Adults with XLH develop a painful mineralization of the tendon-bone attachment site (enthesis), called enthesopathy. Treatment of mice with XLH (Hyp) with 1,25D or an anti–FGF23 Ab, both of which increase 1,25D signaling, prevents enthesopathy. Therefore, we undertook studies to determine a role for impaired 1,25D action in enthesopathy development. Entheses from mice lacking vitamin D 1α-hydroxylase (Cyp27b1) (–/–C) had a similar enthesopathy to Hyp mice, whereas deletion of Fgf23 in Hyp mice prevented enthesopathy, and deletion of both Cyp27b1 and Fgf23 in mice resulted in enthesopathy, demonstrating that the impaired 1,25D action due to high FGF23 levels underlies XLH enthesopathy development. Like Hyp mice, enthesopathy in –/–C mice was observed by P14 and was prevented, but not reversed, with 1,25D therapy. Deletion of the vitamin D receptor in scleraxis-expressing cells resulted in enthesopathy, indicating that 1,25D acted directly on enthesis cells to regulate enthesopathy development. These results show that 1,25D signaling was necessary for normal postnatal enthesis maturation and played a role in XLH enthesopathy development. Optimizing 1,25D replacement in pediatric patients with XLH is necessary to prevent enthesopathy.X-linked hypophosphatemia (XLH) is characterized by high serum fibroblast growth factor 23 (FGF23) levels, resulting in impaired 1,25-dihydroxyvitamin D  The enthesis is a specialized tissue that anchors the musculotendinous unit to bone. It functions to off-load the mechanical stress between muscle and bone and, therefore, is required for normal limb and body movement . The fibPHEX gene (Cyp27b1), resulting in decreased production of 1,25-dihydroxyvitamin D3  (X-linked hypophosphatemia (XLH) is the most common form of inheritable rickets  that results from a mutation in the HEX gene . It is c 1,25D) –8. The mD –8. The  . Entheso  –12; thos  . Common    because   .Hyp mice originate from progenitors that express Sox9 (a marker of chondrocytes) and scleraxis (Scx) , which increases 1,25D levels, prevented enthesopathy in Hyp mice despite dramatic increases in Fgf23 expression  in Scx-expressing cells to determine if 1,25D acts directly on enthesis cells to suppress BMP and IHH signaling. In addition to determining a role for impaired 1,25D signaling due to enhanced FGF23 levels in XLH enthesopathy development, these studies will also define a role for 1,25D in normal postnatal enthesis maturation.To identify the relative roles of impaired 1,25D action and enhanced FGF23 action (both of which are due to high circulating levels of FGF23), and the Hyp mice have an expansion of hypertrophic-appearing fibrocartilage, or enthesopathy, cells (HECs) that stain positive for safranin O (SafO), a marker of cartilage proteoglycans, and alkaline phosphatase (ALP) activity, a marker of mineralization, at P30  (Fgf23 (–/–F) (Hyp mice lacking Cyp27b1 (–/–/HypC) or Fgf23 (–/–/HypF) were analyzed. –/–C mice fed a high-calcium, high-phosphate rescue diet have normal serum calcium and phosphate levels; thus, analysis of –/–C entheses will enable determining whether impaired 1,25D signaling contributes to the enhanced BMP and IHH signaling observed in Hyp entheses and whether 1,25D action plays a role in normal enthesis maturation , which have normal serum calcium and phosphate levels when fed a rescue diet, will differentiate whether impaired 1,25D production due to high FGF23 levels or actions specific to FGF23 lead to XLH enthesopathy. Because FGF23 overexpression is unlikely to be the only consequence of the PHEX mutation (Cyp27b1 and Fgf23 in Hyp mice (–/–F–/–/HypC) will further elucidate if 1,25D- and FGF23-independent effects of the PHEX mutation influence XLH enthesopathy pathogenesis. Like –/–F–/–C mice, –/–F–/–/HypC mice weaned onto a rescue diet have normal serum calcium and phosphate levels   or Fgf23turation . Hyp micD levels . Examina–/– mice  7), wil, wilCyp2mutation , ablatioe levels   and RUNX2  have an expansion of SafO+ HECs with increased immunoreactivity for BMP target p-SMAD1/5/8 and IHH targets PTCH and RUNX2 . BM. BMHyp Aenthesis –18. In snd RUNX2 , confirmnd RUNX2 . Deletioentheses . The perve to WT . Ablatioentheses . Quantit with WT .Cyp27b1, all mice null for Cyp27b1 have an increased percentage of enthesis cells that stain positive for ALP .Our previous results demonstrated that rol mice  and 3. Tactivity . SimilarHyp entheses, there is also a significant increase in mRNA expression of BMP and IHH target genes in Hyp entheses by P14 (–/–C mice demonstrate increased expression of BMP target genes Noggin (Nog) and Ihh, with Ihh expression being slightly, but significantly, higher in Hyp entheses as compared with –/–C entheses. P14 –/–C entheses also have enhanced expression of IHH target genes Ptch1, Runx2, and Gli1, and cartilage proteoglycan marker aggrecan (Acan), whereby the fold increase in expression of these genes relative to WT entheses was similar to that seen in Hyp entheses , which leads to the phosphorylation of SMAD1/5/8 . BMP2 angenitors , and stugenitors . BMP famgenitors , 28–30.Gdf5 was increased in both Hyp and –/–C entheses, whereas Bmp2, Bmp4, and Bmp6 mRNA expression was not elevated in either Hyp or –/–C entheses  from P2 to P30 increased serum phosphate levels, maintained normocalcemia, and prevented the increase in cartilage proteoglycans, BMP and IHH signaling, and ALP activity in entheses, thus blocking the development of enthesopathy in Hyp mice . f/f;ScxCre+Vdr mice and corresponding f/f;ScxCre–Vdr control mice have normal serum calcium and phosphate levels , and of the chondrogenic marker Acan , have enhanced BMP and IHH signaling and ALP activity, thus confirming that impaired 1,25D signaling as a result of increased serum FGF23 levels in Hyp mice leads to enthesopathy.Our previous investigations demonstrated that the treatment of esopathy . Becausepression , these sFgf23in Hyp mice restored normal BMP and IHH signaling in Hyp entheses, suggesting actions specific to FGF23 or actions downstream of FGF23, such as decreased 1,25D production, regulate enthesopathy development. However, deletion of Cyp27b1 in –/–F and –/–/HypF mice (–/–/F–/– and C–/–/F–/–/HypC) resulted in increased BMP and IHH signaling in entheses, similar to that seen in Hyp and –/–C control mice, thus demonstrating that impaired 1,25D production due to elevated FGF23 levels, not actions specific to FGF23, results in XLH enthesopathy. Deletion of Phex in –/–C and –/–/F–/–C mice (–/–/Hyp and C–/–/F–/–/HypC) did not lead to further increased immunoreactivity for BMP and IHH target genes or ALP activity in entheses, suggesting PHEX-specific functions are unlikely to play a dominant role in XLH enthesopathy development. Like mice and humans with XLH, mice overexpressing Fgf23  had normal mineral ion homeostasis as well as Achilles entheses with enhanced BMP and IHH signaling. These results demonstrate that 1,25D acts directly on Scx-expressing cells to inhibit BMP and IHH signaling in entheses.Gdf5-expressing cells  relative to WT mice , suggesting XLH enthesopathy develops in the early postnatal period  in either y in Hyp  and C–/–Hyp mice with intermittent 1,25D combined with phosphate supplementation after enthesopathy has already developed did not suppress ALP activity in Hyp entheses  is not lower, and the expression of BMPRs 1A and 2 is not changed, compared with P14 entheses. Expression of Gdf5 and its preferred receptor Bmp1b is decreased in P60 versus P14 entheses, indicating this could play a role in the decreased BMP and IHH signaling observed with aging.Deletion of the entheses , whereasRt+ mice  did not liferate , thus reHyp and –/–C mice if 1,25D therapy is initiated early in postnatal development, indicating that 1,25D signaling is protective against enthesopathy development. These findings support the beneficial effects of early, consistent, and optimized 1,25D or FGF23Ab therapy. Enhancing 1,25D signaling early in development may prevent the development of XLH enthesopathy.Taken together, our results demonstrate that in XLH, impaired 1,25D action due to high serum levels of FGF23, not the direct effects of increased FGF23, underlies the pathogenesis of enthesopathy. 1,25D acts directly on enthesis cells to suppress BMP and IHH signaling and can prevent enthesopathy in Cyp27b1, Fgf23, or both, with or without the Hyp mutation, were analyzed. Cyp27b1 null mice (–/–C) lack exon 8, a region coding the heme-binding domain  (Fgf23 null mice (–/–F) have a knockin of EGFP following the ATG in exon 1 . –/–C mice were mated to –/–F mice to generate mice null for both Cyp27b1 and Fgf23 (–/–/F–/–C). Hyp mice  were mated to –/–C, –/–F, and –/–F–/–C mice, generating Hyp mice null for Cyp27b1 (–/–/HypC), Fgf23 (–/–/HypF), or both (–/–/F–/–/HypC).All mice were on a C57BL/6J background. Mice of each genotype were backcrossed at least 8 generations into the C57BL/6J background. All mice were maintained in a virus- and parasite-free barrier facility, exposed to a 12-hour light/dark cycle, and housed with up to 5 mice per cage. To examine the relative roles of 1,25D, FGF23, and PHEX in enthesopathy development, mice lacking  Canada) . Fgf23 nVdr in Scx-expressing cells, mice carrying exon 4 floxed alleles   and Scx-CreERt mice  . Tamoxifen  dissolved in sunflower oil was injected i.p. on P30 and P31 into f/f;ScxCreERt+Vdr mice. The enthesis phenotype of f/f;ScxCre+Vdr mice and tamoxifen-treated f/f;ScxCreERt+Vdr mice were compared with that of age- and sex-matched f/f;ScxCre–Vdr and tamoxifen-treated f/f;ScxCreERt–Vdr mice, respectively. Age- and sex-matched WT and Hyp mice that were littermates of each other served as additional controls.To delete the ia, USA)  were matERt mice   were fed a gamma-irradiated rescue diet  with 2% calcium, 1.25% phosphate, 20% lactose, and 2.2 IU vitamin D/g) starting on day 18 to prevent hypocalcemia and hypophosphatemia  at 175 pg/g/d from P2 to P18 and then 80 pg/g/d after P18. The dose of 1,25D was decreased on P18 when mice were weaned onto the rescue diet to prevent hypercalcemia. Male and female Hyp mice were treated with 175 pg/g/d from P30 to P60  at ×5 magnification and isolated using a microsurgical knife by slicing through the proximal end of the tendon and the region adjacent to the distal portion of the calcaneus (-RNA Tissue/Insect Kit (Zymo Research).The site at which the P14 or P60 WT, –8 M 1,25D prior to exposure to recombinant human GDF5 (rhGDF5)  for 30 minutes. For gene expression analyses, chondrocytes were pretreated for 18 hours with 1 × 10–8 M 1,25D prior to exposure to rhGDF5 (200 ng/mL) for 4 hours. Chondrocytes were harvested for protein or RNA  after 72 hours in culture.Primary chondrocytes were isolated from P2 WT murine ribs and cultured as previously described , 48. ChoProtein concentration was quantitated using the BCA protein assay (Pierce) and 7.5 μg of protein was subjected to Western blot analysis. Membranes were blocked in EveryBlot blocking buffer  and then incubated with primary Abs against p-SMAD1/5/9 , SMAD1 , or Actin , followed by incubation in HRP-conjugated secondary Abs . Signals were detected with ECL Plus (Amersham Biosciences).RNA was reverse transcribed with SuperScript II (Roche). Real-time quantitative PCR was performed using the QuantiTect SYBR Green RT-PCR kit (Qiagen) on the Light Cycler 480 II (Roche). Gene expression was quantitated relative to β-actin and then normalized to the corresponding WT entheses of the same age isolated on the same day (for enthesis RNA) or control-treated chondrocytes (for primary chondrocyte RNA) for each sample, using the methods of Livak and Schmittgen .P < 0.05. Because male and female mice have uniform phenotypes within each mouse genotype, analyses are performed with each group including equal numbers of mice of both sexes.All data shown are reported as mean ± SD. One-way ANOVA followed by Fisher’s least significant difference test was used to analyze significance between all control and genotype or treatment groups , 50. SigAll animal studies were approved by the IACUC at Brigham and Women’s Hospital . This study was performed in accordance with the recommendations in the NIH’s Guide for Care and Use of Laboratory Animals.All data presented in this manuscript and in the Supplemental Material are available upon request from the corresponding author.ESL designed the project. RR, JTB, MS, SV, and ESL contributed to mouse colony management. All authors contributed to molecular biology experiments. Histological analyses were conducted by RR, JTB, MS, SV, SA, and ESL; histomorphometric analyses were conducted by RR, SA, and ESL. SA and ESL prepared the manuscript. SJ contributed to the primary chondrocyte experiments.


