Droughts have become more severe and recurrent over the Indian sub-continent during the second half of the twentieth century, leading to more severe hydro-climatic and socio-economic impacts over one of the most densely populated parts of the world. So far, droughts have mostly been connected to circulation changes concomitant with the abnormal warming over the Pacific Ocean, prevalently known as “El Niño”. Here, exploiting observational data sets and a series of dedicated sensitivity experiments, we show that the severity of droughts during El Niño is amplified (17%) by changes in aerosols. The model experiments simulate the transport of boundary layer aerosols from South Asian countries to higher altitudes (12–18 km) where they form the Asian Tropopause Aerosol Layer (ATAL) . During El Niño, the anomalous overturning circulation from the East Asian region further enriches the thickness of aerosol layers in the ATAL over the northern part of South Asia. The anomalous aerosol loading in the ATAL reduces insolation over the monsoon region, thereby exacerbating the severity of drought by further weakening the monsoon circulation. Future increases in industrial emissions from both East and South Asia will lead to a wider and thicker elevated aerosol layer in the upper troposphere, potentially amplifying the severity of droughts. A number of studies attribute droughts to El Niño effects5, which modify the Walker and Hadley large-scale circulations through atmospheric tele-connections. The existing literature shows that most of the Indian droughts are accompanied by El-Niños7. In addition to the increasing frequency of droughts, South Asia underwent a widespread declining trend in its total seasonal monsoon precipitation (~7% from 1951 to the present). This is largely blamed on the anthropogenic aerosols as they generally reduce temperatures over land, resulting in weaker land-sea thermal contrast9. The land-sea thermal contrast is the essential force required to pull the monsoon circulation from ocean to land. Aerosols interact in many ways with the monsoon - the enhancement or suppression of the Indian summer monsoon rainfall depends on its duration and scale (including remote influences), along with its distinct direct and indirect radiative effects10.The century-long systematic monitoring of Indian monsoon records reveals that frequent floods and droughts were not unfamiliar to this nation of 1.3 billion people. Statistical analysis shows that in the second half of the twentieth century the frequency of droughts has increased relative to the first half11 may have a role in controlling the monsoon precipitation. The development of the ATAL is associated with convective transport of aerosols from the lower atmosphere to the UTLS14. Observations from satellites , balloon-borne experiments (BATAL)15, (StratoClim)16, and CARIBIC aircraft  reveal that the ATAL extends over the 12–18 km range and is possibly composed by sulfate aerosols along with black-carbon, organic aerosols, nitrates and dust particles18. The unprecedented growth in Asian aerosol emissions during the past few decades, mainly from China and India, is therefore expected to exacerbate this phenomenon19. Indian emissions have an important contribution to the ATAL due to the vicinity of the centre of Asian monsoon convective updraft20. This region shows a higher growth in sulfate aerosol compared to China21 leading to a pronounced contribution to the ATAL composition. These sulfate aerosols significantly alter the radiative balance of the atmosphere by reflecting incoming solar radiation22 back to space. CALIPSO and SAGE-II observations show a positive trend (+0.004 over the period 1996–2014) in UTLS aerosol optical depth during summer which causes a regional radiative forcing of around −0.1 W•m−2 over that 18 year period12. A previous study23 has indicated the presence of large amounts of fine dust particles and carbonaceous aerosols in the upper troposphere in early monsoon season.In addition to this conventional understanding, the presence of the recently discovered aerosol layer in the upper troposphere and lower stratosphere (UTLS) known as “the Asian Tropopause Aerosol Layer (ATAL)” covering South Asia  during the monsoon season25, often associated with El Niño events, which promote forest fires over many parts of the globe26. For example, widespread forest and peatland fires over maritime southeast Asia in September 2015 released large amounts of aerosols and carbon emissions 28. The smoke lifted by the monsoon circulation and trapped in the anti-cyclone18 also alters the regional chemical budget and the associated radiative forcing17. This usually large contribution from natural sources can mask some of the contribution from anthropogenic aerosol.Fire-generated aerosol is also commonly observed over Asia during summer monthsHere, using satellite observations and model simulations, we explore for the first time the contribution of anomalously high aerosol loadings over the UTLS region during El Niño in worsening drought conditions over the Indian subcontinent.12 of CALIPSO scattering ratio at 532 nm (SR@532 nm) averaged over the Asian region  at 15, 16 and 17 km also peak during the summer of 2009, another El Niño year. However, the 2009 northern hemisphere summer UTLS was also influenced by volcanic aerosols from the Sarychev eruption  in June 200929. While it is difficult to separate volcanic aerosols from ATAL using satellite observations, the use of models allows us to understand the distinct role of ATAL during El Niño years by removing the influence of volcanoes (i.e. the contribution of this eruption on UTLS aerosols is not included in the model). Overall, our model simulations capture the general behavior of the ATAL and the influence of El Niño on its loading and geographic distribution, but have the tendency to overestimate its thickness.Observations from the CALIPSO satellite of scattering ratio at 532 nm, a proxy for mixing ratio in the atmosphere, reveal the presence of the elevated layer of aerosol over South Asia during July-August of 2015 and 2016 Fig. . During 19. Micro pulse lidar at Visakhapatnam and boundary layer lidar observations at Gadanki and Tirupati in India also show elevated aerosol in the lower troposphere (2–5 km) during the monsoon season due to transport from West Asia32. The monsoon convection can lift these Asian boundary layer aerosols and trace gases to the UTLS18. Due to the reduced rain and associated scavenging, the aerosol loading over the Indo-Gangetic Plain and North India is anomalously high during El Niño33  and local carbonaceous componentsposphere –5 km dur−1 to 5E-4 km−1 near 100 hPa) strengthens the layer over the entire North-Indian belt, which is mainly governed by the vertical transport of aerosols from East Asia (~110–120°E). Model simulations for the El Niño years 2009 and 2015 also show higher aerosol loading (3E-4 km−1 to 5E-4 km−1) in the ATAL . The year 2014 was likewise an El Niño year with considerable precipitation deficiency in June and July associated with the warm Pacific SST anomaly, which eventually turns out to be less-deficit/moderate-excess in the following months (August/September) accompanied with the decrease warming in Pacific SST37. Due to the changes in the circulation pattern, especially in the second half of the season, the aerosol uplifting from the east Asian region becomes relatively weak  from North India and East China accumulates during El Niño, while sulfate aerosols (mostly scattering) are transported from East-Asia . While this effect is caused by the aerosol enhancement during El Niño in the free troposphere and UTLS, there is also a considerable contribution from UTLS aerosol changes alone. Using dedicated model experiments, we quantify the effect on solar radiation at the surface due to aerosol changes during El Niño (aeronEL-aeronCL) in both these regions  over the Arabian Sea, north India, and the northern part of East Asia and decreases (−0.25 W•m−2) over the Indian peninsula, Bay of Bengal and the southern part of East Asia. The inhibition of surface solar radiation by the aerosol layer during El Niño has a cooling effect at the surface. The model simulations show an associated surface cooling of 1.5 K to 2 K at most locations except the desert regions of west-Asia and Mongolia , we find that the change in UTLS gases (for 52 gas phase species40) also reduces surface solar radiation (−5 to −15 W•m−2) over North India, comparable to the effect of aerosols (−10 to −20 W•m−2)  leads to mean regional  surface solar radiation changes of −0.6 to −1.1 W•m−2. This suggests that the changes in gases in the UTLS will contribute alongside the aerosol-induced radiative effects at the surface  Fig. . Using oace Fig. . Also, tace Fig. , which p−1. The inclusion of aerosols (aeronEL-aeronCL) amplifies decrease in rainfall by 17% over the central India . We estimate (aeronAMIP-aeroffAMIP experiments) that the aerosol loading in 2009 and 2015 has induced a rainfall deficit of 1–7 mm•day−1 (i.e. ~14%) over the central India .This combined effect produces an aggravated subsidence over the Indian region compared to the individual effect of El Niño. The effect of aerosols weakens the zonal winds and enhances the anomalous subsidence over the monsoon region from the meridional overturning circulation over 15–30°N Figs , S5a,b. iño Fig.  is furthiño Fig. . This weThe El Niño induced atmospheric circulation lifts the boundary layer aerosol from East Asia to the UTLS and then to West Asia into the ATAL region.The added blanket of aerosol reduces the solar insolation, cooling the upper troposphere over the Tibetan Plateau and surface over North India.The resulting enhanced stability leads to a weakening of the monsoon Hadley circulation.The anomalous large-scale subsidence results in amplifying the severity of monsoon droughts.There are additional radiative impacts from changes in UTLS gases also contributing to the increase in monsoon droughts severity.We attribute the increase of drought severity to the following mechanism:46. In a global warming environment, the tropical Pacific is predicted to witness more frequent extreme El Niño events49. Even if we are to follow the more moderate RCP4.5 IPCC scenario50, aerosol loadings over South Asia are still expected to remain large at least until the end of the 2040 s. Thus, the more extreme El Niño events, in a background of rising aerosol emission will have an adverse impact on the severity of droughts over the monsoon region. Another important aspect is that the distinct role of aerosols during the pre-monsoon and the monsoon season in the presence of El Niño. While pre-monsoon aerosol loading over North India and the TP reduces the severity of drought33, aerosol increases during the monsoon enhance it.The ECHAM5-HAMMOZ is utilised to understand the role of the elevated aerosol layer in exacerbating the severity of monsoon droughts during El Niño. The observed changes can be attributed to the changes in aerosol fields. The increase in industrial emissions from India and China adds millions of metric tons of aerosol into the atmosphere51 and already exposed to the vulnerability of hydrological extremes52. The increase in the severity of droughts accompanied with El Niño has a profound societal impact that can dry the whole of Central