Maximum enhancement of methane production was 47–68% compared to raw FVHW when 5% solid content, 1-hour reaction time and 60–100 °C reaction temperature were applied in pretreatments.The increasing population creates excess pressure on the plantation and production of fruits and vegetables across the world. Consumption demand during the whole year has made production compulsory in the covered production system (greenhouse). Production, harvesting, processing, transporting, and distribution chains of fruit and vegetables have resulted in a huge amount of wastes as an alternative source to produce biofuels. In this study, optimization of two pretreatment processes  was investigated to enhance methane production from fruit and vegetable harvesting wastes (FVHW) that originate from greenhouses. NaOH concentration (0–6.5%), HCl concentration (0–5%), reaction temperature (60–100 °C), solid content (1–5%), time of reaction (1–5 h), and mixing speed (0–500 rpm) were chosen in a wide range of levels to optimize the process in a broad design boundary and to evaluate the positive and negative impacts of independent variables along with their ranges. Increasing NaOH and HCl concentrations resulted in higher COD solubilization but decreased the concentration of soluble sugars that can be converted directly into methane. Thus, the increasing concentrations of NaOH and HCl in the pretreatments have resulted in low methane production. The most important independent variables impacting COD and sugar solubilization were found to be chemical concentration (as NaOH and HCl), solid content and reaction temperature for the optimization of pretreatment processes. The high amount of methane productions in the range of 222–365 mL CH The acceptance of the generated wastes as a resource within the scope of the circular economy, recovery, and reuse is the major condition for sustaining life on earth. The increasing population coupled with economic development has resulted in huge agricultural production . Based o2 covered area for the production of fruits and vegetables. South Korea, Spain, and Japan followed China in the ranking. Turkey, holding approximately 772 km2 of covered production area, occupies fifth place across the world for the massive production of fresh fruit and vegetables from greenhouses. It is expected that the global commercial greenhouse market will be 32.31 billion USD with an annual growth rate of 8.8% by 2021. Judging by this enormous production, it is predictable that huge amounts of fruits and vegetables wastes have been generated during the production, harvesting, processing, transporting and distribution chain. Fruits and vegetable wastes are the main components of municipal solid waste. The conventional disposal methods of FVHW from greenhouses are the storage in forest area, uncontrolled burning and landfilling by farmers [China is placed in the first rank of the world with a 27000 km farmers , the deg farmers .2 of greenhouses have been located in Antalya, south of Turkey. It is around 47% of Turkey’s greenhouse production hold in Antalya. Eighty percent of fruit and vegetables, such as tomato, cucumber, pepper, eggplant, courgette, banana, strawberry and any other are grown in the greenhouses. Apart from all these positive contributions, the region has been under heavy pressure in terms of FVHW to be managed. The production area urgently required the implementation of relevant policies for managing the waste disposal and recovering energy from renewable and cost-free lignocellulosic wastes.Maintaining high agricultural production capacities for Turkey, as well as for other countries, has three main objectives. These are increasing agricultural income and its contribution to national income, meeting agricultural and food demand of nationals domestically, and keeping farmers on their lands to secure agricultural production . About 22 and other greenhouse gases [On the other side, fossil fuels have supplied over 80% of global energy needs. However, these fuels are not renewable and lead to climate change by the production of COse gases . Consequse gases ,10. However, the complex structure of agricultural residues is the major limitation, which makes lignocellulosic biomass refractory to anaerobic digestion and yields in low methane production ,12. ThisOn the other hand, FVHW, primarily non-commercial parts of plants such as leaves, bodies, stems as well as unmerchantable fruits and vegetables, have a milder lignocellulosic structure than the other agricultural residues and require less severe pretreatment conditions before AD. There have been very limited studies investigating the methane production potentials of mixed FVHW combined with pretreatment methods in the literature ,15,16,17In this study, it was proposed to combine the cost-effective integrated process of chemical-assisted thermal pretreatment and anaerobic digestion for the improvement of methane production from FVHW. The impacts of sodium hydroxide (NaOH) and hydrochloric acid (HCl) assisted thermal pretreatment on methane production were investigated. In the first stage of the work, impacts of variables  on soluble chemical oxygen demand (sCOD) and soluble sugars (sSugar) were determined with very detailed experimental work for NaOH- and HCl-assisted thermal pretreatments. In the second part of the work, process optimizations were performed for both pretreatment processes; optimum conditions for methane production were discussed and biochemical methane potential (BMP) tests were implemented under the determined conditions. Finally, molecular bond characterization and surface properties of raw and pretreated FVHW were compared. FVHW were obtained from Antalya region. The area of Kumluca has been well known for the extensive production of fruits and vegetables in greenhouses. Kumluca area makes a significant contribution to the fruits and vegetables supplies of Turkey and its trade partners during the four seasons. FVHW included leaves; stalks; stems; roots; and unmerchantable fruits from pepper, cucumber, tomato, courgette, and eggplant. FVHW were sun-dried and milled to 1 mm size for the characterization tests, while size-reduced (4–5 mm) fresh wastes were subjected to chemical-assisted thermal pretreatment experiments and BMP tests. The analyses of total solids (TS), volatile solids (VS) and total chemical oxygen demand (COD) were performed according to standard methods 2540C and 5220B, respectively . The conFresh FVHW were pretreated in a 0.3 L stainless steel reactor  equipped with a temperature controller and magnetically-driven turbine agitator. NaOH and HCl were selected as chemical agents in the chemical-assisted thermal pretreatments experiments to enhance methane yield. Selected independent variables and their ranges for NaOH- and HCl-assisted thermal pretreatment are presented in Chemical-assisted pretreatments experiments were planned by central composite design (CCD) of Response Surface Methodology (RSM). Four independent variables with the three levels were utilized for CCD in Design-Expert® 11 software trial version  for NaOH- and HCl-assisted thermal pretreatments. Fifty-one pretreatment experiments containing three experiments at the center point along with the duplicates of each run were planned for each chemical (NaOH and HCl) assisted thermal pretreatment by CCD. Consequently, 108 values for each response  were used in the optimization of each chemical-assisted pretreatment processes.p- and F- values. The quality of models was concluded via the coefficient of determination (R2) and an adjusted determination coefficient (adj-R2). With the help of the obtained model equations, optimizations of chemical-assisted thermal pretreatment processes were performed by using maximization and minimization criteria for independent and dependent variables [® software program for process optimization. The significance of the goals was chosen as (+++++), the highest, for all variables in software. Design-Expert® software solved all goals for all variables to obtain the desirability function. The optimization module investigated the merging of independent variable values that meet the demands placed on each of the dependent variables. The targets were chosen as maximum sSugar and sCOD production, and optimizations were performed for economical process conditions along with the high sSugar and sCOD production.Results of experiments were modeled and acquired regression models were assessed by the analysis of variance (ANOVA), regression coefficients along with the ariables ,30. The 2/CO2 (70%/30%) gas mixture to provide an anaerobic environment. Triplicate BMP tests were done for all pretreated samples, raw waste, and seed sludge control. BMP tests ended after the 30 days. During fermentation, CH4 and CO2 components formed in the BMP reactors were detected by Varian CP-4900 Micro-Gas Chromatography (Micro-GC) . Varian CP-4900 was supplied with a thermal conductivity detector (online-TCD) and a 10 m PPQ column. Injector and column temperatures in Micro-GC were 110 °C and 70 °C, respectively. High purity grade helium gas was used as a mobile phase with a flow rate of 25 mL/min. For measuring total gas volume, a gas-liquid displacement device was used filled with a saturated pH 1 acidic salt solution prepared according to Standart Method 2720 [4/gVS and then this value was subtracted from the methane amount produced in reactors to normalize the produced methane amount. The impact of chemical-assisted thermal pretreatment was also evaluated by a BMP test. The BMP test according to Carrère et al. (2009) and Us and Perendeci (2012) ,31 was ahod 2720 . The amoScanning electron microscopy (SEM) was utilized to evaluate microstructural changes on the surface of wastes after the application of chemical-assisted thermal pretreatments. Raw and pretreated FVHW were lyophilized and coated with gold-palladium for 120 seconds under 18 mA vacuum and samples were examined by SEM with a Zeiss Leo 1430 electron microscope  at a voltage of 15 kV. −1 and 4000 cm−1 wavelength range with a spectral resolution of 4 cm−1 and with 16 scans per sample.To investigate the impacts of thermochemical pretreatment on waste structure molecular-bond characterization, samples were examined by an FTIR-Varian 1000 Scimitar series FTIR spectrometer . Samples were lyophilized and pelletized with KBr before analyses. Measurements were performed in 500 cmHigh carbohydrate content as cellulose and hemicelluloses was obtained as 56.86% from the FVHW . Carbon −1 was obtained with no NaOH addition, almost at the same concentration level, which varied in the narrow range of 486–629 mgsCOD gVS−1, regardless of the changes of other independent variables. On the other hand, increasing NaOH concentration from 0% to 3.3% and 6.5% boosted the COD solubilization. The average sCOD values of 1112 mgsCOD gVS−1 and 1243 mgsCOD gVS−1 were measured for 3.3% and 6.5% NaOH concentration, respectively, with a moderate fluctuation between 832–1248 mgsCOD gVS−1 and 1034–1356 mgsCOD gVS−1. Furthermore, no remarkable changes with other independent variables were found. The most significant increase in sCOD of 1356 mgsCOD gVS−1 was observed in the pretreatment experiment performed at 6.5% NaOH concentration, 100 °C reaction temperature, 1 h reaction time, and 500 rpm mixing speed, yielding more than a 240% increment compared to raw sCOD of the FVHW. Conclusively, the addition of NaOH resulted in more dissolved organic material, measured as sCOD. Thus, the concentration of sCOD increased with increasing NaOH concentration.The main purpose of NaOH- and HCl-assisted thermal pretreatment of FVHW is to make more cellulose and hemicellulose available for the anaerobic microorganism and to enhance methane production. Since the enhancement of methane production was related to sCOD and sSugar ,32, impa−1 with the average sCOD of 554 mgsCOD gVS−1. A similar trend observed in sCOD by increasing the amount of NaOH was observed by increasing the amount of HCl. However, the measured amounts were at the level of 613–1149 mgsCOD gVS−1 (average 800 mgsCOD gVS−1) and 590–1232 mgsCOD gVS−1 (average 908 mgsCOD gVS−1), for 2.5% and 5% HCl, respectively, used in the pretreatments. Basically, −1, whereas sCOD of the nonexplosively pretreated paper tube at 190 °C with 2% NaOH was found at 2280 mg L−1. The results from Teghammar et al. (2010) [−1 at 1 g NaOH/100 g solid to 12007 mg L−1at 4 g NaOH/100 gr solid. They indicated that the concentration of sCOD increased with the addition of NaOH. Jard et al., 2013 [Palmaria palmata. They also indicated that thermo-chemical pretreatment induced a change in the structure of macroalgae, increasing the cell-wall porosity and thus facilitating the solubilization of COD. The results from our study were found to be consistent with Wang et al. (2009), Teghammar et al. (2010) and Jard et al. (2013) [Teghammar et al. (2010)  investig. (2010)  revealed. (2010)  applied l., 2013  investig. (2013) ,34,35.