Hypomagnesemia was historically prevalent in individuals with type 1 diabetes mellitus (T1DM), but contemporary results indicate an incidence comparable to that in the general population, likely due to improved treatment in recent decades, resulting in better glycemic control. However, a recent study found a significant difference between the serum Mg isotopic composition of T1DM individuals and controls, indicating that disruptions to Mg homeostasis persist. Significant deviations were also found in samples taken one year apart. To investigate whether the temporal variability in serum Mg isotopic composition is linked to the transient impact of administered insulin, Mg isotope ratios were determined in serum from 15 T1DM individuals before and one hour after insulin injection/meal consumption using multi-collector inductively coupled plasma-mass spectrometry. Consistent with results of the previous study, significant difference in the serum Mg isotopic composition was found between T1DM individuals and 10 sex-matched controls. However, the average difference between pre- and post-insulin injection/meal T1DM samples of 0.05 ± 0.13‰ (1SD) was not significant. No difference was observed for controls before (−0.12 ± 0.16‰) and after the meal (−0.10 ± 0.13‰) either, suggesting a lack of a postprandial Mg isotopic response within one hour of food consumption, and that the timing of the most recent meal may not require controlling for when determining serum Mg isotopic composition. Diabetes mellitus is a metabolic disorder that is typically characterized by the loss of the ability of β-pancreatic cells to adequately produce and secrete insulin, leading to hyperglycemia . Accordi−1) was commonly reported in individuals with T1DM . Th. Th26Mg p = 0.79) was observed between the serum Mg concentration before (mean = 22.5 ± 2.9 mg L−1) and after the meal (mean = 22.2 ± 2.5 mg L−1) in healthy controls, suggesting a lack of a postprandial response within one hour of food consumption. Also, no difference (p = 0.23) was observed in the diabetic group between samples taken before (mean = 20.9 ± 3.4 mg L−1) and one hour after insulin administration and consumption of the meal (mean = 19.9 ± 2.2 mg L−1). Seven out of 14 T1DM individuals in which HbA1c was determined had good glycemic control (HbA1c ≤ 7.5%), which is in-line with recent trends . Th. Thr = −−1), and foods containing phytates, polyphenols, and oxalic acid  have the ability to bind Mg and produce insoluble precipitates, reducing Mg bioavailability and absorption [26Mg increasing from herbivores to higher-level consumers  [26Mg compared to vegetarians and vegans. Fluoride is commonly found in drinking water and other dietary staples, and prevents Mg absorption through binding and production of insoluble complexes [A limitation for the present study is that there are a number of factors that can influence Mg homeostasis, complicating the study of serum Mg dynamics . Althougsorption ,49,50,51d chain) ,53. Theromplexes ,55,56. Complexes ,63,64,65omplexes ,24. Otheomplexes ,66.3 and 12 M HCl  were further purified by sub-boiling distillation in a Savillex DST-4000 acid purification system . Ultrapure TraceSELECT® 9.8 M hydrogen peroxide and ACS grade acetone were purchased from Sigma Aldrich .Blood samples were collected in Vacutainer BD SST™ ll Advance Tubes with Gel and Clot Activator (5 mL). Ultrapure water (resistivity > 18.2 MΩ cm) was obtained from a Milli-Q Element water purification system . Trace metal analysis grade 14 M HNO−1) from Inorganic Ventures  for quantification purposes. A 1000 mg L−1 Ga standard solution (Inorganic Ventures) was used as an internal standard for element quantification. A standard solution, CPI Mg , was used as an in-house isotopic standard for monitoring instrument performance and the accuracy and precision of the Mg isotope ratios. The Seronorm™ Trace Elements Serum reference material  was also used for method evaluation.A multi-element solution containing Na, Mg, K, Ca, Fe, Cu and Zn was prepared from single-element standard solutions  and AG50W-X8 strong cation exchange resin  purchased from Bio-Rad  were used for Mg isolation.This study was reviewed and approved (BC-05376) by an independent Medical Ethics Committee associated with Ghent University Hospital and Ghent University, and all subjects signed an informed consent form concerning this study. The study was conducted according to the guidelines of good clinical practice (ICH/GCP) and the Declaration of Helsinki established to protect people participating in clinical studies.Participants were recruited by the Ghent University Hospital. Ten apparently healthy male participants (control group) and 15 males with T1DM  were recruited for this study. The average age of the control group was 35.1 ± 14.6 (1SD) years and the T1DM group was 47.7 ± 16.5 (1SD) years. Participants were asked to consume their usual breakfast at 8:00 and arrive at the Ghent University Hospital at 11:00 to provide their first blood sample. Age, weight, and height were recorded at this time . In the ® PFA beakers using 2 mL of 14 M HNO3 and 0.5 mL of 9.8 M H2O2 at 110 °C for 24 h. After cooling, the vessels were opened and the digests were evaporated to dryness at 90 °C. The residues were then dissolved in 1.2 mL of 0.4 M HCl for Mg isolation using cation exchange chromatography as described previously [3. Following isolation, samples were evaporated to dryness at 90 °C and dissolved in 0.3 M HNO3 for Mg isotopic analysis. Samples were further diluted for Mg quantification and isotope ratios measurement.Samples were thawed and aliquots of ~0.25 mL were digested in closed Savillex−1) was used. The collision reaction cell was pressurized with the NH3/He gas mixture  and pure He . The 24Mg nuclide was monitored in a mass shift approach 3+), whereas 71Ga was monitored on-mass [The Mg concentration was determined using an Agilent 8800 ICP-MS/MS instrument . To correct for signal drift, matrix effects, and instrument instability during element quantification, external calibration with Ga as internal standard (10 μg L on-mass . Instrum26Mg/24Mg and 25Mg/24Mg) were measured using a Thermo Scientific Neptune multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS) instrument  following the protocol reported by Grigoryan et al. [Magnesium isotope ratios . The normality of the datasets was tested using the Shapiro-Wilk’s test. To compare the control and T1DM groups, the Student’s t-test was used for parametric datasets, whereas the Mann-Whitney U test was used for non-parametric datasets. To evaluate any within-group differences before and after insulin injection and/or meal consumption, the paired t-test was used for parametric datasets, whereas the Wilcoxon matched-pairs signed-rank test was used for non-parametric datasets. The significance of relationships between variables was assessed using the Pearson correlation coefficient (parametric data) or the Spearman rank correlation coefficient (non-parametric data). If one of any two datasets being compared was non-parametric, the non-parametric test was employed. p-values of <0.05 were considered statistically significant.Statistical analyses were performed using GraphPad Prism version 10.0.2 for Mac OS, GraphPad Software, Boston, MA, USA, −1). This is comparable rates of hypomagnesemia in the general population [p = 0.18), indicating that the effect of administered insulin is not a cause of the temporal variability in the serum Mg isotopic composition previously observed in T1DM individuals [p = 0.49) was observed for controls before (−0.12 ± 0.16‰) and after the meal (−0.10 ± 0.13‰), suggesting a lack of a postprandial Mg isotopic response within one hour of food consumption, and that the timing of the most recent meal may not require controlling for when determining serum Mg isotopic composition. Future work should investigate whether the large temporal Mg isotopic variations that were previously identified in the serum of T1DM individuals are captured by sampling during the period between insulin injection (when intracellular Mg2+ is increased) and meal consumption [2+ and determination of its Mg isotopic composition to elucidate the short-term Mg dynamics in blood following insulin injection.This study Investigated the effect of administered insulin as a cause of temporal variability in the serum Mg isotopic composition through the comparison of blood serum samples collected before and one hour after insulin injection and meal consumption by T1DM individuals. The serum Mg isotopic response of healthy individuals after meal consumption was also examined. A significant difference in the serum Mg isotopic composition was found between T1DM individuals and controls, corroborating previous results . This supulation ,11,12. Hividuals . No diffsumption . It is asumption . Future


Digital PCR (dPCR) has great potential for assessing gene editing or gene mutation due to its ability to independently inspect each DNA template in parallel. However, current dPCR methods use a fluorescence-labeled probe to detect gene variation events, and their ability to distinguish variated sequences from the wild-type sequence is limited by the probe’s tolerance to mismatch. To address this, we have developed a novel dPCR method that uses a primer instead of a probe to sense gene variation. The enhanced Taq DNA polymerase in the PCR system has a high mismatch sensitivity, which enables our dPCR method to distinguish gene mutations from wild-type sequences. Compared to current dPCR methods, our method shows superior precision in assessing gene editing efficiency and single-base DNA mutation. This presents a promising opportunity to advance gene editing research and rare gene mutation detection. CRISPR/Cas9 genome editing technology has undergone rapid development and is currently being utilized in scientific research on animals ,2,3,4, pDigital PCR (dPCR) technology amplifies and detects individual template DNA molecules in massive droplets or microwells ,30. BeneTo address this issue, we have developed a new digital PCR method, named “get-dPCR” (genome editing test dPCR) to assess gene editing efficiency and gene variation. This method utilizes a specific PCR primer, known as “watching primer” , which iHOXB13 coding sequence , the BRAF c.1391G > T (G464V), and the BRAF c.1799T > A (V600E). According to targeted sequencing information from The Cancer Cell Line Encyclopedia (CCLE) [For this analysis, we chose three gene mutation sites that are commonly used in cancer gene mutation detection ,40. Thesa (CCLE) , they arKRAS 5′UTR region indicated with a HEX fluorescence, and three mutation-specific amplicons for detecting KRAS c.35G > T, BRAF c.1391G > T, and BRAF c.1799T > A, indicated using Cy5, ROX, and FAM fluorescences, respectively. When using wild-type Taq polymerase for the get-dPCR analysis, the wild-type target sequence of Lenti-X 293T cells also produced a Cy5, FAM, and ROX fluorescence which is intended to be specific to the three gene mutations  FBS  and 1% penicillin-streptomycin  according to the manufacturer’s instruction. All the cells were maintained at 37 °C with 5% CO2 and were regularly checked for mycoplasma using a MycoBlueTM mycoplasma detector kit according to the product manual .The SW620 cells , MDA-MB-231 cells , HT-29 cells , and Lenti-X 293T cells  were cultured using Dulbecco’s Modified Eagle’s Medium  supplied with 10%  [® Microtubes.The genomic DNA was prepared from Lenti-X 293T, SW620, MDA-MB-231, and HT-29 cells using the TIANamp Genomic DNA Kit  according to the manufacturer’s instruction, and their cancer mutation information was retrieved from the BioPortal database “er 2022) ,44. The KRAS c.35G > T, BRAF c.1799T > A, and BRAF c.1391G > T, respectively. The corresponding mutation was located at the 3′ end of each watching primer. The primer Tm value was calculated using the online Oligo Calc tool using the ”Salt Adjusted” parameter [For the GEF-dPCR analysis, we used primers and probes that were designed according to a previous report . A speciarameter . All theA typical 30 μL dPCR reaction system included 6 μL of 5 × TaqMan probe buffer, 50 fg of plasmid DNA or 5ng genomic DNA, 6 pmol of each primer, 3 pmol of each probe, and 0.6 μL of Taq polymerase . The PCR reaction systems were then subjected to a digital PCR analysis on a Small Turtle Technology BioDigital QING-digital PCR system. Briefly, the reaction system was loaded into the digital PCR chip using the Fully Automatic Sample Processing System loader S200, and then cycled on the Cycler S200 instrument  with the following program: a preincubation at 50 °C for 10 min, an initial denaturation at 95 °C for 10 min, then 45 cycles of 95 °C for 30 s, 65 °C for 30 s, followed with a final holding at 25 °C. After the PCR reaction, the fluorescence signal was read and analyzed using a Biochip Reader Imager T200 .


