Polymers are intrinsically flammable materials; hence, fire retardance (FR) is required in their most common applications . Recently, it has been reported that cyclodextrin (CD) and cyclodextrin derivatives are beginning to be introduced into Intumescent Fire Retardant (IFR) formulations in place of pentaerythritol, which is used in IFRs that are currently on the market. Since IFRs are of less environmental concern than their hazardous halogen containing counterparts, the use of natural origin compounds in IFRs provides a way to comply with green chemistry issues. BCD and BCD derivatives presence in IFR mixtures promotes a higher yield of blowing gases and char when polymeric materials undergo combustion. Both processes play important roles in intumescence. The key rule to obtain in insulating compact char is the good dispersion of the nanoparticles in the matrix, which can be achieved by functionalizing nanoparticles with BCD derivatives. Moreover, CD derivatives are attractive because of their nanosized structure and their ability to form inclusion complexes with many compounds used as FR components, reducing their release to the environment during their shelf life of FR items. Often, fire retardance performed by BCD and BCD derivatives accompanies other relevant properties, such as improved mechanical resistance, washability resistance, self healing ability, thermal conductivity, etc. The application of CD fire retardant additives in many polymers, such as poly(lactic acid), poly(propylene), poly(vinyl acetate), poly(methyl methacrylate), linear low density poly(ethylene), polyamides, and polyesters are comprehensively reviewed here.  Intrinsically, polymers are highly flammable. Their extensive use in fields, such as construction, transportation, electrical and electronic, textiles, clothing, and so on, causes death or severe wounding of several people in fire events every year. From 2014 to 2016, an estimated 24,000 residential building electrical fires were reported to United States fire departments each year. These fires caused an estimated 310 deaths, 850 injuries, and $871 million in property loss x+·[(A n−)x/n·yH2O]x−, where M2+, M3+, and An− are divalent and trivalent metal cations and interlayer anions, respectively. They are suggested as flame-retardants additives, thanks to their endothermic decomposition at high temperatures. However, the improvement of polymer-based nanocomposites properties closely depends on the distribution of nanofiller in the polymer matrix. Biobased modifiers of the nanofiller with flame-retardant activity are preferably to this goal. Kalali et al. [3O4-CDBS-LDH hybrid into an epoxy matrix not only improved the flame-retardance and mechanical properties of EP, but also slightly increased the thermal conductivity (from 0.220 for EP and CDBS modified EP to 0.270 Wm−1·K−1 for Fe3O4-CDBS-LDH modified EP). These attractive performances were attributed to the unique structures, uniform dispersion states of the hybrids, and to the cooperative effect LDH/organic modifiers. Moreover, hydroxides based-layered double hydroxides (LDHs) are new attractive nanofillers. The chemical formula of LDHs can be [Mi et al.  selectedXuan et al.  preparedTo increase the fire retardance of Poly, PVA, a special modification of BCD was that investigated by Feng et al. : In a PVTGA analyses (carried out in air) showed that, in the presence of Metal-MC, the initial degradation of polyphosphoric acid (PPA) that is obtained from APP through reaction d in The UL-94 rating results indicated that a V-0 rating could be achieved when the total dosage of flame retardant was only 15 %wt. However, it was shown that 0.1–0.5 wt% metal MC in combination with 14.9–14.5 wt % APP was the suitable loading to obtain good flame retarded PVA composites. However, further investigation needs to support these hypotheses. BCD and Silicon containing compounds are attracting attention as new flame retardants. Hence, Wang et al. synthesized BCD containing silicone oligomers (CDS) , who comIn order to obtain a better intumescent flame retardant system, CDS was also used as a synergistic agent in new intumescent flame retardant system containing a triazine polymer as char-forming agent (CFA) . 25% loaWhile some biobased FR components are actually on the market, to now all of the mentioned BCD fire retardance solutions are not scaled up yet, nor produced at an industrial level. Fire performance, easiness of processability, low toxicity, and preservation of other functional properties of polymers are just some of the main criteria that are to be taken in account in considering the convenience of scaling up these biobased FR.Fire performance must be evaluated in respect of fulfillment of fire regulation specifications. In FR development, three main types of tests are usually carried out to characterize the flammability properties: LOI, UL94, and cone calorimetry, from which the main parameters can be extracted and introduced in the normative. As far as BCD based FRs are concerned, the key element is the achievement of the barrier effect through the promotion of a charred layer formation on the burning polymer, hence charring was generally related to lower pHRR and THR. Dripping of flamed droplets must also be avoided; however, UL94 V-2 rating, not a satisfactory rating for many applications, is often achieved with some BCD fire retarded polymers.The protection of human health and environment must be promoted in any industrial business, which must comply with national and over national legislation. Several normatives restrict the use of some substances, the industrial development of new biobased FR must of course cope with these issues and with the REACH  regulation. Moreover, natural products are not safe products by definition; their innocuousness must always be checked and a LCA approach should be used to determine the effective environment impact and the accomplishment of the green chemistry principles of new biobased FR. Due to the capacity of including some FR components of low molecular weight in their cavities, BCD and BCD derivatives reduce the release to the environment of these species, resulting in an increase fire retardance activity and in more environmental friendly behavior. Introduction in FR formulation of relatively high molecular weight maltodextrin endowed with inclusion capacity and relevant charring can also be investigated in view of their fire retardance application .BCD based FRs that are introduced in a polymer matrix often produce a more homogeneous composite, showing better physical properties. Special morphologies, such as double layer films, microcapsules, or grafted systems are able to impose new properties to the resulting polymer composites, such as resistence to washability, thermal conductivity, and so on. Improvement in this field are highly welcomed. Eventually, economic criteria are at the basis of the industrial development of biobased FR. In this respect, low cost raw materials and low cost processes are pursued. Promising cheap raw materials are those that derive from well-established industries. BCD turn out to be more attractive, because of its wide employability in food, drug, and other industries, which make the cost of BCD nearly comparable to that of other biomolecules such as lignin or chitosan. Government policies can also affect industrial costs; for example, Sweden has recently introduced Tax on Certain Chemicals in Electronics , with 50%/90% tax reductions to apply to products that contain no halogenated compounds and phosphorus at load < 0.1 wt % This will enforce the industrial application of effective FR containing very low amount of P. On the other side, pollution charge, like a carbon tax, also supports virtuous environmental comportments. BCD were first used in flame retardants at the turn of the year 2000, but it was from 2010 when BCD was available on the market at a reasonable price, which studies on it very burst.BCD alone is not suitable as a FR for any polymer. BCD and phosphorous-containing molecules  promote fire retardance in polymers containing active functional groups, such as PET, PLA, Polyester elastomers, PMMA, epoxy resins, etc. by promoting the formation of a stable insulating char on the surface of the burning sample through interaction of the polymer and BCD with phosphoric acid that was developed from the phosphorous-containing molecules. Therefore, BCD, a natural origin resource, can replace molecules of fossil origin, such as PER in IFR formulation, hence meeting the green chemistry criteria. Where polymers are traditionally fire retarded with volatile phosphorous species acting in gas phase, the incapsulation of the phosphorous species into the cavity of BCD avoids the release of such compounds into the environment; at the same time, the amount of the residue increases and the load of phosphorous-containing species is reduced. However, fire retardance effectiveness is preserved or even increased and fire retardance turns out from a gas to a condensed-phase mechanism. Therefore, a more limited amount of P/N compounds are required to meet the fire retardance standard. However, due to the earlier radical charring of BCD, polyolefins, such as polyethylene, which contains little unsaturation (due to the synthesis), are not conveniently protected by the char that formed from the P/BDC FR system, at least according to the UL94 and LOI criteria. This is attributed to the occurrence of radical degradation reactions that reduce molecular weight and viscosity of the polymer promoting dripping of flamed droplets. Phosphorilated BCD do not improve fire retardacy very much, unless they are mixed with additional APP. This is due to the low degree of phosphorylation of BCD. On the contrary, fire retardance was enhanced in EVA or WPC composites by microencapsulating APP in a shell of crosslinked BCD. In this way, a better dispersion of APP was attained, as well as a microcrystalline size of the charred residue, which induces a better protection of the burning polymer substrate.BCD-NSs effect on fire retardance of some polymeric matrices, such as polyamides, is able to react with phosphorous degradation moieties but has less effect on polyolefins. In this case, burning rate, THR, HRR, and SEA are diminished, so far reducing the fire risk. However, the UL94 rating is hardly affected, allowing, in most cases, the dripping of flamed polymer droplets. On the other hand, systems containing BCD-NSs often display a better fire retardance behavior than the analogous with simple BCD, because a better dispersion of the additive can be achieved in non-polar polymers. To improve the shielding capacity of the char, the addition of expandable graphite or graphene oxide or nanoparticles proved to be useful. Often, fire retardance accompanies other relevant properties, such as improved mechanical resistance, self healing ability, thermal conductivity, etc. The key rule to obtain an insulating compact char is the good dispersion of the nanoparticle in the matrix that can be achieved by functionalizing nanoparticles with BCD derivatives.


