Results: Treatment with anakinra significantly and transiently increased eosinophils, from 0.2 [0.1–0.3] to 0.3 [0.1–0.4] × 103 cells/µL (p < 0.001) and from 0.3 [0.2–0.5] to 0.2 [0.1–0.3] × 103 cells/µL, with suspension (p < 0.001). Changes in eosinophils correlated with the changes in peak VO2 . Eosinophils were higher in patients with injection site reactions (ISR) , who also showed a greater increase in peak VO2 . Conclusion: Patients with HF treated with anakinra experience a transient increase in eosinophils, which is associated with ISR and a greater improvement in peak VO2.Background: Interleukin-1 blockade with anakinra leads to a transient increase in eosinophil blood count (eosinophils) in patients with acute myocardial infarction. We aimed to investigate the effect of anakinra on changes in eosinophils in patients with heart failure (HF) and their correlation with cardiorespiratory fitness (CRF). Methods: We measured eosinophils in 64 patients with HF , 55 (51–63) years of age, before and after treatment, and, in a subset of 41 patients, also after treatment cessation. We also evaluated CRF, measuring peak oxygen consumption (VO The correlation between continuous variables was assessed using Spearman’s rank correlation coefficient. Categorical variables were compared using chi-square test or Fisher’s exact test, when indicated. The within-group paired differences were compared using the Wilcoxon signed-rank test. A 2-sided 2 was 14.2 (11.6–16.9) mLO2·kg−1·min−1, and the VE/VCO2 slope was 31 (27–35). The duration of on-treatment with anakinra was 4 (2–12) weeks, whereas the time from discontinuation of anakinra to the latest off-treatment assessment was 12 (4–12) weeks. Baseline demographic and clinical characteristics are summarized in p > 0.05).We identified 64 patients with HF who received anakinra treatment and had an available CBC with differential before and during anakinra treatment . Among those, 41 (64%) also repeated the assessment after discontinuation of the treatment . The median age was 55 (51–63) years, and 32 (50%) were biological females and 47 (73%) self-identified as Black/African-American. Baseline CRP was 6.2 (3.0–15.4) mg/L, LVEF was 49 (33–60)%, E/e’ was 14.2 (9.7–19.9), peak VO3 cells/µL to 0.3 [0.1–0.4] × 103 cells/µL, p < 0.001) and this change was reverted after the discontinuation of the treatment , as shown in p < 0.001) and LER  during treatment was observed, and this was reversed after suspension .Treatment with anakinra was associated with a significant increase in eosinophils , and a statistically significant increase in eosinophils  in patients treated with anakinra for 12 weeks  (2. These data show a greater increase in eosinophils with longer duration of treatment.Comparing the patients treated for 2–4 weeks (N = 35) and patients treated for 12 weeks (N = 29), we found no significant changes in eosinophils in patients treated with anakinra for 2–4 weeks (from 0.2 [0.1–0.3] × 10 < 0.01) . The dur2 , DASI score , and negatively with changes in CRP , NT-proBNP , and the E/e′ ratio . No significant correlations were found for changes in the other parameters , as shown in Of note, the changes in eosinophils over time were significantly positively correlated with the changes in peak VO2  and DASI sore , and positively with changes in CRP . On the other hand, only LER correlated with MLHFQ score .Both changes in NER and LER correlated negatively with the changes in peak VO3 cells/µL vs. 0.20 [0.10–0.40] × 103 cells/µL, p = 0.023), higher absolute peak VO2 , larger change in peak VO2 , longer on-treatment exercise time , and lower on-treatment NT-proBNP levels , as shown in ISR were reported by eight (13%) patients (median age of 51 [45–59] years), of which four (50%) self-identified as females and four (50%) as Black/African-American. None of these ISR patients required drug discontinuation. There was no significant difference between patients with or without ISR at baseline; on the other hand, patients with ISR had higher on-treatment eosinophil counts (0.45 [0.38–0.60] × 102).We herein report, for the first time, that IL-1 blockade with anakinra leads to a significant transient increase in eosinophils in patients with established HF, with greater changes with longer duration. These changes are reversed after discontinuation of treatment. Moreover, the changes in eosinophils were inversely related to markers of systemic inflammation  and directly related to greater cardiorespiratory fitness , supporting the concept that an enhanced eosinophilic response to anakinra is associated with clinical improvement. Accordingly, we also found a positive correlation between eosinophils, NER, LER, and the changes in peak VO2 and perceived functional capacity (DASI score). Whether the difference in improvement in CRF was dependent on the different baseline characteristics between patients with or without injection site reactions, or whether it was a biomarker of a greater response to the treatment cannot be determined with the current study and requires additional research. Moreover, whether the change in eosinophils is simply a biomarker or also a mediator of the anti-inflammatory mechanism of anakinra is not known. The inverse correlation between eosinophils and measures of impaired cardiac diastolic function  and the direct correlation with cardiorespiratory fitness (peak VO2) suggest a possible cardioprotective effect of eosinophils.Injection-site reaction during treatment with anakinra is the most common side effect, reported in more than 70% of cases in some series, and this finding can be associated with an increase in circulating eosinophils and dermal infiltrate of eosinophils . We showPatients with HF treated with anakinra experience a transient increase in blood eosinophil counts. The eosinophil count may serve as a favorable prognostic biomarker for IL-1 blockade in HF, and patients with ISR may represent a subset of patients with an enhanced eosinophilic response that experience a greater benefit from the treatment. Furthermore, these data may also open the way to new mechanistic research about the roles of IL-1 and eosinophils in HF.


When used as a supercapacitor electrode, the composite exhibits a high specific capacitance of 270.74 F g−1, with an excellent capacitance retention rate of 98.4% after 10,000 cycles. The symmetrical supercapacitors (SSCs) with two carbon fiber electrodes (CWFZ2) showed a high power density of 2272.73 W kg−1 (at an energy density of 2.46 W h kg−1) and an energy density of 4.15 Wh kg−1 (at a power density of 113.64 W kg−1). Moreover, the SSCs maintained 81.21% of the initial capacitance after 10,000 cycles at a current density of 10 A g−1, which proves that the SSCs have good cycle stability. The excellent capacitance performance is primarily attributed to the high conductivity and N source provided by the zeolite imidazole framework. Because of this carbon material’s unique structural features and N-doping, our obtained CWFZ2 electrode material could be a candidate for high-performance supercapacitor electrode materials.Developing highly porous and conductive carbon electrodes is crucial for high-performance electrochemical double-layer capacitors. We provide a method for preparing supercapacitor electrode materials using zeolitic imidazolate framework-8 (ZIF-8)-coated wood fibers. The material has high nitrogen (N)-doping content and a specific surface area of 593.52 m In the past few decades, the shortage of fossil energy and the deteriorating environment have affected society’s sustainable development. Developing green, low-cost, and sustainable energy storage materials and devices is critical ,2,3. Amo−1 at a scan rate of 50 mV s−1 [−1 [2 g2 and excellent cycle stability [The typically used electrodes in EDLCs are carbon-based because of their high specific surface area (SSA), good electrical conductivity, and nontoxic and harmless characteristics ,10,11,120 mV s−1 . Gao stu s−1 [−1 . Wu preptability . Howevertability ,19.−1 at a current density of 0.5 A g−1 and a good rate performance [−1 at a current density of 0.1 A g−1 and a low resistance (0.53 Ω) [−1 [Therefore, to solve these problems, the current strategy is to increase the secondary carbon source to improve the electrode material’s conductivity, such as carbon nanotubes (CNTs), graphene, carbon aerogels, and conductive polymers. Wang used graphene functionalized with polyetherimide as the base material to anchor the ZIF-8-obtained carbon frameworks, exhibiting a specific capacitance of 218 F gformance . Wang ob3 Ω) [−1 . However−1 at a current density of 0.5 A g−1) and excellent cycle stability . As an electrode material, the obtained CWFZ2 is incorporated into a symmetrical SC (SSC) device exhibiting a high power density of 2272.73 W kg−1 and an energy density of 4.15 Wh kg−1. Accordingly, the CWFZ2 has strong application potential as an electrode material in SCs.Wood fiber (WF) is a kind of natural and renewable polymer material that is normally used in traditional fields such as wood-based panel manufacturing, pulp and paper making, etc. With the development and progress of technology, wood fiber, a material with high carbon content and abundant surface active functional-groups, has been used to prepare carbon-based electrode materials. Inspired by the previous work, we propose a strategy using WF to synthesize concentric shaft-coated structural carbon materials without surface modifiers as electrode materials. The chemical composition of WF is mainly cellulose, hemicellulose, and lignin. The surface is exposed to a large number of active functional groups, such as the hydroxyl and carboxyl groups, which play a crucial role in fixing active substances. Therefore, WF can fix ZIF-8 well and prevent its accumulation during the synthesis process, and also provide a secondary carbon source with excellent conductivity. Simultaneously, ZIF-8 provides a source for N doping for carbon materials and improves its carbon structure, specific surface area, and conductivity. The results show that the obtained carbon