The results revealed a substantial reduction in carrot yield under limited irrigation, reaching about 32.2% lower than under GWR100%. Therefore, under limited irrigation conditions, the combined application of Hgd and K2SO4 resulted in a significant yield increase of 78.9% compared to the control under GWR80%. Conversely, under GWR100%, the highest average yield was achieved by applying either Hsp and Kh or Hsp and K2SO4, resulting in yields of 35,833 kg ha−1 and 40,183 kg ha−1, respectively. However, the combination of Hgd and Kh negatively affected the yield under both GWR100% and GWR80%. Nonetheless, applying Kh in combination with Hgd under GWR80% led to improved nitrogen, phosphorus, potassium, potassium/sodium ratio, and total sugar concentrations, while reducing sodium content in carrot roots. Based on this study, it is recommended to adopt GWR80% and treat plants with a combination of Hgd and foliar K2SO4. This approach can help plants overcome the negative effects of water stress, improve yield and root quality, and achieve optimal water use efficiency.Water stress poses a significant challenge for carrot cultivation, leading to decreased yield and inefficient water use efficiency. Therefore, it is crucial to provide plants with suitable supplements that enhance their stress resistance. In this study, we investigated the effectiveness of humic and potassium applications on carrot growth, yield characteristics, root quality, and water use efficiency under varying irrigation levels. A split-split plot experiment was conducted, with two levels of gross water requirements (GWR) (100% and 80%) assigned to the main plots. The subplots were treated with humic acid through foliar application (Hsp) or soil drenching (Hgd). The sub-subplots were further divided to assess the impact of foliar potassium sources  and mineral applications (potassium sulfate, K Addressing these challenges requires the development of water-saving agricultural practices and improvements in water use efficiency to ensure global food security2. Water stress adversely affects nutrient balance, primary and secondary metabolism, and turgor regulation in plants4 resulting in reduced crop yield and quality5. To mitigate the negative impacts of water stress, the use of organic materials has been considered as an agronomic solution6.Climate change, rapid population growth, and soil degradation pose significant challenges to the agriculture sector−), phenolic (OH−), alcoholic, and carbonyl fractions8. Humic substances are generally classified into humic acids, fulvic acids, and humins9. While humins have a non-degrading fraction and have received less attention, researchers have focused on humic and fulvic acids due to their ability to rapidly improve soil fertility and health10. Numerous studies have reported that humic acid applications offer several benefits, including enhanced plant growth, cell permeability, photosynthetic rate, cell elongation, soil structure, water use efficiency, and nutrient transport and availability15. Furthermore, humic acid has been found to promote root growth and nutrient absorption, making it an excellent foliar fertilizer that positively influences leaf, root, and fruit development18.Humic acid, a key component of plant nutrition, is widely used to supplement synthetic and organic fertilizers. It is a naturally occurring polymeric-heterocyclic organic compound that contains carboxylic (COOHDaucus carota L.) are an important vegetable known for their high content of carbohydrates, carotene, minerals, fiber, and vitamin C20. However, conflicting results have been reported in the literature regarding the effects of humic acid application on plant growth and yield22, Some studies found no significant effects, while others observed negative effects on yield traits and quality24. In this concern, Ampong et10 demonstrated that it is essential to test the effects of humic acid under specific conditions before reliable recommendations can be made. Therefore, we hypothesized that the adoption of potassium applications could lead to a kind of nutritional balance with humic, improving yield quality, in particular under water stress conditions.Given these properties, the adoption of humic acid in carrot plants is expected to increase productivity and improve crop quality. Carrot roots , phosphorus (P), potassium (K), yield and water use efficiency (Iwue) of carrot plants. The studied area lies in an arid climatic province, Table −1 as soil applications in three equal portions initiated after 30 days of cultivation, 60 and 90 days. On the other hand, Hsp took the same previous dates for applying H as foliar applications at a rate of 3 g L−1. While, sub-sub plots were divided into three parts to apply K applications, namely . In the control treatments, carrot plants were sprayed with distilled water. While in Kh and K2SO4 treatments, 2 g L−1 of K as foliar spraying was applied in a Kh and K2SO4 form, initiated after 30 days from cultivation then four times every 15 days interval. The net space (10.0 m long × 4.5 m width) of each experimental unit, accordingly, the experimental work involved 54 plots {2 irrigations levels × 3 H treatments × 3 K treatments × 3 replicates}.In order to achieve the objective of the current study, a split-split plot design was used with three replicates. In the main plots, two irrigation water levels were allocated, i.e., 100 and 80% GWR for carrots. In this concern, before the experiment began, soil water parameters were measured, and allowed for a reduction in soil moisture to 60% of the available water, which was the critical limit on carrot development based on previous studies. Therefore, based on this knowledge, irrigation is carried out every two days. Moreover, the irrigation amounts applied to carrot plants under 80% GWR level were proportionally obtained from 100% GWR, as will be handled later. A set sprinkler irrigation system was used to irrigate carrots and each irrigation plot was equipped with a manometer valve to maintain the operating pressure at 2.5 bar and a flow emitter which was used to control the quantity of the targeted irrigation water at each irrigation level. The distance between the sprinklers was 10 m, and the distance between the lines was 9 m. Furthermore, there were buffer zones between the experimental units to avoid interactions (9 m width). While humic (H) application methods were assigned in the subplots . In the Hgd treatment, H was applied at a rate of 30 L haDaucus carota L., cv. Kuroda Max), were sown on the 2 of November 2019 and the 4 of November 2020 in the first and second seasons, respectively. The experimental site was well prepared and soil tillage. All agricultural practices required for carrot production were followed as commonly used in the region and the Egyptian Ministry of Agriculture recommendations for newly reclaimed soil. Carrot seeds were sown on ridges, with a 75 cm spacing between rows and 8 cm between plants. Carrot seeds were purchased from Takii Seed Co. This cultivar is recommended as a highly yielding-commercial cultivar. Furthermore, this cultivar and the implemented methods in the current study complied with international, national, and institutional guidelines and legislation. The harvest was on the 29 of February 2020 and 5 of March 2021, with a total growing season of 120 and 122 days during the first and second seasons, respectively.Seeds of carrot , and 5.0% potassium. While KH was purchased from Zain Fert Co. (it contains 75% H + 4% fulvic acid + 2% iron (Fe) + 10% K2O).37, which can be calculated as:The reference evapotranspiration (ETo) was determined by directly entering specific data that were obtained from the Toshka agrometeorological station, in the CROPWAT package, version 8.0, and ETo was calculated by Penman–Monteith equation as indicated by−1).ETo = Reference evapotranspiration (mm day−2d−1).Rn = Net radiation (MJm−2d−1).G = Soil heat flux (MJmoC−1).Δ = Slope vapor pressure and temperature curve (kPa −1).γ = Psychrometric constant (kPa °C−1).U2 = Wind speed at 2 m height (mses-ea = Vapor pressure deficit (kPa).T = Mean daily air temperature at 2 m height (°C).38 as the following equation:The crop evapotranspiration of carrot (ETc) was calculated according to−1).ETc = Crop evapotranspiration (mm day−1).ETo = Reference evapotranspiration .GWR = The gross water requirement .Etc = Crop evapotranspiration }, and sodium (Na) in carrot roots were estimated according to43. The total carotenoids and carbohydrates (%) in the roots were determined according to the method described by Boadi et al.44, total sugar (%) was determined according to the method described by Yusuf et al.45. While the total chlorophyll in leaves at harvest was determined according to Molina et al.46.After the harvest (with an average of 121 days), the carrot roots were chopped into small pieces, air-dried, and oven dried at 70°C for five days then ground using stainless steel equipment. Additionally, soil samples were taken from the root ridges in each treatment to measure  according to Estefan et al.−2) were recorded for each plot and then converted to kg ha−1.At the harvest, ten plants were randomly taken from each plot to record the average of the following measurements were recorded: plant height (cm), number of leaves, root length and root diameter (cm), leaves plant fresh weight (g), weight of root (g), and root yield .Iwue = irrigation water use efficiency (kg m−1) and.Y = Yield (kg ha3 ha−1).GWR = The gross water requirement (mp ≤ 0.05 level) as per48.Means of variance (ANOVA) were analyzed in all data to determine any statistically significant differences. Statistical analysis was determined by using the statistical package Costat version 6.303. The least significant difference (LSD) for the average data of the two growing seasons was used to test the differences between treatments  values in the soil under two levels of gross water requirements (100 and 80%) at the end of the experiment are tabulated in Table 2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. Likewise, available K in the soil samples decreased by 21.4% (Kh), 13.1% (K2SO4), 32.2% (Hgd), 42.5% (Hgd + Kh), 26.1% (Hgd + K2SO4), 42.5% (Hsp), 52.8 (Hsp + Kh), and 43.9% (Hsp + K2SO4). Finally, relative to full irrigation (GWR100%), soil organic matter in the control treatment attained a greater increase by 266.7, 233.3, and 200% for Hgd + Kh, Hgd, and Hgd + K2SO4, respectively.Relative to full irrigation (GWR100%), TDS of the control treatment fell by 2.2, 1.1, 3.2, 4.9, 2.7, 5.3, and 6.7% for Kh, K2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. Likewise, available K in the soil samples decreased by 8.3% (Kh), 3.3% (K2SO4), 18.1% (Hgd), 30.0% (Hgd + Kh), 25.7% (Hgd + K2SO4), 6.6% (Hsp), 14.0 (Hsp + Kh), and 10.9% (Hsp + K2SO4). Finally, relative to full irrigation (GWR100%), soil organic matter in control treatment was attained the greater increase by 600 and 500% for Hgd + Kh and Hgd + K2SO4, respectively.On the other hand, relative to limited irrigation (GWR80%), TDS of the control treatment fell by 0.8, 0.3, 4.8, 11.0, 8.7, 2.0, and 4.3% for Kh, KTDS levels attained higher increases by 18.8% (Hsp) 17.6% (Hsp + Kh) and 16.4% (Kh) under limited irrigation conditions (GWR80%), respectively, compared with those under well-watered conditions. On the other side, available K in the soil samples achieved fluctuating results, where it attained greater increases values at (Hsp + Kh) and (Hsp) under limited irrigation conditions by 47.0% and 31.1%, respectively, compared with those under well-watered conditions. Similarly, soil organic matter either achieved fluctuating results, where it attained greater decreases values at (Hsp) and (Hsp + Kh) under limited irrigation conditions by 50.2% and 50.0%, respectively, compared with those under full irrigation (GWR100%).2SO4) on carrots N are presented in , The adoption of GWR80% led to a decrease in N contents of 31.9 and 22.9%, respectively, compared to GWR100%. However, when adopting GWR80%, N content was enhanced than GWR100% by applying sole soil applications of Hgd or a combined application of Hgd + Kh by 17.5 and 11.0%, respectively. While there was a non-significant effect by applying the combined applications of Hgd + K2SO4 and adopting GWR80% than GWR100%. On the other side, adopting GWR100% and applying the combined foliar applications of Hsp + Kh attained the maximum increase of N content (668 mg kg−1), although that significantly equaled the adoption of GWR100% x combined application of Hsp + K2SO4 (645 mg kg−1).Table  in Fig. A. Accord2SO4), as can be seen in , P contents increased by 48.9, 29.8, 36.2, 85.9, 93.5, 14.8, 57.9, and 80.2% for Kh, K2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. The results demonstrated that the highest P contents (53.4 mg kg−1), were obtained with GWR100% and applying combined foliar applications of Hgd + Kh, however, that significantly equaled the adoption of the combined application of Hgd + K2SO4 under the same irrigation level.By comparing the examined applications