Volcanic eruptions can impact the mass balance of ice sheets through changes in climate and the radiative properties of the ice. Yet, empirical evidence highlighting the sensitivity of ancient ice sheets to volcanism is scarce. Here we present an exceptionally well-dated annual glacial varve chronology recording the melting history of the Fennoscandian Ice Sheet at the end of the last deglaciation . Our data indicate that abrupt ice melting events coincide with volcanogenic aerosol emissions recorded in Greenland ice cores. We suggest that enhanced ice sheet runoff is primarily associated with albedo effects due to deposition of ash sourced from high-latitude volcanic eruptions. Climate and snowpack mass-balance simulations show evidence for enhanced ice sheet runoff under volcanically forced conditions despite atmospheric cooling. The sensitivity of past ice sheets to volcanic ashfall highlights the need for an accurate coupling between atmosphere and ice sheet components in climate models. The impact of volcanism on ice sheet melting during the last deglaciation is poorly understood and limited by a lack of suitable proxies. Here, the authors combine annually resolved records of ice sheet melting with numerical models to show that ice sheets are sensitive to high-latitude volcanic eruptions. Moreover, volcanic eruptions can influence the mass balance of ice sheets and glaciers through changes in precipitation and surface ocean and air temperature10.A better understanding of the direct and indirect effects of volcanogenic aerosols on ice sheets is critical in the context of future meltwater contributions to global sea-level rise and ocean circulation. For instance, deposition of aerosol particles on snow and ice can affect the surface energy balance by lowering the snow albedo, thereby accelerating meltwater runoffAlthough the response of present-day ice sheets to volcanism can be well characterized through observations and modeling of their surface mass and energy balance, little is known about the sensitivity of ancient ice sheets to volcanism. Specifically, empirical evidence that directly highlights the response of ice-sheet melting to external forcing is still lacking.Here we achieve this using a new precise 1257-year long chronology from a continuous sequence of annual glacial varves, which record changes in the melting rate of the Fennoscandian Ice Sheet (FIS) during the period ∼13,200–12,000 years BP. Precise synchronization to the Greenland ice-core chronology allows for the first time comparison to ice-core volcanic records at an unprecedented precision and suggests a causal relationship between ice sheet melt events and volcanism.11 in the provinces of Småland and Östergötland13 —using the Vedde Ash isochron13 . The cumulative mismatch between the varve and the GICC05 time scales from the Vedde Ash to the end of Greenland Interstadial 1 (GI-1)/onset of Greenland Stadial 1 (GS-1) (726 GICC05 years) does not exceed 0.15%, with a difference of only 1 year13. In turn, this allows a confident annual-scale comparison of the two time scales (Methods section).The glacial varve chronology is here synchronized to the Greenland Ice-Core Chronology 2005 (GICCO5)13, here presented as a unified mean varve thickness record  (Methods section). The focus of our analysis leans on the older portion of the chronology , which is composed of 56  overlapping varve diagrams42− time series from the GISP2 ice core17 (Methods section), which records past explosive and sulfur-rich volcanic activity  relative to present day (Methods section). The results are used to drive a field-validated physically based energy balance model incorporating snowpack/ice mass balance and to test the sensitivity of ice sheet runoff to ash deposition (Methods section). We simulate one of the largest high-latitude volcanic eruptions in historical time—the Laki eruption —to test the effect of high-latitude eruptions on radiative forcing and climate (Methods section). To simulate the induced-climate impact of the volcanic eruption, we adopt present-day boundary conditions. Recent studies using an atmospheric general circulation model30 suggest that the atmospheric circulation during the last deglaciation may have been similar to today over the North Atlantic, provided that the height of the Laurentide Ice sheet is lower than the Rocky Mountains, such as during the analyzed period31. Other studies33 have also shown the predominant role of topography over sea surface temperature and sea-ice extent in altering North Atlantic atmospheric circulation. However, it is likely that sea surface temperature and sea-ice changes, together with a different strength of the Atlantic Meridional Overturning Circulation, had impacted atmospheric circulation primarily in winter, as suggested by some proxy data34. Our model experiments, on the other hand, focus primarily on the melt (summer) season . Furthermore, to simulate changes in snowpack/ice mass balance in a more realistic fashion, we apply corrections to model output that allow us to drive our runoff model using Younger Dryas (YD) temperature, shortwave radiation, and precipitation conditions (Methods section). We also perform an additional experiment whereby we simulate an identical high-latitude eruption starting in winter and lasting for 4 months for comparison to the summer case where the eruption initiates midway through the year (Methods section).To explore the potential climate feedbacks on the ice sheet induced by volcanic eruptions, we turn to a set of climate simulations performed with two climate models to account for both the volcanic SO35 but it also limits the chemical reactions that form sulphate particles, which further decrease the SWRF. However, the winter experiment highlights that global climate altering sulfate emissions may not have occurred for all eruptions, though maintain the possibility of significant distal ashfall36.Our climate model simulates a summer (JJA) cooling of approximately −3.5 °C over southern Scandinavia  in response to the large summer high-latitude volcanic eruption, whereas no significant cooling is observed in the simulation where the eruption occurs in winter. In large part this difference in cooling is due to the weak solar insolation during winter. This not only leads to a reduction in the net shortwave radiative forcing (SWRF)α = −15%) can lead to increases in runoff that still more than offset these low temperatures at high elevations  can result in a significant volcanic ash distribution over the Atlantic region, both easily reaching Northern Europe and Scandinavia. New findings45 also indicate that volcanic ash clouds from historical mid-size eruptions of North American and Icelandic volcanoes have resulted in frequent ash fallouts over Northern Europe at intervals of <50 years, which are also not necessarily associated with climate altering sulfate emission. More importantly, instrumental observations show that such eruptions can be a major agent of glacier melting via tephra-induced surface albedo changes46. Equatorial eruptions on the other hand, even though they can have a global climate impact, result in very little (if any) ash deposition in Greenland due to their latitude.Critically, recent studies have shown that even moderate-size Alaskan22, which implies that were more likely to be explosive due to interaction with water, and therefore ash will have been transported over relatively longer distances. Finally, reconstructions47 show that such explosive eruptions were mainly associated with mafic events that produced very dark tephra that are more effective at decreasing ice sheet surface albedo.Altogether, more frequent high-latitude volcanism during the final stage of the last deglaciation likely resulted in direct ash deposition across the FIS—predominantly from Icelandic sources just upwind of the ice sheet (due to the prevailing westerly flow). Moreover, these eruptions were likely to have occurred sub-glacially48, which are then more likely to access and evacuate untapped sediment stores underneath the ice sheet48.Results from snowpack simulations demonstrate that significant increases in summer and winter runoff can result from even a slight reduction in snow/ice albedo Tables , 5 and 749. These drainages are known to cause stepwise expansions in the subglacial hydrological network in an upglacier to downglacier direction, causing transient but substantial peaks in the suspended sediment concentration of proglacial meltwater once the supraglacial water connects to the pre-existing network50. This implies that enhanced runoff would allow a more extensive, efficient subglacial hydrological system to form, and hence supply extra suspended sediment to the Baltic Ice Lake to create an ETV.In addition, extra runoff at high elevations will have increased the likelihood of supraglacial lakes forming and draining to the ice sheet bedBy placing runoff response to volcanic forcing into the context of contemporary ice sheet mass balance and subglacial hydrological processes, this also suggests that the timing, style, and duration of eruptive events during the melt season will be crucial as to whether they are recorded in the varve record or not. For example, eruptions that are sustained throughout the melt season are more likely to produce the largest cumulative runoff response . Although speculative at this point, a causative link cannot be ruled out. Thus, further work is required to verify the impact of volcanic aerosol forcing and ice surface albedo changes on the recession of the FIS beyond the spillway threshold in south-central Sweden.As a final remark, we observe that one ETV corresponds with the catastrophic drainage of the Baltic Ice Lake In conclusion, evidence from our glacial varve records indicate that during the last deglaciation volcanism caused more extensive melt and enhanced levels of runoff from the FIS. We suggest that this was primarily an expression of decreased ice sheet albedo owing to deposition of dark volcanic ash. Our results highlight the sensitivity of ancient ice sheets to volcanogenic aerosols and the necessity to employ dynamic and interactive ice-sheet components in climate models that reproduce ice configuration changes in response to external forcing. This study also provides motivation for further investigations with regard to the mechanisms behind catastrophic freshwater surges of the past and their pivotal role on rapid climate change.12, and (iii) the internal chronological consistency verified by statistical analysis, independent14C dating, and well-defined biostratigraphic marker horizons, respectively13. An overall uncertainty (entailing precision and accuracy) of ±0.5% (2σ) has been assigned to the varve chronology13. However, this should be considered as a highly conservative estimate.The chronology provides an annually resolved and continuous record where the potential problem of missing varve years is overcome by cross-dating several overlapping varve-thickness records. The precision of the chronology is verified by the (i) general lack of disturbed layers and good preservation of the varves in all the sequences that compose the unified chronology, (ii) the evenly high lateral consistency of numerous adjacent—and distal—cross-correlated sequencesThe varve thickness record was filtered using a low-pass spline to remove harmonic functions below the 20th degree, which is similar to dividing the time series by a low-pass filter with a cut-off frequency of 1/25 year. The spline was preferred to other filters based on the fitting performance and we observe that the following results are independent of the type of filter used. The time series was then divided by its standard deviation. Finally, we identified anomalous melt events as years characterized by an annual varve thickness that exceeds +2σ .13. This suggests that the two records are evenly synchronous during the interval under analysis and that the age uncertainty due to sampling resolution, and allocated to each SO42− sample, is not higher than the relative mismatch between the two chronologies.Prior to comparison with ice-core volcanic records, we linearly interpolated the volcanic sulfate time series to annual resolution