Method: We assessed the hepatic function by using the dynamic LiMAx® test and biochemical parameters in 21 patients with sepsis or septic shock receiving CytoSorb® in a prospective, observational study. Points of measurement: T1: diagnosis of sepsis or septic shock; T2 and T3: 24 h and 48 h after the start of CytoSorb®; T4: 24 h after termination of CytoSorb®. Results: The hepatic biotransformation capacity measured by LiMAx® was severely impaired in up to 95 % of patients. Despite a rapid shock reversal under CytoSorb®, a significant improvement in LiMAx® values appeared from T3 to T4. This decline and recovery of liver function were not reflected by common parameters of hepatic metabolism that remained mostly within the normal range. Conclusions: Hepatic dysfunction can effectively and safely be diagnosed with LiMAx® in ventilated ICU patients under CytoSorb®. Various static liver parameters are of limited use since they do not adequately reflect hepatic dysfunction and impaired hepatic metabolism.Background: Sepsis is often associated with liver dysfunction, which is an indicator of poor outcomes. Specific diagnostic tools that detect hepatic dysfunction in its early stages are scarce. So far, the immune modulatory effects of hemoadsorption with CytoSorb Sepsis is not only one of the most common diseases worldwide, but it is also a leading cause of death . It is aSepsis and septic shock are frequently accompanied by sepsis-associated liver dysfunction (SALD). However, determining the incidence of SALD is difficult due to the lack of a uniformly accepted definition. As a result, a comprehensive registry is missing and the exact incidence is unknown ,11,12.The liver is strongly implicated in the development and amplification of the multiple organ dysfunction syndrome (MODS). The massive hepatic release of cytokines and activation of multiple immunological pathways further compromise the clinical situation ,14,15,16To assess liver function in sepsis, paraclinical static parameters are mostly used, such as elevated serum concentrations of bilirubin, alkaline phosphatase, or aminotransferases. Using these parameters is currently the best approach because a gold standard or a generally available specific test is missing ,10,12,23®) is a non-invasive procedure and a good predictor of mortality in patients with sepsis [® test has been successfully used in the ICU for monitoring antibiotics and for dosing antimycotics [The maximum liver function capacity test  of the cytochrome P450 group [13CO2. The LiMAx® value is calculated based on the maximal change in the isotope ratio of 13CO2 and 12CO2 compared to the individual baseline (delta over baseline (DOB)). With an elevation of hepatic metabolic efficiency, more 13CO2 is exhaled. As a result, DOB and LiMAx® values increase [The LiMAx50 group ,33,34. Mincrease ,34.® value <140 µg/kg/h is associated with a severely impaired hepatic function [® levels may indicate the early recovery of liver function before the static liver parameters start to normalize [Values > 315 μg/kg/h define the normal range. Between 314 µg/kg/h and 140 µg/kg/h, the hepatic metabolic activity is impaired. A LiMAxfunction ,36. An iormalize ,37.® is a high-tech polymer adsorber with a surface area >45,000 m2. It has a strong binding capacity for a variety of predominantly hydrophobic molecules with a molecular weight <55 kDa, such as pro- and anti-inflammatory mediators, immune response-inducing damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs), and pharmacological substances  [CytoSorbc drugs) ,42,43,44® equally reduces equally elevated levels of pro- and anti-inflammatory cytokines in sepsis in a concentration-dependent manner. Thereover this therapeutic principle does not affect a specific pathway of the immunological network [® can potentially decrease mortality [Previous research addressed the possible effects of adsorption of various DAMPs, PAMPs, pro- and anti-inflammatory cytokines. Hemoadsorption with CytoSorb network ,45. The  network , to down network ,45,47,48 network . CytoSorortality ,50,51,52ortality ,53,54,55® to mitigate or accommodate exaggerated and dysregulated immunological responses .This prospective clinical investigation evaluated dynamic and static liver function in patients with sepsis or septic shock, who received hemoadsorption with CytoSorb® in patients with sepsis and septic shock” (MiHaS-Study). The study is listed in the German Clinical Trials Registry (DRKS00017211). Before including the participants in the study, the patients, the respective caregivers or independent consultants gave their written informed consent.The ethics review board of the medical faculty of the Ruhr University Bochum, located in Bad Oeynhausen, approved the study protocol (file number 2019-440). The research project is called “Change of microcirculation and liver function under CytoSorb® were included between March 2020 and March 2021  was reached [® was performed at four defined points of time:All patients received individualized standard sepsis therapy according to current guidelines  and rena reached . On admi reached . The dyn1: Preadsorber: (−4 h) before connection to hemoadsorption with CytoSorb®.T2: 24 h after the start of CytoSorb® therapy.T3: 48 h after the start of CytoSorb® therapy.T4: Postadsorber: 24 h after completion of CytoSorb® therapy.T1 to T4 as part of routine clinical practice: sequential organ failure assessment score (SOFA) [In addition, the following parameters were collected at Te (SOFA) , blood g® Analyzer  the LiMAx® test was performed. Then, 2 mg/kg body weight of 13C-labeled methacetin was injected intravenously and metabolized to paracetamol and 13CO2. The DOB was measured and quantified over 60 min [13CO2 detector via a special adapter. To avoid interactions  and to create stable measurement conditions, renal replacement therapy with hemoadsorption was interrupted during measurements. In addition, the infusion regime, ventilation parameters, and inspiratory oxygen concentration (FiO2) [® tests was at least 24 h.By using the Flipr 60 min ,34. Expin (FiO2)  were kep®  was used. For an adjunctive hemoadsorption, a CytoSorb® adsorber was integrated into the pre-filter position according to individual indication by the intensivist, in accordance with manufacturer recommendations and internal hospital standards. The adsorber was replaced after 8–24 h, depending on the clinical course of the disease . Following current recommendations, the dialysis dose was between 20 and 30 mL/kg/h [® dose (ABP) a maximum blood flow rate of 200 mL/h under citrate anticoagulation was always targeted, depending on the volume status of the patient and pressure conditions of the CVVHD [Renal replacement therapy was always indicated as CVVHD based on relevant guidelines . The mul mL/kg/h ,66,67. The CVVHD ,68.® values during the observation period from T1 to T4. The secondary endpoints included the course of various static liver parameters, IL-6 levels, and the dose of norepinephrine.The primary endpoints were LiMAxhttps://www.r-project.org (accessed on 5 April 2021)). Data were collected electronically using Microsoft Excel . All statistical analyses were performed with the program R, version 4.0.5  or median plus interquartile range (25th and 75th percentiles), whichever was appropriate. p-values were corrected according to Bonferroni. A two-sided p-value < 0.0125 was statistically significant.Group differences (sepsis vs. septic shock) were evaluated with appropriate tests depending on the scale and data distribution: Fisher’s exact test , T-test for unrelated samples , or Mann–Whitney test. The 1–2, T2–3, and T3–4. A two-sided p-value < 0.0167 after α-adjustment by using the Bonferroni correction was set as the statistical threshold.Subgroups (sepsis or septic shock) were analyzed by using Fisher’s exact test , paired-samples T-test , or Wilcoxon signed-rank test. A sliding baseline was chosen for the four points of measurement, comparison Tn = 15) with 71%, pulmonary (n = 3), or other parts (n = 3). No significant differences in baseline characteristics between sepsis and septic shock were found, except for lactate, which was significantly lower in sepsis (p = 0.00002)  were recruited between March 2020 and March 2021 . The med0.00002) .n = 14) of patients developed a septic shock. APACHE-II and SOFA scores at T1 were: 31.19 ± 4.19 and 14, respectively, p = 0.5 and p = 0.28. Patients with septic shock had a 15% higher SOFA score than patients with sepsis. A total of 95% (n = 20) of patients were mechanically ventilated. Parameters of hemoadsorption showed no differences. The achieved ABP was 13.23 ± 1.71 L/kg for the total cohort . A total of 67% (n = 8) of patients with septic shock did not survive the ICU stay, 50% (n = 7) of them died between the 10th and 19th day, and 57% (n = 8) died within 90 days. The observed 28-day- and 90-day-mortality for the total cohort was 33% (n = 7) and 38% (n = 8), respectively, p = 0.032 and p = 0.018. ICU mortality was equal to 90-day-mortality. In summary, every patient, who had been transferred from the ICU to periphery wards, survived.LOS in ICU was 24.38 ± 15.67 days (range 5 to 59 days). All patients with sepsis survived 90 days. A total of 57% (1 for both groups (p = 0.276). There were no differences between sepsis and septic shock (1), 38% (n = 8) of all patients had limited liver injury, another 57% (n = 12) had a severe liver injury and 5% (n = 1) had a normal liver function according to LiMAx® (Dynamic liver function was significantly impaired at Tic shock . Preadsoo LiMAx® . The following results refer to the cohort with sepsis : n = 3) of participants showed a severe liver damage and 43% (n = 3) an impaired hepatic function according to LiMAx® (® decreased from T1 to T2 by 34% (p = 0.148) and tended to recover slowly but not significantly after 48 h of hemoadsorption . From T3 to T4 the mean values of LiMAx® recovered significantly by 69% (p = 0.003) and exceeded the starting point (T1). A total of 43% (o LiMAx® . The meaThe following results refer to the cohort of septic shock :n = 9) of patients had a severely impaired liver function and 36% (n = 5) had an impaired liver function. There was a significant deterioration in the mean value of LiMAx® from T1 to T2 by 45% (p = 0.003). After 48 h under hemoadsorption (T3), the dynamic liver function tended to improve from T2 to T3 (p = 0.025). This mean value recovered significantly from T3 to T4 by 92% (p = 0.001) 24 h after the end of CytoSorb® therapy. T4 had the highest LiMAx® results (T4: 188.36 µg/kg/h).A total of 64% (n = 3) of patients showed hepatic insufficiency (n = 8) of all patients had impaired liver function at T1, and elevated ALT levels were still observed in six patients at T4 (There were no significant differences in static liver parameters observed between sepsis and septic shock at the four points of time . The cumficiency . When rets at T4 . 