under GWR100%, P content increased when applying combined applications of Hgd x foliar applications of K as  significantly increased K contents when adopting GWR100. Furthermore, GWR100% was pronounced with combined applications of Hsp + K2SO4 or GWR80% with combined applications of Hgd + Kh, for attaining the highest K content in the carrot root. However, applied combined applications of Hgd + Kh under water-limited treatment caused the greatest increase in K content to 735,4 (mg kg−1), an increase of 10.70% compared with that in Hgd + Kh under GWR100%.The results demonstrated that K has a unique role that can prevent serious injury to carrot plants under stressful conditions by increasing plant tolerance. Where, by comparing the control in Fig. C, it was−1) for the control to 385 (mg kg−1) in GWR100% treatment and applying Hsp + Kh , respectively.The Ca content in carrot root increased from 281 , which significantly equaled applying tap water (control). Compared irrigation levels under different examined applications, Na content was increased under GWR80% by 4.0, 8.4, 6.1, 6.1, 3.6, 11.1, 13.3, and 18.7% for control, Kh, K2SO4, Hgd, Hgd + Kh, Hsp, Hsp + Kh, Hsp + K2SO4, respectively.Based on the results of variance analysis, the individual and interaction impacts of examined irrigation levels, K, and H obviously affected Na content mainly by regulating water status. However, the dual interaction effects of K applications and irrigation were insignificant. The content of Na in carrot root was increased by adopting GWR80% compared to GWR100%, Fig. B. Althou2SO4) on the Ca /Na ratio in carrots are presented in , under GWR100%.The impacts of irrigation levels (100 and 80% GWR), H applications as (Hgd and Hsp), and K as Kh and KSO4 on thTo maximize K /Na ratio in the carrot root, either Hgd combined with Kh or Hsp + Kh was applied to provide a protracted, under both irrigation levels Fig. D. Accord2SO4, Hgd, Hgd + Kh, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. While it was increased under GWR80% compared to GWR100% by 10.3% for Hgd + Kh treatment. Generally, full irrigation level (GWR100%) was pronounced with the combined soil Hgd x foliar applications of Kh for attaining the highest chlorophyll content (11.4 mg g−1).To increase the content of chlorophyll during the whole growth period, the carrot plant significantly influenced its chlorophyll formation and metabolic functions depending on irrigation quantities and K nutrition source. Therefore, chlorophyll content in the carrot leaves was enhanced by adopting GWR 100% compared to GWR 80%, Fig. A. Althou2SO4, Hsp + Kh, and Hsp + K2SO4. The highest carotene contents were recorded by applying combined foliar applications of Hsp + (Kh or K2SO4) and adopting GWR100&80%, although that significantly equaled GWR100% x sole application of K2SO4. While the lowest carotene content was obtained under control GWR80% irrigation level (1.97 mg 100 g−1).On the other hand, compared to the control treatments in Fig. B, the co were obtained by applying sole foliar applications of Kh and adopting GWR100. Likewise, adopting GWR80% without any auxiliary applications attains the lowest carbohydrate content (21.3 mg 100 mL−1).According to the results of variance analysis Table , the indFig. C, adopti2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. Likewise, relative to control GWR80%, total sugars increased by 51.4, 20.0, 25.7, 62.9, 45.7, 20.6, 42.9, and 37.1% for Kh, K2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. Collectively, spraying Kh applied as combined with soil application of Hgd under GWR100& 80%, resulted in significantly higher values than the other treatments , although that significantly equaled GWR80% x sole application of Kh (5.3%).Relative to control full irrigation treatment (GWR100%), total sugars in the carrot root in Fig. D increas2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively, indicating that apply full irrigation water under these conditions increased plant height. The highest plant height was obtained with GWR100% irrigation level and applying the applications of Hgd or Hsp applied as combined with foliar applications of (Kh or K2SO4). The lowest plant height was seen under control at GWR80%.Based on the results of variance analysis, the individual and interaction impacts of examined irrigation levels, K, and H obviously affected plant height. However, the dual interaction effects of H applications and irrigation or K applications and irrigation were insignificant. The results in Fig. A indicat2SO4 when adopting GWR100% (14), that were significantly equaled GWR100% x the combined applications of Hsp + K2SO4 (11).By comparing the control in Fig. B, it was, although that significantly equaled GWR100% x combined application of Hgd + Kh (1177 g plant−1). Moreover, the adoption of combined application of Hgd + K2SO4 applications under GWR80% attaining the highest weight of carrot roots (142 g plant−1), although that significantly equaled GWR100% x combined application of Hgd + K2SO4 (121 g plant−1).On the other hand, the superiority of Hgd applications with limited irrigation (GWR80%) is still pronounced compared to Hsp causing increases in carrot root length, shoot fresh weight and the weight of carrot roots as can be seen in Fig. D,E,F. Ac2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. Likewise, relative to control limited irrigation treatment (GWR80%), carrot yield increased by 222.2, 89.2, 261.4, 178.0, 374.0, 144.3, 349.9, and 292.3% for Kh, K2SO4, Hgd, Hgd + Kh, Hgd + K2SO4, Hsp, Hsp + Kh, Hsp + K2SO4, respectively. The highest carrot yield was observed by applying the foliar applications of Hsp + K2SO4 under GWR100% . Likewise, when adopting the GWR80% irrigation level without any auxiliary applications, the carrot yield attained the lowest recorded value (7865 kg ha−1). Furthermore, the combined applications of Hgd + Kh were found to have decreased carrot yield, particularly under GWR 100%.According to the results of variance analysis, the individual and interaction impacts of examined irrigation levels, K, and H obviously affected carrot yield. Compared to the examined irrigation level in control treatment without any auxiliary applications, carrot yield was increased by adopting GWR100% of irrigation level compared to GWR80%, Fig. A. Althou−3, a decrease of 14.2% compared to that in GWR100%. By adopting the GWR80% irrigation level, Iwue increased from 2.4 (kg m−3) for the control GWR80% without any auxiliary applications to 11.4 (kg m−3) for Hgd + K2SO4. Additionally, by adopting GWR80% Iwue increased by 137.0% in Hgd + Kh and by 57.9% in Hgd + K2SO4 compared with that in GWR100%. The lowest Iwue values 2.81 (kg m−3) were recorded by applying combined foliar applications of Hgd + Kh and adopting GWR100%, although that significantly equaled the control of GWR100& 80% without any auxiliary applications. The lowest Iwue values were in control GWR80%, followed by control GWR100% (2.81 kg m−3) or under the same irrigation level (GWR100%) × (Hgd + Kh) at 2.81 kg m−3.As can be seen in Fig. B, under Efficient water management has become imperative for water conservation in many regions worldwide. Implementing appropriate irrigation scheduling and fertigation techniques can help reduce water loss and increase Iwue.51. While N and P are essential for chlorophyll and carbohydrate synthesis, as depicted in  with K2SO4 showed better results in increasing carrot yield. We postulated that under GWR100% irrigation, there is a higher stomatal opening in plant leaves55. Therefore, applying foliar applications of Hsp in conjunction with potassium applications, either as Kh or K2SO4, enhances nutrient absorption due to the synergistic benefits of potassium and humic acid applications. Particularly, Hsp facilitates the direct delivery of sufficient nutrients to the production sites (plant leaves), compensating for the reduced efficiency of the carrot roots under these conditions47, which subsequently leads to increased nutrient absorption as shown in  along with either Kh or K57, formed chelate components with cationic metals59, and enhanced nutrient metabolism and photosynthesis60, it seemed to primarily promote rapid growth of vegetative plant organs rather than reproductive plant organs. This led to a shortened storage period compared to the vegetative period, resulting in reduced root diameter and weight (Fig. 61 indicating that high doses of humic substances can improve soil physical characteristics but have uncertain effects on chemical soil characteristics and crops28. Furthermore, Raheem et al.62 demonstrated that increasing the application rate of humic acid resulted in the lowest total yield of lettuce, and Maibodi et al.63 found that only 100 mg l−1 of humic acid improved various characteristics of perennial ryegrass. Asri et al.64 showed that tomato fruit weight, diameter, and yield increased with increasing rates of humic acid up to a certain threshold, beyond which negative effects emerged. Similar findings have been reported by65 highlighting the variability of organic amendments' impact on crop yield, which is influenced by environmental factors, soil conditions, the composition of the amendment, and crop type. Therefore, our results emphasize the importance of careful fertigation management, particularly when applying organic amendments under full irrigation conditions. This approach promotes accelerated production and storage processes over vegetative processes, resulting in increased carrot yield.In contrast, we observed negative effects on carrot yield when adopting the combined applications of Hgd + Kh, especially under full irrigation conditions. We attribute this to the high doses of humic substances resulting from the simultaneous application of Hgd + Kh under favorable conditions. To clarify, the foliar application of Kh appeared to provide significant amounts of plant nutrient requirements, compensating for the reduced efficiency of the carrot roots, particularly under GWR100%. On the other hand, while the application of Hgd improved soil organic matter contents Table  and soilght Fig. C,F, but ght Fig. A,B,E. Th2SO4 under GWR80% improved carrot yield and achieved the highest Iwue values. We hypothesize that when implementing limited irrigation, plants experience some degree of water stress, which triggers a series of physiological responses. These reductions in water availability seem to enhance root efficiency47. While negatively impacting yield under control treatments. However, applying Hgd improves soil water and nutrient status by increasing nutrient availability, leading to improved water and nutrient absorption from the rhizosphere, as observed in available K values (Table 66, which have shown the indirect effects of humic acids on soil properties such as aggregation, aeration, permeability, water-holding capacity, micronutrient transport, and availability69 , as well as the direct effects on photosynthesis, plant growth, crop performance, and nutrient uptake rate70. Furthermore, the foliar application of K2SO4 provides significant amounts of potassium, which contributes to cell membrane integrity through osmotic adjustment, regulates stomatal movement, catalyzes cell division, enhances yield and quality, facilitates carbohydrate transformation, and promotes sugar synthesis74. Additionally, humic substances and K2SO4 enhance the exudation of organic acids by plant roots (Table 75. These findings are consistent with Rose et al.28, who demonstrated that the combined application of humic and mineral fertilizers forms complexes that release nutrients slowly, leading to increased yield. Consequently, the combined applications result in higher yields with reduced irrigation quantities, leading to the highest Iwue for carrots under these conditions.Based on our findings, the adoption of combined applications of Hgd + Kes Table A, and enThis research highlights the positive effects of supplying stressed carrot plants with auxiliary foliar applications of potassium and humic amendments, leading to improved nutrient uptake and carrot yield. However, the combined application of foliar potassium humate and soil amendments of humic resulted in a decrease in carrot yield, regardless of whether full or limited irrigation levels were applied. These findings challenge the notion that organic applications always enhance yield and suggest that the impact is influenced by factors such as the specific fertigation technique and the quantities of humic substances applied. Further studies are needed to explore this impact on other crops.For regions facing water scarcity, it is recommended to adopt combined foliar applications of potassium sulfate and soil amendments of humic at 80% of the gross irrigation water requirements as auxiliary treatments for water-stressed carrot plants. These combined applications have shown the potential to improve nutrient uptake, carrot yield, and mitigate the effects of water stress. Furthermore, they enable a higher increase in water use efficiency for carrot crops.