Tripterygium wilfordii Hook F., has various biochemical and pharmacological activities, and is now being developed as a promising anti-tumor agent. Inhibitory activity of compounds towards UDP-glucuronosyltransferase (UGT) is an important cause of clinical drug-drug interactions and herb-drug interactions. The aim of the present study is to investigate the inhibition of celastrol towards two important UDP-glucuronosyltransferase (UGT) isoforms UGT1A6 and UGT2B7. Recombinant UGT isoforms and non-specific substrate 4-methylumbelliferone (4-MU) were used. The results showed that celastrol strongly inhibited the UGT1A6 and 2B7-mediated 4-MU glucuronidation reaction, with 0.9 ± 0.1% and 1.8 ± 0.2% residual 4-MU glucuronidation activity at 100 μM of celastrol, respectively. Furthermore, inhibition kinetic study (Dixon plot and Lineweaver-Burk plot) demonstrated that celastrol noncompetitively inhibited the UGT1A1-mediated 4-MU glucuronidation, and competitively inhibited UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameters (Ki) were calculated to be 0.49 μM and 0.045 μM for UGT1A6 and UGT2B7, respectively. At the therapeutic concentration of celastrol for anti-tumor utilization, the possibility of celastrol-drug interaction and celastrol-containing herbs-drug interaction were strongly indicated. However, given the complicated nature of herbs, these results should be viewed with more caution.Celastrol, a quinone methide triterpene isolated from  Tripterygium wilfordii Hook F. is an ivy-like vine, widely used in Traditional Chinese Medicine for hundreds of years to treat fever, chills, edema and carbuncles [Tripterygium wilfordii Hook F. [rbuncles . Celastr Hook F. . Celastr Hook F. ,4,5,6. A Hook F. ,8.Herb-induced adverse effects are a major cause that limits the clinic utilization of herbal medicines. The metabolic behaviour of herbal components plays a key role in herbs’ adverse effects, including metabolic activation-induced herb toxicity and metabolic enzymes inhibition-based herb-drug interactions . For metThe human uridine glucuronosyltransferases (UGTs) are phase II conjugation enzymes that can conjugate various endogenous substances and exogenous compounds . GlucuroTo date, the inhibitory potential of celastrol towards UGT isoforms remains unclear. In the present study, the inhibitory effect of celastrol towards two important UGT isoforms (UGT1A6 and UGT2B7) was investigated. Dixon and Lineweaver-Burk plots were used to determine the inhibition type. In Dixon plot and Lineweaver-Burk plot, we can determine the inhibition kinetic type through evaluating the intersection of the lines in the plots. When the intersection is in the second quadrant and vertical axis for Dixon and Lineweaver-Burk plots respectively, the inhibition type is competitive. When the intersection is in the horizontal axis, the inhibition type is noncompetitive.vs. celastrol concentration showed that the inhibition kinetic parameter (Ki) was 0.49 μM. Different from the inhibition type of UGT1A6, Dixon plot (As shown in xon plot A and Linxon plot B demonstxon plot C.etc.) is exclusively metabolized by UGT2B7.UGT1A6 is a major UGT isoform in human liver that glucuronidates various drugs, toxins, and endogenous substrates, including acetaminophen, benzopyrene, and serotonin ,23. The et al. showed that many environmental factors  could influence the celastrol concentration in Tripterygium wilfordii Hook F. [When celastrol is employed as an anti-tumor agent, the effective dose is approximately 1–10 μM, which is much higher than inhibition concentration of celastrol towards UGT1A1 and UGT2B7. Additionally, as a promising anti-cancer agent, celastrol is likely to be administered with multiple drugs. Therefore, high attention should be given to the celastrol-associated drug-drug interaction due to the inhibition of UGT1A1 and UGT2B7. However, when evaluating the celastrol-containing herbs-drugs interaction, these results should be explained with caution because complicated herbs’ factors might influence the results. For example, processing of herbs and many environmental factors  can influence the quantity of celastrol in herbs, which might strongly alleviate the celastrol-containing herbs-drugs interaction. The previous study performed by Du  Hook F. . TherefoD-glucuronide (4-MUG), Tris-HCl, 7-hydroxycoumarin and uridine 5'-diphosphoglucuronic acid (UDPGA)  were purchased from Sigma-Aldrich . Recombinant UGT1A6 and UGT2B7 expressed in baculovirus were obtained form BD Gentest Corp. . All other reagents were of HPLC grade or of the highest grade commercially available.Celastrol (purity ≥ 98%) was purchased from Weikeqi Biotechnology Co. Ltd . 4-methylumbelliferone (4-MU), 4-methylumbelliferone-β-2, 50 mM Tris-HCl buffer (pH 7.4), and 4-MU in the absence or presence of different concentrations of celastrol. The concentrations of 4-MU were as follows: 110 μM for UGT1A6, and 350 μM for UGT2B7. Celastrol was dissolved in methanol and the final concentration of methanol was 0.5% (v/v). After 5 min pre-incubation at 37 °C, the UDPGA was added to the mixture to initiate the reaction. Incubation time was 120 min for UGT2B7, and 30 min for UGT1A6, respectively. The reactions were quenched by adding 100 μL acetonitrile with 7-hydroxycoumarin (100 μM) as internal standard. The mixture was centrifuged at 20,000 ×g for 10 min and an aliquot of supernatant was transferred to an auto-injector vial for HPLC analysis. The HPLC system  contained a SCL-10A system controller, two LC-10AT pumps, a SIL-10A auto injector, and a SPD-10AVP UV detector Chromatographic separation used a C18 column  at a flow rate of 1 mL/min with UV detection at 316 nm. The mobile phase consisted of acetonitrile (A) and H2O containing 0.5% (v/v) formic acid (B). The following gradient conditions were used: 0–15 min, 95–40% B; 15–20 min, 10% B; 20–30 min, 95% B. To evaluate the order of inhibition kinetics and calculate the inhibition parameters, various concentrations of celastrol  were added to reaction mixtures containing different concentrations of 4-MU . Dixon and Lineweaver plots were adapted to determine the inhibition type, and the second plot of slopes from Lineweaver-Burk plot vs. celastrol concentrations was utilized to calculate Ki value.The probe substrate for all the UGT isoforms tested was 4-MU which is a non-selective substrate of UGTs. Incubation with each UGT isoform was carried out as previously reported . The mixOur study indicates the possible occurrence of celastrol-drug interactions or celastrol-containing herbs-drug interactions due to the strong inhibition of celastrol towards UGT1A6 and UGT2B7. However, given the complicated nature of herbs, these results should be viewed with more caution.


In the ablation areas, the presence of clouds results in a reduction of downward latent heat flux at the snow/ice surface so that much less energy is available for surface melt, which highlights the importance of indirect time-integrated feedbacks of cloud radiative effects.Clouds have been recognized to enhance surface melt on the Greenland Ice Sheet (GrIS). However, quantitative estimates of the effects of clouds on the GrIS melt area and ice-sheet-wide surface mass balance are still lacking. Here we assess the effects of clouds with a state-of-the-art regional climate model, conducting a numerical sensitivity test in which adiabatic atmospheric conditions as well as zero cloud water/ice amounts are assumed , although the precipitation rate is the same as in the control all-sky simulation. By including or excluding clouds, we quantify time-integrated feedbacks for the first time. We find that clouds were responsible for a 3.1%, 0.3%, and 0.7% increase in surface melt extent  in 2012, 2013, and 2014, respectively. During the same periods, clouds reduced solar heating and thus daily runoff by 1.6, 0.8, and 1.0 Gt day Changes in the ice sheet mass  are a function of the surface mass balance (SMB) and ice discharge across the grounding line (D), where the ice starts to float: MB = SMB – D. Before the 2000s, the absolute values of the rates at which SMB was decreasing and discharge was increasing were almost the same4. Recently, however, SMB has played a dominant role in the GrIS’s negative mass balance with more than 80% of mass loss attributed to increased surface runoff7, which is defined as water that flows away from the ice sheet into the surrounding ocean. SMB is a consequence of energy and mass interactions between the atmosphere and the snow or ice surface. The lowest annual GrIS SMB since the 1990s, attributable largely to a record surface melt event8, was recorded in 20127. Moreover, clouds played a key role in this event10 through the cloud radiative effect (CRE)17:Snet and Lnet are net shortwave and longwave radiant fluxes at the snow/ice surface; Snet and Lnet are taken as positive when they are directed into the surface. Typically, at the surface, the shortwave CRE  is negative whereas the longwave CRE  is positive. In polar regions covered with snow, which has a very high albedo, the longwave CRE is sometimes greater than the shortwave CRE (CRE is positive). This situation is known as the “radiation paradox11” where by surface heating can occur even under cloudy-sky conditions, so clouds act as a forcing factor on the climate system.The Greenland ice sheet has lost a significant mass of ice since the early 1990’s11 is qualitatively valid, quantitative impacts of both the CRE and its resultant feedback processes on the changes in  climatic ice sheet physical conditions, namely ice sheet-wide surface melt and the resultant SMB have not yet been fully evaluated. Recently, one study18, which examined climatic trends for cloud fraction and the ice sheet SMB simulated by a regional climate model, found a relation: a decreasing cloud fraction has driven an ice sheet SMB reduction since around 1995. However, the quantitative impacts of clouds, as well as the underlying physical mechanisms by clouds on surface melting have not been sufficiently understood to rank their effects relative to other melt processes, although the importance of changes in the atmospheric circulation pattern over the ice sheet especially during the summer has been highlighted18. Here, we used the regional climate model NHM–SMAP19 to perform a model sensitivity test investigating the role and effects of clouds in which atmospheric conditions were assumed to be adiabatic as well as zero cloud water/ice amounts, thus eliminating the CRE (see Methods for more detail). Then by comparing the results between control  and sensitivity (clear-sky) simulations, we evaluated the quantitative effects of clouds on the ice sheet surface melt area extent and SMB from 2011–2014. Our method has an advantage in that it allows us to consider time-integrated atmospheric feedback processes imposed by instantaneous changes in downward radiative properties caused by the presence or absence of clouds; these time-integrated feedback processes were not taken into account by any previous studies.Although the radiation paradox19 showed that NHM–SMAP successfully reproduced measured GrIS climate conditions and diurnal variations during our study period (2011–2014), which includes the 2012 record surface melt event8. We introduce here the concept of “mass balance year20”, defined as from September of the first year to August of the following year, because it is needed to calculate annual accumulated and averaged values. Simulated cloud fractions for the 2011–2012, 2012–2013, and 2013–2014 mass balance years are listed in Table −2. In the northern part of the ice sheet, as well as in low-elevation areas, the CRE was relatively small. Low albedo mainly due to the exposure of bare ice in the low-elevation areas, where most melt and runoff occur, obviously affected the low CRE, because low albedo enhances the shortwave CRE. Cloud height above the surface might also affect the CRE, which should be investigated further in the future. At Summit station . However, comparison