fiber (CWFZ2) electrode material exhibits high energy storage performance  at 1732 cm−1 and an increase in carboxyl intensity (COO–) at 1593 cm−1 indicate that some carbonyl groups are bound to ZIF-8 in situ. Furthermore, a unique band at 1049 cm−1 indicates cellulose has C–O–C or C–O vibrations. The FTIR spectrum of WF@ZIF-8 shows a band at 758 cm−1, which is related to the out-of-plane bending of the 2-methylimidazole (2-MeIm) ring of ZIF-8 [of ZIF-8 . The WF 2 adsorption–desorption isotherms of carbonized ZIF-8 (CZ) and different CWFZs. The adsorption–desorption curves of CZ and different CWFZs overlap, and the isotherms increase sharply at low relative pressures (P/P0 < 0.01), indicating the existence of microporous structures inside all samples. Simultaneously, CZ and the different CWFZs samples have a hysteresis loop under moderate relative pressure (0.3 < P/P0 < 0.9), indicating a mesoporous structure in the sample [2/g, respectively. Furthermore, the pore size distribution map proves that CZ and the different CWFZs have hierarchical porous structures with micropores and mesopores  and ordered carbon (G-band), respectively. Overall, the intensity ratio of the D-band to the G-band (ID/IG) can confirm the defect density of carbon [D/IG values of CWFZs are lower than CWF (0.958) and CZ (0.873), indicating that there are fewer disordered regions, which is beneficial for energy storage [The crystallographic structures of the different samples were characterized using XRD measurements. The spectrum of the ZIF-8 synthesized in this experiment correlates with the simulated crystal structure spectrum previously published in f carbon . It can  storage .sp2 hybrid graphite-like carbon structure, which means that most of the C atoms are arranged in the conjugate honeycomb lattice. The peak at 285.9 eV is related to the N-sp2 C, which also confirms that nitrogen doping is occurring in the carbon skeleton. The peaks at 287.3 eV and 288.8 eV correspond to the C–O bond and O–C=O bond, respectively. From the results, it is further confirmed that N bonding to the carbon skeleton has a potential contribution to its electrochemical performance [The surface chemical compositions of the samples were further investigated using XPS measurements plotted in formance ,35. The formance . These pformance . N-Q canformance . All theformance ,40. Heteformance .−1 in a voltage window from −0.08 to 0 V  of CWFZ2 is shorter than that of CWF (1.98 Ω), CZ (0.51 Ω), CWFZ1 (0.47 Ω), and CWFZ3 (0.61 Ω). Therefore, the equivalent series resistance (ESR) of CWFZ2 is the lowest, including electrode resistance, the contact resistance between the electrode and the current collector, and the ionic resistance of the electrolyte [y-axis, indicating its ideal EDLC behavior and fast ion diffusion and transfer. The extremely low electrical resistance of CWFZ2 is primarily due to the excellent conductive base provided by the fibers, the partial graphitization of the carbon materials, and the hierarchical porous structure. The microporous and mesoporous structures in the sample can provide large spaces for ion diffusion, which can then improve the rate ability. From the Bode phase diagram in ctrolyte . The str−1, indicating good charge transfer characteristics. −1 at a current density of 0.5 A g−1, which is significantly higher than that of the other carbon-based electrode materials previously reported, such as PC-850 (132.8 F g−1 at 0.5 A g−1) [−1 at 0.5 A g−1) [−1 at 0.2 A g−1) [−1 at 1 A g−1) [T (162 F g−1 at 1.25 A g−1) [−1 at 0.5 A g−1) [−1 (The CV curve shapes g of CWFZ5 A g−1) , GPNC (25 A g−1) , N-HPC (2 A g−1) , CMCMs (1 A g−1) , H-N-N-T5 A g−1) , and PC15 A g−1) . The eleg−1) [−1 i. The sp−1 to 200 mV s−1. All of the CV curves appear similar to those of the rectangles. Furthermore, the GCD curves show similar isosceles triangle shapes at various current densities, indicating good reversibility and EDLC behavior  after 10,000 cycles demonstrates the high reversibility and stable electrochemical behavior of the CWFZ2-based SC 2·6H2O, methanol, ethanol absolute, potassium hydroxide (KOH), polyvinylidene fluoride (PVDF), and acetylene black were purchased from Sinopharm Chemical Reagent Co., Ltd. . All the deionized water in the experiment was made by the laboratory.WF was obtained from poplar wood through thermal grinding. The 2-methylimidazole (2-MeIm), Zn(NO3)2·6H2O, and was continuously stirred for 30 min. Next, 10 mL methanol solution with 2-MeIm was added dropwise into the solution, and the mixture was continuously stirred for 2 h. Finally, the above materials were washed with methanol solution and deionized water and dried overnight at 70 °C in an oven to obtain the WF@ZIF-8 composite (WFZs). A series of WFZs denoted as WFZ1, WFZ2, and WFZ3 were prepared by subjecting the initial mass of wood fibers and the precursor of ZIF-8. The initial mass of components is provided in detail in First, WF was placed in a mixed solution of 10 mL methanol and 10 mL deionized water containing Zn(NO2 at a flow rate of 150 mL min−1. The temperature was increased to 800 °C (heating rate of 5 °C min−1), and this was maintained for 2 h. Finally, the tube furnace was naturally cooled to 25 °C, and the CWFZs were removed. The CWFZs obtained by the carbonization of WFZ1, WFZ2, and WFZ3 were named CWFZ1, CWFZ2, and CWFZ3, respectively.The WF@ZIF-8 carbon fibers (CWFZs) were prepared by placing the WF@ZIF-8 composite in a tubular furnace , which was subsequently flushed with pure NThe structure and morphology of the different samples were characterized using scanning electron microscopy , transmission electron microscopy , and energy-filtered TEM mapping. The sample compositions were analyzed using X-ray diffraction  with a Cu Kα source , Raman (Horiba Scientific LabRAM HR Evolution), Fourier transform infrared spectroscopy , and X-ray photoelectron spectroscopy . The SSA and pore size distribution of the CWFZs were analyzed using the Brunauer–Emmett–Teller (BET) method (QUADRASORB SI).2) as the working electrode . The three-electrode system used a 6 M KOH electrolyte with an Ag/AgCl2 electrode and a platinum plate as the reference and counter electrodes, respectively. Cyclic voltammetry (CV), galvanostatic charge/discharge (GCD), and electrochemical impedance spectroscopy (EIS) were conducted in an electrochemical workstation. From the GCD curves, the specific capacitance of the CWFZs was calculated using Formula (1),t is the discharge time, I is the current, △V is the working voltage, and m is the loading mass of the CWFZs. A simple SSC was assembled using two CWFZ2 electrodes and a separator (cellulose paper). The performance of the SSC was evaluated using a two-electrode system in an electrochemical workstation.The electrochemical performances of the CWFZs were measured in an electrochemical workstation (CHI660E) using a three-electrode system. The acetylene black, PVDF, and CWFZs were made into a slurry (mass ratio of 1:1:8) and then coated onto foamed nickel (1 × 1 cm2 g−1, exhibits a high specific capacitance of 270.74 F g−1 in alkaline electrolyte, and exhibits an excellent capacitance retention of 98.4% after 10,000 cycles. The obtained SSCs show a high power density of 2272.73 W kg−1 at an energy density of 2.46 Wh kg−1. This work provides an efficient way to synthesize hierarchically porous N-doped carbon materials for high-performance supercapacitors.We have successfully developed an efficient method to obtain biomass carbon fiber electrode materials with excellent electrochemical performance. Among the carbon fiber electrode materials, the carbon of the wood fibers builds a conductive network for electron transport, and ZIF-8 provides a nitrogen source to improve the energy storage performance of the carbon material. The as-prepared CWFZ2 composite has a specific surface area of 593.52 m


This work focused on the development of porous scaffolds based on biocomposites comprising two biodegradable and biocompatible biopolymers: a terpolyester, poly (PHBHVHHx), and the bacterial polysaccharide FucoPol. The PHBHVHHx terpolymer was composed of 3-hydroxybutyrate (55 wt%), 3-hydroxyvalerate (21 wt%), and 3-hydroxyhexanoate (24 wt%). This hydrophobic polyester has low crystallinity and can form elastic and flexible films. Fucopol is a fucose-containing water-soluble polysaccharide that forms viscous solutions with shear thinning behavior and has demonstrated emulsion-forming and stabilizing capacity and wound healing ability. Emulsion-templating was used to fabricate PHA-based porous structures in which FucoPol acted as a bioemulsifier. Compared with the scaffolds obtained from emulsions with only water, the use of FucoPol aqueous solutions resulted in structures with improved mechanical properties, namely higher tensile strength (4.4 MPa) and a higher Young’s Modulus (85 MPa), together with an elongation at break of 52%. These features, together with the scaffolds’ high porosity and pore interconnectivity, suggest their potential to sustain cell adhesion and proliferation, which is further supported by FucoPol’s demonstrated wound healing ability. Therefore, the developed PHBHVHHx:FucoPol scaffolds arise as innovative porous bioactive structures with great potential for use in tissue engineering applications. Emulsion-templating is a promising approach for the fabrication of scaffolds with high porosity (up to 99%) and interconnected pores , which aPoly (PHBHVHHx) is considered one of the most promising additions to the PHAs family because it combines short-chain length (scl) and medium-chain length (mcl) monomers into terpolyesters with interesting novel properties ,9. When Haloferax mucosum (DSM 27191) to stabilize emulsions between water and nonpolar solvents such as chloroform and n-hexane ) was also reported to synthesize PHBHVHHx with a monomer composition within the same ranges by cultivation on dodecanoic acid and different odd-carbon fatty acids [Rhodospirillum rubrum ATCC 25,903 (6 wt%) [Aeromonas hydrophila 4AK4 strains (1–13 wt%) [C. necator P(3HB)-4 (3–18 wt%) [The PHBHVHHx