In total, 38 volatile compounds were identified in ‘Fuji’ × ‘Cripps Pink’ population, including 23 esters, 3 alcohols, 7 aldehydes and 5 others. With the combination of aroma phenotypic data and constructed genetic linkage map, 87 QTLs were detected for 15 volatile compounds on 14 linkage groups (LGs). Among them, a set of QTLs associated with ester production identified and confirmed on LG 6. A candidate gene MdAAT6 in the QTL mapping interval was detected. Over-expression of MdAAT6 in tomato and apple fruits showed significantly higher esters accumulation compared to the control, indicating it was critical for the ester production. Our results give light on the mode of inheritance of the apple volatilome and provide new insights for apple flavor improvement in the future.Aroma is an essential trait for apple fruit quality, but the understanding of biochemical mechanisms underlying aroma formation is still limited. To better characterize and assess the genetic potential for improving aroma quality for breeding, many efforts have been paid to map quantitative trait loci (QTLs) using a saturated molecular linkage map. In the present study, aroma profiles in ripe fruit of F Malus × domestica Borkh.) are widely cultivated all over the world because of the highly flavored fruits with unique flavor characteristics (E)-2-hexenal, α-farnesene, estragole, esters like hexyl acetate, hexyl hexanoate and 2-methylbutyl acetate  combined with gas chromatography–mass spectrometry (GC-MS), as a simple, rapid, comprehensive and high sensitivity method for the volatile compounds assessment, has been widely used for the extraction of volatile profiles in apple fruit . With thTo date, several QTL mapping researches have been carried out in cultivated apples in order to detect genomic areas involved in the inheritance of aroma volatiles. Taking advantage of the saturated genetic linkage map, 1 population of ‘Fuji’ × ‘Cripps Pink’ was used to dissect the genetics underlying aroma volatile compounds with HS–SPME/GC–MS and locate the QTL regions to further verify the candidate genes that affect the volatile accumulation in apple fruit. These findings lay a foundation for analyses of the genetic mechanisms underlying aroma volatiles and the breeding of apple varieties with better flavor in the future.In this study, an F1 segregating population with 300 individuals was developed from the cross of ‘Fuji’ × ‘Cripps Pink’. The 300 lines of this population were grafted onto dwarf rootstock M26 and planted at a density of 1.5 m × 4 m at the Baishui Apple Experimental Station of Northwest A & F University, Shaanxi Province, China. Orchard management procedures such as irrigation, fertilisation and pruning, were similar for all apple trees. The apple fruits from 246 and 223 individuals were harvested in two growing years (2019 and 2020). Three biological replicates with six fruits for each individual were collected at the ripe stage. Slices of flesh tissue were separated, immediately frozen in liquid nitrogen and stored at -80°C until use.An FFruit volatile compounds were analyzed according to the method reported by A Thermo Trace GC Ultra gas chromatograph  equipped with an HP-INNOWax capillary column (60 m × 0.25 mm × 0.25 µm) was used to analyze the volatile compounds in samples. The oven programming conditions were: 40°C kept for 3 min, ramped at 5°C/min to 150°C, then increased at 10°C/min to 220°C and held for 5 min. The carrier gas was helium at a flow rate of 1.0 mL/min with the ion source and transfer line temperatures were both set at 240°C. Mass spectra were monitored from 35 to 450 m/z with ionizing electron energy of 70 eV. Volatile compounds were identified by comparison with retention times (RT), retention indices (RI) and the mass spectra of the NIST 14 library (NIST/EPA/NIH). Quantification of volatile compounds was performed using the peak area of the internal standard as a reference based on the total ion chromatogram (TIC).1 segregating population with 300 individuals from the cross of ‘Fuji’ × ‘Cripps Pink’ was utilized to construct a high-density genetic linkage map by whole genome resequencing (WGS) in the previous study .An Fus study . QTL mapvia reverse transcription using the PrimeScript™ RT Reagent Kit  . The qRT-PCR  experiment was performed using an iQ5 Multicolor Real-Time PCR Detection System . MdActin was used for target gene normalization in accordance with 2-ΔΔCt method to calculate the relative expression level. Three replicates per each sample were performed. The gene-specific primers used for qRT-PCR are listed in In order to know more about the expression patterns of candidate gene, total RNA of different development stages  and tissues  in ‘Fuji’ apple was extracted with a Plant RNA Purification Kit  following the manufacturer’s instructions. cDNA was obtained MdAAT6 without the stop codon was inserted into a pCAMBIA2300-GFP vector under the control of the CaMV 35S promoter, and the primers used are listed in Nicotiana benthamiana leaf by Agrobacterium-mediated infiltration according to the method by The coding sequence of Agrobacterium-mediated transient transformation in apple fruits was carried out with minor modifications from the procedure of Agrobacterium tumefaciens strain GV3101. The agrobacteria cell suspension was evenly injected into the apple fruits with a syringe. The injected apple fruits were placed in paper bags and kept in the darkness for 12 h and then transferred to a light growth chamber without the bags at 26°C for 5 days. Fruits were injected with the empty vectors, used as the negative controls. After 5 days, the infiltrated fruits were collected for aroma volatile compounds determination. Three biological replicates with 6 fruits each were used for analysis.Malus domestica cv. ‘Fuji’ was cloned into pCAMBIA2300 vector. Agrobacterium-mediated tomato (Solanum lycopersicum cv. Micro-Tom) transformation was performed as described by To generate transgenic tomato plants, the coding region of candidate gene from P < 0.05).All data were obtained from three replications. Statistical analyses were performed using the IBM SPSS Statistics 21  and Excel 2019 software. The data were analyzed using Student t-test or one-way analysis of variance (ANOVA), and significant differences between groups were assessed by the Tukey test  and male parent (‘Cripps Pink’), including 23 esters, 3 alcohols, 7 aldehydes and 5 others (E)-2-hexenal were the most abundant compounds (average content > 10 µg/kg FW in 2019 and 2020) in ‘Fuji’ apple. While in ‘Cripps Pink’ apple, 2-methylbutyl acetate, hexyl acetate, hexyl butanoate, hexyl 2-methylbutyrate, (E)-2-hexenal, estragole and α-farnesene showed the highest contents (more than 10 µg/kg FW in two years). Moreover, esters were the dominant aromatic compounds in both parents in two years, accounting for 51.14% , 66.28% , 53.56% , 67.45%,  of the total volatile content, respectively -2-hexenal and α-farnesene were the most abundant volatiles (average content > 40 µg/kg FW over two years) in F1 progeny fruits and the mean contents of them were 95.80, 78.81, 43.73 μg/kg FW in 2019 and 128.94, 72.63, 90.30 μg/kg FW in 2020, respectively. Moreover, the relative contents of α-farnesene, estragole, hexyl 2-methylbutyrate and hexyl butanoate were widely distributed among F1 progeny fruits, ranging from 0.20 to 866.93 µg/kg FW, 0.23 to 684.34 µg/kg FW, 0.21 to 431.52 µg/kg FW, 0.11 to 370.66 µg/kg FW, respectively. Additionally, as shown in E)-2-hexenal, nonanal, (Z)-2-nonenal, estragole and (Z)-anethole), with a relative range of variation between 42.96% and 187.16% along the two years. These unsegregated compounds showed continuous variation, typical of polygenic inheritance, although their distributions were generally skewed toward high values. Also, a continuous distribution and considerable transgressive segregation observed in the F1 population demonstrated that both parents contributed alleles and these volatiles were confirmed to meet the requirements for further QTL mapping.In the ‘Fuji’ × ‘Cripps Pink’ population, the volatiles present in both parents all could be detected in the offspring. A total of 15 volatile compounds were unsegregated in F parents Table 2,1 progeny fruits of ‘Fuji’ × ‘Cripps Pink’ population  present in both parents, but separation occurred in the Fpulation Table S21 progeny fruits of ‘Fuji’ × ‘Cripps Pink’ population were represented in heatmaps for 2019 (E)-2-hexenal was observed in the heatmap. Additionally, the content of aroma compounds belonging to other type in this study was highly positive correlation with estragole and α-farnesene.Correlations among volatile compounds in the F1 population were clustered based on their compound classes and chemical properties. For hierarchical clustering, these volatile compounds roughly divided into four groups according to their aroma profiles across the two years (E)-2-hexenal and (E)-2-octenal. Cluster 2 showed enrichment of 2-methyl-1-butanol, pentyl butanoate and 3-methylbutyl butanoate. Cluster 3 mainly consists of some important esters . Cluster 4 was grouped by the compounds with high levels in apple fruit, for instance, α-farnesene, estragole and 1-hexanol. These results suggested that volatile compounds were co-regulated according to specific modules within the F1 population.The correlation analysis also revealed that volatiles in F1 progeny fruits of ‘Fuji’ × ‘Cripps Pink’ population were identified for two years. As shown in 2) explained by these QTLs ranged from 5.1% (qHex19-1) to 23.1% (qHex19-4) with the LOD value varied from 3.05 to 10.34. Among them, 25 QTLs for 4 esters  mapped on LG2, LG4, LG6, LG8, LG11, LG15, LG17 were identified and explained the phenotypic variation from 6.0% to 23.1% (E)-2-hexenal, nonanal, (E)-2-octenal and (Z)-2-nonenal), 12 and 29 QTLs were detected on 5 and 13 linkage groups, respectively . A total of 21 QTLs controlling 4 other volatiles -anethole) were identified on 9 linkage groups with the LOD value from 3.15 to 12.06  located on LG6 showed the high transcript levels in this QTL. Therefore, we further investigated the role of MdAAT6 in influencing volatile ester formation. As shown in MdAAT6 continued to increase and showed highest expression levels at 150 days after full bloom, about 5.5-fold compared to 60 days after full bloom. The tissue-specificity of MdAAT6 expression indicated that its transcript significantly accumulated in peel and pulp  at the ripening stage. In order to determine the subcellular location of MdAAT6, the recombinant GFP fusion protein was transiently overexpressed in tobacco leaves and the green fluorescence signal indicated that MdAAT6 was located in the cytoplasm (MdAAT6 in apple fruit significantly increased the hexyl butanoate (7.38 μg/kg FW), hexyl 2-methylbutyrate (43.47 μg/kg FW) production and hence, esters accumulation (225.82 μg/kg FW), while silencing the expression of MdAAT6 inhibited the production of ester compounds. To verify the regulatory effects of candidate gene in ester synthesis, over-expression of MdAAT6 in ‘Micro-Tom’ tomato was conducted. Three transgenic lines were generated, with no differences in fruit size or color between the wild type and transgenic tomato plants. Compared with the wild type, the esters contents in transgenic tomato fruit were significantly higher (approximately 1.80-fold)  in ‘Royal Gala’ apple fruit  with esters production was validated by QTL analysis and candidate gene mapping in a segregating population obtained from the ‘Fuji’ × ‘Cripps Pink’. Transient transformation of apple fruits and transgenic tomato plants with enhanced MdAAT6 expression produced more esters. Functional characterization demonstrated that the candidate gene MdAAT6 could catalyze the synthesis of esters and produce high levels in ripe fruit, suggesting MdAAT6 also one of the candidate genes responsible for apple fruit ester production besides MdAAT1 and MdAAT2.The ester compounds in apple fruit are thought to be produced from two main different pathways, the fatty acid pathway for formation of straight chain esters and the isoleucine pathway contributing to branched-chain esters . Alcoholg banana , melon  can be found in the text.YS and ZZ designed the study. YJ analyzed the data. YH supervised the project. YS drafted the manuscript. ZZ gave critical suggestions throughout the study and revised the manuscript. All authors contributed to the article and approved the submitted version.