India, affecting agriculture and the livelihood of millions of people. It is well known that reducing aerosol emissions is essential from an air quality perspective, but here we highlight its additional effects on the regional monsoon hydro-climate. These two benefits of decreased pollution levels would promise a better future and would help to avoid negative consequences for millions of lives.While highlighting the complex relationship between droughts, El Niño and aerosols, the present study addresses the potential consequences of simultaneous occurrence of multiple adverse climate drivers (El Niño and aerosols) which may amplify the severity of meteorological phenomena in the present day climate. The plains of North–Central India are under hydrological stress53, a tropospheric chemistry module, MOZ54, and an aerosol module, namely the Hamburg Aerosol Model (HAM)55. The model parameterization and other details are previously documented56. The model set-up consists of nitrates in gaseous form and not in the particulate aerosol speciation. A cirrus cloud scheme has been previously described58.We perform sensitivity experiments using state of the art ECHAM5-HAMMOZ aerosol-chemistry-climate model. The model comprises an atmospheric general circulation module, ECHAM5http://eccad.sedoo.fr/)59. The sulfate, black carbon and organic carbon emission are from AEROCOM-II emission inventory for the year 200060.The model simulations were performed at spectral resolution T42 corresponding to 2.8° × 2.8° in horizontal and 31 vertical levels from surface to 10 hPa with a time step of 20 minutes. To avoid the effect of pre-monsoon aerosols, we started the model integration from 21 May to 31 May to generate ten ensemble members. The model was spun up for one month, and analysis is presented for the monsoon season (July–September). The simulations were performed for the year 2003 since it was a normal monsoon rainfall year and there was no Indian Ocean Dipole (IOD) or El Niño. The anthropogenic and biomass burning emissions are from the year 2000 RETRO project dataset (available at 33). In aeronAMIP and aeroffAMIP experiments (2005–2016) the model is forced with monthly varying SST and meteorology.In four sets of experiments, namely, aeronCL, aeroffCL, aeronEL and aeroffEL the model is forced with a monthly varying climatological SST as a lower boundary condition. The monthly climatological SST is derived from HadISST  for the period 1979–2010. For the aeronEL, and aeroffEL, El Niño experiments, the model is forced with SSTs typical of an El Niño (Table https://disc.gsfc.nasa.gov/datasets/TOMSEPL3_V008/summary), (2) Aerosol Optical Depth (AOD) from Multi-Angle Imaging Spectroradiometer (MISR)61 for July-September during the period 2000–2013 (https://misr.jpl.nasa.gov/getData/accessData/), (3) CALIOP Scattering Ratio (SR) for July - August during June 2006–December 2015 . The TOMS detects aerosols over bright targets (e.g. deserts), while MISR provides aerosol information over the oceans and land63. TOMS aerosol index data during 1979–2005 comprises measurements from two different instruments Nimbus-7 (Nov 1978–May 1993) and Earth Probe (July 1996–2005). The TOMS AI data after 2001 is not recommended for trend analysis due to calibration issues associated with sensor degradation (http://daac.gsfc.nasa.gov/guides/GSFC/guide/tomsl3 dataset.gd.shtml)64. However, this data is considered in the current study since the focus is to study the seasonal variation.Aerosol distribution during the monsoon season is studied from three different satellites (1) Aerosol Index from Total Ozone Mapping Spectrometer (TOMS) for July-September during the period 1979–2005  II data between the late 90’s to 200512. The stratospheric aerosol product dataset is now publically34 available at https://eosweb.larc.nasa.gov/project/calipso/stratospheric_table.The CALIOP lidar makes range-resolved measurements of elastic backscatter at 532 and 1064 nm and of linear depolarization ratios at 532 nm67 the rainfall data from GPCP  for the period 1979–2015 (https://www.esrl.noaa.gov/psd/data/gridded/data.gpcp.html) and TRMM_3B42 (version 7) rainfall estimate for the period 1997–2016 (https://pmm.nasa.gov/data-access/downloads/trmm). In this study, we have considered only strong El Niño years 1957, 1965, 1967, 1972, 1977, 1982, 1991, 1994, 1997, 2002, 2004, 2009 and 2015 when the amplitude of the SST anomalies in either eastern or central tropical Pacific exceed one standard deviation. Also, the Niño-3 index must be more than the threshold value (>0.5) from June to September. In our analysis, we considered monsoon season of the co-occurring El Niño year since lead-lag correlations between all Indian summer monsoon rainfall and Niño-3 index exhibits a maximum negative correlation for zero lag year (details are documented previously)68We used zonal and meridional wind data from National Center for Environmental Prediction (NCEP) reanalysis, available for the period 1948–200942 with six shortwave and nine longwave bands, and a delta-Eddington two-stream scattering solver at all wavelengths. Aerosol scattering and absorption coefficients, together with asymmetry parameters, were calculated for each aerosol size mode and spectral band69. The indirect aerosol effect was not considered here. We also compared the relative radiative forcing in the lower-mid troposphere and in the UTLS by performing additional simulations where changes in aerosols, methane, ozone and water vapour were only applied at 1000–300 hPa and at 300–70 hPa, respectively.The aerosol direct radiative forcing (DRF) in the aeronCL and the aeronEL simulations and the radiative forcing of methane, ozone and water vapour were calculated using the offline version of the SOCRATES radiative transfer model−1, see Fig. In spite of the fact that the spatial variations of the scattering ratio reproduced reasonably well Figs  and 4, t70. It is known that there is a reduced rainout during El Niño71. The tropospheric aerosol column also contributes to an increasing scarcity of rainfall over India. It should be noted that aerosols in the troposphere have an important influence on precipitation by modifying cloud microphysics over shorter timescales. previous study10 has shown that, over the timescale of a week, the rainfall over central India  is positively correlated with the concentration of natural aerosols such as desert dust and sea salt over the Arabian Sea. All these further indicate the importance and complexity of aerosols in modulating the Indian monsoon.The distribution of black carbon and dust aerosols in the troposphere (860 hPa) and mid-troposphere (500 hPa) from aeronCL and aeronEL simulations is illustrated in Fig. Elevated aerosol layer over South Asia worsens the Indian droughts


Traditional elastomers are mostly dielectrics; existing conductive elastomers are conductive composites with electric conductors. Herein, we introduce a series of ionic conducting elastomers (ICE) by salt in polymer strategy. The ICEs possess good stretchability, transparency and ionic conductivity. Moreover, the ICEs exhibit very high stability in air, under high temperature and voltage, with excellent adhesion properties and no corrosive effects to metal electrodes. Touch sensors are fabricated using these ICEs—impedance spectra and impedance complex plane are tested and analyzed to clarify different stimulus of the touch sensors. These ICEs provide possibilities for flexible electronics and soft machines. Conductive elastomers are often made of composite materials and realization of high transparency and high elasticity at the same time is therefore hard to achieve. Here the authors use a salt in polymer strategy to fabricate ionic conducting elastomers (ICE), which show good elasticity and transparency and simultaneously high conductivity. Function elastomers with conductive properties provide tremendous opportunities for flexible electronics and soft machines, which have pushed for a rapid growth in the related research fields these years7. They can serve as stretchable electrodes and wires, components in capacitive or resistive circuit elements, such as sensors, actuators, and cables. Existing conductive elastomers are mostly conductive composites: rigid conductors including metals, carbons, and conducting polymers are filled into or coated on elastomers to obtain conductivity and elasticity13; conductive fluids such as liquid metals16 and liquid electrolytes18 are injected into pipelines of elastomer substrates to implement functionality. Traditional conductive composite elastomers are hard to achieve transparency and elasticity at the same time, which hinders their application in optical related soft electronics. In addition, the manufacturing processes are complex and costly.Elastomers including natural or synthetic rubbers have been studied and utilized for centuries and enabled diverse modern technologies, benefiting from their soft and deformable properties and diverse functionality24. For example, Sun group have demonstrated a highly stretchable and transparent touch panel by using hydrogel electrolytes. The panel can be operated under large areal strain with 98% transmittance for visible light. Such performances are hardly achieved by traditional electronic conductors20.Herein, we intend to introduce a concept of conductive elastomers, ionic conducting elastomers (ICE), achieving functionalities which are extremely difficult or even impossible to realize by traditional conductive composite elastomers. Distinguished from electronic conductors, ionic conductors or so called electrolytes use ions as current carriers. By using ionic conductors, a series of devices have been reportedICE shows good stretchability, transparency, and conductivity at the same time. ICE is synthesized by salt in polymer strategy, achieving conductivity by ionic transportation through polymer chains. On the other hand, since the material is solvent free, it is quite stable in air, shows neither weight loss nor the decay of its stretchability, transparency, and conductivity. Additionally, ICE possesses great high-temperature stability, with a decomposition temperature up to 300 °C, which makes it possible to use ICE at high temperature. Moreover, ICE is not corrosive to ordinary metal electrodes, achieving long-term stability of ICE-metal joints, because it does not contain water and absorb moisture form air (the polymer matrix is hydrophobic). And, its decomposition voltage is several times higher than hydrogels. Therefore, higher voltages can be applied to the ICEs. With such advantages, ICE may be an ideal material for engineering ionic devices. We have demonstrated its application for a touch sensor.26, LiTFSI easily dissolves in varies of solvents and polymers and form electrolytes with high conductivity and transparency. After the success of LiTFSI in the salt in polymer system, several other salts, such as LiClO4 (lithium perchlorate) and LiCl (lithium chloride) were tested, but failed to obtain transparent and conducting elastomers using these salts, due to separation during photocuring process.ICE was fabricated by instant photocuring process. Lithium bis(trifluoromethane sulfonimide) (LiTFSI) was employed as the electrolyte salt, and butyl acrylate (BA), polyethyleneglycol diacrylate (PEGDA), 1-hydroxycyclohexyl phenyl ketone (photo-initiator 184) were used as monomer, crosslinker, and photo-initiator, respectively Fig. . The fac3. The tensile tests were performed on an electronic tensile machine with a 50-N load cell, and the stretching rate was set at 100 mm min−1. As shown in Fig. |Z|) sharply decreased and span several orders of magnitude at high frequency range (10 KHz–10 MHz), while at low frequency range (100 mHz–10 KHz), |Z| remained in the ballpark of the starting point. On the other hand, the negative phase angle (-ϕ) nearly maintained as constant at low frequency range; when the frequency increased from 1 KHz to 10 MHz, −ϕ significantly changed from ~0° to ~80°. The capacitance (Cp’) versus frequency was plotted in Supplementary Fig. Cp’ increased sharply and span several orders of magnitude, from 10−11 F (10 KHz) to 10−6 F (100 mHz). While at high frequency range, Cp’ maintained constant at 10−11 F. Obviously, the testing sandwich contained capacitance and resistance characteristics at the same time. These phenomena were quite different from electric conductors. We measured impedance spectroscopy of a carbon sponge , so did the ϕ (~0°). Thus, when employing ionic conductors, the applying frequency needs to be well considered. We read the bulk resistance (114 KΩ) of ICE from the Nyquist plot of impedance spectrum , calculated by the equation σ = L/SR, where L corresponded to the thickness of ICE, S corresponded to the effective overlap area, and R corresponded to the bulk resistance. ICE exhibited higher conductivity as the temperature increased, as shown in Fig. −5 S cm−1 at 200 °C. This can be explained as follows: higher temperature contributed to more intense movement of polymer chains and ions, resulting in easier ion transport and higher conductivity.Photographs in Fig. 2 and 320 °C in air), which made the operating temperature can even exceed 100 °C. We compared the stability of ICE and hydrogel electrolyte at different constant temperatures  in open air. The hydrogel for comparison experiments was polyacrylamide (PAAm) hydrogel containing 2 M lithium chloride (LiCl) salts. The hydrogel was synthesized via the method in Ref. Because ICE was solvent free, it possessed many special characteristics, which were different from traditional gel electrolytes. Figure −2. And, decomposition voltage of ICE was several times higher than that of hydrogel electrolytes, as shown in Fig. −1 at the range from 0 to 10 V. Detected current of hydrogel electrolyte increased sharply as the applying voltage exceeded ~1 V, accompanied by bubbles generating at the interfaces of electrodes and electrolyte, indicating the decomposition of water by electrolysis. ICE was quite stable through the whole voltage range, indicating higher voltages can be applied on ICE electrode.Practically, ionic conductors have to be connected with metals to implement functions, regardless of receiving electrical stimuli or output electric signals. The stability of electrolyte–metal junctions has to be concerned. Photographs in Fig. −6–10−5 S cm−1. The comonomers were frequently employed for solid-state polymer electrolytes and they enhanced the conductivity. As an example, by introducing PMPEG550A into the polymer substrate helped increasing ionic conductivity. The homopolymer of PMPEG550A with 0.5 M LiTFSI have a ionic conductivity of 6.3 × 10−5 S cm−1. We showed that as the volume percentage of MPEG550A increases, the conductivity of the copolymer increases . We illustrated impedance spectra detecting signals from different types of stimuli. Figure |Z| of the sensor increased and the −ϕ (negative phase angle) changed slightly  to negative value (~−40°) in the same frequency range. Figure ϕ became positive values. Since human can be regarded as an ionic conductor, when finger touched the sensor, human became part of the circuit. Finger conducts currents and introduced new elements into the circuit, resulting in the change of the circuit characteristics. We set a single frequency (f = 20 KHz) to detect signals from different stimulus, as shown in Fig. Touch sensors were fabricated using copolymer ICE  to detect touch signals, as shown in Fig. Interestingly, insulating film covered sensor can still sense finger touch . We controlled the thickness of the ICE by adjusting the thickness of the silicone spacer. For copolymer ICEs, the volume ratio of comonomers and BA were set at 1:1, molar percentage of photo-initiator 184 were set at 1%, no use of crosslinkers.N,N-methylenebisacrylamide (MBAA) 0.06 wt.% and the initiator Ammonium persulfate (APS) 0.16 wt.%, with respect to the AAm monomer, were added. After sonicating and degassing the mixture in a vacuum chamber, N,N,N’,N’-tetramethylethylenediamine (TEMED) 0.25 wt.%, with respect to the weight of the AAm monomer, was added as the accelerator. The solutions were poured into glass molds. The gel was prepared after 1 h.The molar concentrations of Acrylamide (AAm) and LiCl aqueous solutions were 2.17 and 2 M. The cross-linker 3) for tests. The stretching rate was set at 100 mm min−1.The mechanical tests were performed on an electronic tensile machine  with a 50-N load cell. The samples were cut into dumbbell shape . The samples for tests were 1-mm thickness. The wavelength for testing were set from 800 to 400 nm. The reference for measuring transparency was air.Vrms  was set at 1 V. The samples were treated without metal spraying on surfaces. For testing touch sensors, the impedance tests were performed on an electrochemical workstation (CHI660E), testing Vrms was set at 0.5 V.The impedance tests were performed on a broadband dielectric/impedance spectrometer (Novocontrol GmbH). The samples were 1-mm thickness, the testing copper electrodes were diameter of 30 mm, testing −1) under flowing N2 and air by using alumina crucible.The TGA measurements were performed on a TGA Q 5000 via scanning a temperature range from room temperature up to 500 °C (10 °C min−1 on a peel strength tester (GM-718).A 1 mm thick ICE sheet were cut into a shape of 1 mm × 20 mm × 80 mm. A stiff backing (PET film) was introduced to prevent elongation of ICE while peeling. Then ICEs were bonded onto substrates. The 90°-peeling tests were with a constant peeling speed of 50 mm min−1 at the range from 0 to 10 V.Decomposition voltage of ICE and hydrogel electrolyte were tested via Linear Sweep Voltammetry (LSV) on an electrochemical workstation (CHI660E), the samples were sandwiched by two steel round electrodes for tests, the scan rate was set at 0.5 mV s3. Two aluminum electrodes were then attached at both ends of the ICE for testing. Finger touched on the middle of the sensor without pressing for a touched measuring. The sensor was stretched to twice of the original length for a stretched measuring. The insulating film for covered touch sensor was silicone membrane.The ICE (co-MPEG550A) was cut to a shape of 80.0 × 15.0 × 1.0 mmThe data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


To design and evaluate the accuracy of a novel navigation template suitable for posterior cervical screw placement surgery by using 3D printing technology to improve the existing guiding template design.The researchers (including spine surgeons and technicians) used CT to perform thin-slice scanning on 12 cases of normal upper cervical vertebral specimens and defined the screw channels that were completely located in the pedicle without penetrating the cortex as ideal screw channels, then designed the ideal channel of the upper cervical vertebral  pedicle screw by computer software which was regarded as the preset values, and recorded the screw entrance point, transverse angle and sagittal angle of the ideal channel. Then, researchers designed the novel navigation templates for placement pedicle screw according to the ideal screw channel preset values and manufactured them with one for every single vertebra by 3D printer. A senior spine surgeon performed the posterior surgery to implant pedicle screw on the specimens by the novel navigation templates, then performed CT thin-slice scanning on the specimens again after removing the screws, and reconstructed the actual screws channel by computer software, recorded the screw entrance point, transverse angle and sagittal angle of the actual channels which were defined as the actual values and evaluated them according to Kawaguchi’s pedicle screw evaluation standard finally. The differences between the preoperative preset values of ideal screw channel and the postoperative actual values of actual screw channel were compared by a nonparametric paired rank test.48 screws were placed on 12 cases of upper cervical vertebral specimens in total. It showed that the grade 0, I, II, III channels in this study were 47, 1, 0, 0, respectively. The grade 0 channels accounted for 97.92% of the total number of channels. There was no significant difference with regard to the screw entrance point, the transverse angle, and the sagittal angle between the preoperative preset values of ideal screw channels and the postoperative actual values of actual screw channels.To implant pedicle screw assisted with the novel individually navigation template designed by 3D printed in the posterior cervical surgery can improve accuracy of pedicle screw placement and safety of the surgery. Posterior placement of pedicle screw fixation is an important treatment strategy for some upper cervical traumas and diseases, such as fractures, joint dislocation complicated with neurological symptoms. To place the pedicle screw accurately and successfully is the key to the success of the operation. However, the application for posterior placement of pedicle screw fixation is confined by the high risk of vascular and nerve damage due to the complex adjacent structure of the atlantoaxial spine.In recent years, with the application of additive manufacturing (3D printing) technology in the medical field, the placement of pedicle screw assisted by 3D printing navigation templates have been gradually applied to spinal surgery because of its high accuracy –3. HowevThis study was in line with the spirit of the Helsinki Declaration and approved by the Ethics Committee of the Guangdong Provincial Work Injury Rehabilitation Hospital. The human corpse anatomic preservative specimens were obtained from the Institute of Clinical Anatomy of Southern Medical University.The selected specimens should met the following inclusion criteria: (1) age ≥ 16 years old, (2) the neck was free from deformity and injury, and its bone structure was intact.; (3) imaging examination showed that Atala-Dens Interval  ≤ 5mm, or the space Available for the Cord (SAC) ≤ 13mm; (4) The structure itself had not been damaged by surgery or trauma; and (5) X-ray densitometer was normal without osteoporosis.The research enrolled 12 cases of normal atlas and axis anatomical preservation specimens that met the inclusion criteria. From June 2018 to August 2018, posterior screw placement in upper cervical was performed in the laboratory of the Institute of Rehabilitation Medicine of rehabilitation hospital. The specimens included 7 males and 5 females, with the age range from 28 to 63 years. All the instruments and pedicle screws were provided by Kangli Orthopedic Medical Devices Co., Ltd.  and the screw placement was performed by the same surgeon.Imaging examinations were performed by using the SIEMENS 64-slice spiral