biopolymer accumulated by the MMC cells was a scl-mcl-PHA composed of 55 wt% 3HB, 21 wt% 3HV, and 24 wt% 3HHx . Similarectively . The recty acids . Lower 3 (6 wt%) , the rec–13 wt%) ,12,31,32–18 wt%) ,28. For FucoPol was composed of fucose (34.33 ± 2.05 %mol), glucose (33.12 ± 2.02 %mol), galactose (26.21 ± 2.04 %mol), and glucuronic acid (9.93 ± 1.06 %mol), which is the sugar composition characteristic of the polysaccharide FucoPol  [w of 0.9 × 105 Da (5–4.1 × 105 Da) [5–3.5 × 106 Da) [5 Da and 18.4 × 105 Da, were reported for the PHBHVHHx synthesized by different bacteria, namely, the recombinant species of Aeromonas hydrophila 4AK4 and Cupriavidus necator P(3HB)-4   and scl- 106 Da) ,36,37. HP(3HB)-4 . The bioP(3HB)-4 . Such di and PDI .w of 1.40 × 106 Da, together with a PDI of 1.18, which are similar to previously reported values  [w observed in FucoPol gives this biopolymer the capacity to act as a thickening and emulsifying agent to form viscous solutions and stabilize emulsions [On the other hand, FucoPol had an average Mmulsions . MoreoveA. hydrophila 4AK4 with 3HB, 3HV, and 3HHx contents of 71–82 wt%, 7–18 wt%, and 11 wt%, respectively [The diffractogram a shows tectively . For theectively .m1) and at 159 °C (Tm2)  [The DSC curve  for PHBH°C (Tm2) . This phd 173 °C , respectd 173 °C . On the o 148 °C . Interes–170 °C) . Neverth–170 °C) ,47. The g of −3.8 °C  [hydrophila 4AK4 from lauric acid and valerate, which was composed of a lower 3HB content (55 wt%) and higher 3HV and 3HHx contents . The resulting cast film had a lower Young’s modulus (2.0 MPa) together with a significantly lower tensile strength (0.3 MPa) [Higher Young’s modulus values were reported for the cast films prepared with the terpolymers produced by 290 MPa) ,12, whic290 MPa) , increas0.3 MPa) . These Y0.3 MPa) , the skiv/v), resulting in white, stable, and homogeneous emulsions, similar to those reported by Esmail (2021) for emulsion-template PHB and PHBHV scaffolds [The porous scaffolds were prepared by the emulsion templating method, and one of the main challenges of this procedure was producing porous structures with interconnected pores . Given tcaffolds . The porcaffolds .As shown by the SEM analysis , no poroOn the other hand, the scaffolds prepared from the 9.5% polymer solution (w/v) were tested. To determine which continuous phase concentration would result in a more stable emulsion, blends were prepared using 6.7 and 9.5 wt% of PHBHVHHx solutions with a FucoPol solution at 0.1 %w/v. After emulsification, the mixtures were kept at room temperature for 72 h. The emulsion with 6.7 wt% PHBHVHHx and FucoPol at 0.1 %w/v was the only one that showed no phase separation, being thus selected for further testing using two other FucoPol concentrations, 0.5 and 1.0% (w/v).Aiming to improve the emulsions’ stability, FucoPol was tested as a bioemulsifier. For that, different concentrations of PHBHVHHx in the organic phase  FucoPol solution, had a stress-strain curve similar to that obtained for the water emulsion porous scaffold, displaying a Young’s Modulus of 85 MPa, which was slightly higher than the values found for the cast PHBHVHHx films (78.3 MPa) with no statistical significance and for the PHBHVHHx porous scaffold prepared by water emulsion (60 MPa)  . These r(60 MPa) . NeverthThe scaffolds’ mechanical strength is a fundamental property for cell culture since they must support cell proliferation and cell mobility . ConsideThis study demonstrated the valuable properties of the porous scaffolds based on the terpolyester PHBHVHHx prepared by emulsion-templating with an aqueous solution of the bioactive polysaccharide FucoPol. This technique allowed for the combination of the two biocompatible and biodegradable natural polymers, resulting in 3D porous structures with enhanced physical and chemical properties. FucoPol acted not only as a bioemulsifier for emulsion stabilization, improving scaffold production by the emulsion templating technique, but it also enhanced the structural features of PHBHVHHx when compared with cast films. Moreover, incorporating FucoPol’s bioactivity into the PHBHVHHx scaffolds could be an added advantage for their use in tissue engineering. To assess this, cell adhesion and proliferation tests are needed to ensure the scaffold’s applicability in the biomedical area. It could also be interesting to study different methods to obtain scaffolds that combine PHBHVHHx and FucoPol to estimate the best procedures to attain a structured material that gathers the main advantages of both biopolymers.


Kawasaki disease (KD) is an acute pediatric vasculitis affecting genetically susceptible infants and children. Although the pathogenesis of KD remains unclear, growing evidence links genetic susceptibility to the disease.To explore the genes associated with susceptibility in KD, we applied whole-exome sequencing to KD and control subjects from Yunnan province, China. We conducted association study analysis on the two groups.MYH14 and RBP3) through the genotype/allele frequency analysis. A heterozygous variant  of the RBP3 gene was identified in 12 KD cases, while eight heterozygous variants  of the MYH14 gene were identified in 8 KD cases respectively.In this study, we successfully identified 11 significant rare variants in two genes (MYH14 and RBP3 gene may be associated with KD susceptibility in the population from Yunnan province.This study suggested that nine variants in The online version contains supplementary material available at 10.1186/s12969-023-00857-0. Kawasaki disease KD; OMIM611775) is an acute, self-limiting systemic vasculitis syndrome with the main clinical manifestations of fever, oral mucosal changes, rash, cervical lymphadenopathy, conjunctivitisin, and extremity changes, known as mucocutaneous lymph node syndrome (MCLS) 1775 is a. It was IL-18 and IL-1B [inositol 1, 4, 5-trisphosphate 3-kinase C (ITPKC) [Although KD can be diagnosed based on its typical features , while tnd IL-1B , 13, ino (ITPKC) , 15, and (ITPKC) . Meanwhi (ITPKC) –19. Howe (ITPKC) . Most GW (ITPKC) , 21, 22.The case-control sample set used in this study included 93 KD patients and 91 non-KD control cases. All cases were obtained from the Kunming Children’s Hospital, Yunnan province, southern China, and unrelated to each other. Inclusion criteria for KD cases included patients met the criteria for KD , 23, nonWES was conducted using genomic DNA samples obtained from 93 children with KD. The exome sequences were efficiently enriched from 1 µg genomic DNA extracted from the peripheral blood using Agilent liquid capture system  according to the manufacturer’s protocol. Finally, Illumina Novaseq 6000 platform , with 150 bp pair fragments sequencing mode, was used for sequencing the genomic DNA for shotgun library construction. The overall genotyping success rate was 99.5%. Raw image files were processed using CASAVA v1.82 for base calling and generating raw data.Sanger sequencing was performed to confirm the variants identified by WES. PCR was conducted with TaKaRa Taq  under the following conditions: 95 °C for 5 min; followed by 34 cycles at 95 °C for 30 s, 59 °C for 30 s and 72 °C for 30 s; 72 °C for 5 min. PCR products were purified by gel electrophoresis and sequenced using ABI 3730xl DNA Analyzer with the BigDye™ Terminator Cycle Sequencing Kit .p-value < 0.05 was considered statistically significant.Variation frequencies were described as proportions, and SNP allele frequency comparisons between cases and controls were analyzed by Fisher’s exact tests and odds ratios , and 95%p-value < 0.05 according to Fisher’s exact test. The proportion of KD patients with coronary artery aneurysms was 8.6% . The mean age of control group was 2.50 ± 1.76 years (ranged from 3 months to 12 years), and the male to female ratio was 1.6:1 (56/35). In KD and control groups, the proportions of Han were 80.65%  and 84.62%  respectively. The proportions of ethnicity were 19.35%  and 15.38%  respectively. The detail information of the patients was in supplement file In the KD group, there were 87 classic KD patients (93.55%), 8 incomplete KD patients (8.6%) and 2 unresponsive to IVIG (2.2%). The mean age of KD group was 2.40 ± 1.84 years (ranged from 7 days to 12 years), and the male to female ratio was 1.7:1 (59/34). It was slightly higher than the ratio of 1.5: 1 of KD patients generally, A total of 349,054 variants were identified from exome sequence data of 93 KD cases and 91 controls. The filtering steps including: filtering by MAF (MAF < 0.01), Picking of damage variants, case-control analysis. MAF filtering according to the population frequency databases include including 1000G(1000 Genome), ExAC(Exome Aggregation Consortium), and gnomAD (Genome Aggregation Database). Frameshift mutations, terminator mutations, splice mutations, and missense mutation with a Combined Annotation Dependent Depletion (CADD)  scores >TTI1, MYH14, and RBP3) from 46 cases  (Table TTI1 gene (appeared in 26 cases) were excluded as the high allele frequency in control group. A heterozygous variant  of the RBP3 gene was identified in 12 KD cases, while eight heterozygous variants  of the MYH14 gene were identified in 8 KD cases respectively. All of the variants were confirmed by Sanger sequencing.ORs were valued by R package ggplot2, which performed deleterious when ORs > 1. Through association analysis of these 8413 variants, we successfully identified 11 variants in three genes  in exon 1 of RBP3 gene were identified in 12 KD cases, appears to be present at a high rate (12.9%) in the KD group. Eight variants of the MYH14 gene were identified in 8 KD cases respectively. All the allele frequencies were lower than 0.0275%, which indicated both of them were rare variants of genes. RBP3 and MYH14 gene are the first time reported to be associated with KD.In this study, we performed WES to identify rare protein-coding variants responsible for KD susceptibility. Nine variants in RBP3 gene encode interphotoreceptor retinol-binding protein, transport retinoids between the retinal pigment epithelium and the photoreceptors [RBP3 gene variants have been recorded in the HGMD database (https://www.hgmd.cf.ac.uk/ac/index.php), including eleven missense variants, three nonsense variants, one frameshift variant, and one fragment deletion variant . SNHL was reported in approximately 36% patients with Kawasaki [MYH14 gene maybe potentially associate with the symptom.polarity . Sixty gudy Fig. , only c.Kawasaki , rare vaRBP3 and MYH14 were first reported to be associated with KD susceptibility in chinses population. This study’s limitation is a relatively small sample size, so the samples of coronary artery aneurysms and ethnic minorities were limited, unable to conduct more diversified data analysis. Studies on large sample sizes are needed in future to further reveal the relationship between candidate genes and KD.Above all, RBP3 and MYH14) were identified in the case-control analysis . These results provide insights into novel candidate genes and genetic variants that may be involved in KD and related KD complications. Further association studies with expanded KD samples from southwestern China or different ethnic groups are needed to confirm these results.WES was performed on the KD and control group to identify susceptibility genes in patients from southwest China, and two protein-coding gene (Below is the link to the electronic supplementary material.Supplementary Material 1