to avoid smoothing of the ETV record. This allows the sulfate peaks to accommodate a large part of the age uncertainty associated with each potential ETV isochrone , which is thus directly accounted for when testing the significance of the correlation using the Monte Carlo tests described below. A maximum cumulative mismatch of 0.15% between the Vedde Ash and the end of GI-1 is inferred between the glacial varve chronology and the GICC05 chronologyTo evaluate the significance of the correlation between volcanic eruptions and ETVs, we employed three independent Monte Carlo tests. The first approach is a simple permutation analysis whereby the number of original ETVs is randomly shuffled within the time frame of the chronology for 1000 times and for each realization the coherence with the maximum resampled volcanic sulfate anomalies is evaluated. In the second approach the significance of the coherence is inferred via comparison of the GISP2 data to 1000 individual realizations of the varve thickness record with similar red noise spectral characteristics. In the third test the coherence is inferred via comparison of the GISP2 data to 1000 individual Gaussian white noise realizations of the varve thickness record.64 to simulate an extreme high-latitude multistage eruption under present day conditions. NorESM1-M has a horizontal resolution for the atmosphere of 1.9° (latitude) × 2.5° (longitude) and 26 vertical levels. NorESM1-M uses a modified version of Community Atmospheric Model version 4 (CAM4)65, CAM4–Oslo with the updated aerosol module, simulating the life cycle of aerosol particles, primary and secondary organics. The atmospheric model is coupled to the Miami Isopycnic Coordinate Ocean Model—MICOM. A detailed description of the model used in this study can be found in Bentsen et al.63, Iversen et al.64, Kirkevåg et al.66, and Pausata et al.9. The model performance has also been evaluated in these studies from a basic validation of the physical climate63, to the climate response to future climate scnarios64, to aerosol-climate interactions66, and as well as to high-latitude Laki-type volcanic eruptions9.We use the Norwegian Earth System Model (NorESM1-M)2 and dust (median radius = 0.22 μm in accumulation mode), as an analog for ash, mostly into the upper-troposphere/lower stratosphere over a 4-month period. We start the eruption on 1st June in order to replicate the original Laki eruption (details regarding the set-up are provided in refs. 10). We also perform an identical Northern Hemisphere high-latitude eruption, but with start date on 1st December and lasting the full duration of the melt season in order to appreciate the climate effect of such eruption in winter and compare it to the summer case.We mimic the largest high-latitude volcanic eruption in recent history—the Laki  eruption—to simulate the effect of high-latitude eruptions on radiative forcing and climate. We simulate the high-latitude eruption by injecting 100 Tg of SOWe analyze an ensemble of 20 simulations with each member starting from a different year selected from a transient historical simulation (1901–1960). The no-volcano ensemble is obtained by simply considering each of the unperturbed years from the historical simulation corresponding to the same years of the perturbed case.67). We use these data as no-volcano control experiment to force the runoff model, described in the section below. Then we imposed on the YD control simulation the area-averaged volcanically induced anomalies extrapolated from the NorESM coupled simulations. The volcanically induced anomalies inferred using this approach are only meant to be indicative of the climate anomalies during the late deglaciation.To account for the changes in boundary and climate conditions in terms of solar insolation, surface temperature, and precipitation during the late phase of the deglaciation (YD) over southern Scandinavia , we use the output from the simulations performed with the Community Atmospheric Model version 3  that are interpolated to 15 min timesteps. A full description of the model is in Morris et al.68, however a modification in this study is that we account for changes in cloudiness, whereas constant conditions were assumed previously.We apply a field-validated physically based one-dimensional energy balance model of melt, refreezing, and runoff processes that occur within a given snowpack/ice columnRunoff is determined by calculating both energy and mass balances for a 9.5 m column of ice/firn/snow at 1 cm intervals, and determining the potential for melt, refreezing, and percolation through to a dynamically determined impermeable ice layer . For each timestep, melt that does not refreeze within the snow/ice column is lost as runoff.The ability of the model to account for melt, percolation, energy balance, and snow/firn density changes through refreezing represents a much more realistic way of simulating runoff compared to models that calculate melt only. For this study, the distinction between melt and runoff is important, since refreezing within the snowpack means that the former does not necessarily translate to the latter. Similarly, the model accounts for the fact that capacity for refreezing is not constant through time, and will evolve due to density and englacial temperature changes . A threshold temperature of 0 °C is set, at which or below precipitation will fall as snow and contribute to the snowpack rather than fall as rain where it can contribute to percolation, and provide extra thermal energy to the snowpack/ice, freeze, and/or runoff.Daily mean absolute humidity values are provided by the climate model that are interpolated to hourly observations before being converted to relative humidity values using the air temperature values used to drive the runoff model.http://desktop.arcgis.com/en/arcmap/10.3/tools/spatial-analyst-toolbox/points-solar-radiation.htm). This provided hourly SWRF values that are consistent with the time of day, time of year, and the cloudiness conditions given by the climate model. Volcanically forced SWRF is calculated by multiplying the above by the fractional difference between monthly non-volcanic and volcanic SWRF output given by the climate model. To ensure the smooth evolution of SWRF for use in the runoff model, the monthly fractional differences between non-volcanic/volcanic climate model outputs are interpolated to the same temporal resolution as runoff model inputs. This ensures that inputs to the runoff model capture both diurnal variability of SWRF and any effects of volcanic forcing predicted by the climate model. The differences between non-volcanically and volcanically forced SWRF are shown in Supplementary Fig. The climate model provides monthly mean values for SWRF. However, this value will vary substantially over the course of a day, and is non-trivial in providing energy to the snow/ice surface for melting. As such, it was necessary to recalculate SWRF outside of the climate model to capture its diurnal variability. This was achieved using the solar insolation tool of ArcGIS (ESRI) to calculate the daily insolation for a topographically unshielded point at 60° N for a uniform sky. The diffuse fraction value was calculated using the cloud cover values from the climate model, by assuming that at the extremes of cloudiness, zero cloud cover equated to 20% diffuse contribution, while total cloud cover equated to 70% diffuse contribution  and those altered by a Laki-type eruption  occurring (i) in summer (volcanic forcing initiated on 1st June), and (ii) winter (volcanic forcing initiated on 1st December). Each set of simulations had spin-up periods preceding the initiation of the eruption, with the summer eruption simulation beginning on 1st January, and winter eruption on 1st October. These initiation dates were chosen to allow (i) sufficient time for the ice column temperature respond to the atmospheric conditions determined by the climate model output, and (ii) not to include any period of time from the preceding melt season . Consequently no melt and no density changes to the column occurred during the spin-up period. Precipitation was also reduced to zero until the initiation of volcanic forcing or the first day of positive temperatures (whichever occurred earlier). This allowed ice column conditions for volcanically and non-volcanically forced simulations to be directly comparable.−1 to evaluate runoff response at sea level, 500, 1000, and 1500 m elevation, in addition to four different albedo forcing scenarios simulating the effect of ash fall for the volcanically forced scenarios only. The range of albedo changes applied  represent modest absolute reductions compared to initial values that are derived values from Devon Ice Cap, Nunavut, Canada . Consequently, each suite of simulations comprises of 16 different volcanically forced scenarios , and four different non-volcanically forced scenarios (varying elevation only).There are many uncertainties associated with modeling the mass balance of palaeo ice sheets. Rather than avoiding them, our modeling approach seeks to explore these uncertainties in order to characterise the potential range of response for the volcanic and non-volcanically forced scenarios. The scenarios tested involve applying an atmospheric lapse rate of −5.4 °C kmThe large range of uncertainty in initial snow/ice column conditions for each scenario is also explored systematically. This is achieved by running the model for a range of initial snow/firn thicknesses, simulating 0–100 cm thicknesses of each at 10 cm intervals. Each individual scenario is therefore tested for 121 different sets of initial conditions. Consequently, the runoff potential of each ensemble of scenarios is evaluated for 2420 unique combinations of conditions . This allows runoff response to be considered across an elevation range of the FIS, and against different albedo forcing scenarios for a wide range of potential initial conditions.It is also possible to assess the relative contributions to runoff due to each environmental variable  to be evaluated in combination and/or isolation, through comparison to a non-volcanically forced ensemble of simulations. The results of simulations where the effects of temperature and cloudiness are evaluated in isolation are shown in Supplementary Fig. Finally, to test the significance of the variability of cloudiness in controlling runoff, we have also conducted an ensemble of melt/runoff simulations where we add random noise  to the cloudiness data. This aims to evaluate the impact of short-term changes in this input on the overall trends and magnitudes of the runoff results generated by the runoff model. The results of these simulations are presented as the difference between the simulations where the noise has been added to the cloudiness data (Supplementary Figs. In addition, adding noise to the cloudiness values will also change the shortwave radiation fluxes compared to the original simulations. Consequently, for the new simulations the SWRF is recalculated following the same method described above. All remaining data used to drive the new simulations are consistent with those of the original simulations. Supplementary Fig. The varve chronology presented in this study is available along the online version of this article on the publisher’s web-site. All the model data and runoff model codes are available from the authors upon request.Supplementary InformationSupplementary Data 1Peer Review FileDescription of Additional Supplementary Files