CT scanner . At least three segments of the 1st-3rd cervical vertebrae were scanned by CT with the parameters as follows: 1.0 mm in thickness, 0.65 mm in pitch, 1 turn/second in tube speed, 130 kV in voltage, and 104 mA/s in current. The scanned images were saved in a medical format for digital imaging and communication (DICOM).The DICOM images were imported into 3D image generation and editing processing software  to generate a 3D model of the target vertebra. In the sagittal plane, coronal plane, horizontal plane and 3D overall compositions, the cylindrical parts of 3 mm in diameter were imported . ThroughAfter the screw channels were designed, the boffins performed the “wrap” operation on the generated three-dimensional body  , closed The researchers used 3D printed navigation template to assist the screws placement on human corpse specimens. The specimens were placed in the prone position to simulate the intraoperative situation. The scientific personnel performed the median incision on the posterior neck and separated the paraspinal muscles layer by layer to reveal the posterior bony structure of the atlantoaxial , then atThe accuracy of the atlantoaxial pedicle screw was evaluated by a author (C.L.) who did not participate in the surgery. After removing the screws of all of the specimens, CT scans were performed on them again with the parameters consisted with the last time scans. After importing the postoperative CT scan data into Mimics software, The boffins observed the pedicle screw channel position from the sagittal, coronal and horizontal planes , and theThe formed three-dimensional images were processed by "transparency" to observe the actual channel after the screws placement. The scientific research observed the actual screw entrance point and the transverse angle on the horizontal plane from the top view, as well as the sagittal angle from the left and right sagittal planes respectively, and measured them by the Mimics software’s measurement function as postoperative data .IBM SPSS Version 22.0  was used for statistical analysis. Since the data in the research were non-normally distributed, so the nonparametric paired rank test was selected. The difference between the preoperative and postoperative data of sagittal and transverse angle, and the screw entrance point were compared with a nonparametric paired rank test. A two-sided P-value greater than 0.05 was considered statistically significant.The screw placement on all of the specimens was successfully completed by using a novel individualized 3D printed navigation template. The average operative time and screw placement time was 37.67±6.83 minutes and 3.68±0.99 minutes/piece, respectively . The CT All specimens were scanned by CT after the operation. 48 pedicle screws (24 in C1 and 24 in C2) were placed in 12 specimens by using the novel individualized 3D printed screws placement navigation templates. The screw placement accuracy evaluation according to Kawaguchi’s criterion showed tIn many cases, posterior atlantoaxial screw fixation is the gold standard for the treatment of atlantoaxial fractures and dislocations. Due to the complex adjacent structures of the upper cervical spine, traditional surgery has a high risk of vascular and nerve injuries , 7. It iIn spinal surgeries, based on the individual data of atlantoaxial CT imaging, the ideal pedicle screw channel for each patient can be designed by using 3D printing technique, which was regard as "perfect" screw placement. In order to improve the accuracy of screw placement, an ideal pedicle screw placement scheme can be given by using the printed screw navigation templates. 3D printing navigation template has the following advantages: (a) individualized pedicle screw channel design has high accuracy of screw placement, (b) no prior experience is required to assist the operator in performing the "fool" operation, and (c) all the guiding plates are designed independently for every single vertebra, so it will not happen that other or entire pedicle screw channels have error positioning due to the guiding template introperative position change. By using 3D navigation templates, Lu et al  implanteThere are some influential factors in 3D printing screw navigation templates, such as the quality and thickness of the slice of the CT scan, and the process of extracting and modifying the vertebra model on the computer may affect the accuracy of the guiding template or lead to defaults in making it. During the operation the screw channel deviation is prone to occur, if the soft tissue like muscles and ligaments of the posterior structures of the vertebra  are not fully dissected, the guiding template fails to stick closely to the posterior bony surface of the lamina; or the navigation template glides, or the holding is unstable, all theses will lead to the deviation of screw placement. The 3D guiding template is mainly made of photosensitive resin which is brittle and easy to crack, and it will be deformed and invalid when disinfected at high temperature .At present there are some design flaws in the conventional 3D printing navigation templates . First oIn view of the above deficiencies, we have redesigned a new type of navigation template which has not been reported in literature so far, and improved these defects. The novel navigation template canceled the original design of the screw guiding channel, but only retained the position of the screw hole; and replaced the bilateral screw guiding channels with an inward shifting navigation pole. During the operation, the screws were implanted in the screw hole on the navigation template. The navigation pole/ handrail was held to keep the navigation template stable which ensured the direction of drilling and screw implanting parallel to the navigation pole by observing and monitoring from different angles.In the current study, we found that the novel patient-specific 3D printing navigation templates with long inward shifting guiding pole had advantages of more convenient, safer and more accurate when assisting screw placement. (a) After canceling the screw channel in the original design, and only remaining the screw hole, this design kept some flexibility with partial restriction. While indicating clearly the screw implanting point, the chances of adjusting the screw channel subjectively and manually were maintained when the channel was poor. (b) The longer navigation pole would have a larger swing amplitude when the guiding template was displaced or oscillated. By observing the change of the navigation pole, the position change of the navigation template could be more sensitively detected. In this way, it was beneficial for preventing displacement, reducing the screw implanting fault and improving the accuracy of the operation; (c) The inward shifting guiding pole was completely parallel to the screw channel of the original navigation template. The guiding pole was used as a reference for the screw implanting direction. By intraoperative multi-angle observation and monitoring, the direction of screw placement was kept to parallel with the screw pole and the correct screw channel was achieved; (d) A longer navigation pole that acted as a handrail at the same time to be held and secure the navigation template. When the direction of the drill bit was not parallel with the guiding pole, there would be a situation where the drill bit collided with the bottom of the guiding template and generate vibration. At this time, the handrail helped better maintain the stability of the navigation template and reduced its swing and displacement; (e) The inward shifting design of the double-side guiding pole could effectively reduce the volume of the navigation template, thereby minimizing the need for surgical space and soft tissue dissection, reducing bleeding and improving the safety of the operation. Therefore, the screw placement faults could be basically avoided during the operation, as long as attention is paid to exposing the posterior part of the vertebral body, dissecting the soft tissue completely, keeping the guiding template closely adhered to the bone surface, and maintaining the stability of the guiding template.According to relative references, with the help of 3D printing guiding templates, the operation time, blood loss, screw placement time, and intraoperative fluoroscopy times were significantly less compared with the traditional manual screw placement operation. And the accuracy and safety were obviously improved. In our current study, the ideal screw channel data of the preoperative design, had no statistically differences in the transverse angle, the sagittal angle and the screw insertion point, compare with the postoperative CT scan data of the actual screw channel. In addition, postoperative CT scans also showed that up to 97.91% screw channels of all reached the Kawaguchi criterion of grade 0. It could be seen that the use of the new 3D printing navigation templates to assist the screw placement had a high accuracy; at the same time, it suggested that this method is an effective, safe and effective auxiliary implanting screw method, which would result in an actual screw channel that is highly compatible with the theoretically preset ideal screw channel. These are consistent with the results of previous reports ,13, 14.The following precautions should be noted when applying 3D print navigation templates for surgery: (a) Pay attention to completely remove the soft tissue attached to the posterior structure of the atlantoaxial , and hold the navigation pole/handrail stably to ensure the contact surface of the guiding template is close to the bony surface; (b) Avoid the deviation of screw placement caused by the displacement of guiding template and navigation pole. Use the probe to confirm the bone integrity of the bottom and four walls of the screw channel during the operation. Take routine C-arm fluoroscopy to check the screw position; (c) High-temperature disinfection will lead to deformation and lost effectiveness of the navigation template, so epoxy ethane sterilization or low-temperature plasma disinfection is used for preoperative disinfection; (d) Since the guiding template is made of photosensitive resin which is fragile, the operation has to be gentle in surgery; (e) Before designing the screw hole of the navigation template, the drill bit size should be considered, which means the diameter of the screw hole need to be slightly larger than drill bit. At the same time, the navigation pole should be used as a reference, and the direction of the drill bit should be parallel with the pole in surgery. These steps can reduce the collision, cutting and friction between the drill bit and the base of the navigation template.The current study applied a new type of 3D printing navigation template to assist pedicle screw placement in atlantoaxial surgery, which was in accordance with the individualized characteristics of the pedicle. This method was found to have high accuracy and safety, which was beneficial to reduce the risk of operation and has a good clinical application prospects. However, as a preliminary experiment, the sample size was limited. A larger sample size of clinical application is needed in the future to further prove the accuracy and efficacy of this type of the navigation template.S1 Dataset(XLS)Click here for additional data file.