Peripheral neuropathy is a common side effect of cancer treatment with paclitaxel. The mechanisms by which paclitaxel is transported into neurons, which are essential for preventing neuropathy, are not well understood. We studied the uptake mechanisms of paclitaxel into neurons using inhibitors for endocytosis, autophagy, organic anion-transporting polypeptide (OATP) drug transporters, and derivatives of paclitaxel. RT-qPCR was used to investigate the expression levels of OATPs in different neuronal tissues and cell lines. OATP transporters were pharmacologically inhibited or modulated by overexpression and CRISPR/Cas9-knock-out to investigate paclitaxel transport in neurons. Through these experiments, we identified OATP1A1 and OATP1B2 as the primary neuronal transporters for paclitaxel. In vitro inhibition of OATP1A1 and OAT1B2 by glycyrrhizic acid attenuated neurotoxicity, while paclitaxel’s antineoplastic effects were sustained in cancer cell lines. In vivo, glycyrrhizic acid prevented paclitaxel-induced toxicity and improved behavioral and electrophysiological measures. This study indicates that a set of OATPs are involved in paclitaxel transport into neurons. The inhibition of OATP1A1 and OATP1B2 holds a promising strategy to prevent paclitaxel-induced peripheral neuropathy. Peripheral neuropathy is one of the most common side effects of the microtubule-stabilizing chemotherapeutic agent paclitaxel, affecting up to 87% of all treated patients ,2. CurreCurrently, the pathological cascade of how paclitaxel damages nerve fibers is incompletely understood. It is assumed that the multimodal effects of paclitaxel cause neuronal damage, such as axonal transport impairment, mitotoxicity, and inflammation ,12. In tBecause of this plethora of toxic effects, there are currently no clinical treatments to prevent or treat paclitaxel-induced neuropathy. Furthermore, there is experimental evidence that different paclitaxel delivery mechanisms impact the kinetics of paclitaxel in nervous tissue and hence the degree of neurotoxicity ,18. WithIt is assumed that paclitaxel is actively transported into cells by organic anion-transporting polypeptides (OATPs) ,20,21. TThis leads us to our hypothesis that organic anion-transporting polypeptides (OATPs), which are essential for the absorption and tissue distribution of endogenous and exogenous compounds, are involved in paclitaxel uptake into sensory neurons. This study identified OATP1A1 and OATP1B2 as paclitaxel transporters in sensory neurons. Further in vivo experiments showed that inhibiting paclitaxel uptake with glycyrrhizic acid could alleviate paclitaxel-induced neuropathy. This study sheds light on the mechanism of paclitaxel-induced neuropathy and identifies potential targets for prevention and treatment.2 at 37 °C. The respective cell culture medium and concentration of fetal bovine serum (FBS) are listed in All cells for cell culture experiments were maintained in a sterile incubator humidified with 95% air and 5% COIntracellular paclitaxel accumulation in primary sensory neurons and F11  cells was measured via (i) fluorescent paclitaxel exposure  or (ii) liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Cells were either treated with increasing fluorescent paclitaxel concentrations or increasing concentrations of solvent-based Cremophor-ethanol (CreEL) paclitaxel. Cells were treated for 30 min, 1 h, or 2 h with the respective solutions. After incubation, cells were rinsed with PBS, and paclitaxel accumulation was determined by the plate reader at 540 nm when fluorescent paclitaxel was used or by LC-MS/MS when CreEL paclitaxel was used.To investigate the possible uptake mechanisms of paclitaxel, chemicals were used that inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, autophagy, and phagocytosis .Further, 2.5 × 104 F11 cells were seeded in black 96-well plates and cultured in phenol red-free medium for 48 h. Chlorpromazine at 0.0064 µM, 0.016 µM, 4 µM, and 100 µM was applied for 1 h. Indomethacin at 75 µM, 150 µM, 225 µM, and 300 µM was applied for 10 min. Ethyl isopropyl amiloride (EIPA) at 10 µM, 25 µM, 50 µM, and 100 µM was applied for 30 min. Bafilomycin A1 at 10 nM, 100 nM, and 500 nM was applied for 30 min. Phenylarsine oxide at 1 µM, 5 µM, 10 µM, and 100 µM was applied for 30 min. After respective inhibitor treatment, 1 µM of fluorescent paclitaxel was added for 30 min. Cells were then rinsed with PBS before 1:200 Hoechst for nuclear labeling was added for 10 min at 37 °C. Fluorescent paclitaxel and Hoechst’s fluorescence intensity were measured by using the plate reader at 540 nm and 465 nm, respectively.To investigate paclitaxel transport specificity, cell experiments with two paclitaxel derivatives, namely docetaxel and cephalomannine, were performed. Here, 2.5 × 104 F11 cells were seeded in black 96-well plates and cultured in phenol red-free medium for 48 h. Afterward, cells were treated with CreEL paclitaxel, docetaxel, or cephalomannine at 0.1 µM, 0.5 µM, 1 µM, and 10 µM for 24 h. Furthermore, 1 µM fluorescent paclitaxel was added for 30 min. Cells were then rinsed with PBS before 1:200 Hoechst was added for 10 min at 37 °C. Fluorescent paclitaxel and Hoechst’s fluorescence intensity were measured by using the FLUOstart Omega plate reader  at 540 nm and 465 nm, respectively.2O) stock solution -2,5-diphenyltetrazoliumbromid (MTT) assay was performed (i) to determine whether inhibitor pretreatment shows neurotoxic effects; (ii) to assess the neuroprotective effects of transporter knock-out; and (iii) to investigate whether inhibitor treatment sustains the neoplastic characteristics of paclitaxel. To accomplish these objectives, 5 × 103 cells per well were seeded onto a 96-well plate and cultured for 48 h. Cells were incubated for 2 h with inhibitors  before mFollowing the supplier’s instructions, RNA was isolated by using the MasterPureTM Complete DNA & RNA Purification Kit . RNA solution was stored at −20 °C until further use.To synthesize the cDNA of the RNA of all samples, the QuantiTect Reverse Transcription Kit  was used according to the supplier’s instructions. All RT-qPCR experiments were performed in 96-well optical plates with triplets of each sample. Primer annealing temperatures for drug-transport primers  and neurTo obtain samples for the following LC-MS/MS measurements, 5 × 104 overexpressing, knock-out, or F11 cells were seeded into a 24-well plate. Overexpressing and knock-out cells were treated with 0.78 µM, 3.125 µM, or 12.5 µM of paclitaxel for 15 min. For paclitaxel kinetics, cells were treated as described above for 15 min. For the active transport inhibition experiments, cells were treated with increasing inhibitory drug concentrations for 1 h and subsequently for 15 min with 3.125 µM of CreEL paclitaxel. After each treatment, the medium was removed, and cells were washed with ice-cold PBS. To lyse cells, 200 µL of RIPA buffer was added, and cells were incubated for 45 min on ice. Aliquots of the acquired lysates were used for LC-MS/MS paclitaxel concentration measurement and BCA protein assay. Cell lysates were further homogenized via ultrasound sonification for the BCA protein assay (see below).13C6]-paclitaxel), serving as the internal standard. Samples were centrifuged for 10 min at 12,000 rpm. Supernatants were removed and subjected to LC-MS/MS, as described before [For the LC-MS/MS measurements, 50 μL cell lysate was mixed with 50 μL of acetonitrile and 50 μL of acetonitrile solution containing stable isotope-labeled paclitaxel ([d before .y-axis) correlated with the different concentrations that were applied to the cells (x-axis).Paclitaxel concentration of each sample needed to be normalized with the number of cells that it derived from. BCA protein assay  was performed to define protein amounts in the cell lysate sample. Supplier’s instructions were followed; absorbance was measured in a plate reader at 560 nm; and the protein concentrations of the samples were calculated by using the BSA reference absorbance. All results of LC-MS/MS were depicted as ng paclitaxel per µg cells , 0.1% Triton X-100, 0.05% Tween 20 in PBS). Antibody anti-UCHL1  was diluted in 1:200 in 1% BSA and incubated at 4 °C overnight. Cells were again washed with PBS before DAPI in 1:100; the second antibody, AlexaFluor647 , in 1:1000 was added; and cells were incubated for 1 h at room temperature. Cells were washed again, and for analysis, 100 μL of PBS per well was added.2, circularity (perimeter2/4π area) of 1–3, length-to-width ratio of 1–2, average intensity of 800–12,000, and total intensity of 2 × 105–5 × 107. The image masks were then used to quantify the signal of fluorescence-labeled