of CREs calculated by different techniques requires careful consideration21. The observation-based study mentioned above21 considered only instantaneous changes in downward radiant fluxes, whereas in the present study, we considered additional atmospheric feedbacks, which might account for the different result. We discuss the validity of the atmospheric responses of the clear-sky model sensitivity test conducted with NHM–SMAP in the last section of this paper.Previously reported model validation resultsy period 011–2014,22. Accumulated melt areas during JJA were enhanced by the presence of clouds by 5.1 × 108, 4.2 × 107, and 1.2 × 108 km2, respectively  of the southeast, west, and southwest ice sheet shown in Fig. Contrary to the surface melt area extent result, comparisons of the annual accumulated ice sheet SMB within a mass balance year between the all-sky and clear-sky NHM–SMAP simulations show exactly the opposite impact of the CRE Fig. : The ann−1). This result can be attributed to the difference in the simulated MAM surface air temperature due to the presence or absence of clouds; simulated MAM surface air temperatures over the entire ice sheet under all-sky conditions were higher by more than 2 K than those under clear-sky conditions in each year , respectively  showed that surface sublimation during the winter to spring accumulation period was enhanced under all-sky conditions  has ever been conducted25. Therefore, we judge the qualitative atmospheric responses induced by the absence of clouds simulated in this study to be robust. In general, the surface pressure system over the ice sheet is considered to be strongly related to the general circulation pattern (the North Atlantic Oscillation index is a useful indicator of the relationship)29. Our results suggest that atmospheric latent heating due to cloud formation over the ice sheet has a great effect on the general circulation pattern in the area.During JJA, surface pressure around the ice sheet was higher under clear-sky conditions . However, changes in surface wind speed due to the presence or absence of clouds are relatively high especially in the northern GrIS  in the present study is exactly the same as the original configuation18. In the atmospheric part of NHM–SMAP , we employed the “weather forecast mode”, in which the atmospheric profile is initialized every day by referring to parent reanalysis data  to prevent large deviations between the parent (JRA-55) and NHM–SMAP atmospheric fields. In the weather forecast mode calculations, a 30-h-long simulation was carried out every day, starting from 18:00 UTC of the previous day; model outputs from the initial 6 h spin-up period were discarded, and model outputs from the last 24 h were used. In JMA-NHM, the improved Mellor-Yamada Level 3 turbulence closure boundary layer scheme33 is used to couple planetary boundary layer and free atmosphere.We applied a state-of-the-art physically based spatially and temporally high-resolution regional climate model called NHM–SMAP (Non-Hydrostatic atmospheric Model with the Snow Metamorphism and Albedo Process model) to the GrIS19. After that, no initialization of the snow/ice part of the model  was made (“climate simulation mode”) during the calculation period, because no information useable for such an initialization was available.The initial snow/firn/ice physical conditions for the entire ice sheet on 1 September 2011 were prepared by performing a 30year spin-up of the NHM-SMAP model36 in which snow albedo and the solar heating profile in the snowpack are calculated by explicitly considering the effects of snow grain size, light-absorbing impurity (LAI) concentrations in snow/ice, the cloud fraction, and solar conditions. The present study did not take account of LAI (pure snow was assumed), following the original configuration of NHM–SMAP applied to the GrIS19.The SMAP model incorporates the physically based snow albedo modelin situ measurements19. The validation results demonstrated that the model successfully reproduced measured features of the GrIS climate and diurnal variations during that period19.Various aspects of NHM–SMAP were validated for the GrIS from 2011–2014, the same as the study period in the present study, by utilizing 19.The model diagnosed daily melt area extent from hourly snow, firn, and ice surface temperature data and water content profiles. First, the daily maximum surface temperature was extracted at each grid point. If the value reached 273.15 K and the top model layer contained water at the time when the maximum surface temperature was recorded, the grid point was considered to have undergone surface melt19:P is precipitation, SUs is sublimation or evaporation from the surface, SUds is sublimation from drifting snow particles, and RU is runoff. RU is governed mainly by the balance between melt and refreezing rates in the surface snow or ice layer.The ice sheet SMB is calculated by NHM–SMAP as follows37. According to a previous study38, the interaction between clouds and circulation primarily results from three processes: phase changes, radiative transfer, and turbulent transport of air parcels, where condensation and evaporation processes associated with the formation, the maturation or the dissipation of clouds, and the interaction of clouds with solar and infrared radiation, lead to atmospheric heating and cooling perturbations, which stimulate waves and turbulence and which affect the horizontal and vertical distributions of temperature on a wide range of scales. Therefore, in the clear-sky numerical sensitivity test with NHM–SMAP performed in this study, we omitted latent heating in the atmosphere  to account for the impacts of phase change, and also set cloud water/ice amounts to zero to assess the impacts of radiative transfer in the atmosphere. By omitting latent heating, the temperature lapse rate in the atmosphere does not follow the moist adiabatic lapse rate but always follows the dry adiabatic lapse rate. The dry adiabatic lapse rate (9.8 °C km−1) is higher than the moist adiabatic lapse rate because the latter is a result of latent heat release by the cloud formation. By setting cloud water/ice amounts to zero, clear-sky downward shortwave and longwave radiant fluxes can be calculated. At the same time, the model calculates changes in turbulent transport of air parcels in response to changes in the temperature lapse rate and downward radiant fluxes. In general, under the clear-sky condition, upward and downward vertical motions are expected to be suppressed, which would result in higher surface pressure23. Consideration of all the cloud-atmosphere interaction processes38 in the numerical sensitivity experiment is a significant advantage of the present study. However, the same spatial and temporal precipitation rate patterns were used in the sensitivity test as in the control all-sky simulation, because our focus here was on only the CRE and its resultant atmospheric feedbacks.In numerical investigations of the effects of clouds on the changes in atmospheric field, it is not sufficient to modify only atmospheric radiation, because the presence or absence of clouds affects not only atmospheric radiation but also latent heating in the atmosphere (temperature profile in the atmosphere)40.As mentioned above, temporal evolution of atmospheric fields was calculated in weather forecast mode in the control simulation. This model setting was also used in the sensitivity test; therefore, deviations in the atmospheric fields between the all-sky and clear-sky simulations are not expected to be large. Clouds are known as a “fast process” in the terrestrial climate system; therefore, examining the results of the clear-sky numerical sensitivity test on the weather forecast mode time scale can provide insights into the possible atmospheric responses to changes in cloud fraction not only over a period of several days but also on a much longer time scalein situ measurements10 in Supplementary Fig. In the sensitivity test, profiles of snow and ice physical conditions were reset at the beginning of the 2011–2012, 2012–2013, and 2013–2014 mass balance years by referring to the control simulation data. As a result, feedbacks with a timescale of more than a year are beyond the scope of this study. An example comparison between the all-sky and clear-sky downward shortwave and longwave radiant fluxes calculated by NHM–SMAP for the SIGMA-A site, northwest GrIS see Fig. , during Snet is the net shortwave radiant flux, Lnet is the net longwave radiant flux, HS is the sensible heat flux, HL is the latent heat flux, HR is the heat flux associated with rainfall, HG is the subsurface conductive heat flux, and M is the surface melt energy (0 W m−2 when the surface temperature is less than 273.15 K). These fluxes are defined as positive when they are directed into the snow/ice surface.To understand simulated changes in the ice sheet melt area extent and SMB in detail, we introduce the concept of the snow and ice surface energy balance (SEB), defined as follows:The NHM-SMAP source code is available subject to a licence agreement with the Meteorological Research Institute, the Japan Meteorological Agency. Any researchers interested in the code are encouraged to contact the corresponding author , who will assist them in obtaining a licence (by signing a contract) for the code.Supplementary Tables and Figures


Viola biflora L. var. rockiana) range and the warmer range margin, with a temperature difference of ca. 1°C. In addition, turfs were also transplanted outside the range to warmer habitats, simulating two different scenarios of climate warming, +1 and +4°C. Transplanting to warmer sites negatively impacted population growth rates (λ), survival and clonality, but did not affect growth and fecundity, while the productivity of the plant community increased. The reciprocal transplants to the colder habitat showed the opposite effects, for both V. biflora and the plant community, indicating plastic responses of the study species, driven by changes in plant–plant competition. However, the leaf traits underlying the modeled population growth rates were origin-site specific and not affected by the climate-change treatments over the study period, suggesting local adaptation of growth form to competition in the warmer range margin, and to climate adversity in the colder range center. The transplants outside the present species’ range showed consistently stronger reductions in population growth rate and survival, with mortality of 90–100% in the +4°C treatment. This illustrates that climatic changes beyond species’ present climatic ranges pose a serious risk for range contraction and extinction for Himalayan alpine species in the near future. As V. biflora seems mostly limited by competition under warming, its persistence in a future climate may become increasingly dependent on keeping competitive effects from the surrounding community low, for instance by management interventions like grazing and mowing.Climate warming poses considerable challenges for alpine plant species, especially for competitively inferior ones with resource-conservative adaptations to cold climates. The Himalayas are warming at rates considerably faster than the global average, so it is particularly important to assess how and through which mechanisms alpine plant species are affected there. We employed a demographic approach in a climate change experiment, where vegetation turfs were transplanted reciprocally between the central parts of the study species’ ( Under global climate warming, especially alpine plant species with narrow ranges and locally adapted populations face a considerable risk of extinction . In contEnvironmental variation, and hence also experimental alteration, may affect various vital rates in a species’ life cycle, from germination  and survViola biflora L. var. rockiana, referred to as V. biflora from here on, in the experimental turfs to examine whether and how the species responds in both