Chlorella pyenoidose using enzymatic hydrolysis, gel filtration chromatography, and LC–MS/MS. Its lipid inhibition effects indicated that the synthetic peptide PP1 exhibits a good inhibitory effect against porcine pancreatic lipase (PL) (47.95%) at 200 μg/mL, which could be attributed to its hydrogen binding into catalytic sites of PL  by docking analysis. Furthermore, in 3T3-L1 cells, the synthetic PP1 remarkedly decreased the accumulation of intracellular triacylglycerol , which carried a similar consequence as the positive drug simvastatin . Western blot revealed that PP1 inhibited the lipid accumulation and fatty acid synthesis in 3T3-L1 adipocytes in two pathways, primarily: nonalcoholic fatty liver disease (NAFLD) pathway  and AMPK signaling pathway . In short, these results support that PP1 can be used as a potential agent against obesity.A novel lipid inhibition peptide Leu-Leu-Val-Val-Try-Pro-Trp-Thr-Gln-Arg (PP1) (MW 1274.53 Da) was obtained from  Chlorella, a type of single-cell green algae, contains over 50% bioactive proteins and has become a major source for extracting high-yield proteins. It is reported that Chlorella has certain functional properties, such as antioxidant [Bioactive peptides are generally short peptides (2–20 amino acids) that are partially obtained by enzymatic hydrolysis from protein and are important parts in food-science studies. Many different parameters affecting the bioactivity are explored, such as the source of protein, the degree of hydrolysis, peptide structure, amino acid composition, molecular weight (MW), and type of protease . Bioactiioxidant , anti-inioxidant , antihypioxidant , antitumObesity is essentially an excessive energy intake that is stored in the form of triacylglycerols in fatty tissue. Excessive accumulation of triacylglycerols in fatty tissue causes a disorder of lipid metabolism. Presently, obesity is a prevalent disease and has become a major problem in today’s society. Lipases, a subclass of the esterases, catalyze the hydrolysis of fats and oils. For humans, pancreatic lipase is a key lipase for lipid absorption throughout the body and is responsible for the hydrolysis of total dietary fats . PancreaPrevious studies have reported that preventing differentiation of preadipocytes to adipocytes and inhibiting lipid accumulation contribute to anti-obesity . The proC. pyenoidose. Furthermore, its effect on the inhibition of the pancreatic lipase (PL) activity and suppression of fat accumulation in 3T3-L1 cells were investigated.Many recent studies have focused on developing anti-obesity agents from natural substances. However, few reports are available on the lipid inhibition effects of peptides from seaweeds. This research focused on purification and identification of bioactive peptide fractions from enzymatic hydrolysis of C. pyenoidose by using a series of steps [C. pyenoidose, the extracted proteins were enzymatically hydrolyzed with four proteases: pepsin, papain, trypsin, and Alcalase. For the extracted proteins, the percentages of molecular weight (MW) were 40.37% (<3000 Da), 11.02% (<1000 Da), and 29.29% (1000~3000 Da)  , which w50 87.79 ± 5.12 μg/mL, followed by the pepsin hydrolysate, which had an inhibition on PL activity that was IC50 89.65 ± 7.06 μg/mL. In Copper soap assay, the order of PL inhibitory capacities is Alcalase hydrolysate > pepsin hydrolysate > trypsin hydrolysate > papain hydrolysate > protein. To clarify the inhibitory effects on PL, various concentrations of proteins and hydrolysates against PL activities were examined in vitro. In ® G-25, and four fractions (A1–A4) were obtained . The PL inhibitory capacities of different fractions were determined . Resultsobtained b. Data iC. pyenoidose, respectively. Based on top three peptides  with high bioactivity prediction score  by Peptide Ranker (http://bioware.ucd.ie), a short peptide LLVVYPWTQR (PP1) was selected for further study, which can also properly bind to PL surface using Pep-site (http://pepsite2.russelllab.org/). According to MS profile  were performed to analyze the hydrogen bond, electrostatic interactions, and hydrophobic interactions found between the ligands and the active site of PL. The conformations revealed the types of intermolecular interactions between the ligand and the PL pocket, as shown in p < 0.05) at 600 μg/mL  was provided by Professor Zhang Daojing, East China University of Technology, Shanghai, China. BCA Protein Assay Kit and Triglyceride Assay Kit were purchased from Nanjing Jiancheng Bioengineering Institute . Pepsin , papain , and trypsin (1:250 U/g) were obtained from Guangzhou Qiyun Biotech Co., Ltd., Guangzhou, China. Alcalase 2.4 L (P4860), 3--2,5-diphenyltetrazolium bromide (MTT), pancreatic lipase, Orlistat, and simvastatin were obtained from Sigma, San Jose, CA, USA. Primary and secondary antibodies were purchased from Cell Signaling Technology . Gel filtration chromatography Sephadex G-25 was from Whatman Liluo Science & Technology Instrument Co., Guangzhou, China. Other reagents were of analytical grade and were commercially available.C. pyrenoidosa according to how they were extracted in previous studies [The proteins were extracted from  studies . Two graMolecular weight distributions of extracted protein and enzymatic hydrolysates were determined by HPLC. The gel column was TSK gel G2000 SWXL, and the eluate was monitored under λ = 220 nm. The flow phase was composed of phosphate buffer (pH = 7.0) and the velocity of flow was 0.5 mL/min. The enzymatic hydrolysates supernatants were subjected to ultrafiltration through molecular weight cut-off (MWCO) membrane of 5 kDa . Each fraction (<5 and >5 kDa) was collected, and its PL inhibitory activity was measured. The fractions were lyophilized and stored at −20 °C. 2). The column was eluted with distilled water at a flow rate of 0.5 mL/min. The eluate was detected at 280 nm. The eluates at the same peak were collected and then freeze-dried and stored at −20 °C. The PL inhibitory activity of each fraction was measured. The highest active fraction was separated by gel filtration chromatography. Two milligrams of bioactive fractions dissolved in distilled water at a concentration of 40 mg/mL were loaded onto a Sephadex G-25 . Specifically, the sample was diluted 1:5 with eluent A (0.1% formic acid in Milli-Q water) and injected into a C18 column  with a flow rate of 0.3 mL/min and a column temperature of 30 °C. The peptides were separated using the following gradient: 6 ultraperformance liquid chromatography (UPLC) for peptide separation in a C18 column (2.1 × 120 mm) . Ten microliters of each peptide sample (0.4 mg/mL) were loaded for each elution. The elution was made in a linear gradient mode from 0% acetonitrile to 40% acetonitrile (20 min) at a flow rate of 0.3 mL/min. Data of MS/MS were recorded using mascot generic format (MGF) files, which were submitted to Mascot search engine V 2.3 , and sequences of peptides were identified based on a search in the database (Ludwig NR Database). The data acquisition parameters were as follows: electrospray ionization (ESI) source type, positive ion mode, scan region 50–1500 m/z, capillary 3500 V, charging voltage 2000 V, corona 0 nA, nebulizer 0.3 bar, dry heater 180 °C, APCI heater 0 °C, and dry gas 4.0 L/min.http://bioware.ucd.ie) and Pep-site (http://pepsite2.russelllab.org/), the biological peptide LLVVYPWTQR (PP1) was chemically synthesized by GenScript Corporation . The purity of the synthesized PP1 was verified to be higher than 95% by analytical HPLC coupled with MALDI–TOF MS.Based on the bioactivity prediction by Peptide Ranker . The peptide (PP1) was modeled by using Libdock. Orlistat and simvastatin were used as positive controls. The Discovery studio software was used for the docking of PP1, simvastatin, and Orlistat to the PL protein. The potential binding sites  were being identified using the built-in cavity detection algorithm, expanded Van der Waal with 9 Å Probe size. The docking modes were obtained with scoring results about the ligand–receptor combination, which could be used for PL inhibitory activity prediction. Finally, the high score of the poses correctly bound to the active site was evaluated. The 3-D coordinates of the protein complex with the PL (PDB code: 1ETH) were downloaded from the Protein Data Bank were seeded in a 96-well plate for 24 h. After that, the cells were incubated for 6 days in DMEM containing various concentrations (0–1.0 mg/mL) of the synthetic PP1. Cell toxicity was measured by MTT according to the instructions indicated by the manufacturer. For cell differentiation, 3T3-L1 cells were seeded in 6-well plates at a density of 2 × 105 cells/well. Then, the medium was changed by DMEM with insulin , 1 μM of dexamethasone (Sigma), and 0.5 mM of 3-isobutyl-1-methylxanthine. On day 3, the medium was replaced with DMEM containing insulin  and 10% (v/v) FBS every 3 days. Various concentrations of the synthetic PP1 were attendant during the whole process of differentiation induction. Oil Red O staining of the intracellular lipids was carried out, as described previously. Moreover, intracellular triacylglycerol levels were measured by using an Assay Triglyceride Kit. Protein concentrations were determined with BCA Protein Assay.First, 3T3-L1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium containing 10% (v/v) fatal bovine serum (FBS) and 1% antibiotics in a humidified atmosphere of 5% COCells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer for 1 h and then centrifuged at 12,000 rpm for 25 min at 4 °C. The protein concentrations were determined by using a BCA protein assay kit. The supernatants were mixed with 4 × Sodium dodecyl sulfate (SDS) sample buffer and boiled for 10 min. The samples were run on a 4–15% SDS polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% (w/v) bovine serum albumin (BSA) for 30 min and then incubated with primary antibodies at 4 °C overnight. After washing with Tris-Buffered Saline and Tween 20 (TBST) 3 times, the membranes were washed with TBST and incubated with the secondary antibodies. Signals were detected with a scanner , and the fluorescence intensity was measured using Image software. p < 0.05) was determined by a paired t-test. All statistical analyses were performed using SPSS (9.0).Data were expressed as mean ± standard deviation of the number of replicates performed in the experiment. The significance level  possesses inhibitory activity on pancreatic lipase. PP1 can inhibit fatty acid synthesis and fat accumulation in the adipocyte. This suggests the potential of PP1 as a therapeutic agent against obesity.


Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 min) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unknown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of  Fungi are cell factories with important functions for the bioeconomy since they often exhibit superior metabolic and secretory capabilities compared to bacterial and yeast-based production systems . FurtherReversible modifications in protein phosphorylation mediated by protein kinases or phosphatases represents a mechanism that is frequently employed by eukaryotic cells to transmit environmental cues and initiate signal transduction pathways, such as for transcription factor activation , cellulaNeurospora crassa  and is required for GTP-stimulatable activity (The Mitogen-Activated Protein Kinase (MAP kinase) pathway, for example, represents a prototype of a signal distribution channel with phosphorylation being the major switch. In nication , circadinication , phosphanication  as well nication . Phosphonication . This panication  and phosnication . Howevernication  is poorlnication , regulat. crassa . Low amo. crassa . It has activity . Some ofactivity .N. crassa possesses 11 HHKs, one HPT, two RRs  . This munditions . In T. rpression . Howeverpression .N. crassa, such as transcription factors, GPCRs, kinases and F-box proteins. Subsequently, in silico methods enabled us to construct putative proteome-wide protein-protein interaction (PPI) maps. Physiological analyses of several components within the cAMP signaling pathway that have clear substrate-dependent phosphorylation changes indicates that they are involved in metabolic adjustments to new carbon-conditions, including CCR.Despite the importance and widespread occurrence of phospho-modifications as laid out above, the identification of sites of protein phosphorylation is still a challenge , hamperiOverall, this dataset will serve as a benchmark for identification and prediction of phosphorylation reactions during the sensing and signaling of cellulosic carbon sources in filamentous fungi.N. crassa wild type strain (FGSC #2489) were pre-grown for 16 h on 2% sucrose medium, then washed three times with 1× Vogel’s salt solution (no carbon source added) for a total duration of 30 min before transferring to their respective carbon source: 1 mM galacturonic acid  plus 1 mM rhamnose , referred to as GalAR , 2 mM glucose , 2 mM xylose , 0.5% cellobiose  and 2 mM 1,4-ß-D-glucosyl-D-mannose plus 1,4-ß-D-mannobiose . Samples for global proteome and phosphoproteome were incubated for additional 2 min. No carbon condition (NC) was incubated for 1 h after the medium switch . The resulting peptide samples were concentrated and a bicinchoninic acid (BCA) assay  was performed to determine the peptide concentration and samples were diluted with nanopure water for MS analysis. 200 μg peptides aliquots for each sample were dried down for further IMAC enrichment, used for phosphoproteome analysis.3+-NTA agarose bead slurry. Then, the beads containing bound phospho-peptides were washed and desalted using the Stage Tips. After that, the phospho-peptides were eluted from the IMAC beads to the C18 membrane by washing the Stage Tip containing IMAC beads with 70 μl 500 mM phosphate buffer, pH 7.0 and 100 μl 1% FA before being eluted from the C18 membrane with 60 μl 50% ACN/0.1% FA. Eluted phospho-peptides were dried down and stored at −80°C until LC-MS/MS analysis.Immobilized Metal Affinity Chromatography (IMAC) enrichment of phospho-peptides follows a previously established protocol . PeptideTM Hybrid Quadrupole-OrbitrapTM Mass Spectrometer (Thermo Fisher Scientific) in data dependent mode. All mass spectrometry data were searched using MS-GF+  coupled to a Q Exactiveg MS-GF+  to identg MS-GF+  to provig MS-GF+  with theN. crassa via prediction of functional interactions between the identified proteins, using the STRING database , fbx-19 , fbx-20  and fbx-22 . The corresponding deletion strain for cr-1 (NCU08377) is FGSC#11514.The WT and deletion strains were obtained from the FGSC1 . The F-bnidulans : fbx-9   in 24-deep-well plates. VMM media containing 2% D-Xyl (Sigma Aldrich) as control and 0.2% (w/v) 2-Deoxy-D-glucose  plus 2% D-Xyl were used to test 2-DG influence, evaluated after 4 days. To test allyl alcohol sensitivity, 1% D-Glc ± 100 mM of allyl alcohol (Sigma Aldrich) were incubated for 2 days.um (VMM) . Unless D-Glc plus 1% D-Xyl VMM, or 1% D-Xyl VMM for 3 days. The xylanase activity was measured in the supernatant using Azo-xylan  as substrate. Xylanase assays were conducted by mixing 25 μl of culture supernatant with 200 μl of Azo-xylan in 50 μl 1 M sodium acetate, pH 5.0, filling up to a final reaction volume of 400 μl with acetate buffer, and incubated at 37°C for 10 min. Reactions were stopped by centrifugation at 7000 × g for 2 min and by addition of 1 ml of precipitation solution (95% v/v ethanol) to the reaction supernatants. The absorbance was measured at 590 nm.For xylanase activity assays, the strains were grown in 1% fructose VMM during 24 h at 30°C, then transferred to 2% N. crassa was first starved for 30 min and then incubated for 2 min with mono- and disaccharides that are known to act as signaling molecules for plant cell wall-related polysaccharides. This short retention period was deliberately chosen to allow N. crassa to take up inducer molecules, but ensure that the proteome does not have time to significantly change, so that all measured differences are due to post-translational changes as a reaction to nutrient availability and not due to changes in enzyme abundances. A large number of proteins and phospho-peptides were detected by LC-MS/MS analysis in samples from these short-exposure experiments for each induction condition  to analyze the protein distribution across the network. The entire high-resolution network shows that proteins that belong to the same functional class have a tendency to cluster together (A protein-protein interaction (PPI) network was constructed based on identified proteins in the proteomic and phospho-proteomic experiments, and together . NeverthTo visualize the influence of substrates on this PPI network, the proteins that were identified to be phosphorylated under each condition were highlighted within the network . InteresNeurospora to be 182  plus many more for which the localization is less certain. Of these, several are clustered in regions neighboring the GATA-type DNA binding domain (aa positions 742–792) including several that are phosphorylated in different residues. Determining which of the sites (many of which appear conserved in NIT2/AreaA homologs) could be playing regulatory roles, is an interesting question lying ahead.An earlier study had estimated the number of TFs in o be 182 , a numbeo be 182 . Thus, wo be 182 , with nuAnother TF exhibiting the second highest number of modifications is NCU10346 (ADA-13). This protein, with a predicted MYB DNA binding domain (PF00249), showed a total number of 43 phospho-peptides, comprising at least 23 phosphorylation sites.D-GalA + L-Rha and Glucomannodextrins. T366, located less than 20 aa downstream from the VIB-1 DNA-binding domain, appears modified only in D-Xyl.Twenty-seven other TFs presented many identified phospho-peptides each, among which were NUC-1 (NCU09315), ASL-1 (NCU01345) and VIB-1 (NCU03725), all transcription factors important in mounting responses to changes in environmental conditions. Thus, for example VIB-1 has been associated with glucose sensing and carbon catabolite repression , playingNeurospora under different culture conditions remain to be determined.CRE-1 (NCU08807), the yeast Mig1 ortholog that plays a key role in carbon catabolite repression, showed six clearly identifiable phosphorylated residues (with Ascore > 13) in four different regions of the protein (represented by five different peptides) including the extreme N-  and C-terminus. While four phospho-sites  appeared modified in all conditions, some modifications were clearly condition-specific, such as S362  and S209 (exclusively de-phosphorylated in cellobiose). Which phosphorylations may be modifying CRE-1 activity in D-GalA + L-Rha, all of the phospho-sites can be observed, while in no carbon condition only S676 was found to be modified.COL-26/BglR (NCU07788) is a TF that has also been involved in glucose sensing/metabolism, regulation of starch degradation as well as the integration of carbon and nitrogen metabolism , 2017. S2-Cys6 binuclear cluster domain and within the predicted recognition sequence of a cAMP-dependent protein kinase (PKA) phosphorylation site, occurred specifically in the presence of Glucomannodextrins and D-Xyl. This latter phospho-site had already been detected in a previous study  to be mied here . Of CLR-or CLR-2 .Although not a transcription factor, CLR-3 (NCU05846) is still highly relevant for cellulase regulation  and was N. crassa  had 12 regions of phosphorylation and was modified under all conditions. There were two different regions of phosphorylation on protein kinase domain  and one in the AGC kinase, C-terminal domain (S1238) of STK-12.HPT-1 (NCU01489) was not phosphorylated under any conditions in this study. RRG-1 (NCU01895) was phosphorylated under all conditions, with three sites in total. RRG-2 (NCU02413) also had two phosphorylation sites and was not phosphorylated during growth on D-Glc and Glucomannodextrins, with an additional ambiguous position for cellobiose. Phosphorylation of S1223, S153, and S77 was independent of the applied conditions. Response to cellobiose and no carbon resulted in phosphorylation of T1210, while D-Xyl as well as the D-GalA + L-Rha caused phosphorylation of S123. Interestingly, Glucomannodextrins and D-Xyl elicited phosphorylation of an SSTT-motif (S1343-T1346) within a potential PEST domain , suggesting a role for protein degradation in Glucomannodextrins and D-Xyl dependent carbon signaling. Although the AScore for these phosphorylation sites was below 13  phosphorylation of the respective peptides still indicates a relevance of the PEST domain modification.The MAPKKK SskB/OS-4 (NCU03071), representing the first step in the osmolarity pathway, was found to be phosphorylated at S409  in response to D-Glc and D-GalA + L-Rha. Phosphorylation of the serines at positions 77 and other sites adjacent to this was found to be condition-dependent for all conditions except no carbon indicating that this area may be a hot spot for post-translational regulation of carbon response in this kinase. HOG1/OS-2 (NCU07024), the MAP kinase of the osmolarity pathway, showed only two phosphorylated sites, one being a tyrosine. For Y173, phosphorylation was found under all conditions tested. However, phosphorylation of the adjacent T171 obviously required recognition of a carbon source, as no phosphorylated peptide was detected in the absence of carbon.In the MAPKK PBS2/OS-5 (NCU00587), condition-independent phosphorylation occurs at serines 219 and 346, while phosphorylation of S482 was only detected in the absence of carbon. The predicted PKC phosphorylation site at S63 is phosphorylated upon recognition of Glucomannodextrins, D-GalA + L-Rha in combination with other carbon sources for NRC-1. In case of the MAPKK MEK-2 (NCU04612), we found phospho-peptides supporting the presence of both protein variants, with phosphorylation of T490 of variant 2 being present under all conditions tested. Despite detection of several phosphorylated sites, only few carbon-specific ones were found: while D-Xyl specifically elicited phosphorylation of S212, which is close to the active site, the serine at position 351 became phosphorylated upon sensing of D-Xyl, D-Glc and D-GalA + L-Rha. The MAP kinase of the pheromone response pathway, MAK-2 (NCU02393), only showed two phosphorylation sites, of which the tyrosine at position 182 was phosphorylated under all conditions tested. However, the adjacent T180 was phosphorylated only in the presence of D-Xyl and D-Glc.For NRC-1 (NCU06182), the MAPKKK of the pheromone response pathway, several phospho-peptides were detected. Six phosphorylated amino acids reside within predicted casein kinase phosphorylation sites and one in a PKC phosphorylation site. The serines at positions 49 and 558 were phosphorylated under all conditions tested. The predicted PKC phosphorylation site at S189 appears to be starvation-specific (no