1 (1.24) to T2 (1.38) by 11% (p = 0.0002), followed by a non-significant decrease to T4 (1.04). There were no significant changes in other static liver parameters. The median of ALT were below the pathological reference range (<50 U/L) and constantly declined over time from T1 to T4 by 38%. Bilirubin levels decreased  during and after CytoSorb® therapy .The following results refer to the cohort with sepsis : INR incThe following results relate to patients with septic shock :1 to T2, the INR increased significantly by 26% (p = 0.006), followed by a slow decrease up to T4. No significant changes were observed over time for ALT, Bilirubin, and AP. At the starting point (T1) ALT was not pathologically elevated based on median (T1: 33.5 U/L). ALT had an upward  followed by a downward (T4) course during the observation period. The median value at T4 was slightly above the normal range, and the 75th percentile was significantly elevated from T1 to T4. A total of 43% (n = 6) of the patients had pathologically elevated ALT levels at T1 and 36% (n = 5) at T4. Bilirubin was within the normal range related to the median and IQR at all points of time. A total of 21% (n = 3) of the patients had hyperbilirubinemia before the start and after the end of hemoadsorption. AP showed an unremarkable course in the normal range from T1 to T3 and increased after hemoadsorption with CytoSorb® from T3 to T4 by about 35% in the median.From T2 norepinephrine dose was 133% higher in patients with septic shock than in patients with sepsis (p = 0.007)  . Norepinsurement . The following results relate to the group of sepsis : ® from T1 to T2 by about 73% (p = 0.016) and after T2 the values dropped continuously. All participants in this group received norepinephrine therapy and norepinephrine initially increased until T2. A significant decrease became apparent after 48 h hemoadsorption from T2 to T3 by 73% (p = 0.016).A significant IL-6 reduction was detecTable 24 h after initiation of CytoSorbThe following results relate to the cohort with septic shock :1 to T2 by 90% (p = 0.0004). Initially, the norepinephrine requirement increased significantly (T1 to T2) by 154% (p = 0.005). After T2 a consistent and significant reduction in norepinephrine could be observed . After the end of hemoadsorption, the dose decreased from T3 to T4 by 69%.There was a reduction in IL-6 from TSepsis can lead to micro- and macrocirculatory vasculopathy ,69,70,71®, hemodynamic stabilization has already been demonstrated by several research projects [® on dynamic and static liver function in sepsis has not yet been investigated in a comparable setting. Under hemoadsorption with CytoSorbprojects ,77,78,79Typical forms of SALD include hypoxic hepatitis, also known as “shock liver” ,81,82, c® proved to be safe, feasible and reliable under mechanical ventilation. It also had the target of exploring its diagnostic potential during hemoadsorption with CytoSorb®. As shown in our study, this dynamic test is a practical, bedside, noninvasive tool [® in critically ill patients. In the present prospective study, LiMAxive tool  to deter® measurement cycle to exclude any possible interaction or recirculation via CRRT and CytoSorb® represents a significant strength of this study contributing to robust data quality. In contrast, previous investigations had used much shorter interruptions of dialysis (60 s) [Pausing renal replacement therapy during the whole 60 min of the LiMAxs (60 s) ,26. ® achieved promising results for the assessment of hepatic function in liver resection, in liver transplantation [®.LiMAxantation ,34,90,91antation ,29,92 anantation ,28. The ® therapy. A hemodynamic stabilization and the reduction in norepinephrine dose occurred 48 h after the start of CytoSorb®, especially in septic shock. According to these results, rapid control of inflammation and rapid shock reversal can be assumed in both groups. This is one of the primary therapeutic goals of hemoadsorption by CytoSorb®. Other objectives are the prevention or limitation of (multi-) organ failure, improvement of endothelial integrity and reduction in leakages through immunomodulation [We observed initially high IL-6 levels followed by a rapid decrease after the initiation of CytoSorbdulation ,93,94,95dulation ,97,98,99dulation ,73. IL-6 levels correlate with liver dysfunction  and infl® [Furthermore, high IL-6 levels can potentially be used as a criterion of indication for a successful treatment with hemoadsorption ,105,106.® . Primari® ,78,97,98® values at the onset of sepsis and septic shock in the vast majority of patients. Furthermore, our results show maximal impairment of CYP1A2 activity measured with LiMAx® 24 h after the start of CytoSorb® treatment, especially in patients with septic shock and extremely elevated IL-6 levels. Despite rapid shock reversal, a significant hepatic improvement was evident 24 h after the end of CytoSorb®. Thus, the SALD recovered significantly at the subcellular level approximately in a median of 106 h (=4.4 days) after the onset of this life-threatening disease. The metabolic function of the liver was still compromised at T4. A return to normal biotransformation capacity (>315 μg/kg/h) was not detectable even 4–5 days after the start of therapy.The study has shown very low pathological LiMAx® value decreased until the 2nd day, followed by a hepatic recovery on the 5th and 10th day [Kaffarnik et al. came to a similar conclusion: after the onset of sepsis, the LiMAx10th day . Compari10th day  vs. 14.310th day .® value < 100 μg/kg/h indicates critical liver failure or terminal liver failure [® can detect early SALD [In general, a LiMAx failure  and pred failure . Study rrly SALD .n = 20) had a reduced CYP 1A2 activity, either an impaired hepatic function  or a severe liver damage . A similar study had comparable LiMAx® readouts [® values in this study, which refer to hepatic damage or failure.After the onset of sepsis, 95%  in the total cohort. Elevated INR levels are associated with coagulopathy, a common symptom of sepsis, which occurs in up to 70% of patients with sepsis [n = 12) than with LiMAx® .We observed pathological INR with an incidence of up to 57%  of the patients in our study. ALT tended to indicate a possible deterioration of liver function, but it does not correlate with the extent of hepatocellular damage [A similar result occurs with ALT. The hepatic integrity parameter was increased in up to 38%  under hemoadsorption with CytoSorb® in sepsis and septic shock. The intention was to create an approach for future research and to generate new hypotheses.The present study was planned as an observational trial and should provide results regarding the feasibility and interpretability of the dynamic liver function test  require extra organizational efforts in order to avoid any interference.The usability of the LiMAx® on hepatic biotransformation cannot be excluded with certainty because the study period was about 5 days.Despite the prospective study design, certain limitations must be considered. All measurements were performed after the onset of sepsis. Earlier comparative values are missing. Longer-term influences of adjuvant hemoadsorption with CytoSorb® in combination with hemoadsorption by CytoSorb® in mechanically ventilated critically ill ICU patients. It appears that the hepatic biotransformation capacity measured by LiMAx® is massively and significantly impaired at the onset of sepsis. A significant hepatic improvement of metabolic capacity occurred 24 h after the end of hemoadsorption but the liver function was still compromised. It is of utmost importance to choose a liver-protective treatment regime to avoid further damage. Hepatic recovery was paramount during rapid shock reversal facilitated by CytoSorb® with a significant decrease in IL-6 and vasopressor support. Whereas static liver parameters do not adequately reflect the hepatic dysfunction and the impaired hepatic metabolism at the onset of sepsis and during hemoadsorption with CytoSorb®. In addition, due to bilirubin removal with CytoSorb®, common liver function assessments such as the SOFA or MELD score cannot be used. The LiMAx® test can reliably help to assess the severity and the course of hepatic dysfunction at the subcellular level and to make therapeutic decisions faster, individualized, and thus optimized.This is the first prospective study to measure LiMAx


Translocation t occurs in approximately 5% of patients with acute myeloid leukemia (AML) corresponding to 11q23/mixed lineage leukemia (MLL) rearrangement. The AF6 gene on chromosome 6q27 is the fusion partner of the MLL gene on 11q23 in t, which results in a poor prognosis. The case of a patient with 11q23/MLL‐rearranged AML who successfully underwent a third allogeneic stem cell transplantation after treatment with azacitidine (AZA) and venetoclax (VEN) is presented in this article. This report suggests that a combination of AZA and VEN is an effective therapeutic approach for relapsed and refractory MLL‐rearranged AML. The MLL gene on 11q23 fuses with the AF6 gene on chromosome 6q27, which results in a poor prognosis , 48, idem, +add(8)[9], 49, idem, +add(8) × 2 [2], and that of BM cells at relapse showed 46XY, inv(2)(p11.2q13), t[2], and 46XX [18], respectively. The results of the fluorescence in situ hybridization (FISH) for the detection of MLL gene rearrangement indicated the existence of 7% of MLL gene‐rearranged cells. This suggests that additional MLL gene rearrangement was involved in the leukemia relapse. Gemtuzumab ozogamicin (GO) monotherapy (3 mg/m2) and VEN (400 mg/day) was initiated as salvage chemotherapy. As AZA and VEN were well tolerated, no treatment interruption or delay were observed . After three cycles of AZA and VEN, BM aspiration indicated hematological CR with no MLL rearrangement as determined using FISH. Moreover, no MLL::AF6 transcript was detected via RT‐PCR in the peripheral blood sample (BM sample could not be assessed due to low DNA quantity). These results demonstrated the three cycles of AZA and VEN most probably yielded molecular CR as suggested by blood cell evaluation by FISH.BM aspirate indicated normocellularity with 11.6% myeloblasts in the follow‐up after 16 months. The G‐banded metaphase analysis showed a more complex karyotype; 47, XY, add (1)(p11), del(1)(p?), −2, del(2)(q?), t, −5, −6, −7, der(11)?t, −13, +der(?)t, +mar1, +mar2, +mar3, +mar4, +mar5[5]46, and XX [15]. FISH results revealed the existence of 37% of MLL rearrangements, whereas the reverse transcription–polymerase chain reaction (RT‐PCR) revealed no mutation of FLT3 internal tandem duplications. GO treatment was resumed, and then hematological CR was achieved after the first course of the GO treatment. On the other hand, FISH results indicated 7% MLL rearrangement. Subsequently, a combination treatment of AZA , melphalan , and 2Gy (day −1) of total body irradiation followed by the infusion of cord blood cells containing 0.89 × 105 /kg CD34‐positive cells. Graft‐versus‐host disease (GVHD) prophylaxis included tacrolimus (from day −1) and mycophenolate mofetil (from day +1). An interim BM aspiration at day +15 showed the early achievement of complete donor chimerism without residual blasts, and no MLL rearrangement was detected by FISH. Neutrophil engraftment was achieved at day +32. Only grade 1 acute GVHD  which was well manageable was developed. BM aspiration at day +85 showed the CR continuation with complete donor chimerism and no MLL::AF6 transcript by RT‐PCR. The patient has maintained CR 13 months after transplantation  showed a highly expressed BCL‐2 that is directly regulated by MLL::AF4 [GSE13159, Figure The combinations of hypomethylating agents (AZA or decitabine) or low‐dose cytarabine and VEN have been considered the standard treatment strategy for patients with previously untreated or relapsed and refractory AML who are ineligible for allo‐SCT because of its high efficacy and low toxicity rate, especially in patients with adverse cytogenetic abnormality , 5. MLL:MLL::AF4 . MoreoveMLL::AF4 , 8. MLL‐All authors contributed to patient care. Taro Edahiro, Hiroshi Ureshino, and Tatsuo Ichinohe wrote the manuscript. All authors approved the final version of the manuscript.TI received consultant fee from AbbVie, Nippon Shinyaku, Repertoire Genesis, and Nihon Kayaku. TI received grants from AbbVie, Nippon Shinyaku, Repertoire Genesis, Asahi Kasei, Chugai pharmaceutical, CBL Behring, Daiichi Sankyo, Eisai, Kyowa Hakko Kirin, Ono Pharmaceutical, Otsuka Pharmaceutical Sumitomo Dainippon Pharma, and Zenyaku Kogyo. TI received honoraria from Bristol‐Myers Squibb, Eisai, FUJIFILM Wako chemicals, Kyowa Hakko Kirin, Novartis Pharma K.K, Ono pharmaceutical, and Pfizer. No other authors declare potential conflicts of interest (COI).All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The patient provided informed consent to publish this case report.Supporting informationClick here for additional data file.