−1 from the FVHW pretreated at 0% NaOH concentration, 100 °C reaction temperature, 4 h reaction time, and 0 rpm mixing speed, where the maximum increase in reducing sugar was calculated as 67.2%. On the other hand, soluble sugar concentration increased by increasing the HCl concentration from 0% to 2.5%, but the sugars produced by increasing HCl dosage to 5% were transformed. −1 and resulted in a sharp decrease to the average level of 68 mg glucose gVS−1 when the HCl concentration was raised to 5%. It should be remembered that the decrease in sugar concentration necessitates the optimization of the amount of chemical usage in the pretreatment process. The NaOH- and HCl-assisted thermal pretreatment experiments also pursued to observe the alteration of soluble reducing sugars b,d. The F-test with the low value (P model > F = 0.0001) for NaOH- and HCl-assisted thermal pretreatments. To check the significance of the fit of equations for sCOD and sSugar of NaOH- and HCl-assisted thermal pretreatments, ANOVA was made and results are presented in RSM and CCD were applied to derive optimum conditions effective on FVHW for NaOH and HCl-assisted thermal pretreatment. Statistical evaluation was made and the models for sCOD and sSugar were constructed for both pretreatments. Analysis of variance (ANOVA) was employed to investigate the quality of model equations. Quadratic regression models for sCOD and sSugar were significant, as apparent from the Fisher’s R2 and adjusted determination of coefficient, Adj-R2. R2 and Adj-R2 were found to be 0.9503 and 0.9325, and 0.9288 and 0.9033 for sCOD, indicating a high degree of correlation between the response and the independent variables in the quadratic models for NaOH- and HCl-assisted thermal pretreatments, respectively. Signal-to-noise ratio is defined as adequate precision and a ratio bigger than four is favorable. Therefore, the ratios 20.06 and 22.33 have shown adequate signals for the models. Values of Prob > F less than 0.05 indicated that model terms are significant [A), mixing speed (C) and NaOH concentration (D), interactive terms of AD, and square terms of reaction temperature (A2) and NaOH concentration (D2) were significant for the sCOD model of the NaOH-assisted thermal pretreatment, the linear effect of reaction temperature (A), HCl concentration (B) and solid content (C), interactive terms of AB and BC, and square terms of reaction temperature (A2) and mixing speed (D2), were significant for the sCOD model of HCl-assisted thermal pretreatment. The quality of the fit of models was expressed by the coefficient of determination nificant . While tR2 and Adj-R2 were computed as 0.9967 and 0.9956, and 0.9161 and 0.8860 for quadratic models of sSugar of NaOH- and HCl-assisted thermal pretreatments, respectively. Adequate precision with the values of 69.18 and 16.58 was obtained, indicating models can be applied in the design space. Furthermore, the linear effect of reaction temperature (A), reaction time (B), and NaOH concentration (D); interactive terms of AD and BD; and square terms of NaOH concentration (D2) were significant for the sSugar model of NaOH-assisted thermal pretreatment, while the linear effect of reaction temperature (A), HCl concentration (B) and solid content (C); interactive terms of BC; and square terms of HCl concentration (B2) were significant for the sSugar model of HCl-assisted thermal pretreatment. Similarly, the values of p > 0.005). On the other hand, other variables shown in ANOVA analysis presented in −1 and 222 mgsSugar mgVS−1 were predicted for sCOD and sSugar, respectively. To validate the optimization, pretreatment experiments were executed under these proposed conditions. In these runs, 712 mgsCOD mgVS−1 and 236 mgsSugar mgVS−1 were realized for sCOD and sSugar models, respectively. Solubilization of sCOD and sSugar compared to the raw FVHW were found to be 80% and 18%, respectively, for cost optimization conditions.sCOD and sSugar models were operated for the optimization of NaOH- and HCl-assisted thermal pretreatment conditions by Design-Expert® software. During the optimizations of both pretreatment applications, a cost-driven approach was applied. Reaction temperature, reaction time and NaOH concentration were diminished, while sCOD and sSugar were maximized for NaOH-assisted thermal pretreatment. Mixing speed was kept in the ranges. Optimum conditions were obtained with a desirability of 0.681 at 0.67% NaOH concentration, 65 °C reaction temperature, 1 h reaction time, and 500 rpm mixing speed. Under these conditions, 688 mgsCOD mgVS−1 and 553 mgsSugar mgVS−1, respectively. To validate the optimization, specific batch runs were conducted. sCOD and sSugar were determined as 698 mgsCOD mgVS−1 and 438 mgsSugar mgVS−1, verifying the acceptable performance of the models for HCl-assisted thermal pretreatment. The increments of sCOD and sSugar compared to the raw wastes were computed as 76% and 119%, respectively for cost optimization conditions.To optimize the HCl-assisted thermal pretreatment, initial solid loading, sCOD and sSugar were maximized, whereas reaction temperature and HCl concentration were minimized from the point of environmentally sustainable procedures and process costs. Since mixing speed was found to be insignificant for both models, it was set within the range. Optimum conditions were obtained with the desirability of 0.647 at 1.44% HCl concentration, 60 °C reaction temperature, 5% solid content, and 324 rpm mixing speed. Under these conditions, sCOD and sSugar values were estimated as 682 mgsCOD mgVS−1 can be produced when 1-hour reaction time, 0.67% NaOH concentration, 500 rpm mixing speed, and 65 °C reaction temperature conditions were applied in the pretreatment. On the other hand, increasing NaOH concentration resulted in higher sCOD solubilization of approximately 1200–1300 mgsCOD mgVS−1. However, as it is apparently observed from −1 should be produced under the same pretreatment conditions.The impacts of independent variables of NaOH-assisted thermal pretreatment on sCOD and sSugar solubilization are demonstrated in −1 should be produced by the application of 5% solid content, 1.44% HCl concentration, 324 rpm mixing speed, and 60 °C reaction temperature conditions in the pretreatment. As it is clearly seen from The independent variables’ impact on sCOD and sSugar solubilization after the application of HCl-assisted thermal pretreatment is also presented in The most important difference between the two pretreatments was the use of different initial solid content in the pretreatment. Enhancement of methane production from FVHW can be achieved by the usage of 5% solids content along with the 60–80 °C reaction temperature and no or very low chemical agent in the pretreatment.4 gVS−1. The observed methane production profiles for thermal and NaOH- and HCl-assisted thermal pretreatments are presented in Since increasing the NaOH and HCl concentrations from 0% to 6.6% and 0% to 5%, respectively, yielded with the lowly-soluble sugar concentration due to degradation of the produced sugar and possibly the conversion to refractory compounds or inhibitors, the methane productions were investigated with a reduced number of pretreatment experiments. A BMP value of raw FVHW without pretreatment, serving as a comparison, was found as 217 mLCH4 gVS−1 (av. 295 mLCH4 gVS−1) at a range of 60–100 °C reaction temperature and 1–5% solid content. The highest BMP values were 365 and 360 mLCH4 gVS−1 acquired at 60 °C and 100 °C reaction temperature, respectively, along with 5% solid content and 1 h reaction time, as indicated in the red dash line zone.4 gVS−1) was measured from the FVHW pretreated at a NaOH concentration of 6.5%, a reaction temperature of 100 °C, and a reaction time of 4 h. By adding NaOH as 3.3% and 6.5% concentrations, the production of methane decreased from 251–360 mLCH4 gVS−1 (av. 305 mLCH4 gVS−1) without NaOH to 132–133 mLCH4 gVS−1 (av. 133 mLCH4 gVS−1) at 3.3% and further dropped to 121–129 mLCH4 gVS−1 (av. 127 mLCH4 gVS−1) at 6.5%. As it is easily captured from + itself or combined effects of both. In fact, for the sake of clarity, Na+ concentration was approximately 1.8 g Na+/L in the BMP reactor, it reached 2.5 g Na+/L with the supplied NaHCO3 as a buffer in the BMP reactor for the lowest BMP obtained as 121 mLCH4 gVS−1. Despite the fact that slight inhibition concentrations for Na+ are in the range of 3.5–5.5 g/L [+ concentration) caused inhibition.The lowest BMP value  a higher amount of methane production was obtained by the thermal application without using the chemical agent of NaOH or HCl. (ii) Although the chemical usage contributes to the hydrolysis rate of the lignocellulosic structure, the selected high dose for NaOH (3.3–6.5%) and HCl (2.5–5%) should be the probable cause of low methane production. (iii) Maximum enhancement of methane production was 47–68% compared to raw FVHW when 5% solid content, 1 hour reaction time and 60–100 °C reaction temperature were used in pretreatments.2SO4, HCl, H2O2, and CH3COOH) at concentrations of 1, 2, 3, and 4% and three alkali reagents 2 and NH3.H2O) at concentrations of 4, 6, 8, and 10% were applied in the pretreatments at 25 °C during the 7 days. Results revealed that acid and alkaline treatments increased methane production by approximately 10.3% to 115.4% higher yield compared to raw. This phenomenon can be explained by the fact that alkaline and acid pretreatments promote organic solubilization and increase the surface area available for enzymatic accessibility. Monlau et al., (2012) [2O2, Ca(OH)2, HCl, FeCl3 at 4–10 gr/100gr TS concentration along with the 30–170 °C reaction time and 1–24 h reaction time to sunflowers stalks. They found that methane potentials of pretreated sunflower with different chemicals increased to 3–35% compared to the raw sunflower. Although corn straw and sunflower stalks do not seem to be similar to the FVHW at first glance, the cellulose and hemicellulose contents of the sunflower are close to