Avian infectious bronchitis, a highly contagious disease caused by avian infectious bronchitis virus (IBV), is of considerable economic importance to the poultry industry. New IBV TW-like strains have increasingly emerged in China in recent years; hence, evaluating their pathogenicity and developing a specific vaccine to guard against their potential threat to the poultry industry is important. Here, we examined the pathogenicity of a TW-like IBV strain (GD), and evaluated the protective efficacy of the QX-like strain (JS) against GD in challenge infections in chickens. The results revealed that strain-GD-infected birds experienced severe respiratory signs, renal lesions, and 30–40% mortality. The GD virus had extensive tissue tropism, especially in the trachea, lungs, kidneys, and bursa of Fabricius, and was continuously shed via the respiratory tract and cloaca. The QX-like IBV strain JS is able to completely protect chickens from challenge with the TW-like IBV GD field strain, with no clinical signs or gross lesions, decreased tissue replication rates, lower ciliostasis score, and reduced virus shedding. These findings indicate that IBV GD is highly virulent, and that QX-like JS may serve as an effective vaccine against the threat posed by IBV TW-like viruses. Gammacoronavirus genus  is an acute, highly contagious respiratory, and urogenital disease caused by infections bronchitis virus (IBV). The IBV genome consists of a positive-stranded RNA of 27.6 kb in length, and the virus belongs to the Currently, the main method of protecting chickens from IB is vaccination with both live and inactivated vaccines . HoweverA predominant IBV type, QX-like IBV, has been circulating in China since 1998 when the first variant of this type was reported . Over tiHere, we determined the pathogenicity of the TW-like IBV GD strain. We also evaluated the protective efficacy induced by QX-like strain JS against the TW-like field strain GD by observing the clinical signs and gross lesions, and by analysis of the virus distribution, virus shedding, and tracheal ciliary activity in experimental chickens. Our results suggest that TW-like IBV GD is highly virulent, and that QX-like JS may provide effective vaccine protection against TW-like IBV viruses.50) of the JS and GD strains were calculated as 108.02 EID50/mL and 108.03 EID50/mL, respectively.The IBV TW-like GD strain was isolated from IB-affected chickens in the Guangdong Province of China. These chickens had been vaccinated with IBV Mass-type vaccines, and were affected by respiratory and renal disease. The complete genome of the IBV strain GD was sequenced in our previous study . The IBVSpecific-pathogen-free white leghorn chickens were purchased from the Beijing Merial Vital Laboratory Animal Technology Co., Ltd, China. All the animals used in this study were cared for in accordance with established guidelines, and the experimental protocols  were approved by the Animal Welfare and Ethical Censor Committee of China Agricultural University .n = 10 each). Groups C and D were used for sampling (n = 15 each). All the chickens were maintained in isolators under positive pressure in air-conditioned rooms, and food and water were provided ad libitum. Groups A and C were inoculated with 200 μl of allantoic fluid containing 106.00 EID50 of the GD virus via the intranasal and intraocular routes. Groups B and D were maintained as uninfected controls. All the chickens were monitored daily for 14 days. Three chicks from groups C and D were sacrificed at 3, 5, 7, and 10 days post-challenge (dpc). Gross lesions and tracheal ciliary activity were evaluated in the birds. Tissue samples from the trachea, lungs, spleen, glandular stomach, kidneys, and bursa of Fabricius were collected. Some birds died during the clinical observation period, and their tissue samples were also collected. The tissue samples were placed in 10% neutral formalin for histology, or stored at -80°C for viral RNA detection using reverse transcription–polymerase chain reaction (RT-PCR).Fifty 3-week-old SPF chickens were assigned randomly to four groups. Groups A and B were used to observe chicken mortality  according to the manufacturer’s instructions. RT was performed at 37°C for 1 h with 3 μg of total RNA, 1 μl of random hexamer primers , and 0.5 μl of M-MLV reverse transcriptase . PCR was performed in a 20 μl reaction volume containing 10 μl of Taq SuperMix , 10 pmol of each primer, 100 ng of cDNA as the template, and 7 μl of water. Thermal cycling was performed at 95°C for 5 min, followed by 30 cycles of denaturation , annealing , and polymerization , and a final extension step was performed at 72°C for 10 min. A pair of primers  that amplified an 800-bp fragment of the IBV N gene was used in the PCR. The amplified fragments were detected by 1.0% agarose gel electrophoresis.Oral and cloaca swabs were randomly collected at 5 and 7 dpc from 10 birds in challenge group C and five birds in control group D to detect viral shedding via RT-PCR. Viral RNA was extracted with TRIzol® Reagent (Invitrogen) following the manufacturer’s protocol. To transcribe the first cDNA strand, a mixture containing 1 μl of random primers (500 μg/ml), 2.0 μl of dNTPs (2.5 mM), 1.5 μl of RNasin® (50 U/μl), 0.5 μl of M-MLV reverse transcriptase (10 U/μl), and 4 μl of the RNA template was incubated at 37°C for 60 min, and then at 72°C for 5 min. The product was used for PCR, as described above.Total RNA was extracted from the trachea, lungs, spleen, glandular stomach, kidneys, and bursa of Fabricius with TRIzolTo evaluate tracheal ciliostasis, three sections of the upper, middle, and lower parts of the trachea (nine rings per bird) were analyzed. The rings were placed in 96-well plates with Eagle’s culture medium containing 10% fetal bovine serum. They were then examined by inverted light microscopy at a magnification of 400× to determine the degree of integrity and the preservation of ciliary movement in the tracheal epithelial cells. A score of 0 was given if the cilia in the complete tracheal section showed movement; a score of 1 was given if 75–100% of the cilia in the tracheal section showed movement; a score of 2 was given if 50–75% of the cilia in the tracheal section showed movement; a score of 3 was given if 25–50% of the cilia in the tracheal section showed movement; and a score of 4 was given if <25% of the cilia in the tracheal section showed movement or there was no movement at all. The average ciliostasis score was calculated for each group.Tissue samples from the trachea, lungs, spleen, glandular stomach, kidneys, and bursa of Fabricius were fixed in 10% neutral formalin for 48 h at room temperature. These samples were processed routinely, embedded in paraffin wax, and cut into 5 μm sections. The sections were stained with hematoxylin and eosin (H&E) and examined for lesions with light microscopy.7.00 EID50 of the JS strain by intranasal and intraocular administration. The group control and control-GD group were inoculated with the same volume of sterile saline by the same method. Twenty-one days post-inoculation (dpi), groups JS-GD, and control-GD were challenged with 200 μL of the GD strain containing 106.00 EID50 by intranasal and ocular routes. The group control was inoculated with sterile saline using the same method. Birds from all groups were housed with consistent conditions.Forty-eight 6-week-old SPF chickens were randomly divided into three groups of 16 chickens each and then housed in different isolators. Group JS-GD was vaccinated with 200 μL of 10All chicks were observed daily for clinical signs attributable to IB infection for 14 days after challenge. Three chicks from each group were killed humanely and dissected at 5 and 7 dpc. When chicks died during the observation period autopsies were performed in a timely manner. External and internal exceptional situations were recorded in detail and samples of trachea, lungs, kidneys, spleen, glandular stomach, and bursa of Fabricius were collected for virus detection by RT-PCR. Additionally, ten laryngeal swabs from each group were collected for virus detection by RT-PCR by random sampling at 5 and 7 dpc. Tracheal ciliary activity was evaluated at 5 and 7 dpc. RT-PCR and tracheal ciliostasis evaluation were performed as described in the pathogenicity tests. Blood samples from ten chicks in each group were collected at 21 dpi and 14 dpc for IBV antibody analysis using an enzyme-linked immunosorbent assay .t-test in GraphPad Prism version 6.0 for Windows to obtain a statistical analysis of the differences between group control-GD and JS-GD in the protective efficacy test. For the ciliary activity inhibition test, the data collected were analyzed using two-way ANOVA to see whether there was any significant difference between the different groups. Multiple comparisons among the groups were performed using Tukey’s multiple comparison test. Statistical significance was considered as follows: significant at ∗P ≤ 0.05; highly significant at ∗∗P ≤ 0.01; and very highly significant at ∗∗∗P ≤ 0.001.Data were analyzed using an unpaired Figure 1A). The birds in the control groups were alert and active during the experiment.Three-week-old SPF chickens challenged with the GD strain showed clinical signs of infection as early as 3 dpc, and these signs persisted until 10 dpc. The diseased chicks showed signs of coughing, sneezing, tracheal and bronchiolar rales, listlessness, huddling, and ruffled feathers. Four birds in the GD-inoculated group A died during the experiment, beginning at 5 dpc and peaking at 8 dpc, and the mortality rate reached 40% . The bursa of Fabricius showed a heavy exudate of mucus and was in some cases atrophied. No obvious lesions were identified in any other organs in the infection-challenged group or in any birds that recovered from infection with strain GD. No gross lesions were observed in any birds in the control group .At necropsy, obvious lesions were detected in the respiratory tract, lungs, kidneys, and bursa of Fabricius in the chickens infected with the GD strain, including punctate hemorrhage in the throat and trachea and catarrhal exudate in the nasal cavity. The main manifestations in the sampled chicken were swollen kidneys, with urate deposits frequently observed in the tubules and ureters (‘spotted kidney’) at 5 and 7 dpc, and in the dead chickens at 6 dpc . Foci of necrosis, intense multifocal nephritis with lymphoplasmacytic infiltration, and mesenchyme dilation were scattered throughout the kidneys of the GD-inoculated chickens at 3, 5, and 7 dpc, respectively . Hemorrhage, congestion, erythrocytes, and lymphocytes were frequently detected in the bronchial and air capillary lumina in the lungs. Epithelial cell necrosis and lymphocyte emptying were detected in the bursa. No IBV-related lesions were observed in the control birds .Moderate to severe lesions were found predominantly between 5 and 7 dpc in the organs collected from the infection-challenged chickens. The chickens exposed to strain GD had the following lesions: thickening and hemorrhage of the lamina propria of the tracheal mucosa, inflammatory cell infiltrates, and varying degrees of epithelial desquamation and necrosis of the ciliated cells in the trachea at 3, 5, and 7 dpc, respectively (p < 0.0001).Inhibition of ciliary activity in trachea was measured at 3, 5, 7, and 10 dpc. The infection-challenged group showed a maximum average ciliostasis score of approximately 4, whereas that of the control was <1. The difference in ciliostasis between the strain-GD-infected group and the control group was highly significant . Figure 3A). The main clinical manifestations in the dead chicks were kidney swelling, urate deposition on the kidneys, and heavy exudates of flaxen mucus on the bursa of Fabricius . The mental status and appetite in the JS-GD group showed no obvious changes. The organs also exhibited no apparent pathological changes after challenge in the JS-GD group during the observation period.In the control-GD group, four chicks showed signs of depression and tracheal and bronchiolar rales at 7 dpc. The mortality of the group control-GD was 30% during the 14-day observation period (Table 2).Reverse transcription–polymerase chain reaction was used for viral RNA detection in sampled tissues after challenge with IBV GD strain. For the control-GD group, virus distribution in different proportions were detected in all organs at 5 and 7 dpc, in addition to the spleen at 7 dpc. For the immunized JS-GD group, the virus was positive only in the bursa of Fabricius of the chicks at 7 dpc. The proportion of positive samples in the JS-GD group was significantly lower than that of the control-GD group. Furthermore, no virus was detected in any organs of the control group . The difference in the level of ciliostasis was extremely significant between the group vaccinated with the JS strain and the unvaccinated group (P < 0.001).We determined the