Ipomoea batatas (L.) Lam) have not been elucidated. In this study, an MYB transcription factor gene, IbMYB308, was identified and isolated from sweet potato. Multiple sequence alignment showed that IbMYB308 is a typical R2R3-MYB transcription factor. Further, quantitative real-time PCR (qRT-PCR) analysis revealed that IbMYB308 was expressed in root, stem, and, especially, leaf tissues. Moreover, it showed that IbMYB308 had a tissue-specific profile. The experiment also showed that the expression of IbMYB308 was induced by different abiotic stresses . After a 200 mM NaCl treatment, the expression of several stress-related genes  was upregulation in transgenic plants, and the CAT activity, POD activity, proline content, and protein content in transgenic tobacco had increased, while MDA content had decreased. In conclusion, this study demonstrated that IbMYB308 could improve salt stress tolerance in transgenic tobacco. These findings lay a foundation for future studies on the R2R3-MYB gene family of sweet potato and suggest that IbMYB308 could potentially be used as an important positive factor in transgenic plant breeding to improve salt stress tolerance in sweet potato plants.The MYB  transcription factor family plays an important role in plant growth, development, and response to biotic and abiotic stresses. However, the gene functions of MYB transcription factors in sweet potato ( Ipomoea batatas (L.) Lam) is the seventh most important crop in the world and the fourth most important in China [Sweet potato -mediated signaling pathways [AtMYB96 in Arabidopsis, through coordinated auxin and abscisic acid signal pathways, can increase drought tolerance. The overexpression of GmMYB84 enhances soybean drought tolerance by increasing antioxidant enzyme activities and root elongation [OsMYB91, an R2R3-MYB gene of Oryza sativa, was confirmed to be involved in the tolerance to salt stress and plant growth by enhancing the capacity to scavenge active oxygen (ROS) [TaSIM, enhanced salt tolerance when overexpressed in Arabidopsis [FvMYB24 upregulated the expression of several stress-related genes in response to salt stress, thus enhancing the tolerance of transgenic Arabidopsis [MYB genes that participate in environmental stress responses has been inadequate compared with other plant species.Transcription factors (TFs) are key regulators that have crucial functions in response to various stresses . MYB trathaliana ,13,14, Oa sativa ,16,17, ZZea mays ,19,20, Taestivum ,22,23, Bca napus ,25, Maluomestica ,27, Fragananassa , etc. Anof PP2Cs . AtMYB2 pathways . The oveongation . Salt stongation . OsMYB91en (ROS) . An R2R3bidopsis . In strabidopsis . Many R2IbMYB308, from sweet potato, and the expression pattern and function of the gene were analyzed. Moreover, we developed IbMYB308 overexpression lines, and the contribution of IbMYB308 in response to salt stress in transgenic tobacco was analyzed. This study will potentially contribute to the improvement of sweet potato tolerance to abiotic stresses and of the molecular breeding of crops.In this study, we identified and isolated an R2-R3 MYB family gene, cv. Eshu11 was used to clone IbMYB308 and to characterize its function. Wild-type Nicotiana tabacum cv. Wisconsin 38 (W38) was used for plant transformation. Tobacco was grown in a greenhouse . The sweet potato material cv. Eshu11 was also cultivated in a greenhouse .The drought-tolerant sweet potato material TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  . Each 20 μL contained 4 μL 5 × TransScript® Uni All-in-One SuperMix for qPCR, 1 μL gDNA Remover, 1 μg total RNA, and variable amounts of RNase-free water. Then, the process of PCR was as follows: 42 °C for 15 min, then 80 °C for 5 s. The full cDNA sequence of IbMYB308 was obtained based on previous transcriptome sequencing. Specific primers IbMYB308-F/R (IbMYB308 transcriptome sequencing. The PCR reaction system contained 5 μL 10 × PAGE buffer (Mg2+), 8 μL dNTPs Mix, 1 μL primer IbMYB308-F/R, 0.5 μL LA Taq polymerase, 2 μL cDNA, and 32.5 μL ddH2O. The PCR procedure comprised an initial preheating step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s, with a final extension at 72 °C for 10 min. The PCR products were separated via electrophoresis on a 1% agarose gel, and the target DNA fragments were recovered using an EasyPure® Quick Gel Extraction Kit  according to the manufacturer’s instructions. The resulting fragments were cloned into the pMD19-T vector  and sequenced by TIANYI Company .The total RNA of Eshu11 was extracted using TRIzol reagent  by following the manufacturer’s instructions. The genomic DNA of Eshu11 was extracted by the CTAB method, and the genomic DNA was stored at −20 °C. The quality and quantity of the RNA and DNA were visualized by 1% agarose gel electrophoresis, then a Nano-Drop ND-1000 spectrophotometer  was used for quantification at optical densities of 260 and 280 nm, respectively. The first-strand cDNA synthesis was performed using B308-F/R  were desIbMYB308 promoter sequence was cloned using the genomic walking method. IbMYB308 promoter-specific primers IbMYB308pro-F/R (IbMYB308. The PCR reaction system contained 5 μL 10 × PAGE buffer (Mg2+), 8 μL dNTPs, 1 μL primer IbMYB308pro-F/R, 0.5 μL EasyTaq® polymerase, 2 μL genomic DNA, and 32.5 μL ddH2O. The PCR products were separated via electrophoresis, and the target DNA fragments were recovered using an EasyPure® Quick Gel Extraction Kit  according to the manufacturer’s instructions. All the resulting fragments were cloned into the pMD19-T vector  and then transformed into competent Escherichia coli strain DH5α cells and sequenced by TIANYI Company .The 8pro-F/R  were desIbMYB308-F/R primers were used to amplify the genomic DNA with the following procedure: initial preheating step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 60 s, and extension at 72 °C for 90 s, with a final extension at 72 °C for 10 min. The PCR reaction system contained 5 μL 10 × PAGE buffer (Mg2+), 8 μL dNTPs, 1 μL primer IbMYB308-F/R, 0.5 μL EasyTaq® polymerase, 2 μL genomic DNA, and 32.5 μL ddH2O. The PCR products were separated via electrophoresis on a 1% agarose gel, and the target DNA fragments were recovered using an EasyPure® Quick Gel Extraction Kit  according to the manufacturer’s instructions. The resulting fragments were cloned into the pMD19-T vector  and sequenced by TIANYI Company .IbMYB308 sequence was determined using the BLAST-Protein program in the National Center for Biotechnology Information (NCBI) database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome accessed on 7 July 2022). DNAMAN software  was used to compare the homologous sequence of IbMYB308 in sweet potato and other species. Multiple sequence alignments were performed using MEGA X, and the phylogenetic tree was constructed using the neighbor-joining method with the 1000 bootstrap method. Cis-acting elements of the IbMYB308 promoter sequence were analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 7 July 2022). The conserved domain was identified using SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1 accessed on 7 July 2022).The IbMYB308 were analyzed in different tissues of sweet potato. The specific primers IbMYB308-F1/R1 , and 20% H2O2, respectively. There were three biological replicates for each experiment and three technical replicates for each sample. Sweet potato leaves were collected at 0, 1, 3, 6, 12, and 24 h after treatment and quickly frozen in liquid nitrogen. The timing of the stress treatments was determined according to Zhang’s experimental methods [® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  . According to the manufacturer’s instructions, TransStart® Green qPCR SuperMix (50 × 20 μL reactions) includes 2 × TransStart® Green qPCR SuperMix, Passive Reference Dye (50×), and nuclease-free water. Each 10 μL mixture contained 5 μL TransStart® Green qPCR SuperMix , 0.4 μL of each specific primer, 3.2 μL nuclease-free water, and 1 μL cDNA. The qPCR program comprised preheating at 94 °C for 2 min, followed by 45 cycles of denaturation at 94 °C for 5 s, and annealing at 58 °C for 30 s. The expression levels of IbMYB308 were detected using qRT-PCR analysis conducted on the 7500 Real-Time PCR system . IbActin and NtActin genes were employed as the internal reference genes of sweet potato and tobacco, respectively. The specific primers are shown in The expression patterns of 08-F1/R1  used for methods . The firIbMYB308-F2/R2 primers  to further explore the function of IbMYB308. primers  were useIbMYB308 . The resIbMYB308-GFP with CaMV 35S promoter recombinant plasmid into Agrobacterium tumefaciens EHA105 using the freeze–thaw method. The tobacco was transformed by the leaf disc method [IbMYB308 overexpression in the tobacco plants. Genomic DNA was extracted from the leaves of transgenic and wild-type (WT) plants and amplified under the following conditions: preheating at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 1 min, and finally extension at 72 °C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis to confirm the insertion of IbMYB308 into the transgenic plants. Further, qRT-PCR was used to detect the expression of IbMYB308 in transgenic plants, with the NtActin gene used as the internal reference gene of Nicotiana tabacum [Transgenic tobacco overexpression lines (OE lines) were obtained by transferring the pCAMBIA1300- tabacum . TransgeSOD, POD, APX, and the proline synthesis-related gene, P5CS. The genes’ specific primers were designed by Primer Premier 5. The primer sequences are shown in In vitro identification of the salt tolerance of the transgenic tobacco plants was based on the method of Zhang . The trat-test, and the results are presented as the mean ± standard deviation, with the significance level at p < 0.05 or p < 0.01.The experiments were set in three biological replicates for each experiment and three technical replicates for each sample. IBM SPSS Statistics 26 software was used for statistical analysis; the data were analyzed using one-way ANOVA, two-way ANOVA, or Student’s two-tailed IbMYB308 gene by the amplification of cDNA. The 844 bp full-length cDNA of IbMYB308 contains a 768 bp open reading frame (ORF) that encodes 255 amino acids. The predicted molecular weight (MW) of the resulting protein was 28.95 KDa, and the deduced isoelectric point (PI) and instability index were 6.99 and 56.80, respectively. The genomic sequence of IbMYB308 is 1101 bp in length and contains two exons and one intron  domains at the N-terminus, but there was sequence diversity at the C-terminus, indicating that IbMYB308 is an R2R3-type transcriptions factor. To identify the evolutionary relationship of IbMYB308, a phylogenetic tree containing 34 AtMYBs from Arabidopsis thaliana, seven OsMYBs from Oryza sativa, one ItMYB from Ipomoea triloba, one InMYB from Ipomoea nil, and one NtMYB from Nicotiana tabacum was constructed with MEGA X , one light-responsive element (3-AF1 binding site), one element involved in abscisic acid responsiveness (ABRE), one gibberellin-responsive element (GARE-motif), one element involved in salicylic acid responsiveness (TCA-element), three Myb-binding sites, and one element related to meristem expression (CAT-box), indicating that IbMYB308 may be related to plant stress response  and the WT plants were grown both in MS medium with 200 mM NaCl and in stress-free conditions for four weeks. The WT and transgenic lines showed no significant difference in growth status under stress-free conditions, but the growth status of the transgenic lines was better than that of the WT under salt stress (3 U/g·Fw) was twice that of the other lines. The proline content of transgenic lines and the WT were approximately at the same level under normal treatment , and most transcription factor binding sites (TFBS) are in the region of −1000 bp to +200 bp relative to the TSS [IbMYB308 promoter was cloned , and the expression level also differed with the length of processing time [HpMYB48, HpMYB70, and HpMYB102 were upregulated by NaCl and drought at different points. For example, the expression levels of HpMYB70 were highest after 3 h under NaCl treatment [IbMYB308 was induced by abiotic stress, NaCl, H2O2, and drought  and hydrogen peroxide (H2O2) [IbMYB308 overexpression tobacco plants were obtained to analyze the function of IbMYB308. Three transgenic lines  were selected for functional analysis , and a proline synthesis-related gene, P5CS , such as superoxide anion (Oe (H2O2) ,63. SOD e (H2O2) ,65. Prole (H2O2) . MDA cone (H2O2) ,67. The e (H2O2) . In thisanalysis . It was analysis a. In thine, P5CS . Under sne, P5CS b–e. Protne, P5CS f. Furthet stress . Overexpbidopsis . Heterolt stress .IbMYB308 enhanced the salt tolerance of transgenic tobacco.In general, these data revealed that overexpression of IbMYB308, was isolated from Eshu11. The expression patterns and functional characteristics of IbMYB308 were investigated, and the expression levels of IbMYB308 were induced with NaCl, H2O2, and PEG-6000 treatments. The overexpression of IbMYB308 in tobacco improved the tolerance of transgenic tobacco plants to salt stress. These findings suggest that IbMYB308 plays a role in salt stress responses. Moreover, this study is of potential value as a resource for salt-tolerant sweet potato breeding.R2R3-MYB transcription factors are one of the most important classes of transcriptional regulators in plants. In this study, an R2R3-MYB transcription factor gene from sweet potato,


Seeds sense temperature, nutrient levels and light conditions to inform decision making on the timing of germination. Limited light availability for photoblastic species results in irregular germination timing and losses of population germination percentage. Seed industries are therefore looking for interventions to mitigate this risk. A growing area of research is water treated with gas plasma (GPAW), in which the formed solution is a complex consisting of reactive oxygen and nitrogen species. Gas plasma technology is widely used for sterilisation and is an emerging technology in the food processing industry. The use of the GPAW on seeds has previously led to an increase in germination performance, often attributed to bolstered antioxidant defence mechanisms. However, there is a limited understanding of how the solution may influence the mechanisms that govern seed dormancy and whether photoreceptor-driven germination mechanisms are affected. In our work, we studied how GPAW can influence the mechanisms that govern photo-dependent dormancy, isolating the effects at low fluence response (LFR) and very low fluence response (VLFR). The two defined light intensity thresholds affect germination through different phytochrome photoreceptors, PHYB and PHYA, respectively; we found that GPAW showed a significant increase in population germination percentage under VLFR and further described how each treatment affects key physiological regulators. Nicotiana tabacum. PHYA is the sole member of the ‘light-labile’ phytochrome group known to mediate light effects at extremely low light intensities (photon fluences). These are described as a very low fluence response (VLFR), which is irreversibly activated through exposure to both red and far-red light [The seed’s role is to disperse and protect the embryo before determining an appropriate time to germinate in favourable conditions . Seeds aed light ,10. PHYBed light ,12. Thered light . These photoreceptors are integrated within the seed’s molecular network, which regulates the phytohormone ABA’s and GA’s concentration balance . Photo-dDatura ferox seeds, VLFR triggers the activation of endosperm-weakening-related enzymes, endo-beta-mannanase and beta-mannosidase [GA3ox gene, suggesting that the promotion of germination by VLFR is associated with an increase in the synthesis of active gibberellins. Barros-Galvão et al. [ABI4, which in turn repressed NCED6 and NCED9 in Arabidopsis thaliana, thus stunting ABA accumulation. The downstream genes of the phytohormone pathways, which alleviate the mechanical resistance imposed on the micropylar endosperm that dictates germination timing, are strong indicators of dormancy release [EXPA) and xyloglucanendo-transglycosylase/hydrolases (XTH) are up-regulated within the micropylar endosperm of non-dormant seeds through a GA-induced mechanism [EXPA and XTH expression, thus preventing germination.It is known that the PHYA-dependent control of germination and the PHYB-dependent control of germination are spatially and temporally separated in the endosperm and embryo, respectively. It is also known that they occur at different stages of germination, and that PHYB predominantly triggers red/far-red light-reversible seed germination and PHYA mediates distinct VLFR in red and far-red light ,10,13,17nosidase . It also release . Genes fechanism . PIF1, w2O, creating GPAW with a chemistry that depends on the original gas mixture used in the plasma and the preparation conditions [2), and nitric oxide (·NO), as well as longer-life chemical products such as hydrogen peroxide (H2O2), nitrate (NO3−), and nitrite (NO2−). Furthermore, removing nitrogen from the gas mixture and replacing it with an inert carrier gas, such as helium or argon, suppresses the synthesis of nitrogen-based species and enhances the production of reactive oxygen species [Gas plasma technology is a growing sector that is receiving increasing attention for its applications in sterilisation and agriculture, where it has been shown to enhance seed germination and plant development ,22. Amonnditions ,27. Usin species .A. thaliana [To date, the majority of research on how non-thermal atmospheric gas plasma improves seeds germination performance has been focused on direct plasma treatment . More lithaliana , as wellthaliana . HoweverNicotiana tabacum [In this study, we exploited a proven model system of photo-dependent dormancy,  tabacum , to inveNicotiana tabacum has a two-step germination process, in which the ruptures of the testa (seed coat) and the endosperm are separate events  increased by 45% (p < 0.0001) and endosperm rupture (ER) increased by 20% (p = 0.003) compared to the after-ripened control seeds and He/O2 GPAW-treated seeds  intensity using the system described in N. tabacum seeds intended for germination testing. The stack of petri dishes was wrapped in opaque black card to limit light access to the top petri dish. Each petri dish filtered out 55.8% ± 2.4 of photosynthetic active radiation, creating an exponential decay of light availability through each petri dish layer   and low fluence response (LFR), caused significant differences in TR and ER percentages for both GPAW treatments when compared to the control . Both GP < 0.05) a. For ER control b.−2 s−1), Air GPAW treatment resulted in a mean percentage differences with respect to the control of 62.2%, 68.0%, 71.6% and 60.5% at 0.06, 0.16, 0.28, and 0.7 µmol m−2 s−1, respectively. Across the LFR light intensity range, Air GPAW also showed significant improvements at 1.5, 4, 8.8, and 58.1 µmol m−2 s−1 that, respectively, led to significant 56.0%, 40.01%, 19.3%, and 20.4% mean differences with respect to the control. Air GPAW contains a significant concentration of nitrates, which could contribute to the observed response. However, chemically prepared nitrate solutions with concentration similar to that found in Air GPAW (5 mM KNO3−) resulted in smaller differences with respect to the control. In particular, differences of 39.3%, 31.7%, 9.5% and 13.4% at, respectively, 0.06, 0.16, 0.28, and 0.7 µmol m−2 s−1  were observed. No significant improvement was detected in LFR conditions (>1 µmol m−2 s−1)  a.N. tabacum seeds followed the same trend . At higher light intensities (LFR), the effects were not statistically significant. Chemically prepared nitrate solutions only caused a small change in ER at 0.06 µmol m−2 s−1 , but no further significant changes were detected across higher light intensities.The effect of Air GPAW under varying light intensities on the ER of me trend b. Under 2 GPAW also displayed notable effects on dormant N. tabacum seeds across the full range of light intensities. As shown in −2 s−1, there were significant increases in TR of 36.9%, 31% and 54%, respectively. Under LFR conditions, the differences were smaller but remained significant at 1.46, 3.99 and 58.1 µmol m−2 s−1 with increases in the mean TR by 45.3%, 36.3% and 21.1%, respectively. In He/O2 GPAW, the main long-living reactive species found in the solution was H2O2. However, the use of chemically prepared H2O2 solutions (300 µM) led to no significant improvement with respect to the control, although the He/O2 GPAW led to clear improvements in TR performance (He/Oformance a.2 GPAW under VLFR light conditions significantly increased the ER mean (p < 0.05) by 23.6%, 16.6% and 17.5% at 0.06, 0.16 and 0.28 µmol m−2 s−1, respectively, in contrast to the control. Under VLFR conditions, the He/O2 GPAW treatment led to significant ER improvements in contrast to H2O2. Under LFR conditions >1 µmol m−2 s−1, no significant differences were detected between He/O2 GPAW and either the control or H2O.The same pattern of results was demonstrated 5 days post imbibition for ER b. He/O2 GA2ox2 relative gene expression differed across treatment groups 24 and 48 h post imbibition. In total darkness, the expression of GA2ox2 was low, with no statistically significant differences between treatments.The expression of genes closely associated with seed dormancy were analysed across three distinct light conditions (darkness and VLFR- and LFR-intensity white light). GA2ox2 was higher than that in darkness. No significant difference was exhibited in nitrogen-containing treatments at 24 h (Air GPAW and Nitrate); however, the He/O2 GPAW treatment significantly reduced GA2ox2 expression by 49.9%, and H2O2 showed a 25.7% reduction (p < 0.05). Under full light, GA2ox2 expression was down-regulated (48 h) and showed no significant differences between treatments and the control except for H2O2 at 24 h   b.GA3ox2 in darkness, there was no difference in relative gene expression between any