FVHW used in this study to make a comparison. Furthermore, the enhancement of methane production results from this study was found to be coherent with the literature and revealed that pretreatment conditions necessitate optimization and depend on the structure and composition of lignocellulosic wastes. As a result, the selection of a wide of range levels of the independent variables has riveting characteristics to optimize the process in a broad design boundary and to evaluate the positive and negative impacts of independent variables along with their ranges. The results obtained from optimization in the design space have resulted in the suggestion of a low amount of NaOH and HCl usage due to the inhibition effect of the high amount of chemicals on methane production. The selection of a wide range of levels of independent variables has provided an opportunity for evaluating extreme points in optimization.Song et al. (2014)  examined, (2012)  applied 4 production conditions  compared to those of the raw FVHW are demonstrated in SEM images and FTIR spectra of FVHW pretreated at optimized conditions for sCOD and sSugar by NaOH-assisted thermal pretreatments  and HCl-assisted thermal pretreatments , and maximum CHSEM images presented in −1 wavelengths can be apparently observed from the pretreated samples of FVHW by the application of NaOH- and HCl-assisted thermal pretreatments, respectively. Most of the observed modifications in the molecular bonds were found to be similar. While FTIR band intensities were closer to each other at 60–100 °C and 0–0.67% NaOH pretreatment conditions and gave lower density than the raw sample, FTIR band intensities of samples pretreated at 1.44% HCl condition were found to be closer to the raw sample.In the dashed line zone of the FTIR spectra, both NaOH- and HCl-assisted thermal pretreated wastes presented a different spectrum than from raw lignocellulosic wastes. Especially, modifications in spectra at 400–500, 780, 875, 1000–1100, 1150, 1245, 1310, 1380, 1500–1600, 1750, 2300–2400 and 400–500, 775, 875, 1000–1100, 1150, 1280–1310, 1380, 1500–1600, 1750, and 2300–2400 cm−1, FTIR spectra show vibrations of Si–O–Si and NH2 bonds, respectively [−1 wavelength represents the vibration of glucosidic linkage in hemicellulose [−1 [−1 [−1 [−1 and 1380 cm−1 of FTIR spectra indicated CH2 wagging in cellulose and hemicellulose and planar C–H bending in cellulose, hemicellulose, and lignin, respectively [−1 are attributed to aromatic C=C bonds, changes at 1510 cm−1 were found to belong to aromatic ring vibrations of lignin [−1 pointed to C=O stretching vibration in acetyl groups of hemicellulose [−1 may belong to this group. Out of the dashed line zone, pretreated wastes showed a difference only at one wavelength (2400 cm−1) with low densities, compared to raw FVHW. The band at 2400 cm−1 was attributed to C=O stretching within the carbon dioxide [At 470 and 775 cmectively ,44. Si mellulose , and it lose [−1 , C–O–C v [−1 [−1 . Observeectively ). Since f lignin . As chan dioxide .−1.In summary, FTIR analysis revealed that pretreatment caused changes in molecular bond characterization especially in the region of 800–1500 cm−1 was observed at 6.5% NaOH concentration, 100 °C reaction temperature, 1 h reaction time, and 500 rpm mixing speed, yielding more than a 240% increment compared to raw sCOD of the FVHW. The highest sSugar concentration was measured as 335 mg glucose gVS−1 from the FVHW pretreated with no chemicals at a 100 °C reaction temperature, 4 h reaction time, and no mixing, where the maximum increase in reducing sugar was calculated as 67.2% compared to raw FVHW. Increasing NaOH and HCl concentrations resulted in higher sCOD but extremely lower sSugar solubilization. Results of sCOD and sSugar from the NaOH- and HCl-assisted thermal pretreatment experiments were modeled, and statistically significant regression models were developed. The most important independent variables were found to be chemical concentration (as NaOH and HCl), solid content and reaction temperature for the pretreatment processes. FVHW collected from the extensive production greenhouses area of Antalya city was thermochemically pretreated to enhance methane production. The most significant increase in sCOD of 1356 mgsCOD gVS4 gVS−1 methane productions were obtained by the simple thermal application without using NaOH or HCl. Maximum enhancement of methane production was 47–68% compared to raw FVHW at 5% solid content, 1-hour reaction time and 60–100 °C reaction temperature. Two-hundred-and-twenty-two to 365 mLCHThe optimization of chemically-assisted thermal pretreatment processes should be done with the methane production results obtained from the BMP test. On the other hand, increasing the NaOH and HCl concentrations from 0% to 6.6% and 0% to 5%, respectively, resulted in low soluble sugar concentrations. Furthermore, optimization in the design space suggested the use of low amounts of NaOH and HCl due to the inhibition risk of the high amount of chemicals. In addition, BMP tests yielded to low methane production for the samples pretreated with high a amount of chemicals (NaOH and HCl). As a result, it was found that sSugar and methane production results were correlated and sSugar data can be used in optimization in a reliable way for this substrate.Results from this comprehensive study have been encouraging that it serves information to build the potential application and to reduce the waste-related environmental pressure in the production-dense area. Gained optimization solution in the design space resulted in the suggestion of no chemical usage due to the inhibition effect of the high amount of chemicals on methane production. The selection of a wide range of levels of independent variables provided an opportunity for evaluating extreme points in optimization.


HER2)-amplified breast cancers are treated using targeted antibodies and kinase inhibitors, but resistance to these therapies leads to systemic tumor recurrence of metastatic disease. Herein, we conducted gene expression analyses of HER2 kinase inhibitor-resistant cell lines as compared to their drug-sensitive counterparts. These data demonstrate the induction of epithelial–mesenchymal transition (EMT), which included enhanced expression of fibroblast growth factor receptor 1 (FGFR1) and axonal guidance molecules known as neuropilins (NRPs). Immunoprecipitation of FGFR1 coupled with mass spectroscopy indicated that FGFR1 forms a physical complex with NRPs, which is enhanced upon induction of EMT. Confocal imaging revealed that FGFR1 and NRP1 predominantly interact throughout the cytoplasm. Along these lines, short hairpin RNA-mediated depletion of NRP1, but not the use of NRP1-blocking antibodies, inhibited FGFR signaling and reduced tumor cell growth in vitro and in vivo. Our results further indicate that NRP1 upregulation during EMT is mediated via binding of the chromatin reader protein, bromodomain containing 4 (BRD4) in the NRP1 proximal promoter region. Pharmacological inhibition of BRD4 decreased NRP1 expression and ablated FGF-mediated tumor cell growth. Overall, our studies indicate that NRPs facilitate aberrant growth factor signaling during EMT-associated drug resistance and metastasis. Pharmacological combination of epigenetic modulators with FGFR-targeted kinase inhibitors may provide improved outcomes for breast cancer patients with drug-resistant metastatic disease.Human epidermal growth factor receptor 2 ( HER2 becomes amplified and patients with HER2+ tumors can respond to treatment with the monoclonal antibodies pertuzumab and trastuzumab. However, acquired and inherent resistance remains a major clinical problem in this breast cancer subtype, particularly in the metastatic setting. In addition, expression of cytokeratin 5/6 and EGFR are major factors in defining the basal-like subtype of breast cancer and further decrease the already abysmal prognosis of this aggressive form of disease  from the firebrowse website (ed genes . Finallyt tests were used where the data met the assumptions of these tests and the variance was similar between the two groups being compared. P values <0.05 were considered significant. No exclusion criteria were used for these studies.Either two-way analysis of variance or a two-sided Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Table 4Supplemental Figure legendsFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8


Winnie mice, that are characterized by spontaneous chronic intestinal inflammation and ER stress caused by a missense mutation in the mucin MUC2 gene. Idebenone (200 mg/kg) was orally administered daily to 5–6 weeks old Winnie mice over a period of 21 days. Idebenone treatment substantially improved body weight gain, disease activity index (DAI), colon length and histopathology score. Immunohistochemistry revealed increased expression of MUC2 protein in goblet cells, consistent with increased MUC2 mRNA levels. Furthermore, idebenone significantly reduced the expression of the ER stress markers C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6) and X-box binding protein-1 (XBP-1) at both mRNA and protein levels. Idebenone also effectively reduced pro-inflammatory cytokine levels in colonic explants. Taken together, these results indicate that idebenone could represent a potential therapeutic approach against human UC by its strong anti-inflammatory activity and its ability to reduce markers of ER stress.Endoplasmic reticulum (ER) stress in intestinal secretory goblet cells has been linked to the development of ulcerative colitis (UC). Emerging evidence suggests that the short chain quinone drug idebenone displays anti-inflammatory activity in addition to its potent antioxidant and mitochondrial electron donor properties. This study evaluated the impact of idebenone in  Ulcerative colitis (UC) and Crohn’s disease (CD) are the two major forms of inflammatory bowel disease (IBD) which are characterized by chronic and relapsing inflammation of the gastrointestinal tract, with an unclear etiology  that invEmerging evidence points to the potential presence of a pathophysiological circuit consisting of microbial dysbiosis, dysregulated immune response, altered barrier integrity, oxidative stress, genetic mutations and aberrant endoplasmic reticulum (ER) stress that all contribute to the development of intestinal inflammation ,15,16,17Winnie, a mouse model of spontaneous chronic inflammation, recapitulates the symptoms associated with human UC and includes a prominent innate/adaptive/Th17 immune response [Winnie is caused by a single point or missense mutation in the mucin MUC2 gene which leads to mucin misfolding in the ER, resulting in UPR activation-linked ER stress. As a consequence, there is less production of MUC2 from goblet cells, a reduction in goblet cell size and numbers and a diminished mucus barrier, similar to human UC [Winnie mice starts at the age of 6 weeks and progresses to severe colitis by the age of 16 weeks [Winnie is characterized by relapses and remissions [Winnie is more prominent as compared to that in the proximal colon (PC) due to higher epithelial stress and more microbial dysbiosis [Winnie mice revealed a UC-like phenotype that includes crypt abscesses, reduced goblet cell numbers, crypt elongations, mucosal surface erosion, infiltration of neutrophils and a distorted crypt architecture [Winnie model was successfully used to test several clinical drugs such as MCC950, glucocorticoids and