population abundance and underlying vital rates to a climatic change of ca. 1°C, the moderate regional 30-year prediction , and Polygonum viviparum, Potentilla leuconota, Carex laeta, and Carex atrata (site A). Sites L and M lie below the natural tree line, where the study species does not occur naturally. At these sites, the vegetation is dominated by Deyeuxia scabrescens, Halenia elliptica, Pedicularis davidii, Pedicularis sima, Geranium pylzowianum, and Anaphalis nepalensis (site M), and Carex finitima, Carex nubigena, Plantago asiatica, P. leuconota, Trifolium repens, and Clinopodium polycephalum (site L). The study area is located in the transition zone between subtropical humid monsoon climate of eastern China and cold climate of Tibetan Plateau, with a mean annual precipitation of ca. 1000–2000 mm/a concentrated in May to October . While precipitation decreases along this altitude gradient as well, largely due to lower July precipitation in A and HA, air relative humidity and soil moisture do not vary systematically with altitude , a montane site , an alpine site , and a high alpine site . Sites A and HA are located in the natural shrub and grass ecotone, where the study species, In sites HA and A, we established five experimental blocks, and in each block three semi-randomly placed 25 cm × 25 cm plots (they had to contain the study species). In September 2012, two plots from each block of site HA were transplanted to site A and site L, representing a temperature change of +0.9°C within the species range and +3.9°C outside the species range, respectively. In addition, two plots from each block of site A were transplanted to site M and site HA, representing a temperature change of +1.4°C outside the species range and of -0.9°C within the range, respectively. For simplicity, we hereafter refer to these transplants as the “HA warm” , “HA warm+” , “A warm” , and “A cold”  treatment, respectively. The fifth plot was transplanted within the original site and block, as a control . The transplanted turfs measured 25 cm × 25 cm and were 20 cm deep. After excavation, the turfs were placed in wooden boxes and replanted into their respective target sites within 1 day.V. biflora produces long lateral rhizomes, each with multiple flowering ramets, on the same genetic individual. As the below-ground connections cannot be assessed under non-destructive sampling, we used ramets as our working unit , based on a regression model tested on 236 destructively sampled individuals  from sites HA and A in 2012 and 2013 (Supplementary Table V. biflora it is impossible to non-destructively assess how ramets are connected and new clones got therefore the closest ramet (but not seedlings) assigned as “parent.” V. biflora exhibits vegetative dormancy with ramets being able to resprout after having been dormant for up to 4 years  we used integral projection models (IPMs) , 2007. ISome reproductive traits such as the number of seeds per flower, seedling establishment, the probabilities for entering and staying in the seed bank, and seedling size cannot be related to plant size under non-invasive data collection. These values were obtained from another study on the focal species in a climatically similar environment  and usedP-matrix) with size ranges from the observed minimum and maximum sizes minus/plus an increment of 1% of the minimum/maximum size as described in, e.g., Using the R-package IPMpack  we builtWe estimated the uncertainty around the λ’s by bootstrapping. Individual ramets were sampled with replacement to construct a resampled dataset containing the same number of observations as the original dataset. Regression modeling, construction of IPMs and calculation of λ were then repeated as described above using the resampled dataset. Performing this procedure 2000 times generated a set of bootstrap λ estimates, which were used to assess the significance of differences in λ between transplants and controls. Pairwise independent transplant and control bootstrap λ samples were subtracted from each other (control-treatment). A qualitative difference in λ was accepted as significant at the 0.05 level when it occurred in more than 95% of the bootstrap sample pairs.P-matrix, by setting the probability of survival to one for all sizes. The contribution of growth alone could then be calculated using the method outlined above. By subtracting the contribution of growth from the total growth-survival contribution we found the contribution from survival alone.Finally, we used life table response experiments (LTRE) to determine how much changes in the vital rates contributed to the differences in λ between the transplants and their respective controls. The contribution of a given vital rate was calculated as the sum of the differences between the vital rate matrices of the transplant and control treatments multiplied by the sensitivity of a matrix midway between the full IPM matrices of the two treatments  . We sepaTo address origin-related differences in plant architecture we assessed differences in number of leaves, leaf size, and length of the leaf stalk from all plants in all years in our study. For this, we modeled all three variables as a function of origin (fixed effect) and with random intercepts for all treatment–origin combinations and random intercepts for year. We used GLMMs with Poisson errors and log-link for number of leaves, and with Gaussian errors and identity-link for leaf size and leaf stalk length.The difference in community height or cover between transplant plots and controls was analyzed for each transplant treatment by linear mixed effects models with Gaussian error structure. We used year as fixed effect to assess whether or not the difference observed in 2013, 2014, and 2015 were significantly different from prior to transplanting in 2012. The model intercept represents 2012 and indicates whether or not the observed difference in that year is significantly different from zero.V. biflora were stable  throughout the study period and all transplant treatments showed clear and significant differences from this stable state . Transplanting to warmer sites reduced λ in populations originating from both the high alpine and the alpine site, but the magnitude of the effect varied. λ was around 0.7–0.8 in the “HA warm” treatment, 0.7 in the “A warm” treatment, and as low as 0.3–0.5 in the “HA warm+” treatment. As the population size at site A was much smaller than at site HA (see Table 1) uncertainty was higher in the transplants originating from site A, potentially masking statistical significance of the effects of the “A warm” treatment. The transplants from the alpine site to the colder high alpine site led to a λ increase of ∼30% which was significant for all transitions. In the “HA warm+” transplants to the low alpine site, λ was unchanged in the first transition but then dramatically dropped in the following years.Both the high alpine and the alpine native populations of Figures H). Similarly to λ, the negative survival contributions in the warming treatments increased progressively from the “HA warm” treatment, via the “A warm” treatment and to the “HA warm+” treatment. The “A cold” and the “HA warm” treatments were complementary with respect to survival, the respective positive and negative contributions to changes in λ being comparable in effect size.Changes in survival and clonality were the main contributors to differences in λ in all treatments , and by ∼16 cm in the “HA warm+” transplants from the second transition on . In the “A cold” transplants, community height was lower than in the controls by ∼5 cm already in the first transition, the effect size increasing in the following transitions to ∼10 cm . Cover was mainly unaffected by the climate treatments , but was significantly lower in the “A cold” transplants in the last transition . In site HA, cover was highly variable (ranging from 50 to 90%), whereas it was constant and high in the transplants (both to site A and site L) originating from this site .Community height was higher under warming than in the controls in the course of the experiment, by ∼3 cm under both the “HA warm” and the “A warm” treatments . For number of leaves and leaf size, the transplanted populations retained their original trait values even after transplantation to climates differing with ∼1°C . In the “HA warm+” treatment, however, these two traits changed from the “high alpine values” in direction of the “alpine values.”Number of leaves, leaf size, and leaf stalk length had different values in the high alpine and alpine habitats. In the high alpine, the plants produced more, but smaller and lower growing leaves, and in the alpine the plants produced fewer, larger and higher growing leaves , although we acknowledge that our study design does not explicitly test for indirect competition effects vs. direct climate effects. Higher vegetation canopies reduce light availability in the vegetation sward and thus penalize smaller, less competitive species . In the high alpine sites, the plants produce several, small and low standing leaves, a classical growth form strategy in climatically harsh environments . From the second transition  on, mortality in this treatment was very large, reaching 94 and 100% in the last two transitions. Hence, the remaining population in the last 2 years consisted mostly of new recruits, which adopted the above indicated altered growth form as an ontogenetic acclimatization to their environment  need to adjust the photosynthetic temperature optimum under warming of 4°C , the persistence of this V. biflora population at the low site is highly unlikely (V. biflora (In line with g of 4°C . Thus, tunlikely . While n biflora .V. biflora population during the first transition. As both dormancy and clonality may be susceptible to effects of climate change (During this lagged response phase, changes in clonality were, surprisingly, contributing positively to the difference in lambda. In our study, such increase in clonality may also be due to increased re-sprouting after vegetative dormancy. Such re-appearance of dormant plants has been linked to re-mobilization of stored resources , which me change , a separWhile yielding many important insights into climate-change driven responses on alpine plants, our study also has two limitations worth discussing. First, as mentioned above, vegetative dormancy is a factor complicating non-destructive population studies. If the demographic time series is long enough and the temporal extent of the dormant phase short enough, then dormancy can be identified and modeled with satisfactory levels of uncertainty . In shorV. biflora is well supported by other population studies on this species from Europe (Second, our experiment is replicated locally but not regionally, which in principle limits the degree to which our results can be generalized beyond the mountain our study was performed on to alpine systems in general. However, our overall finding of competition-limitation in m Europe , and theV. biflora, clearly is sensitive to climate warming, which is threatening especially populations at the warmer species range margin, where competition already acts limiting for survival and establishment of new recruits in the species. There, the species’ persistence will likely become increasingly dependent on reducing competition from the plant community, e.g., through management interventions like grazing and mowing (The average regional predictions for climate warming in China lie around 1-2°C until about 2050, while extreme models predict an increase by more than 4°C in the second half of the 21st century , which id mowing . Should VV, YY, and GW conceived and designed the experiment. JT designed the demographic study. HC and YY performed the experiment with input from VV. GW and YY provided climate data. JT and HC analyzed the data. JT wrote the paper with contributions from HC and VV. All the authors read and approved the final 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.