paclitaxel.We used a Cellomics ArrayScan XTI microscope with an LED light source to acquire images of stained DRG cultures in 96-well imaging plates (Greiner µclear). Binned (2 × 2) images (1104 × 1104 pixels) were acquired with a 10× (NA = 0.3) EC Plan Neo-Fluor objective  and analyzed using the Cellomics software package. Images of UCHL1-labeled neurons were background corrected (low pass filtration), converted to binary image masks (fixed threshold), and segmented (geometric method), and neurons were identified by using the object selection parameters: size of 80–7500 µmIn this study, 8–12-week-old female and male wild-type C57BL/6 mice  were used. Mice were maintained on a 12 h light–dark cycle, held on sawdust bedding in plastic cages with food and water provided ad libitum. Animal experiments conformed with local state authorities . In total, 43 animals  were used. Three mice were used for primary sensory cell culture, and 40 mice were used to study neurotoxicity in vivo, comparing a paclitaxel treatment group with a glycyrrhizic acid plus paclitaxel treatment group.2O to a total of 100 mM. The general clinical condition was monitored daily, and body weight was assessed on d0 and d7. After treatment, mice were sacrificed by cervical dislocation.To study paclitaxel’s neurotoxic effects and their possible prevention via transport inhibition, 25 mg/kg of CreEL paclitaxel was injected intraperitoneally on d0 and d7. The control group received CreEL paclitaxel injections only, while another treatment group intraperitoneally received an injection of 100 mg/kg of glycyrrhizic acid 3 h prior to CreEL paclitaxel injection. CreEL paclitaxel was obtained via the University Hospital Pharmacy. The glycyrrhizic acid powder was purchased  and resubstituted in HNerve conduction studies of compound motor action potential (CMAP) and sensory nerve action potential (SNAP) were performed blinded on d0 and d11. Recordings of compound muscle action potential (CMAP) amplitudes, sensory nerve action potential (SNAP) amplitudes, and latencies were performed on a PowerLab single acquisition setup , as described before .p-value was at or below 0.05. Four levels of significance are highlighted: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.All data were randomly collected and blindly assessed. For statistical analyses, the software programs MS Excel  and Prism 5.0  were used. Significances were analyzed between different groups by using the Mann–Whitney U test to compare two groups and one-way nonparametric ANOVA  for comparisons between more than two groups. If there were more than two groups that needed to be compared, an additional Dunn’s multiple comparison test was performed. Differences between groups were considered statistically significant when the Recent studies indicate that paclitaxel is taken up into neurons via active transport mediated by different OATPs, particularly OATP1B2 . HoweverTo detect possible passive transport mechanisms, we preincubated F11 cells with inhibitors of phagocytosis , caveolae-mediated endocytosis , micropinocytosis , clathrin-mediated endocytosis , and autophagy  before exposure to 1 µM of paclitaxel. Paclitaxel uptake was not altered by any inhibitor treatment with either phenylarsine oxide, indomethacin, EIPA, or bafilomycin a. InhibiGiven the intracellular accumulation, paclitaxel concentrations rapidly increased over 0.5–2.0 h after exposure to different concentrations of fluorescent paclitaxel b. This aWe subsequently assessed whether the transport of paclitaxel is specific to paclitaxel itself. F11 cells were therefore preincubated for 24 h with paclitaxel or structurally related derivatives docetaxel and cephalomannine  before 1 µM of paclitaxel exposure. Preincubation with paclitaxel and docetaxel, but not cephalomannine, significantly decreased paclitaxel uptake d. PreincOur previously described findings suggest a drug-transport mechanism for paclitaxel. Hence, we examined the expression of OATPs in rat DRG tissue, rat brain tissue, neuronal cell line F11, and pancreatic cancer cell line AR42J a. ComparTo determine whether paclitaxel is actively transported by one of these transporters, we incubated neurons with known pharmacological inhibitors at increasing concentrations. Neuronal cell line F11 was incubated with drug-transport inhibitors  at 0.006On the basis of the expression profile of the described drug transporters and inhibitor experiments, we conclude that OATP1A1, OATP1B2, and OATP2A1 are the most potent candidates for paclitaxel transport. OATP1A1 and OAT1B2 in rodents correspond to OATP1A2 and OATP1B1 in humans, respectively. OATP2A1 is both expressed in humans and rodents. Those transporters were subsequently overexpressed in F11 cells to verify if paclitaxel uptake is altered .An increased intracellular concentration of paclitaxel via LC-MS/MS was observed after the overexpression of candidate transporters OATP1A1 (rodent), OATP1A2 (human), OATP1B1 (human), and OATP1B2 (rodent) a–d. No cSubsequently, we analyzed the uptake of paclitaxel into primary sensory neurons isolated from the dorsal root ganglia (DRG) of adult mice. For this, we applied a high content screening (HCS) microscopy-based approach that provides single-cell data of DRG neurons and non-neuronal cells ,27. For Because glycyrrhizic acid was the most potent inhibitor of paclitaxel uptake, we next used the CRISPR/Cas9-System to knock out the glycyrrhizic-acid-susceptible transporters, OATP1B2 and OATP1A1, in neuronal F11 cells . TreatmeMTT assays were performed to assess whether the inhibitor treatment is able to ameliorate the neurotoxicity of paclitaxel. Neuronal F11 cells were treated with increasing concentrations of inhibitory drugs, as described above, and 1 µM of paclitaxel for 24 h. No changes in cell viability were seen after treatment with taurocholate and ibuprofen. Cell viability further decreased after cotreatment with naringin and niflumic acid at higher concentrations. Treatment with glycyrrhizic acid drastically increased cell viability, to ~97%, at a concentration of 0.16 µM, indicating that higher concentrations of glycyrrhizic acid are able to rescue the cytotoxic effects of paclitaxel. As a positive control, neuronal F11 cells were treated with increasing concentrations of paclitaxel. After 24 h of treatment, paclitaxel concentrations higher than 20 µM resulted in 100% cytotoxicity a.A wide array of toxicity markers was investigated via RT-qPCR to further study the prevention of paclitaxel-induced neurotoxicity. F11 cells were incubated with 1 µM of paclitaxel b or withThe potential interference of transporter modulation with antineoplastic properties of paclitaxel was tested in 11 cancer cell lines, namely A459, CCD-13Lu, CHO-K1, CHOwt, MCF-7, MDA-MB-231, MDA-MB-435, NCI-H460, PC3, and 4T1, which are known to be susceptible to paclitaxel. Cell lines were pretreated with inhibitors and then treated with increasing paclitaxel concentrations to explore a potentially increased resistance to paclitaxel .To present data in a more comprehensive way, a color-coded heatmap of the results was created a. Red inOnly one treatment condition with glycyrrhizic acid in CHO-K1 cells led to no cancer cell death. Otherwise, all the other settings still were able to induce cancer cell death. Overall, the MTT assay results show that pretreatment with drug-transport inhibitors does not increase cancer cell survival b.We were able to inhibit the paclitaxel transport via OATP1A1 and OATP1B2 in immortalized neuronal cultures and primary sensory neuronal cultures. As glycyrrhizic acid is a natural drug that is safe in use and has an anti-inflammatory profile, it seems to be the perfect candidate for in vivo use. Mice were intraperitoneally injected with glycyrrhizic acid 3 h prior to paclitaxel treatment, consisting of intraperitoneal paclitaxel injections once a week for 2 weeks. Behavioral testing, neurography testing, and anatomical testing were performed . An acetInhibition of endocytosis, autophagy, macropinocytosis, and clathrin- or caveolae-mediated transport did not attenuate paclitaxel translocation into neurons. An exception was increased paclitaxel uptake at high inhibitor concentrations, which was likely caused by increased inhibitor cytotoxicity at escalating concentrations. If cells undergo apoptosis and become porous, this could increase paclitaxel influx or the accumulation of fluorescence in the fluorescent paclitaxel that binds to cell structures.Our kinetic experiments demonstrate that peak paclitaxel concentrations in F11 were measured not before 5 h after incubation, which was much longer than one would expect in the case of uptake into the sensory neuron via diffusion. Diffusion is a highly efficient process, and equilibrium is reached after seconds or minutes.Pre-exposure of F11 cells with paclitaxel and its structurally closely related derivative docetaxel prevented paclitaxel uptake, which strongly indicates a saturable, specific transport mechanism. Cephalomannine, on the other hand, was not able to alter paclitaxel influx, probably because of the different hydroxylation sites in the molecular structure, and hence, its induced metabolic differences ,29. FluoWe in detail characterized the transport of paclitaxel into neurons and provided evidence that the inhibition of drug transporters efficiently attenuated the neurotoxicity of paclitaxel without interfering with its antineoplastic properties. In our initial experiments, we assessed a previously unaddressed issue: the possibility of an alternative, transporter-independent route of paclitaxel translocation. Three findings assured us that paclitaxel influx into neurons is a transporter-based process:Among the large number of drug transporters, most evidence indicates that the organic anion transporter polypeptide (OATP) family is critically involved in paclitaxel transport ,22,32. O−/− 1A2tg animals , OATP1A2 has been described to transport paclitaxel, especially into the liver. The OATP1A2 transport of paclitaxel in transgenic mice was described as being higher than that in in OATP1B3 transgenic mice [OATP1A1 is a transporter exclusively expressed in rodents, its human homolog being OATP1A2. In vivo studies on SLCO1a/1bnic mice .In our in vivo study, we used a well-established model of paclitaxel-induced neuropathy ,34. The Glycyrrhizic acid is a naturally occurring component extracted from licorice plants ,38. It hOverall, we here provide evidence that OATP family members are able to translocate paclitaxel. We could modulate this transport to prevent paclitaxel uptake into the neuron and prevent neurotoxic effects while still sustaining the antineoplastic characteristics of paclitaxel . We have