degree of ciliary activity inhibition at 5 and 7 dpc in the trachea. The control-GD group showed a maximum average ciliostasis score of 4, while the average ciliostasis scores for the control and JS-GD groups were below 1 .Serum samples were collected for ELISA antibody tests from 21 dpi and 14 dpc. All chicks inoculated with the JS strain showed positive serum antibody responses at 21 dpi and the average antibody titer was 1966.93. And the average titers of the control-GD and JS-GD groups were 5402.60 and 3632.59 at 14 dpc, respectively. Antibodies against IBV were never detected in the control group (Despite the various live-attenuated and inactivated IBV vaccines derived from large numbers of genetically distinct strains in widespread use across the world, IB outbreaks still occur sporadically because there is little or no cross protection between the different IBV serotypes and new variants constantly appear . Once a The TW-like IBV GD strain was highly pathogenic in SPF chickens, inflicting 100% morbidity and 30–40% mortality. IBV GD caused pale and swollen kidneys, and the renal tubules and ureters were distended with urates. The gross lesions and histopathological changes caused by the GD strain were similar to those described in a report on nephropathogenic IBV strains , thus chThe ciliostasis test is often used to determine the degree of damage to the trachea following growth of IBV in this tissue . We scorThe IBV QX-like strain JS has naturally low virulence and shows good protective efficacy against QX-like virulent field strains. Our findings in this study have further revealed that the QX-like IBV JS strain also provided effective protection against TW-like viruses, with no obvious clinical signs or gross lesions, a clear decrease in ciliostasis, and reduced tissue replication rates and viral shedding in the infected chickens.In summary, our study has revealed that the TW-like IBV GD strain is highly pathogenic in chickens and that the QX-like JS strain provided effective protection against TW-like IBV viruses, indicating that the JS strain may be useful in vaccination programs under field conditions to reduce the economic losses caused by TW-like IBV infections.Conceived and designed the experiments: S-hY, JZ, and G-zZ. Performed the experiments: S-hY, YC, and GX. Analyzed the data: JZ, YZ, and G-zZ. Contributed reagents/materials/analysis tools: G-zZ. Wrote the paper: S-hY, YC, and G-zZ.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.


Although previous research provides converging evidence for the role of posterior regions of the brain  involved in inhibition on creative thinking, it remains unclear as to how these regions influence individual differences in creative thinking. Thus, we explored the relationship between posterior regions , inhibition function, and divergent thinking (DT) in 128 healthy college students. The results revealed that lower inhibition was associated with larger gray matter volume (GMV) in the lingual gyrus, which in turn was associated with higher DT. In addition, GMV in the lingual gyrus mediated the association between inhibition and DT. These results provide new evidence for the role of inhibition in creative thinking. Inhibition may affect the amount of information stored in long-term memory, which, in turn influences DT. Creativity can be defined as the production of a novel, useful, and surprising solution to a complex problem  and is cRecent investigations have consistently linked inhibition function to DT. For instance, creative people with fluency of ideas and associations , are chaOn the one hand, functional imaging studies have repeatedly demonstrated inhibition control cortex plays a crucial role in the creative thinking . ImportaThough little empirical evidence suggests a direct link between inhibition function and TOP regions, it is noteworthy that the inhibition function – a component of executive function – can filter task-irrelevant information that can therefore disrupt goal completion . In otheCollectively, the literature indicates a three-way relationship between inhibition function, TOP, and DT, although the direction of influences is unclear. Therefore, the mediation hypothesis in the present study states that individuals with poorer inhibition function would perform better at DT tasks. Poorer inhibition function may increase the amount information stored in brain regions associated with long-term memory, and the demand for large information storage contributes to increased volumes in these brain regions. In addition, evidence suggests that brain structures represent perceptual- and cognitive-level variables to some degree . More inParticipants comprised 128 right-handed, healthy subjects  who took part in our project to investigate the relationships among creativity, mental health, and brain imaging . All of The Stroop color–word-interference task was used to assess the inhibition of prepotent responses . It is gThe Torrance (1974) Tests of Creative Thinking – based on DT – is the most commonly used test ,b. We usParticipants were given 3 min per task to write all the novel and appropriate responses that they could think of on a personal computer. A consensual assessment technique was used to assess the ideas . All resTo control the impact of general intelligence on brain structure , we measMagnetic resonance imaging (MRI) data were acquired with a 3T scanner . A magnetization-prepared rapid gradient echo (MPRAGE) sequence was used to collect the high-resolution T1-weighted anatomical images .Structural magnetic resonance images were processed with SPM8 software . Specifically, artifacts or anatomical abnormalities were first screened in SPM8. Subsequently, images were manually set along the anterior-posterior commissure (AC-PC) line to get better registration. Next, the new segmentation tool in SPM8 was employed to segment images into gray matter, white matter, and cerebrospinal fluid. The segmented images were submitted for registration, normalization, and modulation using SPM8 plus DARTEL . After tWe extracted six cortical ROIs from the Automated Anatomical Labeling atlas using the WFU PickAtlas Toolbox . The ROIBefore performing a mediation analysis, all data were normalized. The non-interest effect of age, gender, and general intelligence was removed from behavioral data . We used Stroop-effect scores as the independent variable, DT as the dependent variable, and GMV of ROIs as mediators. To further investigate the effects of inhibition and GMV on facets of DT, we set up several models for originality and fluency, wherein originality and fluency of DT tasks were used as dependent variables.Figure 1). After accounting for M, the mediation analysis was determined by assessing whether there is a significant distinction between the total (path c) and direct effects (path c’). When the zero is not encompassed within the confidence interval (CI), a significant difference between the indirect effect (path a b) and zero exists, which indicates that regional structural volume accounts for a statistically significant portion of the relationship between inhibition functions and DT.A simple mediation analysis was employed using SPSS macro with a 0.95 confidence level and 5000-bootstrap sample , 2008. Ir = 0.276, p < 0.01, corrected for multiple comparisons, shown in Table 1) and fluency , and negatively associated with inhibition function .All behavioral data were analyzed using SPSS 18.0 . Partly confirming our first prediction, only lingual gyrus volume was positively associated with originality , only lingual gyrus data were considered for further analysis .Descriptive statistics are presented in p = 0.005; path b = 0.3, p = 0.002; path a × b = -0.069, bootstrapped 95% CI = -0.154, -0.02; Figure 2); (2) GMV of the right lingual gyrus accounts for a significant portion of the relationship between inhibition functions and fluency ; (3) GMV of the left lingual gyrus accounts for a significant portion of the relationship between inhibition functions and originality ; and (4) GMV of the left lingual gyrus accounts for a significant portion of the relationship between inhibition functions and fluency .Inhibition was selected as the independent variable after controlling for intelligence, gender, and age. The right and left lingual gyrus were used separately as mediators to investigate a concrete neural basis for originality and fluency of DT. It was found that (1) the GMV of the right lingual gyrus accounts for a significant portion of the relationship between inhibition functions and originality (path a = -0.231, The present study used mediation analysis to classify the associations between GMV of classical regions located in TOP areas, inhibition function, and DT. The results showed two main findings: poorer inhibition function was associated with increased lingual gyrus volume, which in turn was associated with better DT performance; and the lingual gyrus GMV mediated the relationship between inhibition function and DT.The association between lingual gyrus GMV and DT advances previous structural and functional neuroimaging evidence that suggests that greater lingual gyrus GMV volume is associated with better ideational fluency  and actiThe current findings demonstrate a negative association between lingual gyrus volume and inhibition function. Although there is little experimental evidence for the direct relationship between inhibition function and lingual gyrus, Inconsistent with previous studies showing that inhibition function is positively or negatively associated with creative cognition, we find that inhibition function is not directly associated with DT. Creative thought has been variably associated with focused attention and effective inhibition control , 2016, dThe present study also found that the GMV of the lingual gyrus mediates the relationship between inhibition function and DT. Although previous researches  in behavIn the present study, only the volume of the lingual gyrus was found to mediate the relationship between Inhibition function and DT, compared to the volume of hippocampal, parahippocampal, precuneus, and cuneus regions. This may be because the lingual gyrus is associated with visual memory  and viviWe found that the volume of IFG is not related with inhibition function and DT. Although, these results are inconsistent with previous functional imaging studies which have repeatedly demonstrated inhibition control cortex plays a crucial role in the creative thinking . There aHowever, there are some limitations in the present study. First, it is not sufficient to attribute the link between inhibition and DT to memory processes due to a lack of working memory and long-term memory assessment. In future research, memory measurements should be involved to provide a substantial evidence to examine the mechanism underlying the association between inhibition and lingual gyrus. Second, the Stroop task used in this study may be not an appropriate measurement to test inhibition function, as this version of Stroop task lacks a neural condition. Because of this, we are not sure the difference is due to inhibition function or attention control. Modified task is required to test the relationship between inhibition function and DT in future research.In summary, the present findings – lingual gyrus volume mediated the relationship between inhibition function and DT – are important because they potentially explain the influence of inhibition on DT through the posterior cortex. Limited research has simultaneously examined inhibition, brain structure, and DT behavior to identify their interrelationships and the mechanisms through which inhibition influences DT. The results provide novel evidence for the role of inhibition function in creative thinking by its influence on the amount of information stored in long-term memory. However, longitudinal studies are needed to establish the long-term impact of lingual gyrus volume and the direction of influence in our model.LZ, LQ, QC, JQ, and DY programmed the experiments and analyzed the data; LZ and WY drafted the manuscript and LZ, LQ, QC, WY, MX, and XY provided critical revisions; LQ prepared the figures.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.