Removing vegetation and surface soil resulted in an increase in NO3 concentration of soil solution, and snowmelt strongly affected the NO3 leaching from treated soil and the NO3 restoration process in a snow-dominated region.To clarify the effect of vegetation and surface soil removal on dissolved inorganic nitrogen (N) dynamics in a snow-dominated forest soil in northern Japan, the seasonal fluctuation of N concentrations in soil solution and the annual flux of N in soil were investigated at a treated site  and control sites from July 1998 to June 2000. Nitrate (NO3) concentration in soil solution at the treated site was significantly higher than that of the control in the no-snow period, and it was decreased by dilution from melting snow. The annual net outputs of NO3 from soil at the treated site and control sites were 257 and –12 mmol m year, and about 57% of the net output at the treated site occurred during the snowmelt period. NO


Synaptic plasticity has been considered a key mechanism underlying many brain functions including learning, memory, and drug addiction. An increase or decrease in synaptic activity of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complex mediates the phenomena as shown in the cellular models of synaptic plasticity, long-term potentiation (LTP), and depression (LTD). In particular, protein phosphorylation shares the spotlight in expressing the synaptic plasticity. This review summarizes the studies on phosphorylation of the AMPAR pore-forming subunits and auxiliary proteins including transmembrane AMPA receptor regulatory proteins (TARPs) and discusses its role in synaptic plasticity.  One of the essential features of brain function is the ability to be dynamic in order to express various behaviors. Selective strengthening and weakening of synaptic transmission have been modeled as a critical mechanism for many brain functions including learning, memory, and drug addiction. Long-term potentiation (LTP) and depression (LTD) are well-characterized models of synaptic plasticity, and they can be regulated by changes at presynaptic  and postsynaptic  sites. Importance of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complex has emerged notably in LTP and LTD. In particular, protein phosphorylation is well known to play a pivotal role in the expression of synaptic plasticity, for example, Campal LTP ,2,3. ThiAMPARs are predominantly distributed at excitatory synapses and mediate the majority of fast transmission. The AMPAR complex consists of four pore-forming subunits GluA1–4) and auxiliary proteins including TARP, cornichons-like (CNIH), and cysteine-knot AMPAR modulating protein (CKAMP)/Shisa family in the brain  and auxi,6,7,8,9.To elucidate how phosphorylation regulates synaptic plasticity, extensive research has been conducted to identify phosphorylation sites on the pore-forming subunits . Based oA serine residue (Ser) at 831 in the intracellular region (Ser831) is one of the most attractive phosphorylation sites of the GluA1 subunit. In vitro phosphorylation assays reveal that Ser831 can be directly phosphorylated by CaMKII  or proteA serine residue at 845 (Ser845) is another phosphorylation target site that has been characterized well. In vitro phosphorylation assay with purified PKA identifies direct phosphorylation at Ser845 . In heteIn ex vivo slices, phosphorylation at Ser831 and Ser845 of GluA1 highly correlates with LTP and LTD. To describe their phosphorylation states, phosphorylation site-specific antibodies have been developed and validated by activation of PKC (by phorbol dibutyrate) and PKA (by forskolin and 3-isobutyl-1-methylxanthine) in rat hippocampal slices . Theta bPKC is known to phosphorylate other sites on the GluA1 subunit. Autoradiograph and phosphorylation site-specific antibody combined with purified C-terminal mutants of GluA1 reveal that PKC (but not CaMKII) can directly phosphorylate Ser818 in vitro, and Ser818 phosphorylation increases upon chemical LTP and TBS . Thr840 The phosphorylation studies using heterologous cell systems and overexpression of GluA1 mutants have provided us with valuable mechanistic information. However, it has to be validated in more physiological conditions such as targeted knock-in mouse models . A ‘PentUnlike GluA1, no CaMKII phosphorylation is detected on transiently expressed GluA2 in HEK293 cells . HoweverLTD is abolished in cultured cerebellar Purkinje cells from GluA2 knockout mice, and the abolished LTD can be rescued by transient expression of the wild-type GluA2 subunit . HoweverThe C-terminal ends of GluA2 and GluA3 subunits uniquely have three conserved tyrosine residues . ImmunobTransiently expressed GluA4 subunits are phosphorylated in a PKC-activating (by phorbol 12-myristate 13-acetate) or PKA-activating (by forskolin) condition of HEK293T cells . PhosphoPKA activity is necessary and sufficient for synaptic incorporation of GluA4 subunits in hippocampal slices . The PKAstargazer) show a loss of AMPAR-mediated transmission in cerebellar granule cells [AMPARs exist in the brain as a protein complex with auxiliary proteins  ,46,47,48le cells ,50. The le cells ,51. TARPle cells ,52,53,54SD) shows an increase in synaptic AMPAR activity in cerebellar mossy fiber-granule cell synapses [Phosphorylation of stargazin/TARPγ-2 was firstly described. Both extra-synaptic (Triton X-100-soluble) and post-synaptic density (Triton X-100-insoluble) fractions show phosphorylation of stargazin/TARPγ-2 . Intrigusynapses , consistsynapses . In hippsynapses . Also, Tsynapses .Cm) shows a substantial reduction in hippocampal LTP, suggesting that CaMKII phosphorylation of TARPγ-8 at these two sites is required for LTP [SA) and a triple mutant mouse with StargazinSA knock-in plus knockout of TARPγ-3 and TARPγ-4 [Cm knock-in mice with S277A and S281A show no obvious differences in AMPAR expression compared to wild-type mice [Phosphorylation of TARPγ-8 by CaMKII is shown by in vitro phosphorylation assays . The nin for LTP  Table 2Cm shows  TARPγ-4 . Previou TARPγ-4 . Possiblype mice . In addiype mice .2), and phosphatidylinositol-3,4-5-triphosphate (PIP3) [SD knock-in mice show an increase in synaptic AMPAR activity [The molecular mechanism for how TARP phosphorylation regulates synaptic AMPAR activity is one of the most interesting topics in synaptic plasticity. In the C-terminal cytoplasmic tail of TARPs, the serine sites are adjacent to many arginine residues  . The pose (PIP3) . Fluorese (PIP3) . As disce (PIP3) , and theΔ4) that lacks the last four amino acids, a PDZ-binding motif. The interaction between PDZ-binding motifs of TARPs and membrane-associated guanylate kinase family proteins  is proposed to stabilize AMPAR-TARP complexes at synapses. Consistent with this idea, TARPγ-8Δ4 knock-in mice exhibit ~30% reduction in basal AMPAR transmission in the hippocampus [Δ4 knock-in mice [Since all TARP isoforms commonly have the eight to nine arginine residues adjacent to the phosphorylation sites , it woulpocampus . However-in mice , suggestAccumulating reports have identified more auxiliary proteins of the AMPAR complex, such as CNIH, germ cell-specific gene 1-like protein (GSG1-L), and CKAMP/Shisa ,46,47,48Among the binding proteins to AMPARs ,58, CKAMPhosphorylation of the pore-forming subunits and auxiliary proteins of the AMPAR complex has been extensively studied. The broad spectrum of biochemical approaches such as in vitro phosphorylation assay, phosphopeptide mapping, phosphorylation site-specific antibodies, and Phos-tag polyacrylamide gel electrophoresis has allowed us to identify the actual phosphorylation sites and to describe their states in synaptic plasticity. Combined with the biochemical approaches, in vivo and ex vivo studies including genetically modified mice and electrophysiological analyses have found compelling phenomena and their molecular and cellular mechanisms. However, many questions remain in the field of synaptic plasticity, in particular, in regards to proteomics. Although the native AMPAR complex constituents were recently profiled by proteomics , phospho


How ice sheets respond to changes in their grounding line is important in understanding ice sheet vulnerability to climate and ocean changes. The interplay between regional grounding line change and potentially diverse ice flow behaviour of contributing catchments is relevant to an ice sheet’s stability and resilience to change. At the last glacial maximum, marine-based ice streams in the western Ross Sea were fed by numerous catchments draining the East Antarctic Ice Sheet. Here we present geomorphological and acoustic stratigraphic evidence of ice sheet reorganisation in the South Victoria Land (SVL) sector of the western Ross Sea. The opening of a grounding line embayment unzipped ice sheet sub-sectors, enabled an ice flow direction change and triggered enhanced flow from SVL outlet glaciers. These relatively small catchments behaved independently of regional grounding line retreat, instead driving an ice sheet readvance that delivered a significant volume of ice to the ocean and was sustained for centuries. Ice sheets are vulnerable to changes in the environment where ice discharges into the ocean. Here, the authors show that, in spite of widespread retreat following the last glacial maximum, a sub-sector of the East Antarctic Ice Sheet in the Ross Sea underwent sustained readvance. Although the majority of the EAIS is grounded above sea level, approximately 19 m of sea level equivalent is stored within portions of the EAIS that rest on a bed below sea level and are susceptible to marine forcings9. Understanding the sensitivity and stability of EAIS catchments—for example, the magnitude of their growth and decay over long-term climate cycles, the rates of deglacial retreat and contributions to global ‘meltwater pulses’, and the inward-propagating influence of grounding line dynamics on fast flowing ice streams—is important in assessing the vulnerability of the EAIS to future climate and ocean changes.The East Antarctic Ice Sheet (EAIS) has undergone significant changes in its extent and behaviour throughout the Plio-Pleistocene12. In the western Ross Sea, ice flow was sourced from outlets of the EAIS that breached the Transantarctic Mountains15, terminating at a grounding line 50–150 km inward of the continental shelf-break16  sector of the EAIS in governing, and responding to, ice sheet and ice shelf dynamics in the western Ross Sea.The ice sheet sector of greatest change during the last glacial-interglacial cycle is arguably the Ross Sea sector, where the grounding line advanced by up to 1000 km and the catchment area expanded by ~30% during the Last Glacial Maximum (LGM) relative to the present-day extent. At the LGM, grounded ice streams filled the deep troughs that bisect the Ross Sea continental shelf28. These collectively raise questions of the vulnerability, stability or resilience of different ice sheet sectors to grounding line change, and demand analysis of why different sectors may behave in different ways including, in cases such as we present here, advance for a period of centuries in the face of climate warming and sea level rise.Fringed by the Transantarctic Mountains from the LGM grounding position to the present-day calving front, the western Ross Sea is an excellent location for examining the interplay between source outlets and regional grounding line change. How did upstream EAIS catchments respond to and interact with marine grounded ice flow, grounding line forcings, and the establishment and loss of the floating Ross Ice Shelf? Here we integrate newly acquired geophysical seafloor data with >20 years’ legacy multibeam data from the western Ross Sea  are arranged in a dense field in southern JOIDES Trough, in the forefield of an intermediate-scalelex Fig. . Small-slex Fig. , while llex Fig. . Our newlex Fig.  than hadlex Fig.  and throlex Fig. . These r22. Retreat from northern Drygalski Trough proceeded southward and westward towards David Glacier and the Terra Nova Bay outlets, while small recessional moraines on the NW flank of Crary Bank mark grounding line retreat onto the bank and the partial separation of ice flowing out of the Transantarctic Mountains and off the bank . In the western part of the readvance grounding line, close to the flank of Crary Bank, glacial lineations transition across the grounding zone into subparallel and curvilinear iceberg furrows  would also have been truncated by the SVL readvance set, under this interpretation. However, while the moraines appear to be a continuation of the same field as those that are overprinted, no readvance landforms directly superimpose the southernmost landform assemblages, and therefore evidence for a much greater magnitude readvance remains equivocal. The seafloor deepens here and it is possible that a readvance lobe that grounded in southern JOIDES Trough remained afloat in this deeper zone. On the other hand, continuity of meltwater channel systems in front of and behind the GZW complex31 , the most distal wedge within a large grounding zone complex. Our acoustic stratigraphy reveals a more-or-less continuous buried surface beneath the upper, acoustically transparent unit that comprises the wedge complex , the unit thickness peaks at 19.5 m and has a typical thickness at the GZW1 topset break of 10–14 m. The unit thins to the south although, behind the southernmost GZW2, it maintains a thickness of ~1.4 m. Given the total volume of sediment accumulated in GZW1 and using a range of reported subglacial sediment fluxes that encompass outstanding uncertainties in transport rate and mechanisms , we calculate an upper bound of c. 1000 years on the duration of the stillstand at GZW1, and a lower bound as short as c. 80 years; the duration of the whole grounding zone complex is c. 100–1600 years , we bracket a likely volume of ice discharged through the event’s maximum position ; the upper bound is comparable to discharge from today’s major outlets such as Totten or Pine Island Glaciers (~80–110 Gt a−1)34. This readvance event not only accompanied a reconfiguration of ice flow sourced from the SVL sector of the EAIS, but contributed a potentially significant volume of ice to the ocean.A readvance of ~50 km would propel ~3000–4000 kmion Fig.  to the oFollowing the southern JOIDES readvance event, lineations and grounding line landform assemblages clearly document retreat to Mackay and Mawson Glaciers on the SVL coast, and onto Crary Bank. Grounding line landforms retreat up onto Crary Bank from all sides Figs. . In soutRecessional moraines atop and encircling Crary Bank cannot, however, be linked together to map out a concentric pattern of retreat Fig. . Rather,Glacial lineations and landforms representing east-to-west grounding line movement graze the western flank of Crary Bank  deglaciation of Central Basin prior to SVL readvance raises the possibility