QTLs for growth habit are identified on Arahy.15 and Arahy.06 in peanut, and diagnostic markers are developed and validated for further use in marker-assisted breeding.Arahy.QV02Z8, Arahy.509QUQ, Arahy.ATH5WE and Arahy.SC7TJM. These SNPs and INDELs in relation to peanut GH were further developed for KASP genotyping and tested on a panel of 77 peanut accessions with distinct GH features. This study validates four diagnostic markers that may be used to distinguish erect/bunch peanuts from spreading/prostrate peanuts, thereby facilitating marker-assisted selection for GH traits in peanut breeding.Peanut is a unique legume crop because its pods develop and mature underground. The pegs derive from flowers following pollination, then reach the ground and develop into pods in the soil. Pod number per plant is influenced by peanut growth habit (GH) that has been categorized into four types, including erect, bunch, spreading and prostrate. Restricting pod development at the plant base, as would be the case for peanut plants with upright lateral branches, would decrease pod yield. On the other hand, GH characterized by spreading lateral branches on the ground would facilitate pod formation on the nodes, thereby increasing yield potential. We describe herein an investigation into the GH traits of 521 peanut recombinant inbred lines grown in three distinct environments. Quantitative trait loci (QTLs) for GH were identified on linkage group (LG) 15 between 203.1 and 204.2 cM and on LG 16 from 139.1 to 139.3 cM. Analysis of resequencing data in the identified QTL regions revealed that single nucleotide polymorphism (SNP) or insertion and/or deletion (INDEL) at Arahy15.156854742, Arahy15.156931574, Arahy15.156976352 and Arahy06.111973258 may affect the functions of their respective candidate genes, The online version contains supplementary material available at 10.1007/s00122-023-04327-9. Arachis hypogaea L.) is an allotetraploid legume resulting from the spontaneous hybridization of two diploid species in South America, Arachis duranensis and Arachis ipaensis, followed by natural chromosome doubling , which is primarily driven by the rising demand for edible vegetable oil. In the USA, peanuts are processed into peanut butter, roasted peanuts, confectionery products and snack foods. With a growing global population and limited agricultural land, it is imperative to increase peanut productivity to meet the rising demand.Cultivated peanut , is an important agronomic trait that affects crop yield and facilitates mechanized farming. The plant architecture of cereal crops, such as plant height, number of tillers and tiller angle, correlates directly with grain quality and yield. During the Green Revolution, “Dwarfing alleles” y Hedden . Recent y Hedden . QTLs foy Hedden .Arachis species, such as the diploid progenitors of cultivated peanut, A. duranensis and A. ipaensis, are of the procumbent type, whereas distinct botanical varieties of the cultivated peanut, A. hypogaea, exhibit procumbent (prostrate), decumbent (spreading), semi-erect (bunch) and erect GH traits . Yuanza9102 and wt09-0023 are erect and bunch, respectively. A high level of segregation for the GH trait was observed in the derived RIL population. In May 2021, the F10 RIL population was planted following a randomized block design in three different growth environments, including Yuanyang (sandy loam soil), Shangqiu (loam soil type) and Nanyang (clay soil type). Each plot consisted of two rows spaced 40 cm apart. Each row measured 150 cm in length with 15 cm between plants. The field management adhered to the standard field practice. Before planting, the field was well irrigated and 450 kg per hectare of compound fertilizer  was applied. Plants were irrigated to maintain 60–70% soil moisture throughout the entire growing season.A population consisting of 521 RILs was derived from a cross between Yuanza9102 and wt09-0023, which was described in detail previously  of 521 RILs were investigated in the field about 60 days after sowing. GH was classified into four morphological types, including erect, bunch, spreading and prostrate based on standards established by Pittman . Before The RIL population was genotyped by Restriction Site-Associated DNA sequencing (RAD-seq). After screening for polymorphic SNPs between the RIL parents and further filtration, 24,852 SNPs remained in the dataset. Redundant markers were then removed, and a genetic linkage map with twenty linkage groups (LG) consisting of 5120 markers was constructed by using Joinmap v5.0 , and an INDEL marker for qGHA06 on chromosome 6 was developed by using Primer3Plus (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) (Table S1). A 20 μL PCR was prepared, including 4 μL of 5 × buffer, 1.6 μL of dNTP (2.5 mM), 0.4 μL of PrimeSTAR® GXL (Takara Bio Inc.) DNA polymerase (1.25 U/μL), forward primer (10 μM), reverse primer (10 μM) and 5 μL of genomic DNA (50 ng/μL). PCR was performed under following conditions: initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 10 s, annealing at 57 °C for 15 s, extension at 68 °C for 12 s and a final extension at 68 °C for 7 min. The 200 bp PCR product was sequenced by BGI Tech Solutions Co., Ltd. . The genomic DNAs of 165 RILs and 77 germplasm accessions were extracted by applying the CTAB method .SNPs and INDELs in the QTL region were examined in the peanut resequencing data. Polymorphic markers between RIL parents were selected, and the effects of these variants were predicted by SnpEff version 5.1 . A SNP at 156,931,574 bp on chromosome 15 was located in the coding sequence of Arahy.509QUQ (annotated as MADS-box transcription factor 17-like), resulting in a splicing site variant. In addition, an INDEL variant  located in the coding sequence of Arahy.ATH5WE (annotated as MADS-box transcription factor 6) may lead to a frameshift mutation. On chromosome 6, an INDEL variant in the coding sequence of Arahy.SC7TJM (annotated as homeobox-leucine zipper protein) led to a conservative in-frame deletion of 3-nucleotide (ATT) repeats coding for asparagine on the reverse strand.By leveraging the resequencing data analysis, the effects of SNPs and INDELs in the QTL regions of LG 15 and LG 16 were predicted using the SnpEff package 5 INDEL genotype; and 79 erect RILs carried genotype “A:A”/“C:C”/“TA:TA” on chromosome 15 and either C(ATT)5 or C(ATT)3 as the INDEL genotype on chromosome 6. In the validation panel, 35 prostrate or spreading accessions and 42 peanut accessions with erect or bunch GH were also genotyped by these molecular markers 3 on chromosome 6 (Table S4), whereas the erect/bunch accessions were found to feature the chromosome 15 genotype “A:A”/“C:C”/“TA:TA” and the INDEL genotype C(ATT)5 or C(ATT)3 or C(ATT)4. Furthermore, genotypes with “C:C”/“A:A”/“-:-” on chromosome 15 and C(ATT)4 on chromosome 6 were found to be closely associated with erect or bunch GH trait (Table S4).The three KASP markers on chromosome 15 and an INDEL marker on chromosome 6 were then examined in 165 RILs (Table S3). Among them, 55 spreading or prostrate RILs had consistent genotypes featuring chromosome 15 markers “C:C”/“A:A”/“-:-” and chromosome 6 INDEL marker C(ATT)Previous QTL studies in peanut for height of main stem, length of first lateral branch and number of primary branches were limited to the SSR-based low-density genetic map  was found to correspond to a defective gibberellin 20 oxidase 2 (GA20ox2) , in accordance with LG 16 of the genetic linkage map where the INDEL marker for qGHA06 was assigned to.In this study, we have identified 5 or C(ATT)3 INDEL marker on chromosome 6 were identified in the erect and bunch GH groups. In addition, the genotypes with “C:C”/“A:A”/“-:-” on chromosome 15 combined with C(ATT)3 on chromosome 6 were closely associated with the spreading and prostrate GH. The INDEL genotype on chromosome 6, C(ATT)4, was unique to the erect and bunch peanut accessions in the validation panel consisting of 77 germplasm accessions (Table S4). In the 165 RILs that were selected for genotyping, the C(ATT)4 allele was not identified because of its absence in their parents. Instead, the genotypes of 31 erect peanuts among the 165 RILs were “C:C”/“A:A”/“-:-” on chromosome 15 and C(ATT)5 on chromosome 6, consistent with the parent wt09-0023. However, in the validation panel, “C:C”/“A:A”/“-:-” on chromosome 15 combined with C(ATT)5 on chromosome 6 were solely identified in the spreading and prostrate accessions. Overall, the two QTLs identified in this study have proven to be useful in fine mapping for the GH traits, offering promise for further identification of novel QTLs for GH traits in populations with different genetic backgrounds.In this study, four markers closely linked with peanut GH were developed and validated in both the RIL population and a validation panel. The genotypes of three KASP markers on chromosome 15 were strongly correlated with one another, either with “C:C”/“A:A”/“-:-” or “A:A”/“C:C”/“TA:TA”. Notably, peanut accessions with spreading or prostrate GH were exclusively “C:C”/“A:A”/“-:-” for the three markers on chromosome 15. Peanut accessions carrying the “A:A”/“C:C”/“TA:TA” markers on chromosome 15 combined with C(ATT)qIOPTA15 was found to be adjacent to qGHA15 on LG 15, while qIOPTA06 was colocalized with qGHA06 on LG 16 , Nanyang (NY) and Shangqiu (SQ).Supplementary tablesBelow is the link to the electronic supplementary material.


Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids  using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates.Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type  Crocus (Iridaceae) flowers and reach particularly high levels in the stigmas of Crocus sativus (saffron), the most expensive spice in the world [Crocus stigmas  was replaced by zeaxanthin, a xanthophyll that is normally accumulated in leaves only under high-light conditions . In this report we used both Agrobacterium tumefaciens-mediated infiltration, and inoculation with a custom-built tobacco etch virus , to overexpress the saffron CsCCD2 dioxygenase, which catalyzes the first dedicated step in crocin biosynthesis. Results suggested that depending on the overexpression method used, CsCCD2 showed relaxed substrate specificity in planta, also cleaving lutein and, to a lesser extent, violaxanthin and β-carotene. Remarkably, we obtained a maximum apocarotenoid and crocin yields (1129.6 and 883.7 µg g−1 dry weight (DW), respectively) in genome-edited plants with the highest levels of zeaxanthin, in which CsCCD2 was expressed with the viral vector.Interestingly, expression in planta of the above CCDs is sufficient for crocin accumulation, suggesting that the ALDH and UGT activities are endogenously expressed in plant tissues. Heterologous expression systems include maize endosperm , rice ana leaves ,11,14, ta leaves ,16, and a leaves . Since mN. benthamiana laboratory isolate (LAB) plants [Lycopene ε-cyclase (LCYe) and two Zeaxanthin epoxidase (ZEP) genes  [lcye1lcye2ZEP1zep2) in which only one of the two ZEP genes was inactivated, and HZ-11 (lcye1lcye2zep1zep2) in which both ZEP genes were inactivated.) plants , edited −2 s−1, given from five Lumilux Cool Daylight L 58W/865 and one Fluora L 58W/77 fluorescent lamps, Osram, Germany). Agroinfiltration was performed by inoculating sub-apical leaves of the second and third whorls, as previously described [N. benthamiana Wt and HZ lines were co-infiltrated with A. tumefaciens C58C1 cells containing pBI121:CsCCD2 [Wild-type (Wt) and HZ plants were grown in soil for 5–6 weeks in a growth chamber with controlled temperature (22 °C) and photoperiod  liquid media containing 50 µg mL−1 kanamycin. Cells were brought to optical density (600 nm) of 0.5 in 10 mM MES-NaOH, pH 5.6, 10 mM MgCl2, and 150 μM acetosyringone, and Vir genes induced for 2 h at 28 °C. Using a needleless syringe, cultures were infiltrated in the abaxial side of two leaves of five-week-old N. benthamiana Wt and genome-edited (HZ-9 and HZ-11) plants. After agroinoculation, plants were cultured in a growth chamber at 25 °C under a 12 h day–night photoperiod with an average photon flux density of 240 µE m−2 s−1. Symptomatic systemic leaves were collected at 10 dpi and processed for metabolite analysis.Plasmid pGTEV:CsCCD2 was built using standard molecular biology techniques with the corresponding restriction enzymes  and T4 DNA ligase . The Eco81I-ApaI DNA fragment of pGTEVΔNIb-CsCCD2L  was replLEAN-S48 , were tr−2 s−1) during the entire extraction procedure. Samples were homogenized  with 1.8 mL 100% cold acetone spiked with 50 mg L−1 of internal standard , using the TissueLyser MM300 . Samples were centrifuged , and supernatant was transferred in two new tubes. A quantity of 0.8 mL acetone was directly transferred into a new tube, centrifuged , and 0.6 mL of the supernatant was filtered using Whatman™ Mini-UniPrep™ Amber Syringeless Filter vials  for LC analysis. A quantity of 0.9 mL was directed to saponification, and thus transferred to a 2 mL tube containing 100 mg of Ambersep 900 OH resin , which was pre-treated with ultrapure water  as previously described [g, 4 °C), 0.6 mL of supernatant was removed and transferred into filtered Whatman™ Mini-UniPrep™ Amber Syringeless Filter vials  for LC analysis. Based on previous experience, this protocol results in recovery efficiencies >90%. Analytical HPLC was carried out on a LC-PhotoDiode Array (PDA) system . LC separation was performed using a Carotenoid C30 column   and the column temperature was 25 °C. The elution system was A, MeOH; B, MeOH/water (4:1 v/v) with 0.2% ammonium acetate; and C, tert-Butyl methyl ether (C). The gradient was 0 to 1.2 min 95% A, 5% B; 3.5 min 80% A, 5% B; 6.8 min 30% A, 5% B, 65% C; 16 min 95%, 5% B. PDA data were recorded in the 200–700 nm range [Carotenoids were extracted from 10 mg of ground, lyophilized leaf tissue under dim laboratory light conditions  cold methanol spiked with 0.5 µg mL−1 formononetin  with vigorous agitation for 30 min at 25 °C. Samples were then centrifuged at 20,000× g for 20 min; the supernatant was collected, filtered with HPLC PTFE filter tubes (0.22 µm pore size), and subjected to high performance liquid chromatography-photodiode array detection-high resolution mass spectrometry (LC–PDA–HRMS) analysis using a Q-Exactive mass spectrometer  as previously described [2, 2.5 µm; Phenomenex, Torrance, CA, USA) using as mobile phases water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B) at a total flow rate of 250 µL/min. The separation was developed using 5% B for 0.5 min, followed by a 24 min linear gradient to 75% B. The ionization of polar and semi-polar apocarotenoids was performed using a heated electrospray ionization  source with nitrogen used as a sheath and auxiliary gas, set to 45 and 30 units, respectively. The vaporizer temperature was 270 °C, the capillary temperature was 30 °C, the discharge current was set to 5 μA, and the S-lens RF level was set at 50. The acquisition was performed in the mass range 110–1600 m/z both in positive and in negative ion modes with the following parameters: resolution 70,000, microscan 1, AGC target 1 × 106, maximum injection time 50. All the solvents used were LC-MS grade . PDA data were recorded in the 200–700 nm range. Crocins, crocetin, picrocrocin, and OH-β-cyclocitral were identified in positive ionization mode in LC-MS and LC-MS/MS chromatograms and quantified in PDA chromatograms as follows: crocin and crocetin peak areas were integrated at their λmax = 443 nm, picrocrocin and OH-β-cyclocitral at λmax = 257 nm, and the internal standard formononetin at λmax = 256 nm; the areas were quantified considering the specific extinction coefficients (ε1%) of each compound, using the ε1% values reported for crocins and crocetins [Semi-polar metabolites were extracted as previously described with some modifications . Brieflyrocetins , for picrocetins , and forrocetins . Data weThe Past software (version 3.11)  was usedN. benthamiana tissues from inoculated plants was performed using a Zeiss 7080 Axio Observer  equipped with a C-Apochromat 40X/1.20 W corrective water immersion objective lens. For the multicolor detection of crocins and chlorophylls, imaging was performed using the sequential channel acquisition mode. A 458 nm laser was used for excitation of crocin and chlorophyll autofluorescence, which was detected from 465 to 620 nm and from 690 to 740 nm, respectively. Images were processed using the FIJI software , “http://fiji.sc/Fiji” (accessed on 4 April 2021).Laser scanning confocal microscopy (LSCM) analysis of N. benthamiana [Lycopene ε-cyclase (LCYe1 and LCYe2) and two Zeaxanthin epoxidase (ZEP1 and ZEP2) genes present in the N. benthamiana allotetraploid genome were targeted. Two different edited lines were obtained: HZ-9 (lcye1lcye2ZEP1zep2) in which only one of the two ZEP genes was inactivated, and HZ-11 (lcye1lcye2zep1zep2) in which both ZEP genes were inactivated . In both lines, ε-branch (lutein) and β-branch (violaxanthin and neoxanthin) xanthophylls were completely replaced by zeaxanthin, which is undetectable in Wt lines  was observed in HZ-9 leaves, which contain higher levels of zeaxanthin, the CsCCD2 preferred substrate. In HZ-11 leaves, in spite of the higher levels of zeaxanthin, much lower total apocarotenoid and crocin levels were observed , probably due to the less efficient agroinfiltration process. The relative abundance of different crocin isomers in leaves of Wt, HZ-9, and HZ-11 plants was similar, with trans-crocin 3 being the most abundant isomer, followed by cis-crocin 4. In addition, in the HZ lines we observed an increase in the accumulation of hyperglucosylated crocins, trans and cis-crocin 5, compared to the Wt.A strong increase in total apocarotenoid and crocin levels  cistron (TEVΔNIb) to increase cargo capacity [trans, such as in a transformed N. benthamiana line in which this viral protein was expressed under the control of cauliflower mosaic virus 35S promoter and terminator [N. benthamiana genotypes not carrying the 35S:NIb transgene. In this vector, CsCCD2 is expressed as the first gene product of the viral polyprotein, since this position in the viral polyprotein is crucial to efficiently target the chloroplast [We also investigated V vector  as a seccapacity . Thereforminator . To circoroplast   compared to agroinfiltration . Also in this case, picrocrocin and OH-β-cyclocitral accumulated at higher levels both HZ-9 lines compared to the Wt, and comparable levels were detected between the two edited lines  reduction with respect to xanthophylls .N. benthamiana leaves, followed by lutein, neoxanthin, violaxanthin, and β-carotene; (ii) the higher expression levels obtained via TEV relax the observed substrate specificity of CsCCD2, making it able to cleave neoxanthin and violaxanthin, two xanthophylls that are not cleaved upon agroinfiltration; (iii) the highest crocin levels are reached in HZ-11 plants, indicating that genome editing and a virus-based expression system play additive roles in achieving maximal crocin production.Taken together, these data suggest the following scenario: (i) zeaxanthin is the preferred substrate for CsCCD2 cleavage in The different amenabilities of the different carotenoids to cleavage by CsCCD2 in leaves are probably due to their different structures , but theN. benthamiana leaves.HZ leaves, in which CsCCD2 is expressed either by simple agroinfiltration or using a viral vector, not only show quantitative differences in crocin accumulation, but also qualitative differences in the types of apocarotenoids accumulated . AgroinfN. benthamiana leaves together with CsCCD2 increases the levels of crocins, indicating that endogenous vacuolar transport in N. benthamiana leaves may be rate-limiting for crocin accumulation [N. benthamiana transporters mediating vacuolar localization of crocins are induced by the increased abundance of their substrate. This hypothesis could be tested by transcriptomic analyses of TEV:CsCCD2–infected plants in comparison with an empty vector control.Another factor contributing to accumulation of crocins is their sequestration into vacuoles ,40. Co-amulation . Qualitamulation , the endN. benthamiana is a promising bio-factory system for the production of biopharmaceuticals and small molecules, and new plant-breeding techniques such as genome editing, agroinfiltration, and the use of viral vectors are powerful tools to deploy its full potential [N. benthamiana leaves, as well as to study the in planta substrate specificities of carotenoid cleavage enzymes.otential . In this