treatment groups at 24 h; however, after 48 h of imbibition, Air GPAW increased GA3ox2 transcript abundance by 326.1% (p < 0.0001) with respect to the control and by 160.7% (p < 0.0001) with respect to the nitrate solution. Similarly, in VLFR conditions, only Air GPAW treatment increased the expression of GA3ox2 at 48 h by 200.7% (p < 0.05) with respect to the control and by 60.6% with respect to the nitrate solution (p < 0.05)  c,d. TherGA3ox2 with respect to the control , He/O2 GPAW caused an increase of 81.7 % (p < 0.05), Air GPAW caused an increase of 55.6% (p < 0.05), and H2O2 caused an increase of 34.6% (p < 0.05) (p < 0.05), whilst He/O2 GPAW demonstrated a 35.4% increase with respect to H2O2 (p < 0.05) (2O2 treatment caused a significantly lower relative expression of GA3ox2 in N. tabacum seeds in contrast to the control (H2O2: −43.7%)  c. Theref < 0.05) c. After  −43.7%) d.EXPA4 gene showed a significant increase in expression in the light conditions compared to dark or VFLR conditions across all treatment sets, particularly after 48 h of imbibition  (p < 0.0001). Under full light conditions, there were no significantly different results in EXPA4 expression at either time point. The relative expression of PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) was increased in light conditions compared to darkness after 24 and 48 h, but there were no significant differences between the treatments  b. Furtheeatments c,d. We pdormancy . The proN. tabacum, such as Havana 425, germination is photo-dependent and requires light [N. tabacum dormancy and germination is, in part, under the control of the PHYA and PHYB systems. Recent work by Dong et al. [Nicotiana species are known to have one PHYA gene and, due to duplications during Solanaceae genome evolution, several PHYB genes [Lactuca sativa Grand Rapids; this is a low fluence response (LFR) that requires a photon fluence rate of 1–1000 µmol m−2 s−1. For A. thaliana, it is known that PHYA breaks dormancy and induces germination via the very low fluence response  in wavelengths from across the whole visible spectrum [A. thaliana by interacting with signalling pathways targeting gibberellin and abscisic acid metabolism, as well as the expression of downstream cell wall remodelling genes.In positively photoblastic varieties of es light . Accordig et al.  demonstrYB genes ,37,38,39YB genes ,40,41,42YB genes  using Laspectrum . Recentlspectrum  revealed2 GPAW had a clear positive influence on both TR and ER percentages under VLFR conditions. What is interesting is that we observed germination-stimulating effects from both GPAW samples used in this study, Air GPAW and He/O2-GPAW. Air GPAW has a significant concentration of nitrate  and a low H2O2 concentration  in contrast to He/O2 GPAW, which had a very low nitrate concentration (18 µM) and a relatively high H2O2 concentration (294 µM) [3−), nitric oxide (·NO), hydrogen peroxide (H2O2) and transient species such as hydroxyl radicals (OH·). Reactive oxygen species (ROS) signalling leads to the induction of the GA3ox genes in seeds catalysing the biosynthesis of GA. Apoplastic ROS (aROS) produced in the cell wall are involved in embryo growth and endosperm weakening. Crosstalk between reactive nitrogen species (RNS) and phytohormones plays a prominent role in seed germination.Our results  showed t(294 µM) . It is t3− solution; the concentration of nitrate was similar to that measured in Air GPAW. The same can be said for He/O2-GPAW, which led to significant improvements across the whole range of light conditions in contrast to the H2O2 treatment, with a similar concentration to that measured in He/O2 GPAW. This is evidence that Air GPAW and He/O2 GPAW affect germination across light conditions in distinct ways that are also distinct from the known effects of NO3− and H2O2, respectively. Potentially, GPAW treatments could influence germination performance by influencing mechanisms regulating dormancy that bypass the light requirement for germination, positively influencing the mechanism downstream of phytochromes (including VLFR PHYA activation) and/or affecting the physiological regulation of germination directly, e.g., by releasing the mechanical resistance imposed on the micropylar endosperm by the seed-covering layers [Air GPAW showed improvement not only across a range of VLFR and LFR light conditions compared to the control but also in contrast to seeds treated with a 5 mM KNOg layers ,19,31,44A. thaliana [N. tabacum seeds [A. thaliana that light-activated phytochromes (A and B) interact with and promote the degradation or repression of negative germination regulators such as PHYTOCHROME-INTERACTING-FACTOR 1 (PIF1), REVEILLE 1 (REV1) and REVEILLE 2 (REV2) [GA3ox1 and GA3ox2, two key enzymes that regulate the rate of bioactive GA biosynthesis. Furthermore, PIF1 has been shown to up-regulate the catabolic gene GA2ox2 and two GA repressor (DELLA) genes. PHYTOCHROME-INTERACTING-FACTOR 4 (PIF4) is known to be an integrator of light and temperature cues [N. tabacum seed gene expression pattern analysis does not provide any evidence for PIF4’s involvement in the mode of action of different GPAWs. Depending on the species, plants contain a varying set of PIFs, with eight members in A. thaliana and a similar number in Solanaceous species [Nitrate, nitric acid and nitric oxide (⋅NO) are well-documented as compounds to attenuate dormancy and increase the germination of photo-dormant seeds, a phenomenon that seemingly depends on crosstalk with the abscisic acid (ABA) signalling network. In the case of Air GPAW, bypassing the light requirement for germination cannot simply be attributed to nitrate signalling through the NLP8, CYP707A2 mechanism described for thaliana , becauseum seeds . The GA um seeds . The revum seeds . It has 2 (REV2) . PIF1, Rure cues . However species . Future GA2ox2 showed significant down-regulation under very low fluence light conditions and GA3ox2 was up-regulated in both dark and very low fluence conditions   such as EXPA and XTH would be expected, which is in agreement with our results . There wination) b. Under or EXPA4  except f pathway ,54,55. 2 GPAW was most influential in VLFR light conditions by suppressing GA2ox2 expression at 24 h in contrast to all treatment groups, including H2O2 (2 GPAW through both primary and secondary reactions provide a more oxidative environment that triggers the down-regulation of GA2ox2 under VLFR conditions. This idea is supported by previous work that demonstrated that H2O2 significantly down-regulated GA2ox2 expression in N. tabacum [2O2 and He/O2 GPAW both led to an up-regulation of GA3ox2 expression at 24 h (3 by H2O2 (50 mM) treatment [2O2 is believed to be a major regulator of GA biosynthesis in ROS dormancy-alleviation mechanisms. However, our results suggest that other reactive species are significant factors as well.He/Oing H2O2 a. This c tabacum . H2O2 an at 24 h c, which reatment . H2O2 is2 GPAW likely increased germination performance under VLFR conditions due to ROS signalling; however, it is also possible that GPAW bypasses the requirement for light by directly promoting ER weakening—a limiting factor to seed germination in N. tabacum [2.−, H2O2, and ·OH accumulate in ER during weakening and in the growing embryo to aid cell-wall weakening during seed germination [N. tabacum, a common model for photo-dependent, physiologically dormant seeds. This discovery has many potential applications. Seed dormancy and its regulation is a survival mechanism essential for adapting to and colonising new environments and for sustainably intensifying agricultural production. Our results offer several exciting avenues of research into the details of the underpinning mechanisms that should be pursued further. He/O tabacum ,44. ROS mination . GPAW diNicotiana tabacum L., cultivar Havana 425, seeds were produced through a fresh propagation. Plants were grown in a rotation of 28/20 °C in a 16/8 h cycle, respectively. The harvest of mature seeds took place ca. 40 d after pollination when brown capsules were mature, and then seeds were dried at 15% relative humidity (RH) before being frozen in airtight containers at −20 °C. Seeds were defrosted at room temperature 2 h prior to experiments. For after-ripening, seeds were placed within an environment of 33% RH at 20 °C for specified periods in each assay .Dormant 2 plasmas that were sustained using a dielectric barrier discharge (DBD) reactor [GPAW was produced by exposing distilled water to non-equilibrium air and He/O reactor ,58. A mi2 GPAW treatment, a mixture of 98% helium (BOC N4.6) and 2% oxygen (BOC N5.0) was used at a flow rate of 1 standard litre per second (SLPM); for this treatment, the reactor operated at 8.0 kV 30.1 kHz. For the air treatment, compressed air  was used at a flow rate of 1 standard litre per second (SLPM); for this treatment, the reactor operated at 18 kV 24.3k Hz. The voltage and frequency were measured using a Tektronix P6015A high voltage probe and a TBS 1102B Digital Oscilloscope, respectively.For the He/O2 GPAWs is shown in 2O2 was quantified using a modified method first described by Eisenberg [2O2 standards or GPAW in a 96-well transparent plate . This reacted with H2O2 in solution to form pertitanic acid, which could be optically detected. In particular, the substrate has a light absorption peak at 407 nm, which was recorded using a Tecan Spark 10 M plate reader . A standard curve with a range of 0–10 mM was used to calculate the molar extinction coefficient. The chemical characterisation of the Air and He/Oisenberg . Titaniu2− and NO3− were quantified by a Griess-reagent-based method, as demonstrated by García-Robledo et al. [NOo et al. . A standHydroxyl radicals were quantified using terephthalic acid (4 mM), which could be detected through fluorescence (excitation 315 nm and emission at 425 nm) when hydroxylated to 2-hydroxy terephthalic acid (HTA). The reaction was buffered to pH 6.8 using a potassium phosphate buffer (20 mM), and a standard curve was produced to calculate the molar extinction coefficient. A 45 min reaction time was chosen for all experiments.−2 s−1, as indicated in Germination experiments were conducted in four biological replicates. Approximately 40 seeds were placed in 6 cm petri dishes with two ⌀ 5.5 cm cellulose filter papers  and 1.5 mL of the specified treatment solution. For the incubation in continuous darkness, seeds were plated under a green light condition and wrapped with two sheets of aluminium foil. Germination assays in the full light were carried out under continuous white light  at a fluence rate of 111 µmol mTesta rupture was visually recorded using a microscope. A crack in the dark testa with clear exposure of the endosperm determined a rupture. Endosperm rupture was counted when the radicle protruded the covering endosperm. For statistical comparisons, replicate data points were analysed through ANOVA and Tukey’s analysis. −1 while force and displacement were recorded simultaneously. The micropylar endosperm was measured at 3 h (no TR) and 36 h (TR).Puncture force experiments were performed as described previously . ImbibedFor each sample, 20 mg of seeds were collected at specified times, frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using the RNAqueous™ Total RNA Isolation Kit (Invitrogen) according to manufacturer’s instructions. Quantification and purity were measured via a Tecan 10 M plate reader, and only samples with OD ratios of at least 2.2 (260/280 nm) and 2 (260/230 nm) were used for cDNA synthesis. cDNA was synthesised from 1 µg of total RNA using a Superscript III reverse transcriptase (Invitrogen), as described by Grainge et al. .RT-qPCR was performed as described by Grainge et al.  based on