Upon channel bars or point bars within the lows of the Yellow River, a new sedimentary structure, named ‘silt mushroom’, has been observed. The process of their formation is interpreted to be via the ice process. The name, the silt mushroom comes from their figurative form. This is because they look somewhat similar to mushroom’s in size and shape; being in the range of 1 to 10 cm in diameter, with the medium 3–5 cm, and on average 10 cm in height, occuring generally in groups, and occasionally in isolation in relatively soft silt. They develop in the transition from winter to spring, and are convincingly related to ice processes. Ice-induced silt mushrooms are best examined in association with the many other newly discovered ice-induced sedimentary structures (over 20 kinds). Clearly, up to now, ice processes have been significantly underestimated. With the substantial discovery of the ice-induced silt mushroom, it opens up new questions. This is because its structure mirrors the same sedimentary structures found in rocks, questioning their genesis, and sedimentary environment analysis. This achievement is significant not only in sedimentology, but also in palaeogeography, palaeoclimate, geological engineering, hydraulics and fluviology. The Yellow River, is the second largest river in China, and is well known for the highest levels of mud and silt in the world12456789101112131415171819202122232526272829303133343536373839404142434445464748495051522  is introduced herein to define a relatively small, circular, vertical, mushroom-like, ice-induced sedimentary structure . It mustTo describe the form simply, it visually has a mushroom shape, mostly of a short and thick mushroom . The silThe stem is the main and most important element of a silt mushroom. In general, the stem has an uneven height, a nearly columnar shape with somewhat peculiar curve surface like a mustard stem , and itsThe base is not an important part of a silt mushroom, for it is not often observed. The base is smaller than the stems and its internal structures are not as well developed as cap and stem. Actually, the base does not often develop individually and connects altogether with surrounding sediments. We have carefully observed the base and its surrounding sediments by clearing up and cutting a few important areas at which the silt mushrooms develop very well and have not found any traces formed by freezing-thawing, and nor have found any needle stick phenomenon. A large number of observation shows that the bases and their surroundings sediments are relatively homogeneous . But on (1) Their sizes are commonly larger than that of the cross beddings formed by river flows, which are generally smaller than 2–3 cm in height. Nevertheless, the heights of the cross beddings in the stems are commonly larger than 3 cm ((2) The dips of the cross beddings developed in the stems are not as same as that of the cross beddings formed by usual river flows and even have opposite directions like a funnel in the vertical sections of a stem ((3) We can observe two silt mushrooms with clear inclined beddings in (4) Especially, the cross beddings are circular or arc-like shapes in the horizontal sections of a stem ((5) The continuity of the lamina of the cross beddings in the stem is mostly not as good as that of the cross beddings formed by river flows.The most important sedimentary structures are the beddings developed in the stem. The bedding could reveal the genesis of the silt mushroom to some extent. The observation shows that the vertical variations within an ice-induced silt mushroom are typically attributable to the beddings by changing grain size, composition, color, texture or internal structure. An ice-induced silt mushroom may be heterogeneous, rhythmic, gradational or deformed. Most of silt mushrooms are heterolithic (composed of different sediments) as a result of sorting into repeated interlaminations of sediment of contrasting composition or grain size carried by ice flow . A point worth emphasizing is that some silt mushrooms have well-developed cross beddings in their stems   We directly observe in situ that the covering layer on the stem collapse. (2) We can make the covering layer to collapse to form a silt mushroom in the field. (3) All the silt mushrooms develop within an ice melt depression; (4) The experiment provides strong evidence that the silt mushroom forms really by the ice block melting and the covering layer to collapse and not by freezing and thawing.In order to distinguish if the silt mushroom is formed by needle stick or by covering layer collapse, the key issue is that we should make sure that if the stem rise from the ground or the ground collapse. All the evidence suggests that the stem does not rise and the ground does collapse, so the silt mushroom does not form by needle stick and does form by the ground collapse, all the evidences are as follows: The repeated freeze-thaw cycle may really reform or erode the sediment surface textures4748495052The formation mechanisms of ice-induced silt mushrooms is very important and worthy of study because this new phenomenon has many applications for the academic field of sedimentary environment analysis. In terms of the ice-induced silt mushroom’s genesis, one can infer this is due to the unusual flowing processes of the Yellow River; these occur from the end of the winter to the beginning of the spring. As mentioned above, the ice-induced silt mushrooms are associated with many other ice-induced sedimentary structures. Like these, the distribution boundary of silt mushrooms is strictly controlled by the ice block melt boundary . TherefoAs we know, the Yellow River is unique in the world. Firstly, because the Yellow River located in the relatively dry region of north China; the total water yield is insufficient and during 1990s the Yellow River was often in zero flow. Secondly, for its tremendous sediment load and a very small gradient in the lower, this is especially the case in the section from Binzhou Bridge to the estuary; the lower of the Yellow River becomes narrower and shallower, resulting in frequent flood. Thirdly, the lower of the Yellow river is in a warm temperate zone with four distinct seasons. Every year when it is getting cold in winter, the Yellow River freezes and the mains are obstructed by ice blocks, which form ice flow. So, during the transition from winter to spring, a lot of ice blocks are washed onto the channel bars and point bars to form an unusual sedimentary environment. This provides particularly favorable natural conditions for the formation of sedimentary structures. This includes silt mushrooms.Additionally, the riverbed’s fine texture is both texturally and compositionally important to the formation of the silt mushroom. The uniqueness of the ice layers built upon the mud and silt, and the river’s fine-grained sediment load. These are generated by the Yellow River’s flow and make this a singular phenomenon. Of all the conditions observed, it is the unusual hydrological phenomenon generated by the Yellow River’s ice-run that can be attributed to formation of ice-induced silt mushrooms. These hydrological actions collectively play a key role in the formation of ice-induced silt mushrooms, as ice-melting creates the honeycomb-like holes; these holes are later filled with silt. This thesis, therefore, proposes that there is a formation model of the Yellow River delta silt mushroom that relates to the ice layer formation and melting process.It is really that there are a lot of needle ice blocks on the channel bar or point bar in the lower of Yellow river. The needle ice in the lower of the Yellow river in thicken is extremely compatible with that of Outcalt (1971)(1) Direct observation in situ that the covering layer on the stem collapses. (2) One could make the covering layer collapse to form a manmade silt mushroom that is the same as a natural one (3) All the silt mushrooms, whether in groups or individual, develop within an ice melt depression ((4) The experiment has been repeated to show that the silt mushroom can form through the thawing of an ice block and the collapse of a covering layer on the stem  The ice block is washed onto the channel bar and/or point bar; this takes place during the process of ice flow in the transition between winter and spring ((2) when the sun shines on the ice block, the sections that have silt and mud thaw easily and so the ice block only partially melts; therein, a lot of holes appear, resulting in the formation of a honeycomb-shaped holes, best observed in some of the small ice blocks ((3) When another ice flow happens, the channel bar and\or point bar will be submerged again. Those honeycomb-shaped holes or simple holes will be covered by the mud and silt, sometimes including the ice block, brought on by floods ((4) With the temperature increasing during the transition between winter and spring, the ice block, with its honeycomb-like holes, melts completely; the subsequent silt layer that cover the ice block collapses. This finally results in a complete silt mushroom formation  Witormation . The forIce-induced silt mushroom is a new type of sedimentary structure; its discovery may be significant to complete basic theories upon sedimentology. This achievement is significant not only in sedimentology, but also in geography, geological engineering, hydraulics and fluviology.Ice-induced silt mushrooms could be of value to environment analysis; the phenomenon requires an exposed environment, with the ice flows happening frequently. The frequent ice flows in combination with a great deal of mud and silt, may be responsible for the ice-induced silt mushrooms visible.Extremely strict conditions are needed for the formation of ice-induced silt mushrooms, such as specific circumstances within an areas; hydrology, climate, sedimentary environment and fine material quality. The ice block washed onto the channel and point bar plays a fundamental role in the formation of ice-induced silt mushrooms.The silt mushrooms present in the lower of Yellow river are extremely similar to those produced experimentally by simulated ice block melt. The apparent success of the study of the lower of Yellow river led to a search for similar structures in other similar modern sediments or ancient rocks in the world. The discovery of ice-induced silt mushrooms, may contribute to furthering our understandings of similar sedimentary structures in rocks, questioning their genesis and furthering similar structures sedimentary environment analyses.Further academic significance from our discovery of ice-induced silt mushrooms, may be as follows:This investigation is mainly restricted to field observations and indoor experiments as follows:Ice-induced sedimentary structures, including silt mushrooms, are discussed in terms of their geological features; field observations are therefore highly important. The concept of a silt mushroom is introduced and the conditions of their formation is also presented. A basic aim of fieldwork on silt mushrooms should be to observe them in detail and describe them accurately and concisely.Since the lower of the Yellow River is often seriously flooded by ice–flow, field observation can be dangerous and it is imperative that participants select a safe and suitable time and location to conduct their work. Where possible, fieldwork should aim to measure and record some typical silt mushrooms on the channel bar and/or point bar and to select and to document typical ones judged from preliminary field observation. This enables local variability to be distinguished from any regional variability and ensures that common features are seen from a proper perspective. Participants should make sure to take measurements, statistics and photographs of the silt mushrooms where this is possible. Just because of this, the field observation may be too rather arbitrary to select. Experience over twenty years informs us that the ideal location to investigate silt mushrooms is the section of the river from Binzhou Bridge in Lijin to the estuary. The distance covered is approximately sixty kilometers long. The section near Shengli Yellow River bridge is especially convenient to reach and has a lot of channel bars and point bars that is easy to approach, especial the ice blocks are easy to be washed onto the channel bars and point bars to form a special texture like a sandwich for having a lot of curves that is favorable for ice block to be washed onto the channel bars and point bars to form sandwiches that is of central importance in formation of silt mushrooms.