD) biomarkers. Longitudinal trajectories of SOMAscan Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder affecting the expression of dystrophin protein, an essential protein that maintains muscle fiber integrity and function . The lacMany efforts have been undertaken to investigate the use of biomarkers in DMD ,18,19 an® technique, followed by confirmation of a specific set of PD biomarkers by ELISAs. The objective here is to define PD biomarkers that differ in their response to Pred and Dfz, and investigate if these biomarkers are known to be associated with reported differences in term of side effects between these two drugs.There are ongoing debates among families, clinicians, and regulatory agencies over which GC drug (Pred or Dfz) and regimen is better for DMD boys. GC responsive biomarkers (for both safety and efficacy) have been defined and confirmed in different diseases and multiple cohorts ,24,25, uThe study protocol was approved by Institutional Review Boards at all participating institutions that provided serum samples. These included, the Office of Research IRB administrations at the University of California Davis, Davis, CA, the University of Pittsburgh, Pittsburgh, PA, Children’s National Health System, Washington DC, the Conjoint Health Research Ethics Board, University of Calgary and the Human Subject Research Review Committee at Binghamton University, NY. Informed written consent was obtained from the parents of the participants or their legal guardians for biomarker studies at each site.n = 31 boys, 4 to 10 years old) and used steroid-naïve and steroid-treated paired samples to define 107  PD biomarkers from 1310 serum proteins (SOMAscan® array).The CINRG-DNHS is a natural history study of 440 DMD boys. The CINRG-DNHS investigators enrolled these subjects at 20 centers in nine countries . In thisBased on the above-mentioned study , we pre-A larger data set (confirmation dataset B) contained up to 76 longitudinal samples on 29 ambulatory subjects (17 deflazacort-treated and 12 prednisone-treated) from CINRG DNHS. Note that all samples/subjects from screening dataset A were used in confirmation dataset B as well. For both datasets, we focused on ambulatory patients to minimize any possible confounding issues with the stage of disease (after loss of ambulation). Again, these subjects were age-matched. Summary characteristics of the subjects are provided in A subset of PD serum protein biomarkers, including MMP3, IGFBP-2, and IGF-I, were selected for confirmation analysis using ELISA assays. An ELISA kit from Meso Scale Diagnostics (MSD) was used to measure levels of MMP3. Similarly, ELISA kits from Thermo Fisher Scientific and R&D Systems, Inc. were used to measure levels of IGFBP2 and IGF-I, respectively. These candidates were chosen based on the availability of a validated ELISA assay kit. All three assays were sandwich ELISA, which were highly specific for antigen detection. Dilution factors for serum samples were 1:10, 1:400, and 1:100 for MMP-3, IGFBP-2, and IGF-I, respectively. The three assays were performed following the manufacturer’s instructions. Details about serum samples used in ELISA validation are shown in ® and ELISA measurements was investigated on the subset of samples common to screening dataset A and confirmation dataset B.As depicted in the schematic , we firsn = 7) versus Dfz-treated DMD patients (n = 8) accounting for duration of treatment (time on GC) and interaction between treatment duration and type of GC (Pred or Dfz). In general, Pred and Dfz seem to engage the PD biomarkers similarly and altered their trajectory in the same direction in screening dataset A. This was not the case for 17 PD biomarkers that were found differentially altered in their average levels and/or longitudinal trajectories  in Pred-treated vs. Dfz-treated DMD patients. These 17 differentially altered PD biomarkers are listed in We examined differences in the longitudinal trajectories of pre-specified 107 PD biomarkers in Pred-treated patients (p = 0.040) in the Dfz-treated group, as compared to the Pred-treated group. Similarly, longitudinal trajectories of Ficolin-1  and Annexin A2  also seemingly diverged in addition to their differential levels between the two drugs. p = 0.040). While TNFRSF21  was not found to be differentially affected by the two different drugs in terms of longitudinal trajectory slopes but its mean sera levels was significantly lower in the Pred-treated group compared to the Dfz-treated group (p = 0.005). Similarly, mean levels of FCN-1 were lower in the Dfz-treated group relative to the Pred-treated group (p = 0.038), while MMP3 mean levels were significantly elevated in the Dfz-treated group relative to the Pred-treated group.Two major groups of differentially affected steroidal PD biomarkers were thus identified. The first major group consisted of PD biomarkers that were repressed by GC in general, but exhibited a significantly lower mean level in Pred treated group relative to Dfz treated group. These included leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), tumor necrosis factor receptor superfamily member 21 (TNFRSF21), chordin-like protein 1 (CHRDL1), soluble advanced glycosylation end product-specific receptor (sRAGE), annexin A2 (ANXA2), CD166 antigen (CD166), scavenger receptor cysteine-rich type 1 protein M130 (sCD163), induced myeloid leukemia cell differentiation protein (MCL-1), transmembrane glycoprotein NMB (GPNMB), mitogen-activated