thiopurines, and dietary fibers [Several murine models connect ER stress with intestinal inflammation ,23,35,36response . The chrhuman UC . The inf16 weeks . Like inmissions . The colysbiosis ,24,40. Hitecture . Recently fibers ,42, thatCHOP in the HtrA2 knock-out mice, which was associated with protection against neurodegeneration [Idebenone, a short chain benzoquinone, is described as a mitochondrial electron donor and a potent antioxidant . In the neration .Winnie mouse model of spontaneous colitis. The current study highlights the protective role of idebenone in UC to ameliorate chronic inflammation by simultaneously suppressing ER stress and pro-inflammatory cytokine levels.Based on this evidence and on our previous results , we hypoC57BL/6J and Winnie mice of both sexes were obtained from the animal breeding facility of the University of Tasmania. All the animals were housed in a controlled temperature environment with a twelve-hour day and night cycle. All the animals underwent an acclimatization period of 7 days before performing any experiment. All the mice were caged individually with proper access to autoclaved drinking water ad libitum and normal chow pellets. All the experiments were conducted in accordance with the ethics approval from Animal Ethics Committee of University of Tasmania . All the procedures were conducted in accordance to the Australian Code of Practise for Care and Use of Animals for Scientific Purposes .The C57BL/6J mice (aged 5–6 weeks) and Winnie mice (aged 5–6 weeks) were divided into three groups: (1) C57BL/6J Healthy Controls without drug (HC), (2) Winnie Controls (Winnie) and (3) idebenone-treated Winnie (Winnie + I), with eight animals/group. For drug preparation, idebenone was suspended in 0.5% of carboxymethylcellulose (CMC), 4% sucrose and autoclaved powered chow pellets as mentioned previously [Winnie controls received the drug vehicle in food mash, and idebenone containing food mash was administered to the Winnie plus idebenone-treated group for 21 days daily. Autoclaved normal drinking water was provided to all groups. All the mice were sacrificed on day 21.Equal number of eviously . The wetv/v neutral-buffered formalin for 24 h, and subsequently transferred to 70% ethanol. They were embedded in paraffin wax and 5-µm thick tissue sections were cut using a rotary microtome. Paraffin-embedded tissue slides were then stained with hematoxylin and eosin (H&E) dyes and were used for histopathological grading in a blinded manner, as described previously [Disease Activity Index (DAI) was calculated daily by adding the individual scores of body weight, blood in stool and stool consistency over the whole period of the experiment, as mentioned previously ,41,50. Beviously ,41,51. In = 3 slides/group, in a blinded manner.Immunohistochemical analysis was performed to detect the localization of mucin MUC2 proteins using an HRP/DAB Detection IHC kit , as mentioned previously . BrieflyGRP78 (Assay ID: Mm00517691_m1), CHOP (Assay ID: Mm00492097_m1), ATF6 (Assay ID: Mm01295319_m1), XBP-1 (Assay ID: Mm00457357_m1) and PERK (Assay ID: Mm00438700_m1), MUC2 (Mm01276696_m1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID: Mm99999915_g1).The RNA from colonic tissue was extracted and purified using RNeasy Mini kit  according to the kit manual. The concentration of the extracted RNA was calculated using an Eppendorf Biophotometer. The extracted RNA was reverse transcribed to complementary cDNA using iScript cDNA synthesis kit  according to the reaction conditions, as mentioned by manufacturer. Then, 100 nanogram of cDNA from each sample was added to a PCR reaction mixture containing Fast Advanced Master Mix  and a single gene-specific TaqMan probes or primers set . The primers used were Thermal cycling was performed using a StepOnePlus RT-qPCR instrument . Quantification of gene expression was done using a comparative method (ΔΔCT). The threshold cycle (CT) for each gene was normalized to GAPDH (house-keeping gene) and expressed as relative to the mean of related control group.g to collect supernatant. Next, thiobarbituric acid (TBA) was added to the resulting supernatant, which formed MDA-TBA adduct after boiling at 95 °C for 1 h. The absorbance of colored MDA-TBA adduct was detected colorimetrically at a wavelength of 532 nm in a spectrophotometer. The amount of MDA in the tissue samples was quantified by plotting a graph against the MDA standard calibration curve. The data were expressed as nmol/mg protein.The amount of malondialdehyde (MDA) levels present in colonic tissue was determined using lipid peroxidation colorimetric/fluorometric assay kit  as described previously ,52. The For western blot analysis, frozen distal colon tissues were washed with PBS and lysed in radioimmunoprecipitation assay buffer (RIPA) containing protease inhibitor cocktails . The concentrations of protein in supernatants were determined according to the protocol described by the DC protein Assay Kit from Biorad. Tissue-extracted proteins lysates (20 µg) were suspended in loading buffer and separated on SDS-PAGE polyacrylamide gels  in a 1x electrophoresis buffer run at 100 V for 1 h. Subsequently, proteins from the gel were electro-transferred onto a polyvinylidene difluoride membrane (PVDF) using a wet-transfer system. The membranes were then blocked in 5% skim milk prepared in TBST and incubated overnight with primary antibody against NQO-1 , GRP78  CHOP  and β-actin  at 4 °C. The antibodies were detected by incubating with Horseradish peroxidase (HRP)-conjugated secondary antibody  at room temperature for 1 h. Protein bands were visualized using SuperSignal, West Pico PLUS chemiluminescent substrate . The images were captured, and the band densities relative to housekeeping gene (β-actin) were measured using LAS-3000 image reader version 2.2 .v/v fetal bovine serum  in a 12-well plate, as described previously [Explants from proximal and distal colon tissues from each group were excised, washed with PBS and cultured in RPMI 1640 culture medium containing 1% antibiotics solution  and 10% eviously ,50,53. Ap < 0.0001 (****), p < 0.001 (***), p < 0.01 (**) and p < 0.05 (*).Statistical analysis was performed using GraphPad Prism Software version 8.0, California, United States of America). All the data were presented as average mean value ± standard error of mean (SEM) of independent experiments carried out in triplicate or quadruplicate. One-way analysis of variance (ANOVA) followed by Tukey’s post-test was used to evaluate the statistical difference between the three groups. For analysis of body weight change and DAI, Two-way ANOVA followed by Bonferroni’s post-test was used. Data were considered significant when Winnie mice administered idebenone significantly started gaining body weight as early as day 9 (p < 0.01). In comparison to Winnie animals, which gained 4.5 ± 2.3% body weight, idebenone treatment drastically increased body weight by 17.5 ± 1.3% over the observation period of 21 days, equivalent to C57BL/6J healthy controls (HC)   onwards, DAI was indistinguishable from the start of the treatment.The therapeutic potential of idebenone in spontaneous chronic colitis was determined by measuring the change in body weight, disease activity index (DAI)  and colon length. ols (HC) A. As com4 ± 0.4) B. In ideWinnie; colon was visibly inflamed, thickened and shortened on day 21. In contrast, three weeks of oral administration of idebenone significantly prevented the shortening of the colon (8.5 ± 0.2 cm) when compared to Winnie animals (7.7 ± 0.2 cm) (p < 0.05) (Macroscopic assessment of the colon clearly indicated the severity of inflammation in  < 0.05) C.Winnie model is characterized by severe epithelial surface erosion, distortion of crypt architecture/crypt loss, crypt abscesses, loss of goblet cells, submucosal edema and infiltration of inflammatory cells predominantly affecting distal colon (DC) as compared to proximal colon (PC) , with no signs of colon damage , by reducing the average histology score of untreated Winnie animals from 10.7 ± 0.9 to 7.0 ± 0.6 (p < 0.05) reduced by idebenone (5.0 ± 0.8). Spontaneous inflammation of the colon in the lon (PC) A, as revn damage A. Treatm.0 ± 0.6 C. In the.0 ± 0.6 B, the avWinnie mice is the consequence of a missense mutation in the mucin MUC2 gene [Winnie mice confirmed significantly decreased MUC2 expression in both PC and DC goblet cells as compared to colon samples from HC animals , UPR transcription factor CHOP , UPR signaling molecule ATF6 , transcription factor XBP-1  and UPR signaling molecule PERK   and spontaneous chronic intestinal inflammation in nie mice . To expl.0-fold) A–E.Winnie mice displayed in the DC a significant reduction of CHOP, ATF6 and XBP-1 mRNAs by 4.3-fold (p < 0.01), 4.1-fold (p < 0.001) and 4.2-fold (p < 0.001) respectively, comparable to the levels of healthy control animals. In the PC, a substantial reduction of ATF6 mRNA  was detected, while no significant reductions of GRP78 and PERK mRNAs were detected in either PC or DC compared to untreated Winnie mice.In contrast, idebenone-treated Winnie mice. Idebenone treatment in Winnie prevented CHOP upregulation (p < 0.01) decrease in CHOP expression  and chemokine MIP-1α (macrophage inflammatory protein 1), idebenone treatment considerably suppressed their release in both PC and DC explants of Winnie mice. IL-1α was significantly reduced by idebenone-treated PC tissue (p < 0.01), while this effect did not reach significance in the DC explants (p < 0.01). In addition, idebenone significantly reduced the secretion of TNF-α (p < 0.001) and INF-γ (p < 0.001) in DC tissues (p < 0.01) was more prominent than for TNF-α (p < 0.05). Furthermore, idebenone treatment significantly suppressed MIP-1α (p < 0.05) (p < 0.05) by both PC and DC explants. No substantial effect was seen for several other cytokines  E and IL- < 0.05) F secretiytokines .Winnie model of chronic colitis. Idebenone also improved the histopathology, immune parameters and clinical manifestations of colitis, and increased MUC2 levels.The current study described the therapeutic potential of idebenone for ameliorating spontaneous chronic intestinal inflammation. The most prominent observation was that idebenone inhibited ER stress, which represents the main causative pathological feature of the Winnie mice against surface erosion, crypt abscesses, loss of goblet cells, elongation or distortion of crypt architecture and infiltration of immune cells. Surprisingly, at high doses, i.e., >1 g/day, idebenone has been reported to cause gastro-intestinal disturbances such as diarrhea, vomiting and dyspepsia in some patients [The current study used idebenone at a dose of 200 mg/kg, which was selected based on a previous study where idebenone ameliorated acute colitis, pro-inflammatory cytokines and oxidative stress in a mouse model of acute colitis . It has patients ; these sWinnie model, spontaneous chronic intestinal inflammation arises as a consequence of a missense mutation in MUC2 gene, which leads to MUC2 protein misfolding and significantly decreased mucin biosynthesis [Winnie mice [In the ynthesis . Decreasynthesis . Consequynthesis . In contnie mice . Since tGRP78, PERK, CHOP, XBP-1 and ATF6 in colonic tissues of IBD patients and in Winnie mice [GRP78 and PERK expression, which makes them unlikely targets for idebenone. Therefore, it appears unlikely that GRP78 and PERK were involved in reducing ER stress in the present study.Several preclinical and clinical