carbon) and T872 phosphorylation was only detected on cellobiose. In contrast to the osmolarity pathway, specific phosphorylations were also detected for D-Glc  and D-Xyl  specific sites were obvious. Phosphorylation of S450 was specific to the absence of carbon. Additionally, the region between S477 and S487 appeared to be strongly phosphorylated upon detection of carbon and may hence be a hotspot for carbon dependent regulation. Interestingly, this region overlaps with a poor PEST motif  was found to be broadly phosphorylated under different conditions with up to 27 detected different phospho-peptides with 13 variant residues, albeit some have uncertain localization scores within peptides. In many cases, phosphorylation was not condition-dependent, but both for ST motif , which hIn the MAPKK MEK-1, phospho-peptides were detected for different conditions, suggesting carbon-dependent phosphorylations also for this protein. Additionally, specific phosphorylations were detected for Glucomannodextrins on amino acids T92, S135, S139, and S184. The clustered serines at positions S130, S135 and S139 were phosphorylated in different combinations in various conditions and could thus represent an important area for carbon sensing. As also this region overlaps with a potential PEST motif, a contribution of protein stability or degradation in carbon sensing is further supported.D-Glc  (Phosphorylation of the MAP kinase MAK-1 (NCU09842) was found to occur on up to five sites, two of which being tyrosines. Thereby, phosphorylation of Y189 occurred independently of the conditions tested, while Y185 was only phosphorylated in response to AScores) . T187 beN. crassa genome  appears modified only in starvation condition.Previous studies showed that there are 107 serine/threonine protein kinases in the a genome . AccordiT. reesei , which represents 22 in vivo unambiguously modified S, T and Y, suggesting that these sites may be playing regulatory roles. Similarly, PRK-2/YAK1 (NCU07872), the homolog of T. reesei TrYak1, showed 13 distinguishable phosphorylated residues (23 variants), represented in 12 different phospho-peptides under cellobiose condition. Thus, determining the function of these phosphorylation sites will be a worthwhile target for an increased scientific understanding of cellulase gene expression in filamentous fungi.In eukaryotes, protein kinase A (PKA) and the phosphatidylinositol 3-kinase TOR (NCU05608) are two major kinases involved in regulation of cell growth in response to nutritional signals . Yak1 an. reesei . Our resN. crassa. Previous studies showed that PKAc-1 works as the major PKA in N. crassa, while deletion of pkac-2 does not affect fungal morphology, suggesting that these two isoforms may play distinct roles .There are two catalytic subunits of PKA, namely PKAc-1 (NCU06240) and PKAc-2 (NCU00682), in ct roles . AccordiWe analyzed the six G-protein subunits  , prosducThe phosducin like-protein PhLP1 (NCU00441), which acts as a co-chaperone for β and γ subunits  is phospN. crassa (15). GPR-17 and GPR-35 were phosphorylated under all conditions, with an extra phospho-peptide observed only in the presence of D-Xyl for GPR-17 and during starvation (no carbon source) for GPR-35. GPR-9 was phosphorylated in D-Glc, D-GalA + L-Rha and D-Xyl. GPR-18 was phosphorylated in the presence of D-GalA + L-Rha, cellobiose and Glucomannodextrins, but not D-Glc or starvation, suggesting that this GPCR is phosphorylated only during growth on plant cell wall substrates. Results from RNAseq analysis showed that gpr-18 is also among the most highly expressed of the Pth11-class GPCRs during growth on avicel (We furthermore detected 33 distinctive phospho-peptides for 10 GPCRs. This set contains NCU03238 (GPR-9), NCU03253 (GPR-8), NCU04106 (GPR-17), NCU04931 (GPR-18), NCU04987 (GPR-10), NCU06987 (GPR-14), NCU08718 (GPR-35), NCU09195 (GPR-6), NCU09201 (GPR-37), and NCU09427 (GPR-3). Four of these  are Pth11-related GPCRs, a large class previously implicated in sensing/degradation of cellulose in n avicel .The 10 GPCRs were variously phosphorylated in three different regions of the proteins. GPR-3, GPR-8, GPR-14, GPR-17, GPR-18, GPR-35, and GPR-37 are phosphorylated on residues in the intracellular C-terminal region. The mPR-Like/PAQR GPCRs GPR-9 and GPR-10 are phosphorylated on the first extracellular domain, while Stm1-like GPR-6 is phosphorylated in the large second intracellular loop region.Neurospora was grown under constant light conditions for the present study, the data reveal abundant phosphorylations present in clock components. Crosstalk between gene regulation in response to different nutritional conditions and the light response pathway is extensive  or MAPK (S831 and S1015), without the need of a priming phosphorylation events (in vivo studies have shown to display only one phosphorylation site (S21)  and WC-2 (NCU00902), which also impact regulation of plant cell wall degrading enzymes . WC-1 an of WC-1 , 2019. Sn events . Interesn events  which iste (S21) . Herein,The transcription factor SUB-1 (NCU01154) is predominantly known for its function in development in diverse fungi , but hasAlso for VE-1 , functions in development and secondary metabolism are best known , althougThe finding of clear condition-dependent phosphorylation patterns for both the photoreceptors and their regulators, such as SUB-1 or the casein kinases (see below) adds further support to the crosstalk between the reaction to carbon sources in the environment and sensing of light as well as circadian rhythmicity.N. crassa, casein kinases are mainly known for their function in the circadian clock (see above) with important impact on FRQ phosphorylation and stability  did not display any detectable phosphorylation. In the casein kinase II regulatory subunit beta-1  however, S291 was found phosphorylated under all conditions tested, while S78, S101, S105, S106 and S223 show carbon-dependent phosphorylation specific to certain carbon sources (S101 and S105 for example to cellobiose and no carbon conditions). For the second casein kinase regulatory beta subunit , we could not find any phosphorylated peptides, although the protein was detected under all tested conditions. The C-terminus of casein kinase I  on the other hand is phosphorylated at S313 and S320  independent of the condition tested, while S340  show carbon-specific phosphorylations including the absence of a carbon source.N. crassa is predicted to encode for 42 F-Box proteins. For 16 of these, we were able to detect phospho-peptides under at least one condition. Since homologs for some of these phosphorylated proteins had been implicated in regulatory functions of D-Glc utilization and CCR in A. nidulans  displayed reduced growth on arabinan and pectin.The genome of nidulans , the res sources . ParticuD-Glc that is often used to indicate impaired glucose repression in filamentous fungi  and allyl alcohol (AA) . 2-DG isus fungi . When fu of 2-DG . AA is a. oryzae  and A. nnidulans . When CCsitivity . As obseve to AA . These dA. nidulans . For D-Xyl, specific phosphorylation occurred at T159 in variant 1 and around positions and in a different region at variant 2. Besides the support for two variants of adenylate cyclase by confirmation at the peptide level, these phosphorylation patterns indicate specific functions in carbon sensing and signaling of individual compounds for both variants. Consequently, N. crassa likely distinguishes these carbon sources and triggers specific responses also in terms of alternative splicing and coordinated phosphorylation for their utilization or degradation. Besides CR-1 as the core component, the cAMP pathway further comprises the functionalities of phosphodiesterases, which degrade cAMP in response to environmental signals, and protein kinase A . MCB (NCU01166), the regulatory subunit of PKA, showed phosphorylation in two regions of the N-terminus. In most cases, phosphorylation occured upon sensing of several C-sources or constitutively . However, phosphorylation of S41 was specific for recognition of D-Xyl and cellobiose. Phosphorylation of the catalytic subunit PKAC-1 (NCU06240) occured predominantly in two areas of the protein: from T19 to T32, containing eight putative phosphorylated sites, and from T377 to Y386. Both regions are overlapping with potential or poor PEST regions. In many cases, multiple parallel phosphorylations occured on several of the tested carbon sources. In contrast to PKAC-1, the second catalytic subunit of protein kinase A, PKAC-2 (NCU00682) showed only two phosphorylated amino acids. Albeit both having low AScores, phosphorylation of Y255 specifically occured in the presence of cellobiose and phosphorylation of T260 was specific for recognition of D-Glc.In the adenylate cyclase CR-1 (NCU08377), a central component of the cAMP signaling pathway known to be involved in nutritional sensing , we idenD-Xyl as well as no carbon at S797, a site that overlaps with a predicted cAMP-dependent phosphorylation site. The low affinity phosphodiesterase PUR-1 (NCU00237) was not detected in the proteome.The high affinity phosphodiesterase ACON-2 (NCU00478) showed phosphorylation at several sites, in most cases relatively unspecific with respect to the carbon source (particularly S727 and S218). Nevertheless, carbon-specific phosphorylation was observed for Glucomannodextrins at T226 and D-Xyl-induction, the most significantly enriched class was protein kinase , while cAMP/cGMP-mediated signal transduction  was dominating the Glucomannodextrins-induced subset . For the Cellobiose-induced subset, the top 7 classes were related to activities in the nucleus, such as DNA/RNA synthesis, modification and control , followed by signaling pathways . In the D-Glc sub-network, nucleus regulation, metal binding and serine/threonine kinase activity were overrepresented  plus the protein kinase class, cAMP/cGMP-mediated signal transduction and MAPKKK cascade , protein kinase  and phosphate metabolism  were dominating.Within the PPI, CR-1 is predicted to interact with 67 different proteins that form a complex PPI sub-network with a total of 265 connections . For theumigatus . The indcr-1 deletion strain on different substrates demonstrated a substantially decreased capacity to degrade complex substrates  were used as further controls. This extended set of induction conditions allowed us for the first time to identify signaling events that are specific for each of the cues derived from the individual components of a plant cell wall.Numerous studies have indicated that rapid and reversible protein phosphorylations are involved in many perception pathways of environmental conditions across the tree of life . Also inescribed . In the escribed , but wouescribed , D-Xyl for xylan , D-galacr pectin , and Glur pectin . D-Glc aN. crassa protein interactions at the early stage of substrate recognition. While the principal protein classes that putatively participate in the initial stages of substrate recognition  were found to be clustered across the network, numerous interactions between proteins of different functions are also represented. These interactions include many variances in phosphorylated proteins that can be hypothesized to be part of signaling pathways leading to metabolic adaptations. Detecting the vast changes of phosphorylation patterns across the network in response to different carbon sources strongly indicates that phosphorylation reactions are a central part of the environmental response of N. crassa that can be specifically adjusted to the composition of the substrate.To better visualize the complex interplay of phosphorylation reactions, we constructed a protein-protein interaction (PPI) network with all detected proteins, exhibiting 12,066 predicted interactions based on available information gathered from STRING . The proD-Xyl could help to keep CLR-1 de-activated in non-inducing situations.For transcription factors, kinases and other signaling components, such as CR-1, G-proteins and GPCRs, the phosphorylation profile is clearly affected by the substrate composition, providing novel insights into several well-known signaling pathways. For example, the differential phosphorylation of VIB-1, that acts upstream of CRE-1 , could bp-value for the phospho-site localization <0.05). Regarding the G-proteins, RACK1-homologs have been demonstrated