Citrus changshan-huyou, known as Quzhou Fructus Aurantii (QFA), have been commonly used for respiratory diseases. The purpose of this study was to investigate their active compounds and demonstrate their mechanism in the treatment of upper respiratory tract infections (URTIs) through network pharmacology and molecular docking.  The ripe fruits of  The prominent compounds of QFA were acquired from TCMSP database. Their targets were retrieved from SwissTargetPrediction database, and target genes associated with URTIs were collected from DisGeNET and GeneCards databases. The target protein-protein interaction (PPI) network was constructed by using STRING database and Cytoscape. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched. Visual compound-target-pathway network was established with Cytoscape. The effects of compounds were verified on the inhibitory activities against phosphoinositide 3-kinases (PI3Ks). Finally, the molecular docking was carried out to confirm the binding affinity of the bioactive compounds and target proteins. In vitro assays and molecular docking exhibited that TAN, LUT, and AUR acted as PI3Kγ inhibitors.  Five important active compounds, naringenin (NAR), tangeretin (TAN), luteolin (LUT), hesperetin (HES), and auraptene (AUR), were obtained. The enrichment analysis demonstrated that the pathways associated with inflammation mainly contained PI3K/Akt signalling pathway, TNF signalling pathway, and so on. The most important targets covering inflammation-related proteins might be PI3Ks. γ inhibitors, probably contributing to a crucial role in treatment of URTIs. The results revealed that QFA could treat URTIs through a multi-compound, multi-target, multi-pathway network, in which TAN, LUT, and AUR acted as PI3K Complicpathways –7.Citrus changshan-huyou Y. B. Chang, are commonly used for respiratory diseases (such as dry cough after catching a cold) and digestive system diseases [Quzhou Fructus Aurantii (QFA), formerly known as Changshan Huyou, the ripe fruits of diseases , 9. Accodiseases . Recentldiseases  and couldiseases –14. The diseases , 16. Howin vitro. At last, molecular docking was also applied to observing the binding affinity of ligand-target to confirm the inhibitory active compounds and targets.With the support of the local government, the research and development of QFA are highly expected. TCM is characterized by comprehensive medical effects with complex matrices and multiple therapeutic targets. Therefore, it is difficult to elucidate the underlying molecular mechanisms. Compared with experimental research, computational biology research is an option to identify targets and signalling pathway in less time. In the present study, network pharmacology was used for evaluating molecular mechanisms by analyzing the main active compounds and targets of QFA. The inhibitory activities of main active compounds on targets were verified α (p110α/p85α) was purchased from Invitrogen . PI3Kβ (p110β/p85α) was purchased from Eurofins . PI3Kγ (p120γ) and PI3Kδ (p110δ/p85α) were purchased from Sigma-Aldrich . ADP-Glo Kinase Assay Kit was purchased from Promega . PIP2 was purchased from Life Technologies . Dimethyl sulfoxide (DMSO) and ethylenediaminetetraacetic acid disodium salt (EDTA) were purchased from Sigma-Aldrich . PI103 (Lot number: 3 A/122414), a multi-targeted PI3K inhibitor, was purchased from Tocris Bioscience .Naringenin , tangeretin , luteolin , hesperetin , and auraptene  were purchased from Chengdu Push Bio-technology Co., Ltd. . PI3Khttps://pubchem.ncbi.nlm.nih.gov/) was used for retrieving the 2D chemical structure. Traditional Chinese Medicine Systems Pharmacology Database with Analysis Platform  provided pharmacokinetic properties for natural compounds involving oral bioavailability, drug-likeness, intestinal epithelial permeability, blood-brain barrier, and aqueous solubility. By entering the molecule name contained in QFA in the search box to search, the compounds with oral bioavailability (OB) ≥20% and drug-like components ≥0.18 were displayed. Then the active compounds were analyzed to identify biological target genes in the SwissTargetPrediction database (https://www.swisstargetprediction.ch/).The PubChem database (https://www.disgenet.org/home/) and GeneCards database (https://www.genecards.org/) were used for collecting URTIs encoding genes with “upper respiratory tract infection,” “bronchitis,” and “pharyngitis” as keywords as described in the literature. Venny 2.1.0 (bioinfogp.cnb.csic.es/tools/venny/) was used to map drug targets of QFA to the disease targets of URTIs.The DisGeNET database (https://www.string-db.org/) to obtain the targets PPI network. Cytoscape 3.7.1 was applied to constructing the PPI network and Compound-Disease-Target network. Network Analyzer, a network topology analysis plug-in in Cytoscape, was used for topological analysis of PPI networks.The potential targets were input into STRING database (https://metascape.org/) and selected the species as “Homo sapiens.” To perform GO, threshold was set to a p value <0.01, a minimum count of 3, and an enrichment factor >1.5 [p value <0.01, a minimum count of 3, and an enrichment factor >1.5 were selected.The potential targets were input into Metascape databases  were used for the assay. Compounds in DMSO solution and PI3 Kinases solution were diluted and transferred to assay plate by Echo . Test compounds were diluted to the highest concentration (40 μM) and then diluted to the final concentrations of 20, 10, 5.0, 2.5, 1.25, and 0.625 μM, respectively. 50 nL of each of them was transferred to a 384-well plate as assay plate. Kinase solutions of PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ were diluted to concentrations of 0.30, 0.60, 5.00, and 2.40 μg/mL , respectively. 2.5 μL of kinase solution was added to each well of the assay plate except for control wells (2.5 μL of 1 × kinase buffer was added instead). After shaking the plate, 2.5 μL of substrate solution was added to each well to start reaction, and the final concentrations of PIP2 and ATP were 50 μM and 25 μM, respectively. The assay plate was covered and incubated at room temperature for 1 h. 5 μL of ADP-Glo reagent was then added to each well to stop the reaction. Subsequently, the mixture was treated briefly with centrifuge rotor , shaken slowly, and equilibrated for 120 min. Then, 10 μL kinase detection solutions was add to each well, followed by equilibration for 30 min before reading on a plate reader for luminescence. Finally, conversion data was collected on Envision  and relative light unit (RLU) values were converted to inhibition values using the formula of (sample RLU-min)/(max-min) × 100. Herein, “min” means the RLU of no enzyme control and “max” means the RLU of DMSO control. The results were shown as graph in percentage of kinase inhibition in comparison with positive and negative control, and the IC50 values were calculated.The ADP-Glo™ Kinase Assay is a luminescent kinase assay that measures ADP caused by a kinase reaction. ADP-Glo™ Kinase Assay Kit and PI3 Kinases (PI3Khttps://www.rcsb.org/). The docking calculations were performed by the Flex Dock program within SYBYL-X 2.0. PyMOL software  was used for performing ligand/receptor analysis.The 3D structure of active substance of QFA was modelled by utilizing the sketcher toolbars within SYBYL-X 2.0 software. The receptor was searched from the RCSB PDB database  and drug-likeness (DL) criteria, six compounds were retrieved: naringenin (NAR), tangeretin (TAN), luteolin (LUT), nobiletin (NOB), hesperetin (HES), and auraptene (AUR) . Based oSix compounds corresponded to 92, 103, 103, 9, 103, and 113 targets from the SwissTargetPrediction database, respectively. After removing the duplicated genes, a total of 287 targets were finally retrieved from the SwissTargetPrediction database.1951 possible treatment targets were retrieved after removing duplicated targets from DisGeNET and GeneCards database. Further, by Venny 2.1 drawing software, the 287 druggable targets were mapped to 1951 URTIs-related disease targets. Finally, 117 target proteins of corresponding active compounds of QFA were identified to be potential targets in treatment of URTIs. Compound-Disease-Target network is shown in The 117 hub genes were further analyzed on the STRING online data platform and network establishment, and then the data was input into Cytoscape 3.7.1 to obtain the PPI network , which cp value parameter (p ≤ 0.01). Among them, a total of 1797 GO Biological Processes (BP), 92 GO Cellular Components (CC), and 140 GO Molecular Functions (MF) were enriched. The top 10 terms, which are remarkably enriched, are shown in Figures GO terms were selected according to the κB signalling pathways.To investigate integral regulation of URTIs-associated inflammation by QFA, a total of 299 pathways were obtained from database. The top 10 channels are displayed in The compound-target-pathway network was performed by Cytoscape3.7.1 software, which contained 88 nodes  and 325 edges in total . It showin vitro. The IC50 values of five compounds towards Class I α, β, δ, and γ isoforms of human PI3 kinases were determined in the range of 0.625 to 40 μM by using Promega ADP-Glo Kinase Assay Kit. As shown in α, β, δ, and γ isoforms. HES and NAR had no inhibitory activities with IC50 value > 40 μM against PI3Kα, β, δ, and γ isoforms. LUT displayed concentration-dependently inhibitory activities against PI3Ks with IC50 2.49 μM for PI3Kα, 2.95 μM for PI3Kβ, 5.79 μM for PI3Kγ, and 1.55 μM for PI3Kδ  through six hydrogen bonds, in which the oxygen atom at the pyran  are the most intensively studied compared with the Class II PI3K isoforms  and a single Class III PI3K, which consists of a p85 regulatory subunit and a p110 catalytic subunit coded by PIK3CA. Class I PI3K isoforms can be further classified into Class IA isoforms PI3Kα, β, and δ  and Class IB isoform PI3Kγ (p110γ) according to different activating models [α and β are ubiquitously expressed, and PI3Kγ is expressed in granulocytes, monocytes, and macrophages, whereas the PI3Kδ isoform is found in B- and T-cells.PI3Ks are the negative regulators involved in regulating anti-inflammatory responses, immune response, neurodegenerative diseases, cardiovascular diseases, and tumor . Among tg models . As we kγ is expressed at very low levels under physiological conditions, while it is significantly upregulated after stress. After stress in pathological state, PI3Kγ is constitutively enriched in leukocytes. In the airways, PI3Kγ behaves as a trigger or a target in a plethora of respiratory diseases, especially in airway inflammation including ALI, pulmonary fibrosis, asthma, and cystic fibrosis [γ knockout in mouse models of respiratory diseases led to strengthening immunological function and improving airway inflammation, suggesting that PI3Kγ is a therapeutic target in inflammatory-driven respiratory diseases. Multiple lines of evidence demonstrated that inhibition of PI3Kγ activity attenuated ventilator-induced lung damage, idiopathic pulmonary fibrosis, and asthma [γ has been proved to play a pivotal role in respiratory tract inflammation.Class IA PI3K isoforms are particularly implicated in human cancers . Class Ifibrosis . PI3Kγ kd asthma ,27. In s50 2.49 μM for PI3Kα, 2.95 μM for PI3Kβ, 5.79 μM for PI3Kγ, and 1.55 μM for PI3Kδ. It is the first time that LUT has been reported as a pan-PI3K inhibitor at kinase level. PI3Ks were considered to be the molecular target for the antimetastatic effect of LUT, because LUT can directly inhibit PI3K activities and subsequently attenuate phosphorylation of Akt [Among the active compounds from QFA, LUT has been described as an anticancer agent, as well as a potent antioxidant and a neuroprotective agent , which wn of Akt . Moreoven of Akt .