protein kinase 14 (MAPK14), and neural cell adhesion molecule L1 (NCAM-L1). The second (minor) group consisted of PD biomarkers that increased in their levels following GC treatment and consisted of two subgroups. The first subgroup consisted of those that were significantly elevated in serum samples of Dfz relative to the Pred-treated group such as hemojuvelin (HJV), stromelysin-1 (MMP3) while the second group consists of those biomarkers that were significantly elevated in serum samples of Pred relative to Dfz-treated group such as insulin-like growth factor I (IGF-I), ficolin-1 (FCN1), and cGMP-inhibited 3′,5′-cyclic phosphodiesterase A (PDE3A). One interesting PD biomarker is insulin-like growth factor-binding protein 2 (IGFBP-2); this showed diverging longitudinal trajectories (p = 0.006 for difference in trajectory slopes over time) compared to those treated with Pred (p = 0.112) and BMI (p = 0.08) over time between patients treated with Dfz and Pred , IGFBP-2 neared significance , while IGF-I did not validate the SOMAscan® signal. ® data and ELISA data with the IGF-I measurements having a substantially worse Pearson correlation than MMP-3 and IGFBP-2.For the reported data above, we had a small sample size and we did not adjust for multiple testing, thus a follow-up confirmation analyses on a subset of PD biomarkers was carried out. Unfortunately, from the list of PD biomarkers identified in GCs are and continue to be the standard of care for several chronic inflammatory and auto-immune diseases including DMD ,21,25. IA close examination of the 17 differentially altered PD biomarkers (from screening dataset A) between Dfz and Pred in term of their levels, longitudinal directionality and their potential physiological function led to the following interpretation. Among these 17 PD biomarkers, 12 were significantly reduced in the Pred-treated group relative to the Dfz-treated group after adjusting for treatment duration (the samples were age matched). These 12 decreased PD biomarkers by Pred can be classified into subgroups with the first subgroup consisting of inflammatory and immune associated proteins such as LILRB1, TNFRSF21, sRAGE, CD166, and sCD163. Previous studies have suggested that Dfz is a stronger immune suppressant than Pred . In contHowever, the differential decrease in the mean levels of MCL-1, MAPK14, and CHRDL1 by Pred relative to Dfz could be considered to be evidence of possible efficacy in DMD. Indeed, MCL-1 and MAPK14 were previously reported by us  to be elp = 0.0507; near significance). Note that, in our ELISA confirmations, we had the smallest number of additional samples available for IGFBP-2. IGFBP-2 binds to IGF-1 and regulates growth. While IGFBP-2 was decreased by Dfz treatment, IGF-1 was relatively increased by Dfz over Pred. Unfortunately, we were unable to validate the IGF-1 SomaScan® signal using ELISA; nevertheless, an increase in IGF-1 by GC treatment might be associated with antiinflammation efficacy [Another relevant PD biomarker that was differentially altered over time between the two drugs is IGFBP-2. It sharply decreased in its longitudinal trajectory in Dfz-treated group compared to the Pred-treated group. This observation was further validated by ELISA assay , another glucocorticoid, on short-term growth and bone turnover confirmed that both drugs affected short-term bone turnover and growth . HoweverMMP3, also known as stromelysin-1, was dramatically increased in the blood circulation following GC treatment, as previously shown by us  and othe® signal. IGFB2 had a decreasing longitudinal trend associated with Dfz, at odds with the observation for Pred. This may be associated with differential growth stunting seen between the two drugs. The dramatic increase of MMP3 by Dfz relative to Pred remains to be interpreted. Further studies are needed to test the physiological significance of these Dfz and Pred differentially affected PD biomarkers. The study’s limitations include the small sample size, no adjustments for multiple testing, and that the data come from a natural history study with lots of observed variability. We have tried to overcome these limitations by performing lab validation using ELISA and using a larger sample size and longer treatment duration. For biological validation, an external cohort is needed to validate our findings. Note also that we did not investigate direct associations of biomarkers with efficacy/safety outcomes; larger sample sizes are required for such studies and are the objective of ongoing research. While preliminary, this study identified serum proteins that were altered between the Dfz and Pred groups using SOMAscan® array data and we successfully validated two biomarkers using ELISA that may be associated with adverse effects. However, further studies using larger sample sizes collected from well controlled cohorts enrolled in ongoing and future clinical trials comparing the safety and efficacy of Dfz versus Pred are needed to validate and test these differentially altered pharmacodynamic biomarkers identified herein.Here, we investigated less invasive and objective PD biomarkers that might prove useful to monitor disease progression and response to GC therapies in DMD. We identified differences in serum levels of PD biomarkers between Pred- and Dfz-treated subjects, some of which may be associated with safety. Such blood accessible biomarkers in DMD could play an important role in clinical trials and decision making . In our