Lack of appetite is a common problem in elderly people which could lead to the risk of malnutrition. Soup-based product formulation and supplementation for the elderly is an interesting and convenient way to maintain nutritional status. Hence, this study aims to develop ready-to-eat (RTE) soup and instant soup powder using common agricultural commodities. The results indicated that among all formulations, the F7 formula comprised brown rice (15 g), pumpkin (32.5 g), sweetcorn (12.5 g), red tilapia (17.5 g), rice bran oil (1.0 g), and water (21.5 g) with energy ratio (C:P:F) of 58:23:20 receiving the highest sensory scores. The selected formulation (F7) was also transformed into instant powder and both RTE soup and instant powder were evaluated for nutritional composition and storage stabilities at 5 °C and 25 °C, respectively. The nutritional composition analyses indicate that 100 g of RTE soup consists of 13.8 g carbohydrates, 4.9 g proteins, 1.8 g fats, and 1.5 g dietary fibers; the soup is also a rich source of antioxidants and β-carotene. Storage studies suggested that the content of β-carotene and antioxidant activity of both (ready-to-eat and instant powder) types of soup decreased with increasing storage time, while a slight increase in yeast and mold count (<50 cfu/g) was noted. Most importantly, no pathogenic bacteria were detected in ready-to-eat and instant soup during the storage study of 6 weeks at 5 °C and 6 months at 25 °C, respectively. In terms of the high nutritional composition and functional value of the product, 4 weeks of storage at 5 °C and 4 months of storage at room temperature were suggested for ready-to-eat and instant powder soup product, respectively. The world’s aging population keeps increasing, and WHO predicts that one in six people will be aged over 60 years by 2050 and will reach 2.1 billion . The avaHealthy eating is simply eating an adequate amount of macro and micronutrients and well-balanced meals to support the body’s needs. A proper meal should contain 45–60% of carbohydrates, 10–15% of proteins, 20–25% of fats, and about 30 g/day of dietary fibers . NeverthSoup-based product is a more interesting food preparation solution for the elderly since it can fortify any functional ingredients or micronutrient deficiency to meet the body’s requirements. This will be more beneficial for vulnerable elderly populations and worth preventing and promoting health than curative care. The conventional method and simple technology such as pasteurization and drying process are combined to maintain the quality and stability of food during storage and be accessed for small and medium enterprises including household cooking. Consequently, utilizing the agricultural commodities and common thermal process to develop elderly soup products based on recommended macronutrient requirements was the objective of this research. In addition, the changes in quality, microbiological safety, and antioxidant activity were followed during storage.(Oryza sativa L.) , yellow sweet corn kernel (Zea mays saccarata) , yellow flesh pumpkin (Cucurbita moschata) (Doikham™ brand Food Products Ltd.), high oryzanol rice bran oil  and fresh red tilapia fish (Oreochromis sp.)  were purchased from grocery supermarkets in Bangkok, Thailand. Inulin was obtained from Food Ingredient Technology Co., Ltd., Bangkok, Thailand.Brown jasmine rice The raw materials were separately cleaned with running tap water. Skin, seeds, and inedible parts of the pumpkin were removed. The pumpkin flesh was cut into pieces of about 1 inch in thickness. Corn kernels were removed from the sweet corn cobs. Fish flesh in fillet form was prepared. The prepared materials were steamed over boiled water for approximately 15 min for pumpkin pieces and about 10 min for corn kernels and fish fillets. Only brown jasmine rice was cooked using an electric rice cooker. After that, the cooked materials were cooled to room temperature at 25 °C, and then kept at 5 ± 2 °C for further use.Before the formulation of ready-to-eat soup, all ingredients were analyzed for the content of carbohydrates, protein, fat, dietary fiber, ash, and moisture . The INMn = 30). The soup sample that received the highest score for overall acceptability was selected. The proximate compositions, β-carotene content, antioxidant activity, physicochemical quality, and microbial safety were determined. The stability of ready-to-eat soup products was followed during storage at 5 ± 2 °C for 6 weeks.The cooked pumpkin, corn kernel, fish, and brown jasmine rice including rice bran oil and water were weighed according to the soup formula (F1–F7). All ingredients were ground together by a homogenizer  at a high sheer speed for 20 min. The mixed soup was pasteurized at 100 °C for 20 min and hot filled in a wide-mouth glass bottle with sealing closure (200 mL), then immediately cooled and kept at refrigerator temperature (5 ± 2 °C) for analysis. All F1–F7 formulas evaluated the degree of enjoyment of product by sensory analysis  for further study. The proximate compositions, dietary fiber, β-carotene content, antioxidant activity, physicochemical quality, and microbial safety were determined. The stability of instant soup powder was examined during storage at 25 ± 2 °C for 6 months.The selected soup formula (F7) was further developed into the instant soup powder. As in the previous study in the laboratory, an optimum of 15% inulin was added to improve the mouthfeel of reconstituted dry powder soup. The blended soup was prepared and then applied onto a pilot-scale double-roller drum dryer  30 cm in diameter, 45 cm in length, with a 0.1 mm roller gap, a 1 rpm roller speed and an 8–9 kg/cmThe protein was analyzed using the Kjeldahl method as mentioned by AOAC 992.23 . Total fw) was measured by a portable water activity meter  at room temperature (25 °C). The pH value was measured using a pH meter  at room temperature (25 °C). Relative viscosity was determined by a rotational viscometer  with an RV spindle no.2 at a speed of 1 g under room temperature (25 °C). The color was determined using a colorimeter  with a pulsed xenon lamp as a light source. The color values were expressed in the CIELAB parameter, where L*, a*, and b* are the lightness, redness, and yellowness, respectively.Water activity . Although there are various antioxidative mechanisms and assays reported in the literature, ORAC has been widely accepted as a standard assay method to determine antioxidant capacity in food, nutraceutical, and pharma industries . Three sEscherichia coli counts were determined by a presumptive test and then confirmed by using EC broth by incubating at 35 °C for 48 h . DuThe nutritional components such as the presence of carbohydrates, dietary fiber, proteins, and fats for any food product are important in terms of health benefits to the consumer. Besides this, accessibility and affordability of the raw materials to prepare healthy food are a vital component. Hence, in this study, we have used common agricultural commodities which are easily available in the market to prepare ready-to-eat soup for the elderly. The proximate analyses of all raw materials indicated that the highest carbohydrate content was presented in sweet corn kernels (35 g/100 g) followed by brown rice (30 g/100 g) and pumpkin (11 g/100 g) . The hign = 30) voluntarily participated in the soup product liking test. The outcome indicated that F1 had a pale white color and a moderately thick, rice odor. F2 presented the vivid yellow, moderately thick, intense pumpkin flavor and slightly sweet taste. F3 exhibited a yellow color, a mildly thick, sweet smell, and tasted like sweet corn. F4–F6 indicated the yellow color, smooth texture, and slightly sweet taste and odor, similar to the characteristics of their combined carbohydrate ingredients, while F7 revealed a bright yellow color, moderately thick, smooth texture, and sweet odor similar to a combination of distinctive attributes of brown jasmine rice, pumpkin, and sweet corn altogether. Among all formulations, F7 formula received the highest scores for overall acceptability, taste, odor, and texture, which scored over 4.5, meaning a score from “like moderately” to “like very much”, excluding color. F4 was the second most liked formula and had a score of most attributes slightly over 4 (“like moderately”), excluding color score, which was the highest. F5 and F6 revealed the liking score of quality attributes close to 4 or a little bit higher than 4, whereas F1–F3 had the lowest score for all quality attributes compared to the other formulas. Using a combination of carbohydrates in soup products may impact liking score more than using a single carbohydrate source. It is likely that an appropriate blending of both types and the number of carbohydrates may boost the distinctive characteristics, enhance the flavor perception, and mask the sharp odor and strong flavor [Based on the recommended energy distributions for elderly food, g flavor ,27. Basep < 0.05). The β-carotene content (1561 µg/100 g) from day 0 decreased to 1293 µg/100 g at 6 weeks of storage. The ORAC value decreased from the start of the day (3074 µg TE/100 g) to 2324 µg TE/100 g after 6 weeks of storage. There was no significant (p > 0.05) change noted in the pH value (6.51–6.52) of soup during 6 weeks of storage at 5 °C. Soup viscosity seemed slightly reduced, which was about 1.8 × 104 cP at the beginning and revealed 1.6 × 104 cP at the end of storage (p ≤ 0.05). Reduced-viscosity soup may be influenced by the rearrangement of amylose molecules since the starch retrogradation occurs during prolonged storage time [Changes in the quality of the product during storage are well correlated to the product expiration or best-before date. Following analysis of physicochemical properties and microbial safety during storage was crucial for an index of product stability. age time . The ligE. Coli (MPN/g) and pathogenic bacteria such as Salmonella, S. cereus, B. cereus, and C. perfringen were not detected in ready-to-eat soup products during prolonged storage at 5 °C for 6 weeks. The microbial analyses indicate that the ready-to-eat soup product was safe for consumption over the storage period in compliance with the Food Notification [The microbiological indices were followed to assess product safety. Yeast and mold started at < 10 cfu/mL at week 0 and continually