γ-selective inhibitor, which might be a therapeutic drug to treat URTIs. Other studies reported that TAN could impede human respiratory syncytial virus infections and inflammation [TAN, a flavonoid derived from citrus, has been demonstrated to exhibit neuroprotective, antidiabetic, antioxidant, and anti-inflammatory effects –34. Accoammation  and haveammation . Moreoveammation .in vitro inhibition of PI3Ks showed that AUR was a PI3Kγ-selective inhibitor which exhibited stronger activity than TAN, suggesting that AUR has a therapeutic effect on URTIs-associated inflammation. Other studies had confirmed that AUR suppressed inflammatory responses in activated RAW 264 macrophages [γ) and NF-κB, and reducing nitric oxide (NO) production in phytohemagglutinin (PHA) stimulation [AUR, a dietary coumarin in citrus fruits, has been found to exert valuable pharmacological properties as anticancer, antibacterial, antiprotozoal, antifungal, anti-inflammatory, and antioxidant agent , 39. Furrophages , 41. It mulation .α level was upregulated in asthmatic patients [According to the KEGG enrichment analysis, TNF signalling pathway was also the related pathway involved in URTIs-associated inflammation. TNF is a key proinflammatory cytokine and has been implicated in many inflammatory lung pathophysiology, including asthma, chronic bronchitis, chronic obstructive pulmonary diseases (COPD), ALI, and ARDS . Severalpatients , 45. Fropatients . When thpatients . Our stuκB signalling pathway was also involved in URTIs-associated infection. NF-κB is a primary transcription factor which presents in the cytoplasm with no transcriptional activity in the resting state. After stimulation, phosphorylation of IκB by IκB kinase results in nuclear translocation of NF-κB, which regulates immune, inflammatory, cell proliferation, and apoptosis responses [β, and TNF-α in LPS-induced RAW 264.7 cells by inhibiting the NF-κB signalling pathway [κB signalling pathways.In addition to the above two signalling pathways, NF-esponses . Li et a pathway . On the in vitro assays and molecular docking results confirmed that TAN, LUT, and AUR, as PI3Kγ inhibitors, played a crucial role in respiratory tract inflammation. This paper preliminarily released the material basis and mechanism of QFA in treating URTIs and provided some guidance for the further research on the treatment of URTIs.By using the method of network pharmacological analyses, five main active compounds  of QFA were found to act on many target proteins of URTIs, which reflected the characteristics of multi-compound, multi-target, and multi-action pathways of TCM. The inflammation-related proteins like AKT and PI3Ks played a dominant role in the PPI network and in the compound-target-pathway network, suggesting that the mechanism of QFA for the treatment of URTIs was closely related to PI3K/Akt signalling pathway. The result of


Asthma is among the most common chronic diseases worldwide, creating a substantial healthcare burden. In late-onset asthma, there are wide global differences in asthma prevalence and low genetic heritability. It has been suggested as evidence for genetic susceptibility to asthma triggered by exposure to multiple environmental factors. Very few genome-wide interaction studies have identified gene-environment (G×E) interaction loci for asthma in adults. We evaluated genetic loci for late-onset asthma showing G×E interactions with multiple environmental factors, including alcohol intake, body mass index, insomnia, physical activity, mental status, sedentary behavior, and socioeconomic status. In gene-by-single environment interactions, we found no genome-wide significant single-nucleotide polymorphisms. However, in the gene-by-multi-environment interaction study, we identified three novel and genome-wide significant single-nucleotide polymorphisms: rs117996675, rs345749, and rs17704680. Bayes factor analysis suggested that for rs117996675 and rs17704680, body mass index is the most relevant environmental factor; for rs345749, insomnia and alcohol intake frequency are the most relevant factors in the G×E interactions of late-onset asthma. Functional annotations implicate the role of these three novel loci in regulating the immune system. In addition, the annotation for rs117996675 supports the body mass index as the most relevant environmental factor, as evidenced by the Bayes factor value. Our findings help to understand the role of the immune system in asthma and the role of environmental factors in late-onset asthma through G×E interactions. Ultimately, the enhanced understanding of asthma would contribute to better precision treatment depending on personal genetic and environmental information. Asthma is one of the most common chronic diseases worldwide . The gloh2g = 10.6%) than for early-onset asthma , and the genetic correlation (rg) between late-onset and early-onset asthma is 0.67 (h2g) than that by twin studies indicates that hypothesis-free approaches, such as genome-wide association studies of previously unknown genetic loci associated with asthma, should be used.Asthma is a heterogeneous disease in both children and adults. Dissecting the heterogeneity of asthma could reveal the subtype-specific phenotypes and pathogenesis and lead to the development of new therapeutic strategies. In the classification of asthma based on the age of onset, late-onset asthma refers to onset as early as 12 years of age or as late as over 65 years  and diff is 0.67 . The low2, gas cooking, or endotoxin exposures  were suggested to interact with active tobacco smoking for late-onset asthma with potential regulatory functions linked to gene expression regulation in the lung tissue  were identified as significant pharmacogenomics loci on asthma by joint analysis of genome-wide interaction results from discovery and replication populations (Very few genome-wide interaction studies have focused on G×E interaction loci for asthma susceptibility in adults . A genomg tissue . Anotherg tissue . Two SNPulations .This study was conducted to identify loci of late-onset asthma susceptibility showing G×E interactions with multiple environments. We conducted gene-by-single environment (G×singleE) interaction analyses using a fixed effect logistic regression model, PLINK  and genehttps://github.com/Nealelab/UK_Biobank_GWAS): principal component (PC) analysis calculation filter for selecting unrelated samples; sex chromosome filter for removing aneuploidy; filtering of PCs for European sample selection for determining British ancestry; and filters for selecting self-reported ‘white British,’ “Irish,” and “White”. The total unrelated European participants amounted to 356,536.The United Kingdom Biobank (UKB) resource was used for the discovery set of G×singleE and G×multiE interaction analyses, and for one replication set of G×multiE interaction analysis. The UKB is a population-based cohort that recruited over 487,409 individuals aged 40–69 years in the United Kingdom during 2006–2010 . The inihttp://www.hra.nhs.uk/), which allows applicants conduct research on UKB data without obtaining separate ethical approvals. Access to UKB data was granted under application no. 56987: “Classification of asthma patients and identification of group-specific genetic variants”.All participants provided signed consent to participate in the UKB . The UKBN = 20,817) were determined as those that had been diagnosed with asthma by a doctor and had checked for age of onset (≥16 years). Participants who had been diagnosed with chronic obstructive pulmonary disease (COPD) were excluded from the study. Controls  had not been diagnosed with asthma, rhinitis, eczema, hay fever, allergy, emphysema/chronic bronchitis, and COPD . Cases with late-onset asthma . A total of 5,664,578 SNPs was retained for further analysis.At baseline, imputation data of 93, 095, 623 SNPs were available for 487,409 participants using the UKB Axiom Array and the United Kingdom BiLEVE Axiom Array from Affymetrix  . Genotypwww.r-project.org). To draw the Manhattan plots, the “qqman” package was used; “corrplot” and “ggplot2” were used to prepare correlogram and violin plots, respectively. Analysis of variance and association analysis were performed using the R stats package.We performed association analysis, correlogram, Manhattan, bar, and violin plottings, one-way analysis of variance, and correlation analysis (Spearman’s rank correlation) using the R stats package .Of SNPs that were identified in the discovery analysis, independent SNPs were selected through linkage disequilibrium clumping  population-based cohort obtained from the Korean Genome and Epidemiology Study (KoGES)) .N = 191,567), cases with late-onset asthma (N = 513) were determined as those that had been diagnosed with asthma by a doctor and had checked for age of onset (≥16 years). Participants who had been diagnosed with chronic obstructive pulmonary disease (COPD) were excluded from the study. Controls  had not been diagnosed with asthma, rhinitis, eczema, hay fever, allergy, emphysema/chronic bronchitis, and COPD  at the discovery test for three novel SNPs to spare samples per SNP. Among seven, the following environmental factors for each SNP were included in the replication as follows: for rs117996675, four factors ; for rs345749, five factors ; and for rs17704680, four factors . Following exclusion of samples with missing information of environmental factors, the final sample size for rs117996675 was 57,921 ; for rs345749 was 57,817 ; and for rs17704680 was 101,791 .For the second set of replication, we used the Health Examinees (HEXA) population-based cohort obtained from the Korean Genome and