(1) clearing up of a field site and getting a serial of vertical sections by cutting some silt mushrooms and their bases ((2) it is also extremely helpful to photograph as many silt mushrooms and their bases as possible, from a series of positions and at different distances. Fortunately, this is very easy to do in the study area.Field investigation may not go to schedule as a result of not being exposed of the channel bars and point bars for ice flowing or overlain by ice layer for too cold. An effort should always therefore be made to wait for the proper opportunity and to secure the proper location. Once the proper location has been selected, participants should first clear the site carefully and then make a detailed observation including careful description, strict measurements and photographs. The author hereby refers to a working method that is three-dimensional and carefully thought out: ir bases  and 8. Iir bases ; (2) it (1) the formation process of the ice-hole within an ice block; (2) the formation process of the ice-induced silt mushroom.In order to re-create the formation process of a silt mushroom, it can be helpful to design a simple laboratory experiment. The experiment has two aspects: (1) Preparing an ice tank and plastic box. Switch on the ice-tank and put the plastic tank into it. Fill the tank with water to the level of 10 cm. After all the water is frozen, take the plastic tank out of the ice tank and cut the ice block to be 30 length, 20 cm width and 10 cm thicken and put a lump of silts on the surface of the ice block ((2) Wrapping its bottom and sides with a cotton quilt to keep warm in order to prevent the ice-layer from melting too fast, expose the ice layer to the sun to the complete formation of a hole ((3) Put the ice layer into the ice tank after the holes have formed ((4) Pour moderate silt into a plastic box and stir. Slowly add the mixture of water and silt every minute with a spool into the plastic tank. Repeat the process until the silt levels 10 cm in height. Meanwhile, pat the water surface with your hands in order to make the silt closely packed. (5) Leave the sediments to freeze for one night; (6) Take the plastic tank out of the ice tank and wait until the ice layer has completely melted to observe the silt mushroom that has formed  and then a smaller plastic tank . The authors then gathered around 100 kilograms of loose silt from the Yellow River. The experiment followed a number of steps: ce block ; (2) Wraf a hole . (3) Pute formed , fillings formed . The proHow to cite this article: Jianhua, Z. et al. A New Unusual Ice-induced Sedimentary Structure: the Silt Mushroom. Sci. Rep.6, 36945; doi: 10.1038/srep36945 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC) and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin) that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126) of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and an inhibitor (LY294002) of phosphoinositide 3-kinase (PI3K) failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant  or diphenylene iodonium (DPI), PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.In this study, phosphorylation of c-Jun N-terminal kinase (JNK) by the prothoracicotropic hormone (PTTH) was investigated in prothoracic glands (PGs) of the silkworm,  Recombinant B. mori PTTH (PTTH) was kindly provided by Dr. David R. O'Reilly; it was produced by infection of Spodoptera frugiperda-SF21 cells with the vWTPTTHM baculovirus as described previously , and diphenylene iodonium (DPI) were purchased from Sigma-Aldrich . Grace's insect cell culture medium was purchased from Invitrogen . A MAPK/ERK kinase (MEK) inhibitor (U0126), a PI3K inhibitor (LY294002), A23187, and thapsigargin were purchased from Calbiochem . All other reagents used were of analytical grade. [23, 24-In vitro incubation of PGs and the RIA of ecdysteroids followed protocols described in a previous study  (#9251), anti-phospho-ERK (#9101), anti-phospho-4E-BP (#9459), anti-total ERK (#9102), and anti-α-tubulin (#2144) antibodies were purchased from Cell Signaling Technology . The anti-JNK1/3 antibody (sc-474) was purchased from Santa Cruz Biotechnology . Horseradish peroxidase (HRP)-labeled anti-rabbit immunoglobulin G (IgG) was purchased from PerkinElmer Life Sciences .g for 3 min to remove any particulate matter. One percent protease inhibitor cocktail  and 1% phosphatase inhibitor cocktail  were added at the time of homogenization. Aliquots of the supernatants were loaded onto SDS gels. Following electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using an Owl  Bandit™ Tank Electroblotting System, and then washed with Tris-buffered saline (TBS) for 5 min at room temperature. Blots were blocked at room temperature for 1 h in TBS containing 0.1% Tween 20 (TBST) and 5% (w/v) nonfat powdered dry milk, followed by washing three times for 5 min each with TBST. Blots were incubated overnight at 4°C with primary antibodies against phospho-SAPK/JNK , phospho-ERK , phospho-4E-BP , JNK , ERK , or α-tubulin  in TBST with 5% bovine serum albumin (BSA). Blots were then washed three times in TBST for 10 min each and further incubated with an HRP-linked second antibody in TBST with 1% BSA. Following three additional washes, immunoreactivity was visualized by chemiluminescence using Western Lightning Chemiluminescence Reagent Plus from PerkinElmer Life Sciences. Films exposed to the chemiluminescent reaction were scanned and quantified using an AlphaImager Imaging System and AlphaEaseFC software .Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as described previously . Subsequently, 200 μl of gland lysate was incubated with 20 μl of c-Jun fusion protein beads overnight at 4°C, and then a kinase assay was performed following the manufacturer's instructions . The reaction was carried out for 30 min at 30°C and was stopped by boiling the samples in 3x SDS sample buffer. Levels of phospho-c-Jun were measured by Western blotting as described in the section “Western blot analysis”.To measure JNK activity, control or treated PGs (each of a pair of glands for control and treatment from 10 larvae) were lysed in JNK lysis buffer , which removes phosphates from serine, threonine, and tyrosine residues . The real-time qRT-PCR was performed using the following primers: JNK forward, 5′-ACCAAGACTGATATTGTGATAC-3′ and JNK reverse, 5′-AAGTTGTCCGTGTCCATA-3′; Jun forward, 5′-ATGACGAGCAATATCCTC-3′ and Jun reverse, 5′-GATCCGAGCTTCAACATT-3′; and rp49 forward, 5′-CAGGCGGTTCAAGGGTCAATAC-3′ and rp49 reverse, 5′-TGCTGGGCTCTTTCCACGA-3′.Total RNA from PGs was extracted using the TRI Reagent  according to the manufacturer's protocol. Purification of total RNA, reverse transcription and real-time qRT-PCR were performed as described previously  followed by Tukey's test for more than two groups. A p < 0.05 was considered significant.Data are shown as the mean ± standard error of the mean (SEM). Statistical comparisons between different groups were done using either Student's Drosophila. B. mori JNK (BmJNK) was cloned as previously reported by Katsuma et al. (Drosophila DJNK (Basket) and human JNK1  in mammals, only one JNK (DJNK) exists in a et al. . BmJNK wManduca and Bombyx PGs . Results showed that the PTTH injection greatly increased the JNK phosphorylation level, compared to that of the controls, verifying the in vitro stimulation of JNK phosphorylation  and Jun (http://silkworm.genomics.org.cn/: BGIBMGA004164) mRNA expression levels upon PTTH treatment. As shown in Figure JNK or Jun transcription levels between control glands and those treated with the PTTH during the 1- or 2-h incubation periods after in vitro treatment with PTTH.We further conducted an experiment to determine changes in B. mori PGs. In vitro activation by the PTTH of JNK phosphorylation in B. mori PGs was also confirmed by in vivo experiments: a PTTH injection into day-6 last instar larvae greatly augmented JNK phosphorylation of PGs. The kinase assay of JNK in PGs confirmed its stimulation by the PTTH. However, the PTTH did not affect JNK or Jun mRNA expressions. Although ERK/MAPK signaling was previously demonstrated to be involved in PTTH-stimulated ecdysteroidogenesis in PGs of both M. sexta . U73122 was also found to block PTTH-stimulated JNK phosphorylation. These results clearly indicate that PTTH-stimulated JNK signaling is both PLC- and /Ca2+-dependent.The MAPK superfamily was found to mediate intracellular signaling of extracellular agonists and play important roles in various cellular functions including proliferation, differentiation, and apoptosis in a variety of cells (Roux and Blenis, 2+ signaling are together involved in PTTH-stimulated ecdysteroidogenesis (Hsieh et al., Our previous study demonstrated that mitochondrion-derived ROS signaling and CaB. mori PGs during a short-term incubation period, it is not clear whether or not PTTH-stimulated JNK signaling has additional roles. In mammalian cells, it was revealed that growth factors and oncogenes induced sustained activation of JNK to mediate their effects on survival, proliferation, and transformation (Davis, In larval insects, the PTTH is the major stimulator of ecdysteroid secretion in PGs. Early studies showed that the three-dimensional structure of the PTTH resembles those of mammalian growth factors including PDGF, NGF, and TGF-β (Noguti et al., n Davis, . Activatn Davis, . Furthern Davis, . Like ERn Davis, . A wide n Davis, . These dn Davis, . In the In summary, our study demonstrates that JNK represents another class of signaling molecules which reside downstream of the PTTH receptor and which may be necessary for ecdysteroid biosynthesis. Understanding the roles of JNK in signaling and identifying mechanisms that regulate JNK activation in response to PTTH and its downstream signaling pathways are fundamental to a clearer understanding of signaling events that regulate ecdysteroidogenesis in insect PGs.S-HG: designed and conducted the experiments, and wrote the manuscript; GL and SL: provided some ideas and contributed important resources, techniques, and reagents; H-YH and P-LL performed the experiments.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.