increased to <50 cfu/mL at the end of storage. Coliform was < 1.1 MPN/100 mL through storage for 5 weeks and increased to <2.2 MPN/100 mL at the last week of storage. Meanwhile, fication ,37. AlthThe common drum dryer traditional technique was used to produce dehydrated soup powder. Inulin (15%) was added to improve the texture and food palatability. Additionally, it provided dietary fiber and increased yield percentage from about 21.5% (0% inulin) to 30.2% (adding 15% inulin). Inulin used in this study was extracted from Jerusalem artichoke tubers. Generally, the amount of inulin present in any food is safe for consumption. However, added or supplemented inulin portion should be regulated to minimize the side effects. It was reported that the intake of 8–18 g of inulin per day is safe for up to 24 weeks . The druE. coli of less than 1.1 MPN/g. Meanwhile, no pathogenic bacteria including Salmonella, S. aureus, B. cereus, and C. perfringens were detected that complied with the food standard of semi-processed food [The proximate analyses revealed that 100 g of dehydrated instant soup powder contained 41.4 g carbohydrate, 14.7 g protein, 5.4 g fat, 30.8 g total dietary fiber (insoluble fiber 5.1 g and soluble fiber 25.7 g), 5.1% moisture, and energy of 273 kcal . The enesed food  and pathsed food , respectReconstitution of dehydrated soup powder was conducted by dissolving 55 g of instant soup powder in 150 mL of boiled water and stirring until dissolved. The reconstituted soup of 100 g contained about 15 g of carbohydrates, 5.6 g of proteins, 1.9 g of fats, 6.7 g of total dietary fibers (insoluble 1.1 g and soluble 5.6 g), 84.9% of moisture, and 100 kcal of energy . The fibE. coli presented < 1.1 MPN/100 g at the beginning until 4 months, after that an increase in number up to <2.2 MPN/100 g was reported until the end of storage. However, no pathogenic bacteria including Salmonella, S. aureus, B. cereus, and C. perfringen were detected during 6 months of storage at room temperature. These microorganism results were within the accepted limit and conformed to Food Notification No. 416 (2020).p ≤ 0.05), but no significant (p > 0.05) changes in pH, a* and b* values were reported. Increasing moisture content during storage may occur due to the penetration of moisture vapor from the atmosphere through the microscopic pores of packaging material. In raged to the health benefit, it was suggested to keep the instant soup powder product for about 4 months at room temperature (25 ± 2 °C).The β-carotene content and ORAC value were the highest at day 0 which were about 4919 µg/100 g and 8160 µgTE/100 g and continually reduced to 3960 µg/100 g and 7305 µgTE/100 g for 6 m of storage, respectively. The decrease in β-carotene and antioxidant activity during extended room storage could be related to temperature conditions and the oxidation process. The remaining oxygen in the package, product moisture, room temperature, and storage time may positively affect the acceleration of the oxidation reaction . FurtherThe developed elderly soup of both ready-to-eat soup and dehydrated soup powder per serving (about 200 mL) provided calory density of approximately 1 kcal/mL based on recommended macronutrient distributions. Meanwhile, 4.91 kcal/g and 1 kcal/g of caloric values were reported in the elderly soup developed by Irwan  and RiyaThe commonly available agricultural commodities were utilized to prepare ready-to-eat soup and dehydrated soup products for the elderly. The RTE soup formulation which received higher sensory scores consists of brown rice (15 g), pumpkin (32.5 g), sweetcorn (12.5 g), red tilapia (17.5 g), rice bran oil (1.0 g), and water (21.5 g). The developed elderly soup product content includes 105.5 g/100 g of calories, 58:23:20, C:P:F energy distribution ratio, and 1.58 g/100 g of dietary fibers. In addition, both soup types contain a sufficient amount of β-carotene and antioxidant activity. Importantly, the prepared soup products are microbiologically safe for consumption. Moreover, ready-to-eat soup can be stored in refrigerated condition for 4 weeks, whereas instant soup powder can be stored at room temperature for 4 months without losing nutritional value. Hence, the developed ready-to-eat and instant soup powder are a good alternative for the elderly with required daily energy intake. The developed elderly soup product supplement is suggested for consumption at 1–3 servings per day to satisfy the energy requirements. In addition, the developed soup products were prepared with affordable prices and familiar flavors and tastes. A future study will be conducted on soup fortification with added functional ingredients such as folic acid, vitamin B12, vitamin D, etc., that help to promote health or reduce the risk of disease in seniors.


Herein, this study presents a synthetic alternative toward efficient, durable, and recyclable, Fe3C-nanoparticle-encapsulated nitrogen-doped hierarchically porous carbon membranes (Fe3C/N–C). By employing a simple one-step synthetic method, we utilized wood as a renewable and environmentally friendly carbon precursor, coupled with poly(ionic liquids) as a nitrogen and iron source. This innovative strategy offers sustainable, high-performance catalysts with improved stability and reusability. The Fe3C/N–C exhibits an outstanding peroxidase-like catalytic activity toward the oxidation of 3,3′,5,5′-tetramethylbenzidine in the presence of hydrogen peroxide, which stems from well-dispersed, small Fe3C nanoparticles jointly with the structurally unique micro-/macroporous N–C membrane. Owing to the remarkable catalytic activity for mimicking peroxidase, an efficient and sensitive colorimetric method for detecting ascorbic acid over a broad concentration range with a low limit of detection (~2.64 µM), as well as superior selectivity, and anti-interference capability has been developed. This study offers a widely adaptable and sustainable way to synthesize an Fe3C/N–C membrane as an easy-to-handle, convenient, and recoverable biomimetic enzyme with excellent catalytic performance, providing a convenient and sensitive colorimetric technique for potential applications in medicine, biosensing, and environmental fields.Fe Ascorbic acid (AA), also called vitamin C, is a crucial water-soluble vitamin. Its antioxidant properties are effective for developing the immune system, preventing and treating some diseases such as cancer, and creating collagen in various tissues in the human body ,2. Since3C, a member of the iron carbide family, indicated outstanding peroxidase-like catalytic activity because of its high redox capacity, a large number of active sites, and superior surface reactivity , and mK represent the initial velocity, maximum reaction velocity, substrate concentration, and Michaelis constant, respectively. The molar attenuation coefficient of TMB at 652 nm was 39,000 M−1cm−1. The tests were carried out in a colorimetric dish of 1 cm in thickness.Detection of ascorbic acid. AA detection was studied as follows: 15 µL of H2O2 sample, 60 µL of TMB solution, and 20 µL of catalyst dispersion in acetate buffer (pH 4.0) with a final volume 500 µL reacted for 15 min at room temperature, and the blue color of oxTMB was observed. Then, the ascorbic acid samples at different concentrations of 0–200 µM were individually added to the blue solution, and the UV-Vis absorbance at 652 nm was monitored. Similarly, the experiment was repeated by replacing AA with various reagents such as sulfite, citric acid, glycine, serine, alanine, aspartic acid, tartaric acid, arginine, and saccharides like glucose, lactose, and fructose.Characterization. Phase structure was examined on an X-ray diffractometer PANalytical X’Pert Pro  applying Cu Kα radiation (λ = 1.5418 Å) between 5° and 80° at a scan rate of 0.2°/min. Chemical bonding characterizations were monitored through ESCALAB 250Xi X-ray photoelectron spectroscopy (XPS) (USA). The morphology of samples was investigated using a JEOL 7000F  scanning electron microscope (SEM) operated at 10 kV. Samples were sputtered by a thin gold layer for 60 s before the examination. Energy-dispersive X-ray (EDX) mapping was taken on the SEM using an EDX spectrometer. The particle size was determined through transmission electron microscopy (TEM) using a JEOL JEM-2100  operated at 200 kV. The nitrogen sorption isotherms at 77 K were performed using the Micromeritics ASAP 2020 . Before the sorption experiments, all samples were degassed for 7 h at 373 K under vacuum. The Brunauer–Emmett–Teller (BET) equation was used to calculate the specific surface area. Thermogravimetric analysis (TGA) experiments were carried out at a heating rate of 10 °C min−1 from 50 °C to 900 °C under air flow using a TA Instruments Discovery TG. Raman spectroscopy was performed on a Horiba Labram HR system with 532 nm laser excitation. 1H-NMR spectra were performed at room temperature using a Bruker DPX-400 spectrometer operating at 400 MHz. DMSO-d6 was employed as an NMR solvent for the measurement. Additionally, the catalytic attributes were examined through ultraviolet-visible  measurement.3C nanoparticle-functionalized nitrogen-doped porous carbon membranes, termed Fe3C/N–C, were synthesized via a facile pyrolysis of Balsa wood in the presence of a mixture of poly(ionic liquid) and FeCl3 as N/Fe sources. It is known that the chemical nature and microstructure of precursors strongly affect the physiochemical properties and chemical composition of heteroatom-doped carbon products  represents the substrate concentration. It is known that the catalytic power of a sample depends on the mK and maxV values ) from 0 to 200 μM is presented in 2: 0.993)  b. The li: 0.993) .To evaluate the selectivity of detection of AA by monitoring the absorbance of the reaction systems, vitamin C was replaced by various biologically relevant species such as sulfite, citric acid, glycine, serine, alanine, aspartic acid, tartaric acid, arginine, and saccharides like glucose, lactose, and fructose. The significant change in the absorbance intensity at 652 nm was not noticed after adding other interferential substrates. 3C nanoparticles were introduced into a hierarchically porous nitrogen-doped carbon membrane derived from a thin wood slice pre-coated by PIL and FeCl3 before carbonization. Owing to the unique interconnected and oriented porous structure, ultrafine nanoparticles, and nitrogen doping, the as-prepared Fe3C/N–C catalyst demonstrated outstanding intrinsic peroxidase-like catalytic activity with favorable stability and recyclability, which could be utilized to sensitively detect ascorbic acid over a broad concentration range with a low limit of detection (~2.64 µM). The detection system exhibited a high selectivity and anti-interference capacity to ascorbic acid. This study proposes a straightforward and effective way for preparing metal-containing heteroatom-doped porous carbon membranes, which can be generalized to synthesize various other functional carbonaceous materials such as artificial enzymes, revealing great potential in medicine, biosensing, and environmental fields.In summary, uniformly dispersed Fe