that this age may pre-date the reconfiguration. Indeed, the Coulman High core lithostratigraphy comprises a short period of open water conditions, from which the carbonate sample was recovered, prior to a regrowth of ice shelf cover36. The carbonate age therefore indicates either that the whole retreat-readvance-final retreat sequence took place prior to 8.6 cal ka, and the Coulman High lithostratigraphy represents a later calving line oscillation that our grounded ice reconstruction does not capture; or that the first retreat of both the grounding and calving line progressed at least as far as Coulman High, and that the grounded ice readvance we reconstruct around southern Crary Bank was accompanied by ice shelf re-growth over the deep waters, shortly after 8.6 cal ka.We identify several instances of ice flow and grounding line reconfiguration during the deglaciation of the SW Ross Sea. Relative to one another, we infer that the southern Drygalski flow switch is dynamically linked to the JOIDES grounding line readvance, which is clearly supplied by the younger, south and eastward flow from Mackay and Mawson outlets; reconfiguration of Crary Bank ice rise would have occurred during (a) final phase(s) with a restricted grounding line extent. The absolute timing of these events is, unfortunately, difficult to constrain, despite much previous work into western Ross Sea deglaciation sequences38. To the south, the grounding line retreated into Mackay Glacier trough at 6.8–6.0 10Be ka23 and seasonally open marine conditions began ~7.5–6 cal ka40. Within the available window of time, we tentatively suggest that the readvance of SVL ice into southern JOIDES Trough occurred sometime around 8.5 cal ka; the advanced grounding line was sustained for a few centuries; and the ensuing retreat of both the grounding and calving lines was accomplished within ~1500–2000 years. This time window is consistent with sediment flux-based estimates of GZW formation time, but a refined chronology is nonetheless imperative to better constrain the scale and rate of ice margin change and ice margin stability, and to refine understanding of sediment fluxes and GZW formation processes. The Crary Bank Ice Rise was the final remnant of grounded ice in the western Ross Sea.Terrestrial ages from the SVL coast constrain the final deglaciation of the western Ross Sea, and are therefore minimum limiting ages on all of the events we report here. Raised shoreline ages north of Drygalski Ice Tongue suggest this coast was at least seasonally free of ice  by 8 cal ka17, in terms of its timing , style  and the controls governing ice retreat. Halberstadt et al.12 propose a pattern of deglaciation characterised by complex bank-trough relationships and embayment-driven ‘unzipping’ of different sub-sectors of the formerly grounded ice sheet in the Ross Sea Embayment, in a heterogenic fashion. We find the pattern of retreat shown by glacial landforms in the western Ross Sea entirely consistent with this style, and in keeping with growing recognition that this sector experienced shifts in its ice flow configuration21, that the SVL outlet glaciers are sensitive to offshore dynamics23, that coastal and deeper marine deglaciation may be decoupled36 and that this sector’s deglaciation is decoupled from general southward grounding line retreat into the southern Ross Sea embayment32. Encouragingly, recent numerical ice sheet models7 echo this behaviour, demonstrating unzipping of grounding line embayments, ephemeral flow direction shifts (notably of David Glacier), retreat-readvance events and the development of residual bank-top ice caps or ice rises.The deglaciation of the Ross Sea has proven controversialWe have found traces of ice sheet reorganisation during deglaciation of the western Ross Sea, associated with at least one readvance that delivered a significant and sustained volume of ice to the ocean. We link this readvance to relatively enhanced ice flux from the outlet glaciers of SVL and the local reversal of flow direction witnessed in southern Drygalski Trough. These observations couple ice flow behaviour with dynamics of grounding line change, and it is therefore important to consider what mechanisms triggered grounding line retreat and readvance, and drove SVL outlets to dominate flow geometry late in the deglaciation of the western Ross Sea.46 with ensuing progradation of a large wedge. However, this would require progradation of a wedge at least 50 km downstream of its seeding location, a distance towards the upper limit of previously reported GZWs47. This scenario would also need to explain the initial stillstand that would have allowed a GZW to seed. This stillstand position would be located where the reverse bedslope had in fact steepened after the initial retreat, and where JOIDES Trough widens into the Central Basin , or a buoyancy threshold feedback with isostatic rebound53, none of these mechanisms logically explains why a grounding line advance was accompanied by a flow reconfiguration, rather than simply regrowth that maintained the initial flow geometry.Grounding zone self-stabilisation due to sediment progradation may present a conservative explanation of our observations of moraine burial: that a stillstand in the initial retreat enabled a period of GZW growth, such that the grounding line became less sensitive to buoyant forces and stabilised54. Basal crevasse squeeze ridges within the recessional moraine field of JOIDES Trough  were gridded without further processing at 30–40 m. These data were complemented with transit survey lines from three RVIB Oden cruises, which collected data with a Kongsberg EM120 (OSO0708) and EM122 , and EM122 survey data from RV Araon . All surfaces were gridded in UTM58S projection  as the volume of sediment deposited  per metre of grounding line divided by the annual sediment volume flux  over the same unit width, i.e.,We calculate event duration  complex in southern JOIDES Trough. Acoustic stratigraphic reflection horizons corresponding to the seafloor and to a widely identifiable subbottom reflector defining the base of the GZWs were mapped in IHS Kingdom. The two-way travel time (TWTT) difference between the two horizons defines the thickness of the GZW and reveals its spatial variability across the complex. TWTT was converted to sediment thickness using two sound velocity choices—1500 and 1750 m s68 and the volume of sediment transported in basal ice70. Despite the assumptions required to approximate these variables, and genuine spatial and temporal contrasts in both transport mode/rate and sediment supply, flux estimates reported from a variety of research approaches converge around 1–2 orders of magnitude. Here our approach encapsulates the range of uncertainties revealed by previous studies yet without specifically quantifying each source of error, in order to place upper and lower bounds on the possible duration of the readvance event.The local palaeo-sediment flux is unknown and reliable analogue fluxes are hampered by poor constraints on the mode(s) of subglacial sediment transport, particularly the thickness of a mobile till layer3 a‒1 m‒1 (refs. 71) we calculate a range of durations for the build-up of the GZW complex following Eq. 3 a‒1 m‒1 (refs. 72), bracketing the formation time for GZW1 as c. 80–1000 years, and for the whole grounding zone complex as c. 100–1600 years. This is, to an order of magnitude, consistent with using a catchment-wide sediment yield from ref. 32, although these modelled potential yields should be used as indicative only, rather than at face value.Using subglacial sediment fluxes ranging from 50 to 8000 m−1, based on highly elongate glacial lineations that we interpret as a signature of fast flow. We thereby derive an along-flowline distance that would be discharged through the grounding line in each case and, using the glacial lineation distribution, we outline a corresponding zone of the ice sheet behind the readvance grounding line position that was likely discharged during the event  = c.x½ (ref. 73), inland from the mapped grounding line. We use values for constant c from 2.0–3.0 (ref. 74) and satisfy a condition of grounded ice in all grid cells within the defined catchment. The parabola with constant c = 2.5 gives a comparable total ice volume to output from the ice sheet model described in refs. 75, for a timeslice whose ice margin and surface profile most closely match the configuration indicated by geomorphological evidence. For flowline lengths > 500 km  the catchment definition becomes highly speculative. Scaling ice volumes similarly to our other scenarios gives a result well within the range of annual discharges (Gt a−1) for better constrained scenarios, though these extreme case values should not be considered robust.To estimate the range of possible ice volumes discharged during the interpreted southern JOIDES readvance event (Supplementary Table ent Fig. . Within www.marine-geo.org and from http://oden.geo.su.se. Integrated multibeam grids are available from the corresponding author upon request.Raw acoustic data from NBP1502A and legacy data displayed in Figs. Supplementary InformationPeer Review File


Cedrus libani A. Rich.) stands in Southern Turkey. Using data from nine plots established at the western Taurus Mountains, diameter, height, and crown radii of saplings were compared, and spatial point pattern analyses were performed. We found significant differences for the mean diameter and height, and crown radii of saplings among the plots. Univariate pair correlation function showed that sapling pattern was regular only at small scales (r < 0.4 m) but was predominantly random. Bivariate pair correlation function revealed no evidence of spatial interaction between tall saplings and short saplings. Univariate mark correlation function revealed that strong intraspecific competition was detected at small scales (up to 0.55 m). This distance is reasonable for the juvenile age tending of Taurus cedar saplings and should be under consideration during silvicultural treatments to use the site productivity more efficiently.Better understanding of the competitive interaction at the early development stages of the stand is crucial to help schedule silvicultural treatments for young stands and for the better management of the future stands. We used scale‐dependent analysis to improve our understanding of sapling dynamics in the pure Taurus cedar ( Cedrus libani saplings, we used scale‐dependent analysis to explore this interaction. This is significant because we could not find any previous work on this conifer species.To understand and measure the intraspecific competition of  This is promoted not only by genetic inheritance but also by environmental factors  forests, which are the characteristic fire‐prone ecosystems of thermo‐ and meso‐mediterranean belt of Eastern Mediterranean Basin  How do size attributes change between cedar saplings? (b) Does local crowding reduce plant growth? (c) How do these interactions differ by site class? (d) Is there an intraspecific competition between cedar individuals as expected? Therefore, are there any cleaning operations required for these cohorts? (e) To what scale are these interactions lasting?22.1The study was conducted at three pure Taurus cedar stands at the sapling stage in the western Taurus Mountains in Southern Turkey, which represents one of the densest distributions of Taurus cedar  was used. Third, the spatial correlations of the mark “crown radius” based on sapling locations in the nine plots were determined using the mark correlation function (MCF). Although other growth characteristics were measured in the study, crown radius was selected for MCF, because it better represents competition . In the nine plots, there were 1,339 individuals in total and 222 of them were not taller than the mean height of all saplings  but it was predominantly random. In P5, AS were randomly distributed except for the distance interval 0.6–0.7 m where saplings were clumped. The bivariate pair correlation function for TS versus SS indicated no evidence of a significant spatial interaction between TS and SS at all plots except P7.The pair correlation function of all univariate and bivariate patterns approached to their asymptotic value of approximately one within 2.5 m Figure  by beingr = 0.55 m) in all plots  are smaller) while in P1, P2, P4, P5, and P6 stronger  are larger).We compared scale‐dependent competition of saplings with mark correlation function applied to the mark “crown radius” for Taurus cedar saplings. For the AS, strong intraspecific competition was detected for small scales  in any of the studied nine plots. The number of individuals per hectare at the study sites changes between 9,600 and 25,000, which conforms to the findings of Yılmaz et al.  and KantAs is known, site index of a stand is determined according to the height of the tallest trees (H100) in a mature stand at the thinning development stage and the number of trees is higher at stands with better sites then poor ones. Despite all site classes having saplings of the same age, the H100 values of P4 and P6, which are in site class IV, were much higher than those of all our plots in site class III. Although site index determination is carried out for mature stands, growth characteristics of young stands can be informative for the site conditions. Site index of the study sites was obtained from the management plan of the related forest districts. But the results revealed that young stands represent different site class value than the management plan. This could be explained by the potential and realized measures of forest site quality .Spatial distribution patterns and size of trees are not independent from each other, and are commonly affected by competition (Getzin et al., 5In the study, Young Taurus cedar stands showed different spatial point patterns and competitive relationships due to local changes. Competition between individuals ends in distances from 25 cm to not more than 1 m. This knowledge makes valuable contribution to silvicultural treatment at young Taurus cedar stands. Therefore, it is reasonable to offer that the distance between individuals can be arranged as 50 cm for the juvenile age tending and 1 m after first precommercial thinning as an easy application while the classical principles of young stand tending for Taurus cedar should also be taken into consideration. This tending should also focus on short saplings since the Taurus cedar favors establishing single layer stands during the later stages due to its light‐demanding character. In order for this knowledge to be applied to all pure Taurus cedar stands, similar studies should be conducted in different site conditions.None declared.All authors contributed to study design and data collection. O.Y.Y. and O.S. conducted analysis and wrote manuscript in collaboration with A.K. and H.B.T.