Epidemiology Study (KoGES) . The KoGWe used several approaches to evaluate the functional relevance of the identified SNPs. Haploreg (v.4.1) was used to search for the effect of the identified SNPs on the transcription factor-binding site motif and to perform enhancer enrichment analysis . CombineN = 231,145) and for G×multiE interaction analyses , respectively in The basic characteristics of UKB participants in this study are described for G×singleE interaction (p < 0.05/7 = 7.14 × 10–3) in the following order: BMI (p = 1.55 × 10–124), neuroticism score (p = 8.60 × 10–62), insomnia (p = 5.30 × 10–48), TDI (p = 8.79 × 10–21), alcohol intake frequency (p = 4.74 × 10–18), time spent watching TV (p = 2.01 × 10–14), and MET score (p = 3.37 × 10–5) (p = 1.41 × 10–228), insomnia (p = 8.17 × 10–32), neuroticism score (p = 1.77 × 10–67), and time spent watching TV (p = 5.75 × 10–18), whereas it was positively correlated with alcohol intake frequency (p = 3.31 × 13–10) and TDI (p = 2.05 × 10–35).Of the numerous factors associated with asthma, seven factors were selected for G×E interaction analyses based on relatedness, including alcohol intake frequency, BMI, insomnia, MET score, neuroticism score, time spent watching TV, and TDI . We exam × 10–5) . Categor × 10–5) . Additio × 10–5) . The METp < 1.00 × 10–6), with no SNP satisfying the threshold of genome-wide significance (p < 5.00 × 10–8/7 = 7.14 × 10–9)  . Manhattp = 4.25 × 10–8; rs345749, p = 4.41 × 10–10; rs17704680, p = 1.26 × 10–8)   and 17 s × 10–8) . The thr × 10–8)  (Purcell × 10–8) .To explore which environmental factors are the most relevant to individual G×E interaction signals, we estimated the BF values for each environmental factor using a statistical method that assesses the comparative power of each model comparing between the full model and model without an individual environmental factor . A BF vap-values of G×singleE interaction were ranked in the same order or similarly as follows: for rs117996675, BMI > time spent watching TV > alcohol intake frequency > MET score > insomnia, neuroticism score, and TDI; for rs345749, insomnia and alcohol intake frequency > time spent watching TV > BMI, neuroticism score, TDI, and MET score; lastly for rs17704680, BMI > alcohol intake frequency > time spent watching TV > TDI > neuroticism score > MET score, and insomnia , rs345749 was within the intron of FMN1 (15q13.3 of Chr15), and rs17704680 was in an intergenic region near HS3ST4 (16p12.1 of Chr16) (r2 ≥ 0.8) with three loci from publicly available eQTL data sets (GTEx v8) and the pQTL resource  . Becauseresource .RIN1, BRMS1, ACTN3, CTD-3074O7.5, RP11-867G23.8, and C11orf80, and its proxies (r2 ≥ 0.8) were associated with genes such as B4GAT1 and RP11-867G23.8 in the whole blood, stomach, skeletal muscle, fat, testis, nerve, and pancreas. The other lead SNP, rs345749, was associated with FMN1 expression in the thyroid, pituitary, and esophagus mucosa, and its proxy (r2 ≥ 0.8) was associated with GOLGA8O in the skeletal muscle (Two sentinel SNPs (rs117996675 and rs345749) and their proxies were associated with variable expression of nine genes in one or more tissues . A sentil muscle . The lasl muscle .r2 ≥ 0.8) were enriched in transcriptional enhancers and promoters with slightly different patterns in 5 epigenomes (r2 ≥ 0.8) with rs345749 exhibited high CADD scores (>5) and 2 SNPs, rs345862 and rs345867, scored 10.06 and 14.64, respectively, suggesting that they are likely pathogenic.Because genetic variation may affect the modulation of transcriptional enhancers and promoters, we examined data available from the United States National Institutes of Health Roadmap Epigenomics Program  Supplem. We focuigenomes , indicatigenomes . NotablyFor the first set of replication in another European samples, we used Europeans of UKB that were not used in the discovery analysis as explained in the Methods. We performed a replication test of 3 novel SNPs  in 104,701 European participants . Since the sample size was very small, we excluded environmental factors that showed low BF (<1) at the discovery test for each of three novel SNPs . For rs1p = 3.93 × 10–3) that passed the threshold of significance (p = 1.67 × 10–2 = 0.05/3). However, rs117996675 and rs345749 did not showed any significant interaction   . We summs345749) . Among 4s345749) . For rs1p = 3.01 × 10–3), that satisfied the threshold of significance (p = 1.67 × 10–2 = 0.05/3), whereas rs345749 showed no significant interaction (p = 1.45 × 10–1) (We performed a replication test of 2 SNPs (rs345749 and rs17704680) in 37,460 East-Asian samples  using StructLMM. Female frequency and smoking status differed significantly between cases with late-onset asthma and controls . For rep × 10–1) . We also × 10–1) . Among 4p < 5.00 × 10–8/7 = 7.14 × 10–9). However, in the G×multiE interaction study, we found three independent genome-wide significant SNPs: rs117996675, rs345749, and rs17704680. We further identified the most relevant environmental factor per individual variant via BF values.In this study, we performed two types of genome-wide G×E interaction studies of late-onset asthma in European individuals. The environmental factors used in this study included alcohol intake frequency, BMI, insomnia, MET score, neuroticism score, time spent watching TV, and TDI. For G×singleE interactions, we did not detect any genome-wide significant SNPs (A recent study estimated the significance of G×E interactions using the polygenic risk score for obesity-related traits from the UKB and found that the G×E effect of GWAS SNPs significantly contributed to the phenotype variance of BMI by 1.9% in addition to the 15% of polygenic risk score effect . Anotherh2g = 10.6%) than of childhood-onset asthma (h2g = 25.6%) (The development of childhood-onset (or early-onset) asthma is often attributable to perinatal factors, atopy, viral respiratory-tract infections, and the microbiome , whereas= 25.6%) , the higPC contains eQTLs such as RIN1 (whole blood), BRMS1 (stomach), and ACTN3 , which are all involved in immune system regulation. RIN1 (Ras and Rab interactor 1) is an RAS effector that binds to ABL1 and BCR-ABL1, promoting the cytoskeletal remodeling properties of ABL proteins and serves in part to regulate epithelial cell functions, including adhesion and migration (RIN1 in late-onset asthma. BRMS1 (breast cancer metastasis suppressor 1) is associated with nuclear factor kappa light chain enhancer of activated B cells (NF-κB) that is highly related to inflammation and allergic diseases and promotes immunity by controlling the expression of genes involved in inflammation  near igration . Interesigration . Our finammation . The resammation . This inACTN3 locus  was reported as a significantly shared locus (Pmeta = 4.09 × 10–9) by meta-analysis for cross-trait associations between non-atopic asthma and BMI, indicating that our SNP (rs117996675) interacts with BMI to affect asthma, likely through ACTN3 (PC (pyruvate carboxylase), containing rs117996675 in its intron, has been established as an important regulator of hepatic gluconeogenesis and tricarboxylic acid cycle function  and asthma are associated, as 58% of patients with asthma reported impaired sleep quality , BMI was the most relevant environment. Previous association studies of obesity with asthma suggested that obesity-associated asthma consists of two forms of severe asthma, one form (late-onset) with a non-type 2 phenotype and the other form (early-onset) with a variation of type 2 asthma, complicated by the development of obesity . These rN = 14,089) of late-onset asthma and controls  evaluated to date (p < 5.00 × 10–8) for detecting novel interaction loci  of suicidality that interact with 4 substance dependences such as opioid, cocaine, nicotine, and polysubstance dependences   with BMI, and BF analyses showed that six environmental factors, except for carbohydrate intake, contributed to the interaction with this SNP on BMI  . We prev cohort) . This prP on BMI .p-values of the G×singleE interaction. These results were partially supported by functional and regional annotations such as its associations with ACTN3 by eQTL and its location in the PC locus.An advantage of this StructLMM is that it incorporates multiple environments as additive, random effect components into an interaction test. Therefore, StructLMM accounts for simultaneous interactions between various environments and a trait. In this study, we employed seven environmental factors previously shown to be associated with asthma and were associated with asthma in the UKB samples. By estimating the BF values, we prioritized environmental factors for individual variants. For rs117996675, BMI was the most relevant environment as evidenced by BF values of StructLMM and by This study also had some limitations. First, the study was based on a cross-sectional dataset, presenting the possibility of reverse causality. To limit this design bias, we focused on late-onset asthma  rather than on early-onset or total asthma. The individuals included in this study were limited to 40–69 years old. Moreover, the association directions between asthma and six factors  correspond to the findings of previous studies , suggestNext, the StructLMM is based on linear mixed models (LMM) which are not designed to analyze binary traits and can have inflated type I error rates, especially in the presence of unbalanced case-control ratios. A score test called the generalized linear mixed model (GLMM) has been developed to fit logistic regression of binary traits.. UnfortuFinally, we attempted two sets of replications using Europeans that were unused in the discovery set and East Asian samples. Interestingly, rs17704680 showed a nominally significant G×multiE interaction in both replication samples. Moreover, in the replication set of European samples, the best BF factor for rs17704680 was attributed to BMI, consistently supporting the discovery result of European samples. However, because of the limited availability of the factors surveyed in East Asian samples, we used only 4 environmental factors and replaced 2 of these factors with similar factors . Thus, the results obtained for East Asian samples may not strongly support these interactions. Therefore, our findings imply that the replication for G×multiE interactions may require the homogeneity between the replication sample and the discovery sample regarding environmental factors and allele frequencies. Hence, further validation is warranted using another asthma cohort.In conclusion, we identified three novel, genome-wide interaction signals in late-onset asthma that interact with up to seven environmental factors. These three loci are associated with immune system regulation; additionally, rs117996675 may primarily interact with BMI to affect late-onset asthma. Although further confirmation is needed, our findings improve the understanding of the role of the immune system and genetic interactions with environmental exposures in late-onset asthma and may be applied in precision treatment of asthma.