Ruditapes philippinarum clams exposed to the hormonal contaminant 17-α-ethinylestradiol (EE2), at 17 °C and 21 °C. The results reveal that exposure at 17 °C triggers a weak response at low EE2 concentrations, suggestive of a slight increase in membrane rigidity, followed by lipid metabolic stability at higher EE2 concentrations. On the other hand, at 21 °C, lipid metabolism begins to respond at 125 ng/L EE2, with antioxidant docosahexaenoic acid (DHA) helping to tackle high-oxidative-stress conditions, in tandem with enhanced storage of triglycerides. Exposure to 625 ng/L EE2 (highest concentration) enhances phosphatidylcholine (PtdCho) and polyunsaturated fatty acid (PUFA) levels, their direct intercorrelation suggesting PUFA incorporation in new membrane phospholipids. This should lead to increased membrane fluidity, probably aided by a decrease in cholesterol. PUFA levels, considered a measure of membrane fluidity, were strongly (and positively) correlated to intracellular glycine levels, thus identifying glycine as the main osmolyte entering the cells under high stress. Membrane fluidity also seems to elicit the loss of taurine. This work contributes to the understanding of the mechanisms of response of R. philippinarum clams to EE2 in tandem with warming while unveiling novel potential markers of stress mitigation, namely high levels of PtdCho, PUFAs (or PtdCho/glycerophosphocholine and PtdCho/acetylcholine ratios) and linoleic acid and low PUFA/glycine ratios.This paper reports on an NMR metabolomics study of lipophilic extracts of  Ruditapes philippinarum clams [Lampsilis fasciola mussels [Mytilus galloprovincialis mussels [Pharmaceutical compounds (PhACs) are contaminants of emerging environmental health concern which, if not removed efficiently from urban wastewaters by conventional wastewater treatment plants (WWTPs), may lead to adverse health effects on non-target aquatic wildlife ,2,3. Amoum clams ; oxidati mussels ,11; and  mussels ,13. In a mussels ,12,14.Saccostrea glomerata, to estrogenic mixtures. A significant reduction in phospholipids (PLs) and phosphatidylcholine (PtdCho) was associated with a reduction in available and/or an induction of reactive oxygen species (ROS) via estrogen metabolism, which may cause lipid peroxidation and lower availability of phospholipids. NMR lipidomics strategies have also been applied to study the effects of metal(loid)s on mosquitofish [Oxidative stress is one of the early responses to chemical toxicity in marine organisms, which may lead to the oxidative damage of DNA, proteins and lipids . Lipid puitofish  and of duitofish ,28.1H NMR was applied, for the first time to our knowledge, to measure the impact of temperature rise , in tannowledge  that sucnowledge  and lipo1H NMR spectrum of the lipidic extracts of clams, at 21 °C with no exposure to EE2 (control), visually similar to those recorded in other conditions of temperature (17 °C) and EE2 exposure. Peak assignment  occurring at 17 °C at lower EE2 concentrations (5 and 25 ng/L) and then at 625 ng/L (Q2 = 0.52). At 21 °C, changes compared to controls only seemed to occur at the highest concentration  induced slight decreases in FA methyls, GPLs and a ceramide species b. The hi2)n resonance at δ 1.27 ppm, 2)n ratio was used to investigate such conversion. This ratio tended to increase at 125 ng/L EE2, particularly at 21 °C , as a p samples ,36, althR. philippinarum does not exhibit any low EE2 concentration effects and only begins to respond significantly at 125 ng/L EE2 (with decreased DHA and increased TGs). DHA decrease is consistent with higher oxidative stress levels measured at higher EE2 concentrations [R. philippinarum clams exposed to long-term daily rhythms of air [At 21 °C, the lipid metabolome of trations , as thiss of air ; furthers of air . We hypos of air  . Howeves of air , while gs of air , the lats of air .Undoubtedly, it is the 625 ng/L EE2 concentration that triggers the strongest response in bivalves’ lipidome at 21 °C . We propR. philippinarum clams exposed to EE2 at 21 °C. The absence of meaningful correlations between membrane fluidity and the increasing levels of homarine or trigonelline suggest that these compounds are either naturally present in cells (not needing to cross the membrane) and/or are involved in a variety of roles (which would diffuse any existing correlation with membrane characteristics). Homarine has been reported as important to regulate ionic strength and maintain intracellular osmotic pressure [r ca. 0.5) correlation with PUFA levels (membrane fluidity). This would suggest that taurine might be crossing the membrane to exit cells, and, indeed, taurine levels decrease at 625 ng/L compared to 125 ng/L EE2 which suggests either its loss due to membrane degradation and/or its lesser production through hypotaurine oxidation, although the latter explanation is somewhat inconsistent with the high oxidative stress levels reported for 625 ng/L EE2 at 21 °C [Haliotis diversicolor shellfish (muscle/liver) to hypoxia and warming [The above-discussed membrane fluidity increase was here demonstrated to lead to a large increase in intracellular levels of glycine (the osmolyte that more strongly correlated (positively) with membrane fluidity, as viewed by PUFA levels), compared to taurine, homarine or trigonelline. Glycine is one of the key FAAs involved in osmoregulation and cell volume adjustment in hard clams, with reports describing its continuous increase under hypersalinity stress , and we pressure ,43, whilpressure , being kpressure . The rolat 21 °C ,14. Taur warming , similarRuditapes philippinarum were collected in the Ria de Aveiro  in September 2019, and organisms with similar size  were selected for the study. For depuration and acclimation to laboratory conditions, clams were kept in artificial seawater for 10 days , at 17 ± 1 °C, under continuous aeration and a natural photoperiod. The artificial seawater was renewed every 2−3 days. After this period, the clams were subjected to a 28-day chronic toxicity test : 0 (control group), 5, 25, 125 and 625 ng/L. To evaluate the combined effects of temperature rise and EE2 exposure, two different water temperatures were considered: 17 ± 1 °C  and 21 g, 4 °C, 10 min). The supernatant was transferred into a microtube to which 300 µL of chloroform and 300 µL of water were added. The mixture was vortexed, centrifuged again  and left in ice for 5 min. The bottom (apolar) layer was collected and transferred into glass amber vials, dried under a nitrogen flow, and stored at −80 °C until NMR analysis.The detailed clam metabolite extraction procedure has been described before , followiThe dried lipidic extracts were re-suspended in 600 µL of deuterated chloroform  containing 0.03% tetramethylsilane (TMS) for chemical shift referencing. After vortex homogenization, 550 μL of solution was transferred to 5 mm NMR tubes. For each sample, one unidimensional (1D) 1H NMR spectrum was acquired on a Bruker AVANCE III 500 spectrometer , operating at 500.13 MHz for 1H observation, using a 5 mm inverse probe, at 298 K, using a standard 90° pulse sequence, “zg” pulse sequence (Bruker library). A total of 512 scans were collected into 32 k data points, with a spectral width of 7002.8 Hz, acquisition time of 2.34 s and relaxation delay of 3 s. Prior to Fourier transformation, each free-induction decay was zero-filled to 64 k points and multiplied by a 0.3 Hz exponential line-broadening function. The spectra were manually phased, baseline corrected, and chemical shift referenced to TMS signal, at δ = 0.00 ppm. Peak assignments were based on existing literature ,49,50,511H NMR spectra were converted to data matrices , aligned using the recursive segment-wise peak alignment method [1H NMR data, upon unit variance (UV) scaling . The obtained models were considered satisfactorily robust for predictive power (Q2) values approaching or above 0.5. For univariate analysis, each identified metabolite or relevant resonance peaks were integrated into the raw spectra  and normalized to the total spectral area (excluding the water and chlorofom regions). For each pairwise comparison, effect size (ES) values [p-values) were assessed by the Wilcoxon test . Benjamini–Hochberg false discovery rate (FDR) [Prior to multivariate analysis, full-resolution 1D t method  (Matlab t method  and supet method  models w) values ,58 and ste (FDR)  correctite (FDR) . Boxplotte (FDR) . STOCSY te (FDR)  was perfRuditapes philippinarum clams, when exposed to 17-α-ethinylestradiol (EE2), under warming conditions (17 °C and 21 °C), using NMR metabolomics. Whereas the control temperature only triggers a weak response at low EE2 concentrations, subsequently reaching lipid metabolic stability (at higher EE2 concentrations), when the temperature is raised to 21 °C, lipid metabolism begins to respond significantly at 125 ng/L EE2, with selected PUFAs playing an antioxidant role, while enhanced storage of triglycerides at the expense of saturated FAs is noted. The highest EE2 concentration tested (625 ng/L) induces increases in PtdCho and PUFA biosynthesis, probably leading to increased membrane fluidity, aided by a decrease in cholesterol. This effect was found to strongly correlate with intracellular glycine levels, identifying this amino acid as the main osmolyte entering the cells under high stress. Other osmolytes seem to behave differently, with the apparent loss of taurine due to membrane instability, while homarine and trigonelline exhibited no clear dependence on membrane fluidity.We report on the adaptations of the lipid metabolism of R. philippinarum clams exposed to EE2 and warming conditions, this work unveils novel potential markers of stress mitigation mechanisms, such as high levels of PtdCho and PUFAs , as well as low PUFA/glycine ratios. Follow-up studies will investigate the relationship between these biochemical changes and potential mechanisms of R. philippinarum transformation and survival under environmental stress.As well as contributing to the understanding of the response mechanisms of