Major rooibos flavonoids—dihydrochalcones, aspalathin and nothofagin, flavones—orientin and vitexin, and a flavonol, rutin, were investigated to determine their influence on the activity of adrenal steroidogenic enzymes, 3β-hydroxysteroid dehydrogenase (3βHSD2) and cytochrome P450 (P450) enzymes, P450 17α-hydroxylase/17,20-lyase (CYP17A1), P450 21-hydroxylase (CYP21A2) and P450 11β-hydroxylase (CYP11B1). All the flavonoids inhibited 3βHSD2 and CYP17A1 significantly, while the inhibition of downstream enzymes, CYP21A2 and CYP11B1, was both substrate and flavonoid specific. The dihydrochalcones inhibited the activity of CYP21A2, but not that of CYP11B1. Although rutin, orientin and vitexin inhibited deoxycortisol conversion by CYP11B1 significantly, inhibition of deoxycorticosterone was <20%. These three flavonoids were unable to inhibit CYP21A2, with negligible inhibition of deoxycortisol biosynthesis only. Rooibos inhibited substrate conversion by CYP17A1 and CYP21A2, while the inhibition of other enzyme activities was <20%. In H295R cells, rutin had the greatest inhibitory effect on steroid production upon forskolin stimulation, reducing total steroid output 2.3-fold, while no effect was detected under basal conditions. Nothofagin and vitexin had a greater inhibitory effect on overall steroid production compared to aspalathin and orientin, respectively. The latter compounds contain two hydroxyl groups on the B ring, while nothofagin and vitexin contain a single hydroxyl group. In addition, all of the flavonoids are glycosylated, albeit at different positions—dihydrochalcones at C3' and flavones at C8 on ring A, while rutin, a larger molecule, has a rutinosyl moiety at C3 on ring C. Structural differences regarding the number and position of hydroxyl and glucose moieties as well as structural flexibility could indicate different mechanisms by which these flavonoids influence the activity of adrenal steroidogenic enzymes. Flavonoids are a diverse group of plant-derived polyphenols that occur naturally in fruits, vegetables, teas and herbs. These secondary plant metabolites have been shown to exhibit potent anti-oxidant activities, scavenging reactive radicals that cause cellular damage associated with many diseases and clinical conditions. Flavonoids, often referred to as phytoestrogens due to their structural similarity to estrogen and ability to bind the estrogen receptor, may have implications in various clinical conditions and hormone-dependent cancers ,2. AlthoAspalathus linearis, has traditionally been used to aid in the alleviation of sleeplessness, anxiety and nervous tension, ailments related to stress and physiological conditions generally associated with high cortisol levels. Our previous studies showed that rooibos and specific flavonoids inhibit cytochrome P450 (P450) enzymes and cortisol production [Rooibos, a polyphenol-rich herbal tea prepared as an infusion from the stems and leaves of the plant oduction ,11.In vitro studies in COS-1 cells have shown that unfermented rooibos extract as well as the two rare dihydrochalcones, aspalathin and nothofagin, significantly inhibited P450 17α-hydroxylase/17,20 lyase (CYP17A1) and P450 21-hydroxylase (CYP21A2), while also significantly reducing the levels of cortisol in forskolin stimulated adrenal H295R cells. Even though aspalathin and nothofagin are the two most abundant flavonoids in unfermented rooibos, they did not in all cases reflect the same inhibitory effect on steroid levels in H295R cells as brought about by the rooibos extract, suggesting that other compounds contribute to the overall effect of rooibos on adrenal steroidogenesis [ogenesis .In the present study we continued our investigation into the influence of rooibos and polyphenolic compounds, belonging to three distinct classes of flavonoids, on adrenal steroidogenic enzymes. The five major rooibos flavonoids used in this study included the dihydrochalcones, aspalathin and nothofagin, their flavone analogues, orientin and vitexin, as well as the flavanol, rutin . Due to It has been widely reported that polyphenols exhibit a diverse range of beneficial biological effects, and as such, their consumption as part of the daily diet cannot be ignored. Although teas and herbal infusions contribute to the daily intake of dietary flavonoids, the pharmacological actions of these extracts cannot directly be attributed to single compounds within these beverages, unless identified and assayed in isolation. Flavonoids modulate steroid hormone biosynthesis and metabolism due to their interaction with steroidogenic enzymes, either inhibiting or stimulating specific enzymes ,8. We reCYP17A1 plays a central role at the branch point in adrenal steroid biosynthesis, and together with 3βHSD2, determines the shunt of steroid metabolites in the glucocorticoid, mineralocorticoid and adrenal androgen pathways .CYP17A1 catalyses the 17α-hydroxylation of pregnenolone (PREG), yielding 17OH-pregnenolone (17OH-PREG), which is in turn converted to dehydroepiandrosterone (DHEA). These three metabolites are substrates for 3βHSD2, which catalyzes their conversion to progesterone (PROG), 17OH-progesterone (17OH-PROG), and androstenedione (A4), respectively, thereby shunting metabolites into the respective pathways. In addition, CYP17A1 also catalyses the conversion of PROG to 17OH-PROG and 16OH-progesterone (16OH-PROG), with the latter not being further metabolized in the adrenal. PREG conversion by CYP17A1 was inhibited by all the flavonoid compounds assayed, with the compounds exhibiting similar inhibitory effects A. Althoucat, 2-fold less than that of deoxycortisol conversion, clearly indicating that a single hydroxyl group influences the enzyme’s affinity for its substrate, as well as the substrate turnover. It was suggested that the presence of the C17 hydroxyl group of deoxycortisol obstructs the entrance of deoxycortisol into the enzyme’s active pocket [CYP21A2 catalyzes the conversion of PROG and 17OH-PROG to DOC and deoxycortisol, respectively. In contrast to CYP17A1 and 3βHSD2, only the dihydrochalcones inhibited the activity of CYP21A2 significantly, with the flavones and rutin not influencing the conversion of PROG by CYP21A2 C. In thee pocket .While the rooibos extract exhibited the greatest inhibitory effect on CYP17A1 and CYP21A2, inhibition of the other enzymes was negligible. The inhibition of PREG conversion by CYP17A1 A as wellIn H295R cells, the dihydrochalcones and the flavones decreased the total steroid output under both basal and stimulated conditions, while rutin’s effect was only detected in the presence of forskolin. Forskolin mimics the effects of ACTH by activating cAMP pathways in adrenal cells , stimulaOrientin, the flavone analogue of aspalathin, significantly increased the steroid flux in the mineralocorticoid pathway, increasing basal levels of DOC, CORT and 18OH-CORT, suggesting that inhibition of CYP17A1 resulted in more metabolites being channeled into the mineralocorticoid pathway. The data obtained clearly shows that the flavones inhibit CYP17A1, since PREG and PROG levels increased, while 16OH-PROG and 17OH-PROG levels decreased .The inhibitory profile of vitexin was very similar to that of orientin, however, vitexin exhibited a greater inhibitory effect, reducing the total steroid output 1.3-fold under both basal and stimulated conditions, while the effect of orientin on total steroid production was negligible. Interestingly, under stimulated conditions, during which aldosterone synthase (CYP11B2) expression is upregulated , the effWhile the dihydrochalcones and their flavone analogues reduced basal testosterone levels significantly, it was the former compounds which had the greatest effect, suggesting inhibition of 17βHSD. However, the adrenal is not the primary site for 17βHSD expression, as is also the case for 11βHSD2, which catalyses the formation of cortisone and 11-dehydrocorticosterone (11-DHC). The detected levels of these metabolites were very low, with the flavones reducing cortisone levels under basal conditions, while under stimulated conditions, it was inhibited in the presence of nothofagin only. Although 17βHSD and 11βHSD2 are expressed at very low levels in the adrenal [In a recent pharmacophore-based virtual screening study investigating inhibitors of 17βHSD type 3 and 5, key enzymes involved in adrenal androgen production, it was shown that the ligand binding domain of these enzymes is able to accommodate structurally highly diverse ligands which bind to different regions of the active site. An enlargement of the binding cavity was observed in the crystal structure of 17βHSD3/5 when rutin was bound to these enzymes. Furthermore, the X-ray structure of rutin bound to 17βHSD5 showed that water molecules formed a hydrogen bonding network with rutin bound at the base of the ligand binding domain .Rutin, one of the more stable flavonoid compounds in both fermented and unfermented Rooibos, had no significant effect on any of the steroid hormones under basal conditions. However, upon forskolin stimulation, rutin had the greatest inhibitory effect of all the flavonoid compounds assayed, reducing the total steroid output 2.3-fold. In the mineralocorticoid pathway, CORT and 18OH-CORT levels were significantly lower, suggesting inhibition of CYP11B2. In the glucocorticoid pathway, significant reductions in the levels of 17OH-PROG, deoxycortisol and cortisol indicate inhibition of CYP17A1 and CYP11B1, as was also shown in COS-1 cells . UpstreaThe data obtained in the H295R cell line did not in all cases reflect results obtained in COS-1 cells. The expression levels of the individual enzymes assayed in COS-1 cells are comparable, as the cells were transiently transfected with equal concentrations of the appropriate cDNA. Furthermore, the conversion of substrates in COS-1 cells allowed assays to be conducted under very specific conditions. A fixed substrate concentration and no competition from other enzymes for the same substrates, as is the case in the adrenal H295R cell model, allowed us to investigate the effect of flavonoids on the catalytic