Nanoparticles exhibitinggeometrical and chemical anisotropieshold promise for environmentally responsive materials with tunablemechanical properties. However, a comprehensive understanding of theirinterfacial behaviors remains elusive. In this paper, we control theinterfacial anchoring orientation of polystyrene nanodumbbells byadjusting interparticle forces. The film nanostructure is characterizedby the orientation angle analysis of individual dumbbells from cross-sectionalEM data: dumbbells undergo orientation transitions from a distinctivehorizontal bilayer to an isotropic anchoring when electrostatic repulsionis suppressed by either an ionic strength increase or surface amine-modification.This anchoring orientation influences the film’s mechanicalproperties and foam stability, as investigated by a 2D isotherm anddark/bright-field microscopy measurements. Our findings highlightthe potential for precise control of supra-colloidal structures bymodulating particle alignment, paving the way for smart delivery systems. By this, they serve as steric barriers7 and make the interface highly resistant to coalescence.9 Another unique advantage of NPsis their programmability: their geometry and chemical properties canbe tailored throughout synthesis and modification processes, oftenanisotropically.12Pickering stabilization,a phenomenonwhere solid nanoparticles (NPs) stabilize a fluid–fluid interface,has been utilized in drug delivery,14 The shape and surface chemistry  of anisotropic nanoparticles (ANPs) collectivelyorchestrate their interfacial characteristics, by influencing thetrapping energy, equilibrium orientation, and 2D packing structures.18Recent research has emphasized the impact ofgeometrical and chemicalanisotropy of NPs on their interfacial behaviors.11 Also, strategies to controlthe behaviors of a particle with given anisotropy profiles remainunderexplored. Adding another dimension of complexity, the modulationof “supracolloidal” level characteristics  of ANPs by tuning the underlying interparticleforces, can provide valuable insights for a comprehensive understandingof the interfacial properties of ANPs. This knowledge can also beused to leverage the stability and programmability of ANPs in emulsificationand delivery systems.However, most existing studies have primarily focused on individualparticle level.21 Bulk-scalesynthesis with uniform size distribution has also been achieved.24 We hypothesize that changes in interparticle force would alter theinterfacial orientation of nanodumbbells and subsequently modulatethe mechanical properties of ANP films.Herein, we investigate the effect of interparticleforces on theinterfacial behavior and film formation of polystyrene (PS) nanodumbbells.Dumbbell shape was chosen due to its versatility in controlling thesize ratio and surface chemistry of each lobe.26 we synthesized symmetric nanodumbbells and a budded lobe. Usingan amino-silane coupling agent, we amine-modified the seed lobes of db1 and obtained charge-anisotropic Janus dumbbells. Usingthe Grahame equation and zeta potential values (SI text).28Using two-step emulsionpolymerization technique,U = VvdW + Vel; 29 At I = 1.7 mM, long-range electrostatic repulsion dominates, resultingin a high (∼104kBT) energy barrier . However, for db2, althoughthe average energy is repulsive, attraction occurs in ∼50%of the approaching directions , suggesting the possibility of db2 clustering even at low salt.Based on the shape and charge profiles, we calculated pairwisepotential energy of the three NPs anchored at the air–waterinterface as the sum of van der Waals attraction and electrostaticrepulsion of 4.4 and 3.7 nm. This implies that two distant particles will bedrawn together only until they reach an equilibrium spacing at Deq. In the case of db2, attraction is dominant and secondary minimum is absent. At higher I, the Ubarrier values of db1 and sp subsequently decreasedand became zero at I = 0.24 M (db1) and 0.46 M . At I = 1.0 M, repulsion is almostcompletely suppressed, and attraction becomes dominant for all threeparticles , while both nanodumbbells successfully stabilized foams. While db2 formed foams even at I =0 M  foams appeared at I = 0.17 M. This instability may be due to their equilibriumspacing, making the foams “leaky”. Under higher I (≥0.25 M), db1 stabilizedfoams as effectively as db2. These dumbbell-stabilizedstable foams exhibited remarkable resistance, lasting over one month.Weexamined the foaming ability of the NPs 2A,B. Typ = 1.0 M S5, whiledb2 foams remained, while db1 foams disappeared to a greater extent (>80%).This ion-robustness of db2 foams agreeswith their interparticle attraction in a wider ionic regime. We examinedthe cross-section of dumbbell-stabilized foams with cryo-SEM (db1 and ∼1 μm for db2). Whilespherical PS latex typically forms monolayer on foams,31 our dumbbell-stabilized foams were bi- or multilayers, which explainstheir structural robustness.cryo-SEM 2D,E. Thec . The sp formed a well-defined monolayer at I = 0.17 M anda bilayer or trilayer at I = 1.0 M .We investigated the internal structureof the NP films from theircross sections at the planar interface. I = 0.17 M, db1 forms a horizontal bilayer,in which dumbbells are orientedhorizontally as two separate monolayers, and those two monolayersare stacked . In contrast,visual inspection showed that dumbbells in both db1 film at 1.0 M  and a dumbbell’slong axis vector (θd and φd) weredetermined. Then we obtained the angle between the two vectors . db1 at 0.17 M, the horizontalorientation is predominant, showing the highest occurrence at the0–5° bin. When electrostatic repulsion is reduced by eitherionic strength change or surface modification ,the OA distributions widened, corroborating the aforementioned visualinspection. However, for all three conditions, horizontal anchoring(0–30°) was preferred to vertical anchoring (60–90°).This preference is due to the high (5.5 × 105kBT) rotational energy barrier, which makes a dumbbell particleunlikely to rotate vertically if a particle is initially anchoredhorizontally upon deposition. Nonhorizontally oriented particles mayhave originated from particle clusters formed right after deposition.Since tight clustering is energetically stable, once stably clustered,particle orientation would be preserved throughout the lateral compression.For a precise analysis, we extracted the orientationangle  ofindividual dumbbells from cross-sectional SEM images. Briefly, theangles defining the 3D directions of the plane film’s normalvector  and also showed polydisperse thickness .Dark-field microscopy (DFM) revealed self-assembled clusterstructuresof nanodumbbells at zero surface pressure. The clusters 4B. At 1.hickness 4D. The sAp) isotherm curves of the NPs. The x-axis was rescaled with interfacial adsorption affinity (accountingfor particle submersion upon deposition) and close-packing area  and the pressure (πc) were quantified.By calculating ACP/Ac, we estimated the effective number of layers , π increased monotonically up to <10 mN m–1, without sign of collapse. This suggests that db1 particles adsorbs only weakly and submerge upon compression.30 At 0.05 M, collapse occurs near ACP, suggesting the formation of monolayer. In the rangeof 0.1–0.2 M, the film thickness increases to bilayer  πc. In contrast, the πc of db1 abruptly decreased from35 to 21 mN m–1 at I = 0.3 M. The sp also showed a sudden decrease from 40 to 23 mN m–1 at I = 0.5 M. These thresholds alignwell with the energy barrier decrease (S4). Underthe presence of equilibrium distancing, the repulsive force mighteffectively dissipate the lateral compressive stress throughout thefilm. Therefore, the film might withstand stronger lateral compression,leading to higher πc. If attraction is dominant , particle clusters assembled right afterdeposition are jammed upon compression. Cluster–cluster boundaryregions might be structurally weaker, making the entire film moreheterogeneous in thickness and mechanical strength.32 When stress is concentrated at those weaker regions, thefilm may collapse at a lower πc. This πc decrease may also explain the thinning of the db1 film from 2 to 1.5 layers at I ≥0.3 M. Because collapse occurs at a lower πc, thefilm thickness at Ac may also have decreased.decrease 5A and S4c, NP films undergo a wrinklingphase transition, as revealed by bright-field microscopy . For db1 at 0.17 M, three local minima are observed in the 1D autocorrelationcurve  as λ = 5.2 μm (db1 at 0.17 M), 6.5 μm (db1 at 1.0 M), and 7.8 μm (db2). Fromthese values, the bending rigidity of the films was estimated. Thetotal energy of a film on a fluid substrate is minimized when thebending energy and the substrate deformation energy are balanced.34 Thus, the relationship between the equilibriumwrinkle wavelength (λ) and the bending rigidity (B) is given as B = ρg(λ/2π)4, where ρ is fluid density and g isthe gravitational acceleration.34 The calculated B values are 1.12kBT (db1 at 0.17 M), 2.73 kBT (db1 at 1.0 M), and 5.66 kBT (db2). The horizontal bilayer structure of db1 at 0.17 M has relatively weak layer–layer interactionbecause most particles lie horizontally and therefore do not engagein interlayer anchoring  oriented, increasing the number of interlayer anchoringpoints, which can explain increased rigidity. The db2 film exhibits an even wider OA distribution, indicatinggreater rigidity. These demonstrate that interparticle energy profileand subsequent anchoring behavior of the ANPs can impact the film‘smechanical properties.From 1D autocorrelationcurves, we quantified the wrinkles’spatial wavelengths . The boundary between regimesII and III is 0.24 M, the condition where Ubarrier becomes zero and equilibrium distancing disappears. This is alsowhere πc abruptly decreases in the 2D isotherm andthe foam formation ability of db1 becomessimilar to that of db2.From the energy calculation and experimentalresults, we couldspecify three interparticle force conditions of repulsive, weaklyattractive, and strongly attractive, each corresponding to a specificionic regime for db1 5A. Thesedb1 in regimeI), dumbbells exhibit low surface affinity and do not form foams orfilms . At weakly attractive conditions (db1 in regime II), dumbbells spontaneously approach up to theequilibrium distance (Deq), forming thinand weak clusters. Because vertical rotation is energetically unfavorable, particles transition to abilayer arrangement while maintaining horizontal anchoring, whichwe have termed the horizontal bilayer. This structuremay be “leaky”, i.e., gaps may be present between individualparticles, as evidenced by low foam stability. At strongly attractiveconditions (db1 in regime III and db2), repulsion is greatly reduced, further enhancingspontaneous cluster assembly, which leads to a broader distributionof orientation angles. Upon lateral compression, intercluster jammingoccurs, resulting in lower collapse pressure and higher bending rigidity.At repulsiveconditions  of anisotropic nanodumbbells are uniqueto our work.db1 and db2: db1 is ion-responsive, which can switch its filmstructure upon an ionic change. On the other hand, db2 exhibits ion-robustness, which maintains stable filmstructures under ionic changes. This can be implicated in the deliverysystem and emulsion stabilizers.We have also highlighted the distinct characteristics of Future perspectives includeexploring more geometrical and chemicalanisotropies, such as size ratio, charge density, and polymer grafting,to manipulate a wider variety of interparticle interactions. We anticipatethat this will enable innovative strategies for optimizing nanoparticleperformance, increasing their applicability across industries.