studies support a central role of ER stress in the pathogenesis of UC ,57,58. Dnie mice ,24,25 suWinnie mouse model [CHOP expression in a mouse model of mitochondrial disease, which was associated with improved motor function [CHOP gene expression by idebenone in mutant myocilin cells, which was associated with antiapoptotic effects [CHOP mRNA and its protein levels. In addition, it adds to previous studies by providing a mechanistic clue that idebenone also downregulates the expression of ATF6, which is the upstream signaling molecule of CHOP. This effect was confirmed by the fact that idebenone also reduced XBP-1 expression, which is also controlled by ATF6. A previous study reported that glucocorticoids also reduce the expression of CHOP, XBP-1 and ATF6 in Winnie mice, as well in an in vitro model of tunicamycin-induced ER stress, which parallels our results in the present study [ATF6/CHOP/XBP-1 axis. However, this study did not clarify whether idebenone targets CHOP directly or indirectly by affecting genes upstream of CHOP; this will have to be addressed in future studies.The upregulation of ATF6-induced transcription factor CHOP plays a major role in mediating ER stress-induced apoptosis and epithelial cells proliferation , which ise model . Interesfunction . Moreove effects . The prent study . OverallWinnie mice. In line with this result, NQO1 levels of and SOD activity were indistinguishable between healthy control and Winnie mice, which indicates that the Winnie model in the early stages is not associated with excess levels of ROS. Consequently, idebenone did not affect the basal levels of lipid peroxidation and NQO-1 in Winnie mice. Overall, our results therefore indicate that idebenone either utilizes different protective pathways in models of acute versus chronic colitis or that a common, as yet undiscovered mode of action is responsible for initiating different protective responses.Excessive accumulation of ROS disrupts cellular functions by the peroxidation of lipids, a process that can lead to gastrointestinal disturbances . Under pWinnie mice displayed substantially elevated levels of IL-1α, IL-1β, TNF-α, INF-γ, IL-17 and MIP-1α as compared to C57BL/6J healthy controls, which is in alignment with previous reports [Winnie colon. This idebenone-induced anti-inflammatory property was also observed in multiple disease models including DSS-induced acute colitis, LPS-mediated neuroinflammation in BV2 microglial cells and in renal damage induced by titanium dioxide in rat, where idebenone suppressed certain pro-inflammatory cytokines and chemokines [Winnie further support the reports mentioned above regarding the anti-inflammatory action of idebenone.Immune system dysregulation due to abnormal secretion of pro-inflammatory cytokines and chemokines is the hallmark of UC pathogenesis . Intesti reports ,40,41,46emokines ,48. Our GRP78 in Caco2 intestinal cell lines [CHOP and GRP78 in LPS-induced lung injury [Growing evidence points to a direct intersection between immune response and ER stress in inflammatory disorders ,25,78,79ll lines . Furtherg injury . On the g injury ,84,85,86MUC2 expression and the simultaneous reduction of increased levels of pro-inflammatory cytokines  will be required to identify the specific pathways targeted by oral administration of idebenone. The results of the present study establish the possible importance of idebenone therapy, i.e., in targeting multiple factors linked to UC pathology including epithelial ER stress, mucin production and inflammatory markers. Therefore, idebenone could be considered a promising therapeutic alternative to treat the chronic phases of multifaceted UC.To our knowledge, the present study is the first to highlight the potential of idebenone to ameliorate chronic intestinal inflammation by effectively reducing ER stress. The beneficial effects of idebenone in this model of spontaneous chronic colitis were associated with the suppression of ER stress, the restoration of ytokines . It can


ID1 are upregulated in ENZ-resistant cell lines and these results are further confirmed in patient-derived xenografts (PDXs) and patient specimens. Consistent with the finding that ARBS-gained loci are highly enriched with H3K27ac modification, ENZ-resistant PCa cells, organoids, xenografts and PDXs are hyper-sensitive to NEO2734, a dual inhibitor of BET and CBP/p300 proteins. These results not only reveal a noncanonical AR function in acquisition of ENZ resistance, but also posit a treatment strategy to target this vulnerability in ENZ-resistant CRPC.Resistance to next-generation anti-androgen enzalutamide (ENZ) constitutes a major challenge for the treatment of castration-resistant prostate cancer (CRPC). By performing genome-wide ChIP-seq profiling in ENZ-resistant CRPC cells we identify a set of androgen receptor (AR) binding sites with increased AR binding intensity (ARBS-gained). While ARBS-gained loci lack the canonical androgen response elements (ARE) and pioneer factor FOXA1 binding motifs, they are highly enriched with CpG islands and the binding sites of unmethylated CpG dinucleotide-binding protein CXXC5 and the partner TET2. RNA-seq analysis reveals that both CXXC5 and its regulated genes including  Resistance to second generation anti-androgen therapies such as enzalutamide (ENZ) can emerge in prostate cancer patients. Here, the authors identify an ENZ-resistant mechanism driven by AR-dependent transcription of noncanonical targets that make resistant cells susceptible to dual inhibition of BET and CBP/p300 signaling. Acting as a transcription factor (TF) either alone or in concert with pioneer factors such as FOXA1, AR operates transcription programs to promote PCa growth and survival by binding to the full or half ARE or a composite motif consisting of ARE and FOXA1 binding element (FOX/ARE)5. Because of the dependency of most PCa on the AR signaling, androgen deprivation therapy (ADT) has been the mainstay treatment for advanced PCa. Treatment-induced reduction in the level of PSA, which is encoded by the well-studied ARE-driven gene KLK3, has served as a reliable surrogate for the effectiveness of ADT. Unfortunately, the disease usually relapses and becomes metastatic CRPC. The importance of AR re-activation in CRPC is reflected in United States Food and Drug Administration approval of next-generation AR signaling inhibitors such as enzalutamide (ENZ) and abiraterone (ABI)8. While these inhibitors are initially effective, they fail in the majority of CRPC. Different mechanisms related to ENZ and ABI resistance have been identified, which include aberrant glucocorticoid receptor (GR) upregulation9, AR splice variants (ARVs)11, AR gene mutation12, somatic acquisition of AR gene enhancers, and AR gene duplication15. These mechanisms highlight the central role of AR and other nuclear receptors in the development of ENZ and ABI resistance.The androgen-AR signaling axis is the major driver of AR-positive prostate cancer (PCa)18, expression of ARE-dependent canonical AR target genes such as KLK3 is often downregulated or completely suppressed19, implying the acquisition of an AR-indifferent phenotype after next-generation AR inhibitor therapy. Additionally, other studies suggest that AR-negative or -low PCa cells or clones pre-exist in untreated primary tumors and they tend to become accentuated in metastatic foci of the CRPC patients, stressing a role of AR-independent mechanisms in therapy resistance and disease progression23. Considering that a large percentage of CRPC remain AR positive21, in the present study we were interested to define AR-dependent mechanisms of ENZ-resistance. Through genome-wide AR chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) analyses in ENZ-resistant (ENZ-R) AR-positive CRPC cells and meta-analysis and immunohistochemistry studies in patient samples, we demonstrated a previously unrecognized noncanonical AR function in ENZ-R CPRC.A noticeable consensus from various studies is that while most AR-targeted therapy-resistant CRPC continue to express full-length AR (AR-FL)CON, LNCaPCON, LAPC4CON, and VCaPCON) and ENZ-R  cells were maintained by continuous treatment of vehicle and ENZ, respectively. Using C4-2ENZ-R cells as a model, we further confirmed ENZ-R growth of C4-2ENZ-R cells in vivo , TMPRSS2, and NKX3.1 were transcriptionally downregulated in ENZ-R cells compared to control cells  treatment of vehicle (DMSO) or ENZ  we identified, 12,652 ARBS were lost (ARBS-lost) in C4-2ENZ-R compared to C4-2CON while 6908 ARBS were gained (ARBS-gained) and 40,220 ARBS were not significantly altered (ARBS-NSA) , a histone mark of both active enhancer and transcription start site (TSS) regionsSA) Fig. . Cis-regSA) Fig. . These roci Fig. . Becauseers Fig. . These d27 analysis revealed that GC-rich DNA motifs were frequently detected at ARBS-gained loci, but not at ARBS-NSA and ARBS-lost loci  located at CpGis was also much higher than that of androgen-dependent AR-occupied regions (AD-ORs) in C4-2B CRPC cells , actin cytoskeletal regulator PFN2 and ID339 are among the top of the upregulated genes in ENZ-R cells compared to control cells  as well as AR and FOXA1 binding and H3K27ac levels at these gene loci were dramatically downregulated in C4-2ENZ-R cells, and no obvious CXXC5 or TET2 binding peaks were detected at these loci  although the P value of the association was slightly greater than 0.05 , including ID1 and PFN2  of these inhibitors between C4-2ENZ-R and control cells, we found that C4-2ENZ-R cells were highly sensitive to the CBP/p300 inhibitor CPI63742 , CRPC (n = 16), or ENZ-R PCa (n = 13). We found that AR protein was readily expressed in all specimens except one ENZ-R and two CRPC cases  models. We have generated and maintained CRPC and ENZ-R PDX models as previously publishedWe first tested NEO2734 in organoids established from ENZ-R and CRPC PDXs. We found that CRPC organoids were sensitive to both ENZ and NEO2734 treatment, while ENZ-R organoids were highly sensitive to NEO2734 but resistant to ENZ up to a very high dose Fig. . To expl49. These studies further show that several AR-independent mechanisms  and double-negative PCa (DNPC) substantially increases after treatment of the next-generation AR signaling inhibitors including ENZ and ABIsms Fig. , includiRPC Fig. . These fRPC Fig. .Fig. 7A A unique feature shared by the noncanonical AR target genes we identified in ENZ-R cells is the paucity of ARE and FOX/ARE elements. Instead, these loci are located in the GC-rich promoter and non-promoter regions with significant overlap with the CpGis. In agreement with these observations, our genome-wide RNA-seq and ChIP-seq analyses show that CpG dinucleotide-binding protein CXXC5 is largely upregulated in ENZ-R CRPC cells and that CXXC5 and its binding partner TET2 occupy at the noncanonical AR target gene loci. We further demonstrate that TET2 interacts with AR and that AR is indirectly recruited into the CpGi-rich regions by CXXC5 via its interaction with TET2 Fig. . These f52. In agreement with these results, we show that chromatin occupancy of CXXC5 and H3K27ac levels are positively correlated at noncanonical AR target loci and that the BET-CBP/p300 dual inhibitor treatment inhibits CXXC5 protein expression and decreases BRD4 and p300 binding and H3K27ac levels at these loci. These mechanistic bases provide a strong rationale to clinically investigate the anticancer efficacy of bromodomain inhibitors and BET-CBP/p300 dual inhibitors in ENZ-R CRPC