to function as an alternative Gβ in several fungi and are well-known for their additional role as scaffolding proteins for numerous processes in eukaryotes  is required for proper expression of numerous amino acid biosynthetic enzymes in response to starvation for a single amino acid . Genetic evidence suggests that phosphorylation of RACK1 on S122 and T162 negatively regulates its functions  were identified within the RR domain, with S1338 and S1359 being phosphorylated under all conditions. NCU01823 was phosphorylated in different regions with two sites in the Orc1-like AAA ATPase domain, S722 and S723. Regarding NIK-2 (NCU01833), one phospho-peptide was detected at the extreme 3′ end of the first PAS/PAC domain being phosphorylated under all conditions. NCU03164 displayed four phosphorylation sites and is the only HHK with a site (S966) in the HK domain, being phosphorylated in presence of D-GalA + L-Rha, cellobiose, Glucomannodextrins and no carbon.Also for the histidine kinase two-component systems, the identified phospho-sites within the domain structure of the proteins provide novel information that might be helpful to better understand their molecular setup. We found that a few of the thaliana . One of thaliana . In N. cN. crassa. The osmosensing (OS) MAPK pathway has been shown to act as a negative regulator of cellulase production in N. crassa, with the suggestion that osmolarity serves as a proxy for soluble sugars  often results in autoubiquitination (by the SCF) and self-destruction. However, phosphorylation of fungal F-box proteins can also affect other cellular functions, such as localization . This stnditions . It remaVisualization of the entire network can show differences in phosphorylation patterns across conditions, but cannot give a precise idea about the proteins involved in the interactions. We therefore decided to do a detailed analysis of the interactions of CR-1, since this enzyme is essential to the cAMP-dependent protein kinase pathway that is involved in cellulase and xylanase production . EspeciaSimilarly intriguing, the identifiable sub-networks of CR-1 were found to have a specific design according to the interacting proteins with condition-specific phosphorylations. Extending the previously described importance of adenylate cyclase in development and growth , our resN. crassa. The identification of protein phosphorylation followed by the prediction of protein-protein interactions allowed us to visualize the condition-specific phospho-protein distribution within the network. Many of the identified phospho-proteins are associated with sensing and signaling functions in the cell and thus appear to be involved in the regulatory networks governing the specific metabolic adaptation to plant cell wall polysaccharide degradation. Our protein-protein-interaction network analysis highlights that these changing patterns might lead to vast alterations in the respective sub-networks, as is the case for the adenylate cyclase CR-1, identified as a central hub with variances in phosphorylation patterns in the tested conditions. In many cases, the analysis provided amino acid level resolution, allowing to make assumptions about carbon source-specific structure-function relationships. Overall, the taken approach can therefore greatly help to identify novel functional interactions related to substrate signaling, and the experimental data can serve as a resource for future efforts to elucidate the underlying molecular mechanisms.The present study reveals fundamental changes in global protein phosphorylation during the initial moments of substrate recognition in The datasets generated for this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013964 and 10.6019/PXD013964.MH, NT, CT, LL, AS, and JB conceived the study, and each designed and supervised substantial parts of it. MH, NT, YG, KB-J, CN, MG, ML, and LA performed the experiments and acquired the data. All authors helped to analyze and interpret the data, read and approved the final manuscript. MH, NT, KB, MS, LL, LFL, and JB drafted the manuscript, which was critically revised by LA, CT, GB, AS, and GG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The optimal reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction time on the yield of product were investigated, and the straightforward strategy assembles the yield of butein up to 88%. Butein has been found to inhibit xanthine oxidase (XO) activity. Herein, the inhibitory mechanism of butein against XO was discussed in aspects of inhibition kinetic, fluorescence titration, synchronous fluorescence spectroscopy, and molecular docking. The inhibition kinetic analysis showed that butein possessed a stronger inhibition on XO in an irreversible competitive manner with IC50 value of 2.93 × 10−6 mol L−1. The results of fluorescence titrations and synchronous fluorescence spectroscopy indicated that butein was able to interact with XO at one binding site, and the fluorophores of XO were placed in a more hydrophobic environment with the addition of butein. Subsequently, the result of molecular docking between butein and XO protein revealed that butein formed hydrogen bonding with the amino acid residues located in the hydrophobic cavity of XO. All the results suggested that the inhibitory mechanism of butein on XO may be the insertion of butein into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and inducing conformational changes in XO.Butein  belongs to the chalcone family of flavonoids and possesses various biological activities. In this study, butein was synthesized through aldol condensation catalyzed by thionyl chloride (SOCl Hyperuricemia characterized by an elevated level of uric acid in human blood is a critical cause of gout ,2,3. It Butea monosperma, Dalbergia odorifera, Semecarpus anacardium, and Toxicodendron vernicifluum  are Stern–Volmer dynamic quenching constant and quencher concentration, respectively. Kq is the quenching rate constant of biomolecule (Kq = SVK/τ0). τ0 is the average biomolecule lifetime, and its value is 10−8 s [SVK can be obtained from linear regression plot slope of F0/F versus [Q].s 10−8 s . KSV canF0/F versus [Q] at three temperatures , and the relevant Kq values were given in 10 L mol−1 s−1  gave the Ka values for the butein-XO complex at 298, 301, and 310 K F0F0−nd 310 K . The Ka eraction .Kb and the number of binding sites n can be calculated from the following equation  (c): Yellow power, 1H NMR (acetone-d6) δ: 13.62 , 8.11 , 7.77 , 7.68 , 7.35 , 7.22 , 6.91 , 6.47 , 6.37 . 13C NMR (acetone-d6) δ: 192.8 (C=O), 167.6 (C-4′), 165.6 (C-2′), 149.3(C-4), 146.5 (C-3), 145.5 (C-β), 133.3 (C-6′), 128.2 (C-1), 123.4 (C-6), 118.3 (C-α), 116.4 (C-2), 116.0 (C-5), 114.5 (C-1′), 108.7 (C-5′), 103.8 (C-3′). ESI-MS m/z 273.08, [M + H]+.−1, pH = 7.5) to a final concentration containing less than 1% DMSO (v/v). In the 0.8 mL reaction system, a series of assay solutions prepared in phosphate buffer  consisting of a fixed concentration of XO (0.024 U mL−1) and various concentrations of inhibitor solutions were incubated at 25 °C for 30 min, and the reaction was initiated by adding the substrate xanthine (300 × 10−6 mol L−1) to the complex solutions. The assay mixture was incubated at 25 °C for 30 min. The reaction was stopped by the addition of HCl (1 mol L−1). The absorbance of uric acid level was determined by an HPLC method using a Diamonsil ODS-C18 column  on an Agilent 1260 system equipped with a G1311C quaternary pump, a G1329B autosampler, a G1316A thermostatted column compartment, and a G1314F variable wavelength detector coupled with an analytical workstation . Averages of three replicates are presented and the allopurinol was used as a positive control.The XO inhibitory activity assay was performed according to the method modified by our group . The but−6 mol L−1), where butein was at diverse concentrations  and xanthine at a fixed concentration. The involved inhibition mode of the butein on xanthine oxidase was analyzed from Lineweaver–Burk plots, and Ki value was identified by the software GraphPad Prism 5.0 . For competitive inhibition, the Lineweaver–Burk equation can be written in double reciprocal form as follows:The assay was investigated applying the XO activity assay methodology in the absence and presence of butein with a series of concentrations of substrate xanthine  and (7). v denotes enzyme reaction rate in the absence and presence of butein. [I] and [S] are the concentrations of the inhibitor and substrate, respectively. Kmapp presents the apparent Michaelis-Menten constant. The secondary plot of Kmapp versus [I] is linearly fitted, indicating a single inhibition site or a single class of inhibition site [A secondary plot can be plotted from:ion site .−1) was titrated with different concentrations of butein from 0 to 36.36 × 10−6 mol L−1 and then the solutions were incubated 5 min to equilibrate. Afterwards, fluorescence emission spectra of the solutions were measured using a spectrofluorometer at different temperatures  in 300–500 nm wavelength range upon excitation 280 nm. The widths of both the excitation and emission slits were set at 5 nm. The background fluorescence of the buffer  was subtracted from butein–XO complexes.Fluorescence titration assay was conducted by a spectrofluorimeter  equipped with a thermostat bath and a 150 W xenon lamp to characterize the interaction between butein and XO. Briefly, 2.0 mL sample of XO solution  at 15 and 60 nm over a wavelength range of 260–330 nm and 250–330 nm, respectively.Fc and Fm represent the corrected and measured florescence. A1 and A2 are the absorbance of butein at excitation and emission wavelengths, respectively.All the fluorescence-quenching data were corrected for absorption of excitation light and reabsorption of emitted light by the following relationship :F = Fmehttp://www.rcsb.org/pdb). All water molecules and ligand in XO were removed prior to the docking program running, after which the polar hydrogen atoms and Gasteiger charges were added to the macromolecule file. The 3D structures of butein were obtained in Chem 3D Ultra 8.0. The active site with a fixed grid spacing (0.375 points) was enclosed by a rigid dimension of docking center (100 × 100 × 100 points). The best scoring docked model of the ligand was chosen to represent its most desirable binding mode predicted by AutoDock. The PyMol molecular graphic system was used to visualize the conformations and interactions between the butein and the target proteins.The potential binding site of XO-butein was researched on AutoDock . The X-ray crystal structure  of XO used for the docking studies  was down2/EtOH was conducted for the first time in the present study. The optimal reaction conditions including the molar ratio of reactants, the dosage of catalyst, and the reaction time were investigated, and its potential inhibition of XO was discussed in aspect of inhibition kinetics, fluorescence titrations, synchronous fluorescence spectroscopy, and molecular docking. The butein can be prepared in good yield from 2′,4′-dihydroxyacetophenone and 3,4-dihydroxybenzaldehyde based on the catalytic system SOCl2/EtOH, without protecting the hydroxyl groups. In addition, the inhibitory activity on XO showed that butein could significantly reduce uric acid concentration and the IC50 value of restraining XO was found as 2.93 × 10−6 mol L−1 in vitro. Additionally, an attractive inhibition mechanism of irreversible competition is worth further exploration, as it might be helpful in treating gout. The intrinsic fluorescence of XO was quenched statically by butein, and butein bound spontaneously into the active cavity of XO to form the butein–XO complex with one high-affinity binding sites. Besides, hydrophobic interaction is the main driving force of butein to binding with XO. The molecule docking revealed butein is a competitive XO inhibitor and has a high affinity binding site on XO. The effectiveness of butein in reducing uric acid concentration in the blood is required to evaluate by the further in vivo studies. This study has supplied significant insights into the inhibitory mechanism of butein on XO, which will promote butein to be a leading compound by structural modification on the treatment of gout.In summary, a simple and effective method for the synthesis of butein by aldol condensation with the catalytic system SOCl