activity of specific steroidogenic enzymes. In contrast, enzymes are expressed at different levels in adrenal H295R cells and vary from enzyme to enzyme under both basal and forskolin stimulated conditions, with specific enzymes being upregulated when cells are stimulated. It was recently shown that under basal conditions in H295R cells, CYP11A1 is expressed at the highest levels, followed by CYP21A2, CYP17A1, SULT2A1, 3βHSD2, CYP11B2 and CYP11B1, while under forskolin stimulated conditions, CYP17A1 is expressed at the highest levels, followed by CYP21A2, CYP11A1, 3βHSD2, SULT2A1, CYP11B2, and CYP11B1 .Overall, from these results it is clear that orientin and vitexin had the greatest effect on the mineralocorticoid pathway under both basal and forskolin stimulated conditions. The dihydrochalcones had no significant effect on any of the metabolites in the mineralocorticoid pathway, while rutin’s effect was only detected under stimulated conditions, with significantly reduced levels of ALDO precursors, CORT and 18OH-CORT. The influence of rutin was most evident in the glucocorticoid and adrenal androgen pathways under stimulated conditions, with the other flavonoids decreasing metabolites under both basal and stimulated conditions in these pathways.et al. that the isoflavone, diadzein, was able to competitively inhibit the binding of DHEA and PROG to 3βHSD2 and CYP21A2, respectively. The authors suggested, having assayed a range of flavone compounds, that it was the hydroxyl groups at C6 on ring A and C4' on ring B which played an important role in the inhibition of steroidogenic P450 enzymes. They showed, in cAMP-stimulated H295R cells, at a concentration of 12.5 µM, that 6-hydroxyflavone, 4'-hydroxyflavone and apigenin, which contains hydroxyl groups at C5, C7 and C4', inhibited cortisol production significantly [Analyses regarding the influence of structural differences of flavonoid compounds on their ability to inhibit steroidogenic enzymes are hampered due to the uncertainty of the manner in which these compounds would interact with the active site of these enzymes. As the type of inhibition of the steroidogenic cytochromes P450 by the flavonoids is not known, it is possible that binding could also occur at sites other than the active pocket. Furthermore, molecular docking studies reporting on structural aspects of flavonoid compounds impacting on substrate inhibition in terms of steroidogenic enzymes are limited. It has, however, been shown by Ohno et al. [In a more recent study by Hasegawa et al. , it was et al. .et al. [et al. [From our data it appears that the enzymes 3βHSD2 and CYP17A1 are most susceptible to inhibition by the rooibos flavonoids assayed in this study. The natural substrates for these two enzymes, PREG, 17OH-PREG and DHEA contains a hydroxyl group on C3 of the steroid backbone structure involved with substrate binding in the active pocket ,20. It iet al. , investi [et al.  investigThe compounds assayed in this study all contained glucose moieties on either ring A or C, which would, if bound in the active site of the enzyme, affect the orientation of the molecule, impacting on the mechanism of inhibition. It would seem that while a hydroxy group on B ring plays a role in flavonoid inhibitory activity, a second hydroxy group does not significantly affect inhibition or contribute towards increasing inhibition, possibly due to rotation between rings A and B resulting in a degree of flexibility. It should, however, also be noted that aspalathin and nothofagin have a more flexible structure compared to rutin, orientin and vitexin. It is interesting to note that the dihydrochalcones inhibit the catalytic activity of CYP21A2 towards both substrates, while exhibiting no inhibitory effect on CYP11B1, regardless of the substrate. In contrast, the inhibition of the activity of these enzymes towards their substrates differs in the presence of flavone/flavonol compounds, with the inhibitory effect of these compounds being greater when the substrate is hydroxylated at the C17 position.® AX plasmid preparation kits were purchased from Machery-Nagel . Mirus TransIT®-LT1 transfection reagent was purchased from Mirus Bio Corporation . Penicillin–streptomycin, fetal calf serum and trypsin-EDTA were obtained from Gibco BRL . Deuterated cortisol  was purchased from Cambridge isotopes . DMEM/F12 and gentamicin were purchased from Invitrogen/Gibco . Cosmic calf serum was supplied by HyClone®, Thermo Scientific . A bicinchoninic acid (BCA) protein determination kit was purchased from Pierce . The UPLC BEH C18 column was purchased from Waters . The Kinetex PFP column was obtained from Phenomenex . All chemicals were of the highest quality and supplied by reputable scientific suppliers.Unfermented rooibos plant material was provided by the South African Rooibos Council . Aspalathin and nothofagin were obtained from Prof. W.C.A. Gelderblom . Orientin and vitexin were purchased from Extrasynthese . Rutin, steroid metabolites, forskolin, Dulbecco’s modified Eagle’s Medium (DMEM) and an MTT assay kit were purchased from Sigma-Aldrich . Nucleobond2 in DMEM containing 0.9 g/L glucose, 0.12% NaHCO3, 10% fetal calf serum and 1% penicillin streptomycin. The cells were plated into 12 well dishes , 24 h prior to transfection. Cells were transiently transfected with 0.5 μg cDNA  and 1.5 µL ®Mirus TransIT- LT1 transfection reagent according to the manufacturer’s instructions. Co-transfections with baboon CYP11B1/pTarget and human ADX/pCIneo cDNA were carried out using 0.25 µg of each plasmid. Control transfection reactions were performed using the pCIneo vector containing no cDNA insert. Cells were incubated for 72 h prior to substrate addition. Substrate conversion (1 µM) was assayed in the absence and presence of aspalathin, nothofagin, rutin, orientin and vitexin, assayed at 10 µM, and unfermented rooibos extract, assayed at 4.3 mg/mL. The methanolic extract of unfermented rooibos plant material used in this study was prepared and analysed as previously described [g for 5 min and the medium removed via aspiration. The dichloromethane phase, containing the steroid metabolites, was dried under N2, resuspended in 150 μL methanol and stored at −20 °C prior to analyses. COS-1 cells were grown at 37 °C and 5% COescribed ,11. At s2 in DMEM/F12, supplemented with l-glutamine, 15 mM HEPES, pyridoxine, 1.125 g NaHCO3/l, 1% penicillin streptomycin, 0.01% gentamicin, and 10% cosmic calf serum (growth medium). The cells were plated into 12 well dishes  and incubated for 48 h. The medium was subsequently replaced with experimental medium  and cells were incubated for a further 12 h prior to substrate addition. Steroid metabolism was assayed in the presence of selected flavonoid compounds, aspalathin, nothofagin, rutin, orientin and vitexin (10 µM), under both basal and forskolin (10 µM) stimulated conditions. After 48 h, the medium (500 µL) was removed and 15 ng D4-cortisol added as an internal standard. The steroids were extracted as described in H295R cells were grown at 37 °C and 5% CO5 cells/mL) and incubated with selected flavonoid compounds (10 µM) for 8 h. H295R cells were plated out in a 96 well plate  and incubated with selected flavonoids (10 µM) and forskolin (10 µM) for 48 h. Cell viability was assayed using an MTT toxicology assay kit according to the manufacturer’s instructions. Color-standardized controls (media containing either 1 mg/mL or 4.3 mg/mL Rooibos extract) were included to compensate for the possible interference of the Rooibos color with the assay. None of the abovementioned test compounds had a significant effect on COS-1 or H295R cell viability.COS-1 cells were plated out in a 96 well plate  linear least squares regression. Data was collected with the MassLynx 4.0 software program. The data obtained in assays conducted COS-1 cells are depicted as % inhibition of substrate conversion, while the data obtained in H295R cells are expressed as absolute values (nM) ± SEM.All steroid metabolites were analysed and quantified using UPLC–MS/MS . Steroid metabolites from conversion assays in COS-1 cells were separated using a Waters UPLC BEH C18  column, while steroid metabolites produced in H295R cells were separated using a Phenomenex UPLC Kinetex PFP  column, as previously described . A Xevo p < 0.05 was considered to be statistically significant . All results are representative of two independent experiments, performed in triplicate, with the data being expressed as the mean ± SEM. After each assay a protein determination by the Pierce BCA method was performed in order to normalize steroid levels to protein concentrations. Statistical analyses were calculated with GraphPad Prism (version 5) software  using a one-way ANOVA, followed by a Dunnett’s multiple comparison test. A value of This study included five of the major flavonoid compounds present in rooibos. These flavonoids, regardless of structural differences, all inhibited the activitiy of CYP17A1 and 3βHSD2, branch point enzymes in adrenal steroidogenesis. The dihydrochalcones and the flavone/flavonol compounds, however, showed marked differences in their inhibition of the activity of downstream enzymes, CYP21A2 and CYP11B1, expressed in COS-1 cells. In addition, these compounds also exhibited distinct differences in terms of specific substrates. It is clear that structural differences regarding the number and position of hydroxyl and glucose moieties, and structural flexibility, impact on the inhibitory effect of these flavonoids, possibly indicating different mechanisms. Rooibos extract, which contains a wide spectrum of flavonoid compounds, some of which are present at higher levels than was assayed, did not in all cases exhibit the same degree of inhibition. It is thus plausible that, of the array of compounds present in rooibos, some may inhibit while others may stimulate or have no effect at all. Overall, it is clear that, although the mechanism of inhibition is uncertain, the major rooibos flavonoid compounds assayed in this study significantly influence adrenal steroidogenic enzyme activities and steroid hormone levels.