which are addicted to the noncanonical AR activity for survival  prebound with antibodies at 4 °C overnight. Immunoprecipitation antibodies were AR , FOXA1 , H3K27ac , CXXC5 , TET2  and Rabbit IgG . Precipitated protein-DNA complexes were eluted and cross-linking was reversed at 65 °C for 12 h. ChIP-seq libraries were prepared using previously described methods54. High-throughput sequencing  was performed using the Illumina HiSeqTM4000 platforms at the Mayo Clinic Genome Core Facility. All short reads were mapped to the human reference genome (GRCh37/hg19) using bowtie2 (version 2.1.0) with default configurations55. Reads mapped to multiple positions greater than 2 were discarded, and the remaining reads were then used for peak calling by MACS2 (version 2.0.10) with a p value cutoff of 1e-5 56. Peaks located on curated blacklists such as centromere regions were removed (https://sites.google.com/site/anshulkundaje/projects/blacklists). ChIP-seq tag intensity tracks (bedGraph files) were generated by MACS2, and converted into bigWig files using UCSC “wigToBigWig” tool. Genomic distribution of peaks with regard to TSS, and the association of peaks to target genes was performed by Homer package58. The de novo-motif discovery was conducted using STEME, an efficient EM algorithm to find motifs in large data sets59. Heat maps were drawn by deepTools 2.0, and customized python scripts were used for other analyses as needed.For ChIP experiment, chromatin was cross-linked for 15 min at room temperature by adding 11% formaldehyde/PBS solution in cell culture medium60. GSEAs were carried out using the signature scores per gene (fold change) in pre-ranked mode with default settings61. Gene expression was determined by real-time quantitative PCR (qPCR) using Power SYBR Green . Primer sequences used for qPCR are listed in Supplementary Data For RNA-seq, libraries were prepared using Illumina TruSeq RNA prep kit and standard protocol. The RNA libraries were sequenced as 51 nt pair-end reads at one sample per lane of an Illumina HiSeq 2500, generating an average of 265 million reads per sample. All reads were aligned to the human reference genome GRCh37/hg19) by TopHat 2.0.9 using default options. Gene expression counts were generated using RSeQC and expression differential analysis was conducted using edgeR (version 3.6.8)7/hg19 byZ-scores by Z = (x − μ)/σ47, where x is the raw log2(FPKM), μ is the mean and σ is the standard deviation across all samples of a gene. Finally, the Z-scores were summed across all genes to represent the expression score of noncanonical AR target genes. The RNA-seq and clinic data of metastatic CRPC (SU2C/PCF Dream Team)41, treated with ENZ and ABI, was derived from cBioPortal 62.The expression values (log2(FPKM)) of noncanonical AR target genes . C4-2, LNCaP, VCaP, and LAPC4 cells were maintained at 37 °C and 5% CO2 in RPMI 1640 containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Thermo Fisher Scientific). 293T cells were maintained in DMEM medium with 10% FBS. To establish ENZ resistant cell lines, C4-2, LNCaP, VCaP, and LAPC4 cells were cultured in medium containing ENZ. Concentrations of ENZ were gradually increased to 30 μM for C4-2, VCaP, and LAPC4 cell lines except that LNCaP was up to 5 μM ENZ, while control cell lines were cultured in medium with the same amount of vehicle without ENZ. Mycoplasma contamination was tested by the PCR Mycoplasma Detection Set . All cell lines are negative for mycoplasma contamination.AR, CXXC5, TET2, ID1, ID3, or PFN2 gene-specific shRNA  and cultured in medium containing 10% FBS. Cells were fixed at different time points (day 0–5) and cell growth was measured using sulforhodamine B (SRB) assay63.293T cells were co-transfected with control shRNA, ENZ-R cells (3 × 106) were mixed with Matrigel (in 50 μl of 1× PBS plus 50 μl of Matrigel (BD Biosciences)) and injected subcutaneously into the right flank of 6-week-old castrated mice. After xenografts reached a size of ~100 mm3 and then animals were randomized into one of five treatment groups (n = 5–10 per group) including vehicle , ENZ (10 mg per kg body weight), CPI637 (10 mg per kg body weight), JQ1 (50 mg per kg body weight), NEO2734 (10 mg per kg body weight) or combination of CPI637 and JQ1. For the ENZ-R PCa PDX study, PDXs previously generated in the laboratory of Liewei W at Mayo Clinic45 were divided into small pieces (~1 mm3) and injected subcutaneously (s.c.) into 6-week-old castrated mice. After xenografts reached a size of ~100 mm3, animals were randomized into one of five treatment groups (n = 8 per group) in a manner similar to the C4-2ENZ-R xenograft study. Treatments were administrated individually 5 days per week by oral gavage and growth in tumor volume was measured in a blinded fashion using digital calipers. Tumor volume was calculated using the following equation: tumor volume = length × width × width × 0.5.Male SCID mice were generated in house and used for animal experiments. All mice were housed under standard conditions with a 12 h light/dark cycle and access to food and water ad libitum and maintained under pathogen-free conditions. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) at the Mayo Clinic. C4-264 were embedded in 40 μl of Matrigel and cultured in FBS-free DMEM/F-12 medium supplemented with growth factors EGF (50 ng ml−1), FGF2 (5 ng ml−1), FGF10 (10 ng ml−1), B27 (2%), Prostaglandin E2 (1 μM), SB202190 (10 μM), Y-27632 (10 μM), Nicotinamide (12 mg ml−1), N-acetylcysteine (1.25 mM), R-spondin (500 ng ml−1), and Noggin (100 ng ml−1). The volume of organoids (diameter >5 μm) was measured using Image-Pro and the average of organoid volumes for different treatment were calculated. Cell viability assays for organoids were conducted by plating 2000 organoid cells per well of a collagen-coated 96-well cell culture plate in 100 μl of medium with vehicle (DMSO), ENZ or NEO2734 as indicated. Viable cells were counted using a CellTiter-Glo (Promega) Luminescent Cell Viability Assay Kit.For organoid studies, organoids established from PDXsENZ-R xenograft tumors were deparaffinized, rehydrated and subjected to heat-mediated antigen retrieval. Sections were incubated with 3% H2O2 for 15 min at room temperature to quench endogenous peroxidase activity. After antigen retrieval using unmasking solution (Vector Labs), slides were blocked with normal goat serum for 1 h and then incubated with primary antibody at 4 °C overnight. IHC analysis of tumor samples was performed using primary antibodies for CXXC5 , AR , ID1  and PFN2 . The sections were then washed three times in 1× PBS and treated for 30 min with biotinylated goat-anti-rabbit IgG secondary antibodies . After washing three times in 1× PBS, sections were incubated with streptavidin-conjugated HRP . After washing three times in 1× PBS for 5 min each, specific detection was developed with 3,3′-diaminobenzidine . Images were acquired using a Leica camera and software. IHC staining was scored on the basis of the ‘most common’ criteria. Staining score = staining intensity × staining positivity. Staining intensity was graded into four categories: 0, 1, 2, and 3. Specifically, 0 = no staining, 1 = weak staining (staining obvious only at ×400), 2 = medium staining (staining obvious at ×100 but not ×40), and 3 = strong staining (staining obvious at ×40). For staining positivity, 0 = no positive cells, 1 ≤ 10% of positive cells, 2 = 10–50% positive cells, 3 = 51–70% positive cells, 4 ≥ 70% positive cells.PCa specimens used for IHC were obtained from the Tissue Registry of Mayo Clinic  and the Prostate Centre at the University of British Columbia . The studies were approved by the Institute Review Board of Mayo Clinic. Informed consent was obtained from the study participants. All specimens were de-identified from patient information. FFPE tumor samples from patients, PDXs or C4-2Cells were lysed and boiled for 10 min in sample buffer . Equal amounts of protein (50–100 μg) from cell lysate were denatured in sample buffer (Thermo Fisher Scientific), subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher Scientific). Primary antibodies were against AR , CXXC5 , CXXC4 , TET2 , TET3 , TET1 , ID3 , PFN2 , BRD4 , p300 , ID1 , FOXA1 , Flag  and V5  and ERK2 .For the His-tag pulldown assay, purified His-tagged CXXC5 or His-tag control lysis were incubated with Flag-AR, V5-TET2 alone, or Flag-AR and V5-TET2 combination in binding buffer containing 10 mM imidazole for 4 h at 4 °C. Ni-NTA beads (Qiagen) for 3 h at 4 °C, washed with wash buffer containing 10 mM imidazole, and eluted with SDS sample buffer. Antibodies for protein purification were Flag  and V5 . The AR, TET2, and its mutant proteins were produced by TNT Quick Coupled Transcription/Translation System  following the manufacturer’s instructions.Cells were treated with proteinase K and RNase A and genomic DNA was isolated by phenol/chloroform extraction and sonicated. 4 µg of fragmented genomic DNA was immunoprecipitated with antibodies for 5hmC  or 5mC  at 4 °C in IP buffer  for 4 h. Protein A/G beads were added to each reaction and incubated at 4 °C for 2 h and then washed with IP buffer for three times. The beads were suspended in proteinase K digestion buffer  and incubated at 55 °C for 3 h. Samples were extracted using QIAquick PCR Purification Kit (Cat No. 28106). The immunoprecipitated DNA was quantitatively measured by real-time PCR (qPCR) analysis using Power SYBR Green . The primers are same as those used in ChIP-qPCR listed in Supplementary Data 2) and blocked with 5% skimmed milk powder for 1 h at room temperature and incubated with anti-5hmC antibody  at 4 °C overnight. The membrane was subjected to immunoblot analysis using HRP-conjugated IgG secondary antibody  for 1 h at room temperature. To ensure equal spotting of total DNA on the membrane, the blot was stained with 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2).For 5hmC detection, genomic DNA samples were denatured and twofold serial dilutions were spotted on a nitrocellulose membrane . The blotted membrane was UV-cross-linked (70,000 μJ/cmP values from unpaired two-tailed Student’s t tests were used for comparisons between two groups and one-way ANOVA with Bonferroni’s post hoc test was used for multiple comparisons. For treatment time course experiments, two-way ANOVA followed by post hoc test was applied. Statistical analysis is indicated in each figure legend. P value < 0.05 was considered significant.GraphPad Prism 7 was used for statistical analyses with the qPCR, cell proliferation analysis, tumor growth analysis, and IHC quantification data. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Human memory retrieval is the core cognitive process of the human brain whenever it is processing the information. Less study has focused on exploring the neural correlates of the memory retrieval of scientific concepts when presented in word and picture modalities. Fewer studies have investigated the differences in the involved brain regions and how the brain dynamics in these regions would associate with the accuracy of the memory retrieval process. Therefore, this study specifically focused on investigating the human brain dynamics of participants when they retrieve physics concepts in word vs. pictorial modalities, and whether electroencephalogram (EEG) activities can predict the correctness of the retrieval of physics concepts. The results indicated that word modality induced a significant stronger right frontal theta augmentation than pictorial modality during the physics concepts retrieval process, whereas the picture modality induced a significantly greater right parietal alpha suppression than the word modality throughout the retrieval process spurred by the physics concept presentations. In addition, greater frontal midline theta augmentation was observed for incorrect responses than the correct responses during retrieve physics concepts. Moreover, the frontal midline theta power has greater negative predictive power for predicting the accuracy of physics concepts retrieval. In summary, the participants were more likely to retrieve physics concepts correctly if a lower amount of theta were allocated during the maintaining period from 2,000 ms through 3,500 ms before making responses. It provides insight for our future application of brain computer interface (BCI) in real-time science learning. This study implies that the lower frontal midline theta power is associated with a lower degree of cognitive control and active maintenance of representations as participants approach a correct answer. The memory encoding and retrieval processes are the essential cognitive processes of the human brain whenever it is processing any type of information. Memory retrieval is particularly important in determining whether or not humans can successfully retrieve the relevant information they have encoded into their long-term memory (LTM). That is whether they can reactivate and access the information stored in the LTM and make further connections among different pieces of information. If more associations or more links can be established to specific information, the information can be consolidated more efficiently and retrieved more rapidly and easily Lang, . Memory Miller and Cohen  suggesteThe aforementioned studies shed light on the potential and importance of studying which brain regions and what types of brain dynamics are associated with the memory retrieval of scientific concepts spurred by the presentation of word and pictorial stimuli. Thus, this study aimed to investigate whether and how the human brain dynamics involved in the successful memory retrieval of physics concepts are presented in different modalities (words vs. pictures). In particular, we examined oscillatory electroencephalogram (EEG) patterns during the retrieval of physics concepts presented in the picture and word forms. The objectives of this study are first, to explore whether the brain dynamics of physics concepts retrieval processing and the responsible brain regions are different when such concepts are presented in the picture vs. word modalities; second, to investigate whether the brain activities before correct and incorrect responses are different during the process of retrieving physics concepts spurred by different presentation modalities; and third, we wanted to explore whether EEG activities can be used to predict the correctness of the retrieval of physics concepts.A total of 51 undergraduate students  aged 18–22 years participated in the study. All of the participants were right handed and had a normal or corrected-to-normal vision. They were informed about the experiment procedure, read and signed a consent form, and paid for their participation. The Institutional Review Board of the China Medical University Hospital approved this study. All of the students who volunteered to take part in the study majored in science and had learned the physics concepts used in the present study in high school. After EEG pre-processing, four participants’ EEG data were excluded from further analysis because they contributed fewer than two independent components to the component clusters. That is, the EEG data from only 47 of the 51 participants  were used in the study.To explore the cognitive process of the retrieval of physics concepts presented in different modalities, 90 physics concepts items related to the topics of mechanics, optics, electromagnetics, and thermodynamics were selected from secondary school physics textbooks and presented either in pictorial modality (45 physics concept items) or word modality (45 physics concept items). For the picture modality, each trial started with an 800-ms fixation, followed by a 2,000-ms presentation of the physics concept item, a 2,000-ms blank, and then a “go” signal. Each subject had to provide an oral response as soon as possible after the go signal was presented on the monitor, and each trial lasted a total of 6.8 s. In this study, all 42 trials of the physics concepts in each modality were presented by the STIM2 software, and EEG data from each participant were recorded continuously with a Neuroscan SynAmps2 amplifier  with 66 electrodes mounted in an elastic cap. The EEG signals were filtered using an analog band-pass filter from 0.01 to 100 Hz, and the sampling rate was 1,000 Hz. Inter-electrode impedance was maintained below 5 kΩ. The electrode locations followed the international 10-20 system, and all the electrodes were referenced to the linked mastoids. Four separate bipolar electrodes recorded the vertical (vEOG) and horizontal (hEOG) eye movements.−7 or 1e−8. The fast Fourier transformation was then used to transform the EEG activities into power spectrum density in the following frequency bands: θ (4–7.9 Hz), α (8–12.9 Hz), and low β (13–18 Hz).The recorded EEG data were first digitally band-pass filtered using the EEGLAB toolbox  were characterized into brain or non-brain components. We then used the boundary element head model, as implemented in the DIPFIT toolbox , other measures were compressed into a 10-dimension vector, resulting in a 53-dimensional feature space. Each IC with a 53-dimensional cluster position vector was further compressed by principal component analysis into a 10-dimensional vector. To normalize the variance from the different measures in the cluster position vector, the dipole location was given a weight of 20, ERSP was given a weight of 9, and other measures  were given a weight of 1. Any ICs whose distance to the cluster centroid was greater than three standard deviations away from the cluster centroid were removed. The K-means clustering algorithm, a particular modeling technique involving the grouping of homogeneous ICs across subjects by taking various measures of spectra, ERP, ERSP, inter-trial coherence, and scalp topography and dipole location, was used to group the ICs into 42 clusters (with only three dimensions). Subsequently, visual inspection was applied to remove improper ICs from each cluster according to their dipole locations, ERSPs, scalp maps, and power spectra.Before clustering, the component spectrum power, event-related potential (ERP), event-related spectral perturbation analysis (ERSP), inter-trial coherence, and scalp topography of each independent component were computed. With the exception of the dipole source location that was featured in only three dimensions  using three-cycle Morlet wavelets in a frequency range between 3 and 50 Hz Makeig,  implemenentation , 3H–K.The right frontal, left frontal, right parietal, left parietal, right occipital, and left occipital cluster recruited 27, 34, 30, 29, 28, and 30 independent components from 27, 34, 30, 29, 28, and 30 subjects (out of 45 subjects), respectively. The ERSP was employed to visualize the mean event-related spectral changes of the selected clusters over time, which showed the neural activities in the right hemisphere were very similar to those in the left hemisphere. However, the activities in the right hemisphere were considerably stronger than those in the left hemisphere; thus, the following results specifically focus on the right hemisphere.p < 0.01; The average scalp maps and the dipole locations of the clusters in the right frontal cluster are shown in p < 0.01) during the entire retrieval process, including the stimulus presentation and retrieval stages  of the physics concept retrieval for the word presentations showed that the ERSP of the frontal midline cluster exhibited a greater theta power increase in the incorrect responses than in the correct responses . In addip < 0.001]. p < 0.001]. The results of the ERSP and the power mean plots in The study results demonstrated that the retrieval of physics concepts spurred by both the pictorial and word modalities elicited strong theta power in the right frontal region across the entire physics concept retrieval stage until the presentation of the go signal. Previous studies also reported that a strong increase in theta oscillations during the working memory task and that the increased theta activity was sustained during the retention period until information had been retrieved (Raghavachari et al., The results also showed that the right parietal region exhibited significantly more alpha suppression during the retrieval of the pictorial presentations of physics concepts when compared with the word presentations. Klimesch  pointed In addition, our findings also revealed that the occipital theta power increases sharply after the onsets of either word or pictorial presentations of physics concepts, which is followed by a marked alpha suppression. This finding is consistent with those of previous studies (Lai et al., The study results also indicated that the frontal midline cluster revealed a significantly greater theta power and alpha power in the incorrect trials than in the correct trials for both the word and pictorial modalities. In addition, the results of the correlational analysis of the picture modality revealed that the frontal midline theta and alpha power from 2,000 to 3,500 ms was negatively correlated with the correctness of the retrieval performance. In short, the higher the frontal midline theta power, the more likely the subjects were to make an incorrect response in the picture modality. Moreover, the logistic regression analysis further demonstrated that both theta power and alpha power had a negative impact on the students’ likelihood of accurately retrieving physics concepts. The students were more likely to retrieve physics concepts correctly if a lower amount of theta and alpha power were allocated during the maintaining period from 2,000 ms through 3,500 ms before making responses. The natural logarithm of estimated odds and predicted probability of retrieving physics concepts increased when the frontal midline theta and alpha power decreased. Compared with the frontal midline alpha, the frontal midline theta power was a better predictor of participants’ retrieval physics concepts. Klimesch et al.  have sugWhen retrieving physics concepts presented in a pictorial modality, the participants exerted more mental effort to generate incorrect answers than correct answers and to maintain the operation of the retrieval of the physics concepts in the working memory. Our results supported the previous conflict-monitoring theory, which suggests that the timing of the ACC activations reflects that conflicts for correct and incorrect responses should differ (Botvinick et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board China Medical University Hospital/DMR100-IRB-221. The patients/participants provided their written informed consent to participate in this study.H-CS: conceptualization. C-PL, H-CS, L-YH, W-CC, and S-CC: design and methodology. L-YH, W-CC, and S-CC: data collection. C-PL: data analysis. H-CS: supervision. H-CS, C-PL, L-YH, and T-PJ: validation. H-CS, C-PL, L-YH, and T-PJ: writing—reviewing and editing.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.