Isoforms in the PDE3 family of cyclic nucleotide phosphodiesterases have important roles in cyclic nucleotide-mediated signalling in cardiac myocytes. These enzymes are targeted by inhibitors used to increase contractility in patients with heart failure, with a combination of beneficial and adverse effects on clinical outcomes. This review covers relevant aspects of the molecular biology of the isoforms that have been identified in cardiac myocytes; the roles of these enzymes in modulating cAMP-mediated signalling and the processes mediated thereby; and the potential for targeting these enzymes to improve the profile of clinical responses. Cyclic nucleotide phosphodiesterases regulate intracellular signalling by hydrolysing cAMP and/or cGMP. Enzymes in the PDE3 family of phosphodiesterases are dual-specificity enzymes with high affinities for both cAMP and cGMP but much higher turnover rates for cAMP ,2,3. In Several isoforms of PDE3 are expressed in cardiac myocytes, and PDE3 inhibitors are used therapeutically to potentiate cAMP-mediated signalling in patients with heart failure. In the short term, these agents have the desired action of increasing myocardial contractility, but their long-term administration has been shown in several clinical trials to increase cardiovascular mortality ,7,8,9,10Cyclic nucleotide phosphodiesterases comprise a superfamily of enzymes. As of now, more than 50 mammalian isoforms have been described and classified into eleven gene families (PDE1 through PDE11) defined on the basis of sensitivity to pharmacologic inhibitors, kinetic activity, and regulatory mechanisms ,13. The Enzymes in the PDE3 family are transcribed from two genes, PDE3A and PDE3B ,15. In tCyclic nucleotide-mediated signalling is highly compartmentalised in cardiac myocytes, and the roles of individual phosphodiesterases in regulating cAMP- and cGMP-mediated signalling depend upon their intracellular localisation. While PDE3A and PDE3B are both expressed in cardiac myocytes (PDE3A more abundantly ), their The precise intracellular localisation of PDE3 isoforms depends upon their interactions with anchoring, scaffold, and adaptor proteins that recruit the enzyme to multiprotein signalling complexes ,31,32,33L-type Ca2+ channels, ryanodine-sensitive Ca2+ channels, and SERCA2 [PDE3 has long been known to be associated with the sarcoplasmic reticulum of cardiac myocytes ,36. Confd SERCA2 ,41,42,43d SERCA2 . The rolFurthermore, while PDE3 isoforms regulate the phosphorylation of other proteins through the cAMP/PKA pathway, they are themselves substrates for protein kinases that modulate their catalytic activity and protein–protein interactions ,45,46,474(b) receptor, for example, while PDE3A2, PDE3A3, and PDE3B do not [The absence of the S292-4 site from PDE3A2 and PDE3A3 establishes a PDE3A1-specific mechanism for recruitment of PDE3A to the SERCA2 complex. The protein–protein interactions of PDE3 isoforms are distinct in other ways as well, as has been described for the phosphorylation-dependent interactions of PDE3A1, PDE3A2, and PDE3B with 14-3-3 ,48,49,50B do not . These uL-type Ca2+ channels [In cardiac myocytes, PDE3A is also part of a multiprotein complex containing the unconventional AKAP PI3Kγ . While Pchannels ,54  pore opening, oxidative stress, and apoptosis contribute to injury [2+-induced opening of the MPT pore, and associate with caveolin-3-enriched membrane subfractions containing cardioprotective proteins. The recruitment of these cardioprotective proteins to these subfractions is PKA-dependent, and can be reproduced in wild-type mice by PDE3 inhibition [In contrast to PDE3A, PDE3B is mainly located in caveolin-3-rich areas of the plasma membrane and, in cardiac myocytes, in the T tubules . This ino injury . In hearhibition . 3-adrenergic receptor agonists increases the phosphorylation of PDE3B associated with caveolin-1 in caveolae, promoting interactions with β3-adrenergic receptors, PKA-RII (PKA regulatory subunit), and hormone-sensitive lipase [The nature of the localization signal that keeps PDE3B in such spatially and functionally distinct cellular subdomains remains unknown. Various reports indicate that PDE3B can be recruited to different multiprotein complexes with distinct properties. In adipocytes, for example, treatment with insulin increases the phosphorylation of PDE3B associated with internal membranes, promoting its interactions with IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 (p85-subunit of phosphoinositide 3-kinase), PKB (protein kinase B), HSP-90 (heat-shock protein 90), 14-3-3, and a 50 kD protein ,56,59,60e lipase ,61. Whete lipase ,62. Nonee lipase .Of note, however, is that PDE3B can be phosphorylated not only by PKA but also by PKB, the main effector of the PI3K pathway. Although the weak interaction between PI3Kγ and PDE3B reported in the heart awaits further experimental confirmation, severe reduction of the G protein-coupled receptor-driven PI3K pathway by the concomitant loss of PI3Kγ and PI3Kβ has been shown to lead to reduced PDE3B phosphorylation and activity in neurons . Nonethe2+ transients. Experiments in mice with selective ablation of PDE3A and PDE3B indicate that inotropic responses to PDE3 inhibition are attributable specifically to inhibition of PDE3A. Phospholamban phosphorylation, SERCA2 activity, intracellular Ca2+ cycling, and contractility are increased—and inotropic responses to PDE3 inhibition are eliminated—in Pde3a−/− mice; none of these effects are observed in Pde3b−/− mice [Pde3b−/− mice than in Pde3a−/− mice [As discussed above, PDE3A is part of a multiprotein complex in the sarcoplasmic reticulum through which phospholamban phosphorylation and SERCA2 activity are regulated, and PDE3 inhibition potentiates the stimulatory effects of cAMP on SERCA2 activity in microsomes from cardiac muscle. These effects would be expected to increase contractility by increasing the amplitude of intracellular Ca−/− mice . Further−/− mice . Total i−/− mice , indicat−/− mice .) Pde3a ablation in mice lead to increases in the phosphorylation of cAMP response element-binding protein (CREB) and consequent increases in the expression of inducible cAMP early repressors (ICER’s), promoting apoptosis [PDE3 inhibition in rats and poptosis ,67. Inhipoptosis . In factpoptosis . PDE3 acpoptosis , and it PDE3 inhibition has pro-hypertrophic actions in neonatal rat ventricular myocytes . These e2+ transients [With respect to cardiac disease, PDE3 has been of interest principally as a target for increasing contractility in patients with heart failure, a condition in which decreases in β-adrenergic receptor density and increases in Gαi and β-adrenergic receptor kinase activity in cardiac myocytes attenuate cAMP generation lead to decreases in cAMP content, protein phosphorylation and the amplitude of intracellular Caansients ,79,80,81ansients ,86,87,88L-type and ryanodine-sensitive Ca2+ channels may be pro-arrhythmic [This short-term benefit, unfortunately, is outweighed by an increase in mortality from sudden cardiac death of ~3% per year when these drugs are administered chronically ,7,8,9,10rhythmic ,41,42,43The fact that PDE3A and PDE3B have different roles in cardiac myocytes, with PDE3A controlling the pathways responsible for inotropic effects, raises the possibility that targeting PDE3A selectively—or, perhaps even better, selectively targeting one of its three known variants—might improve contractility without increasing sudden cardiac death . The ca2+ [2+ cycling and increase contractility without the deleterious pro-arrhythmic, pro-apoptotic, and pro-hypertrophic consequences (Another possibility is that of targeting individual isoforms not through their active sites but through the protein–protein interactions by which they are localised intracellularly. As noted above, PDE3A1 and PDE3A2 are recruited, by phosphorylation within their N-terminal sequences, to a SERCA2/phospholamban/AKAP/PKA complex in the sarcoplasmic reticulum ,93. As a2+ . Experim2+ . These f2+ ,67,68. Bequences . On the other hand, the protein–protein interactions that localise PDE3B intracellularly may be targets through which the cardioprotective effects of PDE3B ablation can be elicited without the adverse pro-hypertrophic and pro-arrhythmic consequences of PDE3A inhibition. The feasibility of this approach has been demonstrated in experiments using peptides that block the protein–protein interactions of cyclic nucleotide phosphodiesterases. In one example, peptide-array scanning identified a cell-permeant peptide based on a sequence in the N-terminus of PDE3B through which it is incorporated into an Epac1/PI3Kγ complex in vascular endothelial cells. This peptide has been shown to block the integration of PDE3B into this complex, increasing the activation of Epac1 by cAMP and promoting intracellular tubule formation, cell adhesion, and cell spreading . Several isoforms in the PDE3 family are expressed in cardiac myocytes, where they have important roles regulating signalling pathways involved in inotropic, pro-apoptotic, and pro-hypertrophic responses. Inhibition of myocardial PDE3 activity is an established therapeutic strategy for increasing contractility in patients with heart failure, but an increase in sudden cardiac death in patients treated chronically with existing PDE3 inhibitors has limited the benefits. Targeting individual PDE3A isoforms in specific intracellular targets could potentially yield inotropic responses without this adverse effect, while targeting PDE3B could have beneficial actions in ischaemia/reperfusion injury. Recent discoveries open the possibility of selectively inhibiting PDE3A or PDE3B at their catalytic sites or, probably more interestingly, of targeting their unique protein–protein interactions to yield compartment-specific effects on intracellular signalling. Time will tell.


Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing atherosclerotic process by blocking the proliferation of VSMCs.Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a ( Cardiovascular diseases (CVDs) are the most common causes of death. Atherosclerosis, a chronic inflammatory disease of the vessel wall, is the principal underlying pathology of CVD . Atheros2+ concentration ([Ca2+]i) and myofilament Ca2+ sensitivity  assay. VSMCs were pretreated with ketamine (50–500 µM) in a serum-free medium for 30 min and then stimulated with PDGF-BB (10 ng/mL) for 24 h. PDGF-BB treatment significantly increased the toxicity of the VSMCs up to 1.2 folds, compared with the untreated cells A. PDGF-Bp < 0.05) suppressed the PDGF-BB-induced cell proliferation  assay revealed that ketamine at a maximum of 500 µM significantly  siRNA. The results showed that pp2a siRNA suppressed the basal level of the PP2A catalytic subunit (PP2A-C)  C. Moreoved VSMCs C. These pp2a siRNA more profoundly reversed the ketamine-inhibited ERK1/2 phosphorylation -null mice [Among the mitogen-activated protein kinase (MAPK) family, ERK1/2 has been majorly involved in the growth of various cell types . Akt, a ull mice . These aull mice . Moreoveull mice . In someull mice . Hence, PP2A is one of the major Ser/Thr phosphatases associated with the regulation of various cellular processes . PP2A copp2a siRNA alone or combined with ketamine significantly inhibited the PDGF-BB-induced PP2A-C expression. Moreover, this study also found ketamine significantly and concentration-dependently suppressed the phosphorylation and demethylation of PP2A in VSMCs. These notable results further support our finding that ketamine inhibits PDGF-BB stimulated proliferation in VSMCs by the regulation of PP2A.PP2A activity is regulated by several molecular events, such as post-translation modification, auto-regulation, and substrate protein interaction. PP2A catalytic efficiency is reported to be controlled by two major alterations: phosphorylation  and methOur previous study had demonstrated that various concentrations of ketamine (100–500 μM) used to treated microglia did not cause cytotoxicity, and, at the higher concentration (500 μM), significantly inhibited TNF-α production . Shibakal-glutamine, penicillin, streptomycin, fetal bovine serum (FBS), 3--2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich . Okadaic acid was obtained from Calbiochem , and 3-O-Methyl-sphingomyeline (3-OME-SM) was purchased from Biomol . The anti-phospho-p44/p42 extracellular signal-regulated kinase (ERK) (Thr202/Tyr204) was purchased from Cell Signaling . The anti-phospho-Akt (Ser473) and anti-Akt monoclonal antibodies (mAbs) were purchased from Biovision . The anti-phospho-PP2A-C mAb, and anti-demethylated-PP2A-C mAb were purchased from Santa Cruz Biotechnology . The anti-α-tubulin mAb was purchased from Neo Markers . The horseradish peroxidase conjugated donkey anti-rabbit immunoglobulin G (IgG) and sheep anti-mouse IgG were purchased from Amersham . The Hybond-P polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminescence (ECL) detection reagent were purchased from Amersham. Ketamine was dissolved in 0.1% DMSO, and stored at 4 °C.The Dulbecco modified Eagle medium (DMEM), optiMEM, trypsin (0.25%), l-glutamine at 37 °C in a humidified atmosphere of 5% CO2. The growth medium was changed every 2 to 3 days until cells were confluent. A trypsin-ethylenediaminetetraacetic (EDTA) solution was added, and the monolayer was incubated at 37 °C for 2 min. Containing 10 mL of DMEM, cells were detached and centrifuged at 900× g for 7 min before being resuspended in DMEM. For all the experiments, the cells from passages 4 to 8 were used. The animal study was performed under protocols sanctioned by Taipei Medical University’s Animal Care and Use Committee . Male Wistar rats (250 to 300 g) procured from BioLASCO , and maintained in agreement with standards published by the US National Institutes of Health  following their Guide for the Care and Use of Laboratory Animals. To harvest VSMCs, thoracic aortas from Wistar rats were removed, and stripped of the endothelium and adventitia. The VSMCs were acquired by a modification of the combined collagenase and elastase digestion method . The har4 cells/well) were seeded and cultivated in DMEM containing 10% FBS for 24 h. VSMCs were pretreated with ketamine (50–500 µM) or solvent control (0.1% DMSO) for 30 min, followed by the addition of PDGF-BB (10 ng/mL) for 24 h. An MTT assay, which measures the mitochondrial activity, was used to determine cell viability [In 24-well plates, the VSMCs (2 × 10iability . The rat5 cells/dish) were seeded in a 96-well microplate for 24 h and then serum-starved for 24 h. Following preincubation of ketamine (200 and 500 µM) or LY294002  and PD98059  in serum-free medium for 30 min, the cells were treated with PDGF-BB (10 ng/mL) for 48 h. DNA synthesis was assessed using BrdU incorporation assay kits  according to the manufacturer’s instructions. DNA synthesis in VSMCs was assessed by the incorporation of BrdU. VSMCs  for PP2A suppression analysis. The coding regions of the mouse PP2A catalytic subunit (PP2A-C) mRNA targeting the siRNA oligonucleotide were sequenced as pp2a siRNA, 5′-ccauacuccgagggaaucatt-3′. The negative control siRNA comprising a 19-bp scrambled sequence with 3d topicscape (3dT) overhangs was also purchased from Ambion.As described previously , target p value of <0.05 was considered statistically significant.The results are presented as means ± standard errors from at least three independent experiments. The differences between the experiments were assessed using one-way analysis of variance (ANOVA). The statistical significance of the difference between means was determined using a Newman–Keuls test. A The present study showed that ketamine inhibited PDGF-BB-induced VSMC proliferation, which is critical in atherosclerosis. PP2A is an essential and miscellaneous phosphatase in the cellular system. An increasing body of evidence indicates that PP2A is known to regulate the activity of more than 30 different kinases, including PI3K, Akt, and ERK. In this study, we report the first preliminary evidence on the antiproliferative mechanism of action of ketamine in VSMCs. This inhibitory effect could be attributed to the PP2A-mediated inhibition of PI3K, Akt, and ERK, resulting in the overall inhibition of VSMC proliferation. Taken together, our findings support the notion that PP2A activation by ketamine may be an innovative means for therapeutic strategies to inhibit VSMC proliferation, as it plays a vital role in vascular diseases such as atherosclerosis and restenosis.


In this focus review, we summarize recent efforts towards identification of the active sites in MoSx-based electrocatalysts for the hydrogen evolution reaction (HER). We also discuss recent synthetic strategies for the engineering of catalyst structures to achieve high active site densities. Finally, we suggest ongoing and future research challenges in the design of advanced MoSx-based HER electrocatalysts.Hydrogen has received significant attention as a promising future energy carrier due to its high energy density and environmentally friendly nature. In particular, the electrocatalytic generation of hydrogen fuel is highly desirable to replace current fossil fuel-dependent hydrogen production methods. However, to achieve widespread implementation of electrocatalytic hydrogen production technology, the development of highly active and durable electrocatalysts based on Earth-abundant elements is of prime importance. In this context, nanostructured molybdenum sulfides (MoS The edge site of MoS2 was initially identified as the active site for the HER, which then triggered the development of new HER catalysts that can maximize the density of active edge sites, thereby boosting HER activity. The basal surface of MoS2, which were previously believed to be inert towards the HER, can be converted into HER active species via appropriate structural engineering, which include phase transformation, heteroatom doping, defect site generation, and strain engineering. In addition to the crystalline MoS2, amorphous MoSx has also been extensively studied as an efficient electrocatalyst for the HER. Amorphous MoSx can possess abundant active edge structures originating from the building blocks of [Mo3S13]2− clusters, however, the multiple chemical states of sulfur in such species hamper identification of the actual active sulfur states. A comprehensive and systematic study to reveal the key active sites in different MoSx structures still remains a challenging task. Understanding of active sites would enable high-performance MoSx elecrocatalysts for the HER.In this review, we have highlighted recent major achievements regarding elucidation of the active sites in MoS