This study quantifies the impact of atmospheric rivers (ARs) on rainfall in New Zealand. Using an automated AR detection algorithm, daily rainfall records from 654 rain gauges, and various atmospheric reanalysis datasets, we investigate the climatology of ARs, the characteristics of landfalling ARs, the contribution of ARs to annual and seasonal rainfall totals, and extreme rainfall events between 1979 and 2018 across the country. Results indicate that these filamentary synoptic features play an essential role in regional water resources and are responsible for many extreme rainfall events on the western side of mountainous areas and northern New Zealand. In these regions, depending on the season, 40–86% of the rainfall totals and 50–98% of extreme rainfall events are shown to be associated with ARs, with the largest contributions predominantly occurring during the austral summer. Furthermore, the median daily rainfall associated with ARs is 2–3 times than that associated with other storms. The results of this study extend the knowledge on the critical roles of ARs on hydrology and highlight the need for further investigation on the landfalling AR physical processes in relation to global circulation features and AR sources, and hydrological hazards caused by ARs in New Zealand. Firstly described by Newell et al.2, ARs are synoptic-scale filaments representing the 4 or 5 largest water vapour fluxes that occur between the midlatitudes and the tropics on any given day, and the length of an AR is generally five times its width. ARs often linked to extratropical cyclones and fronts5, the key mechanisms of precipitation9 and play a critical role in extreme precipitation events10 in the midlatitudes. As part of extratropical cyclones and fronts, ARs have significant hydrological impacts when making landfall for most mid-latitude regions globally, including extreme precipitation events15 and the frequency of the occurrence of droughts and floods16.Atmospheric rivers (ARs) are global phenomena primarily occurring in the midlatitudes, distinguished by filamentary regions of strong horizontal water vapour transport in the atmosphere18, the central United States19, western Europe20, southern south America21, and South Africa22. ARs can contribute to more than 50% of annual or seasonal precipitation totals (cool season in particular) for these regions. Further, there is a strong connection between the heavy precipitation events and ARs in these regions25. For example, storm events  in California between the 1998 and 2008 water years are shown to be all associated with ARs17.ARs significantly contribute to precipitation totals and produce heavy orographic precipitation when passing mountainous regions in the midlatitudes. Notable contributions of ARs to precipitation totals have been documented on the west coast of North America19. Generally, this combined effect determines the location and intensity of heavy rainfall events across the country, commonly on the western side of mountainous areas and northern New Zealand26. For the South Island of the country, orographic precipitation is the major precipitation mechanism27. The highest annual precipitation totals in New Zealand  occur in the Southern Alps28 as this extended mountain range provides a significant barrier to the prevailing airflows30 all year round. For the North Island, frontal systems and extratropical cyclones are the dominant precipitation mechanisms32, yet mountain ranges and volcanic mountain peaks in the North Island also provide obstacles to the prevailing airflows19.Rainfall caused by the combined effect of extratropical cyclones and their associated fronts with orographic lifting is typical in New Zealand19: the westerly wind belt35 to the south and the band of subtropical anticyclones19 to the north. As such, the seasonality of these global circulation features strongly influence the rainfall seasonality in the country37. For example, the overall seasonal pattern of rainfall in the North Island sees higher rainfall in winter (JJA) due to the increased occurrence of frontal systems when the country experiences more westerlies, and lower rainfall in summer (DJF) as the belt move towards the south37. Conversely, the South Island sees the opposite rainfall seasonal pattern19.Because of its geographic location, New Zealand’s climate is governed by two dynamical global circulation features38 and the frequency of drought and flood occurrences16. However, these global studies are unable to reflect regions highly impacted by ARs, especially considering the diverse orography in New Zealand. Additionally, AR frequency and strength is projected to increase by 60% and 20%, respectively, under the most severe future climate scenario in the southern midlatitudes39. To this end, the hydrological impact of ARs is likely to be altered in the country. Therefore, evaluating the impact of ARs on rainfall at a local scale in New Zealand is essential, given the local climate features, the critical role that ARs play in the global water cycle and midlatitudes climate and hydrology, and the changing climate.For New Zealand, several global AR studies have noted a considerable occurrence of landfalling ARs and their association with heavy precipitation eventsIn this study, we hypothesise that this evaluation will be useful for better understanding the causes of both the spatial distribution of annual and seasonal rainfall totals and heavy rainfall events across the country to provide more useful information for better assessment of climate change impacts on hydrology. The objectives of the present study are (1) to investigate the climatology of ARs in New Zealand; (2) to evaluate the fraction of seasonal and annual rainfall totals linked with ARs; (3) to compare the intensity of AR rainfall and non-AR rainfall, and to investigate the association between the ARs and heavy rainfall events.38. An automated AR detection algorithm and six 6-hourly reanalysis datasets  exceeds predefined thresholds with a length greater than 2,000 km and length/width ratio greater than 2ts Table  with var25. A more detailed description of the datasets, AR detection method, and methods to quantify ARs' impact on rainfall totals and daily rainfall intensity for each 654 rain gauge is provided in the Methods section.The assessment of AR impact on rainfall totals and daily rainfall intensity was based on daily rainfall records from 654 rain gauges with a mean length of 19 years from 1979 to 2018. Rain gauge locations are shown in Fig. This section presents the climatology of ERA-interim ARs and characteristics of the detected landfalling ARs at a 0.125° × 0.125° horizontal grid resolution over New Zealand from 1979 to 2018. Results from other databases are shown in the supplementary materials, where the spatial resolution of the other datasets examined is lower than that of ERA-interim. Note that the analysis in this section only represents ARs at some moment of their life cycle as the temporal resolution of all reanalysis datasets is 6 h.The frequency of AR days and the coefficient of variation (CV) of AR days for each grid cell are shown in Fig. Geometric characteristics, mean landfalling AR direction and AR landfall frequency, and seasonal landfalling AR intensity and mean landfalling AR direction are shown in Fig. Landfalling ARs commonly have a northwest direction, the median of the mean direction is about 121.5° (~ 123°) for ERA-Interim Fig. –S6d. MorThis section examines the impact of detected ERA-interim ARs  on rainfall totals and the frequency of wet AR days across New Zealand. Average annual and seasonal rainfall totals are shown in Fig. The average fraction of annual and seasonal rainfall totals linked with ARs for each rain gauge are shown in Fig. The ratio of the median daily AR rainfall over non-AR rainfall at the annual and seasonal scales are shown in Fig. The connection of the most intense rainy days between ARs is explored by considering those days where rainfall exceeds the 90th percentile values of the series of daily rainfall. Figure 38. Overall, ARs are found to have a strong hydrological impact over western areas of mountain ranges and mountain peaks, and northern New Zealand. Note that the discussion on the impact of ARs on rainfall only focuses on these areas.The investigation of the impact of ARs on rainfall in this study is a major step toward understanding the hydrology and extreme rainfall events in New Zealand. In this study, the impact of ARs on rainfall was evaluated using a high-resolution atmospheric reanalysis dataset and a network of station rainfall data, combined with an automated AR detection algorithm introduced by Guan and Waliser38. The filamentary synoptic feature is notable whereby the median length of landfalling ARs is more than 5000 km (over 4700 km) for ECMWF products , and the median length/width ratio is over 8 for all reanalysis datasets  than in winter (JJA)  Figs. . Most laA) Figs. –S6e,f, tA) Figs. –S6f.Overall, 50–65% of the annual and 40–86% of seasonal rainfall totals accumulated only within approximately 30–82 and 5–31 wet AR days respectively Fig. . The res41. As a result, the western areas of the country’s mountain ranges receive higher rainfall amounts26. North Island rainfall is relatively high in winter (JJA) as the frontal systems’ occurrence increases when the country is covered more by the westerly wind belt37. However, the belt moves further south during the summer (DJF) months and leads to lower rainfall in the North Island37. Consequently, the seasonal pattern of rainfall in the two islands differs19. Further study on the landfalling AR physical processes related to global circulation features and source of ARs around New Zealand is needed.Rainfall in the country is strongly influenced by the interaction between the orography and the prevailing airflows10, although the 99th threshold was used in their study.AR storms are more intense than non-AR storms all year round and found to produce 2–20 times the non-AR rainfall depending on the rain gauge location and season Fig. . Particu42. Under the changing climate, the frequency and magnitude of floods are likely to increase as a result of the increased frequency and intensity of extreme rainfall in New Zealand43. Climate change is expected to also lead to increases in the frequency of droughts in vast areas of the North Island44, including regions where ARs are the major contributor to water resources. Moreover, climate change is suggested to result in a 60% and 20% increase in the AR frequency and strength in the southern midlatitudes, respectively39. Therefore, further work on investigating the connection between hydrological hazards and ARs focusing on regions exposed to both ARs and these hazards is needed for a better assessment of climate change impact on natural hazards.Further, results in this study suggest a potential connection between hydrological droughts and floods in northern New Zealand and where the orography dominates the weather progression in the country Figs. –S6f. Dro45 and the most recent reanalysis dataset version ECMWF, namely the ERA-5 reanalysis project46. Atmospheric reanalysis data between 1979 and 2018 from ERA-interim and ERA-5 at 0.125° × 0.125° and 0.25° × 0.25° horizontal grid resolution were retrieved, respectively. Data used are 6-hourly specific humidity and zonal wind and meridional wind components at 20 vertical pressure levels (300 hPa to 1000 hPa), and land-sea mask. Additionally, detected ARs based on ERA-Interim, the Climate Forecast System Reanalysis47 (CFSR), and the Modern-Era Retrospective Analysis for Research and Applications48, Version 2 (MERRA-2) were retrieved from a global AR database38. Note that the AR detection period based on ERA-Interim, CFSR, and MERRA-2 is 1979–2018, 1979–2015 and 1980–2019, respectively, with a 6-hourly temporal resolution at 1.5° × 1.5°, 0.5° × 0.5°(and 1.5° × 1.5°), and 0.5° × 0.625° horizontal grid resolution, respectively. Table 49 as:q, -1), zonal and meridional vectors (m s-1), respectively; g is the gravitational acceleration (9.81 m s−2), and dp (Pa) is the pressure difference between two adjacent atmospheric pressure levels. Note that all parameters in Eqs. .It is common to employ more than one dataset to assess the robustness of AR detection techniques. Datasets used to identify ARs are the European Centre for Medium-Range Weather Forecasts (ECMWF) Interim (ERA-interim)Station-based daily rainfall totals were obtained from New Zealand’s national climate database web system hosted by the National Institute of Water and Atmospheric Research (NIWA). Note that the periods of records differ among sites, and the observation times of the day are 2100 UTC (2000UTC) during the wintertime (summertime). Sites with less than 10 years of daily rainfall records between (September–August) 1979 to 2018 and completeness less than 100% were excluded because the lack of daily rainfall data could potentially lead to under- or overestimations in the hydrological impacts of ARs. Given these conditions, a total of 654 stations with the minimum, maximum, and mean length of 10, 39, and 19 years respectively were selected over the whole country. Figure 38. ERA-interim and ERA-5 ARs were detected based on the AR detection algorithm introduced in Guan and Waliser38. In the algorithm, IVT and IVT threshold (monthly 85th percentile within a 5-month window centred at that month) for each grid cell and the land-sea mask from the two datasets are the inputs. Note that the land-sea mask values were adjusted only to contain grid cells represent New Zealand landmasses . Briefly, criteria of the AR detection algorithm include the IVT intensity at each grid cell within a contiguous region being above the IVT threshold and a fixed limit (IVT > 100 kg m−1 s−1) for that grid cell, the appreciable poleward direction of the AR (IVTy > 50 kg m−1 s−1), and the length of an AR greater than 2000 km with a length/width ratio greater or equal to 2. Outputs from the algorithm include the variables of shape, axis, and landfall location of the ARs in NetCDF files and detailed characteristics of landfalling ARs in text files. The AR shape variable was used to compute the AR area, the axis was used to calculate the AR length, and the width was calculated as the area divided by the length. The landfall location was marked as the first grid cell that the AR axis intersects the coastlines alone the IVT direction and that AR was labelled as a landfalling AR. The detailed description of the AR detection procedure and criteria of this technique are in Guan and Waliser38. Figure CFSR and MERRA-2 ARs were obtained from a global AR database21. Seasonal AR frequency is expressed as the anomalies from the mean seasonal AR days. The coefficients of variation (CV) for annual and seasonal AR days were calculated to evaluate the interannual variation of AR frequency. The mean monthly percentage of landfalling AR occurrence was computed as the mean of the number of landfalling ARs detected for that month over each year within the period of each dataset.The AR frequency was calculated as the average number of days per year that an AR was detected for each grid cell. The calculation was based on the shape of detected ARs. For example, at least one of the four reanalysis time steps per day meeting the AR detection criteria for it to be counted as an AR day21 and rainfall in the next day50  considered as AR-precipitation event, only AR conditions presented at 4 grid points and rainfall occurred in the same day were considered as AR-generated rainfall in the present study.Consider a given station with daily rainfall records (measured at 2000 UTC or 2100 UTC) on 15 June 2000. To determine if the rainfall should be associated with an AR, the first step is to search for AR conditions (based on AR shape) at the four grid points that enclosed the station site between 2000 UTC (2100 UTC) 14 June 2000 and 2000 UTC (2100 UTC) 15 June 2000. If any of the four ERA-interim time steps that AR conditions (based on AR shape) were found in the 4 grid points, the daily rainfall (> 0 mm) recorded between this period is considered to be AR-generated rainfall, and that day herein considered as a wet AR day. Unlike previous studies that any of the four grid points that AR conditions were present21 . Further, the fraction of annual and seasonal highest 10% rainfall events that are attributed to ARs were also investigated.Due to positive skewness in the daily rainfall data (not shown), daily rainfall intensity under AR and non-AR conditions were compared by the median daily rainfallSupplementary Information.