A98V, rs1800574). The HNF1AA98V exhibited reduced DNA binding. To test function, the HNF1A variant was introduced into the mouse genome by CRISPR/Cas9, and the mice were placed on either a high-fat diet (HFD) or high-sugar diet (HSD). Only 1% of the HNF1A mutant mice developed polyps on normal chow; however, 19% and 3% developed polyps on the HFD and HSD, respectively. RNA-Seq revealed an increase in metabolic, immune, lipid biogenesis genes, and Wnt/β-catenin signaling components in the HNF1A mutant relative to the WT mice. Mouse polyps and colon cancers from participants carrying the HNF1AA98V variant exhibited reduced CDX2 and elevated β-catenin proteins. We further demonstrated decreased occupancy of HNF1AA98V at the Cdx2 locus and reduced Cdx2 promoter activity compared with WT HNF1A. Collectively, our study shows that the HNF1AA98V variant plus a HFD promotes the formation of colonic polyps by activating β-catenin via decreasing Cdx2 expression.The incidence of early-onset colorectal cancer (EO-CRC) is rising and is poorly understood. Lifestyle factors and altered genetic background possibly contribute. Here, we performed targeted exon sequencing of archived leukocyte DNA from 158 EO-CRC participants, which identified a missense mutation at p.A98V within the proximal DNA binding domain of Hepatic Nuclear Factor 1 α (HNF1A  Colorectal cancer (CRC) is the third most common cancer diagnosed in both men and women in the United States. In 2022, it is estimated that ~150,000 new cases of CRC will be diagnosed, causing about 52,580 deaths (Early-onset CRC (EO-CRC) is defined as CRC diagnosed at age < 50 and possesses unique clinical, genetic, and epigenetic characteristics , 4. AccoA98VHNF1A, rs1800574) by performing targeted exon sequencing of archived leukocyte DNA from EO-CRC individuals. The current understanding of HNF1A function has emerged primarily from studies of this transcription factor in the context of mature-onset diabetes of the young 3 (MODY3) and the risk for diabetes mellitus (DM). HNF1A, also known as T cell factor 1 (TCF1), encodes a transcriptional regulatory protein that is required for the expression of several liver specific genes , 158 of the remaining 382 samples were sequenced using the Qiagen NextGen sequencing Multigene Cancer Panel  converts alanine to valine at amino acid residue 98 and was identified in 13 of the 16 cases; while the remaining 3 mutations were located at C241Y, P341S, and Q625E  show that 15% of the participants had diabetes, while at least 30% had dyslipidemia or were obese.Given the 4-fold higher frequency of this gene variant compared with its expression in the general population (2%), we analyzed the function of this variant. The patient demographics summarized in HNF1A and A98VHNF1A variant expression vectors were transfected into the HCT116 colon cancer cell line, which expresses minimal endogenous HNF1A protein. After 2 days, nuclear fractions were prepared (HNF1A consensus DNA binding site in electrophoretic mobility shift assays (EMSAs)  . An unlathe site . Flag anA98VHNF1A variant represented a single-nucleotide mutation resulting in a conservative amino acid change, we tested its function in vivo by introducing the variant into the mouse genome by CRISPER/Cas9 technology on a C57BL/6J genetic background. Since HNF1A is involved in glucose and lipid metabolism and essentially no polyps developed in the A98VHnf1a mice on normal mouse chow, after weaning, +/+Hnf1a, A98V/+Hnf1a, and A98V/A98VHnf1a mice were placed on 3 diets —normal mouse chow , a HFD, and a high-sugar/fructose diet (HSD). All mice had free access to normal water, and the mice were followed for 12 months. Both the A98V/+Hnf1a and A98V/A98VHnf1a mice developed polyps by 6 months of age  or HSD (<3%)  . Since pSD (<3%) , the Hnfthe diet . Among tthe diet . InteresA98V/+Hnf1a and A98V/A98VHnf1a mice at 6 and 12 months showed a loss of normal gland architecture, increased cellularity and increased Ki-67 staining; however, there was no change in HNF1A expression . Fo. Foccnd2and fgf2  and was activity . Therefo+/+ mice . The mRNfunction . In partfunction . β-Catenfunction .A98V/+Hnf1a and A98V/A98VHnf1a colons showed elevated expression of Ki-67 in both the polyps and the normal adjacent mucosa , we tested whether increasing the percentage of sugar or fat in the food would induce a phenotype. Accordingly, we found that both the A98V/+Hnf1a and A98V/A98VHnf1a mice developed colonic polyps when fed a HFD, demonstrating that a loss of HNF1A function contributes to diet-related tumorigenesis.HNF1A is a transcription factor initially identified as a regulator of liver development. It is expressed in a variety of tissues and organs, including liver, pancreas, kidney, and intestine , 46. Numto MODY3 , 47–49. A98V and HFD results in colonic polyp development, these observations warrant further investigation into the broader implications on human CRC.According to the ClinVar database, a total of 812 variants of the HNF1A gene have been reported. The majority of mutations were single nucleotide (639/812). While prevalent, the importance of most single-nucleotide polymorphisms (SNP) remains unknown. Previous studies have identified A98V in the general population with a minor allele frequency of 2.7%, and it is associated with a slight increase in T2DM risk . While wA98VHNF1A participants with EO-CRC, 15% and 30% of these patients carried a diagnosis of DM and dyslipidemia/obesity, respectively. Collectively, the epidemiologic association of metabolic derangements (MAFLD) with colon cancer , suggesting a potential protective effect of estrogen. Moreover, human studies have shown that sexual dimorphism exists at multiple levels in CRC. First, the age-standardized incidence of CRC rate in men is 45% higher compared with women according to the Global Cancer Observatory , 62. SecCurrent studies suggest that modifiable lifestyle factors, such as diet composition, play important roles in the occurrence and progression of CRC . A multic-myc and cyclin D1 in CRC  or lymphoid enhancer factor (LEF) family members and induces target genes such as 1 in CRC –76. In o tissues , 78. Rec tissues , 80. CRC tissues . HNF1A bet genes . An addiet genes .A98V is a loss-of-function missense mutation prevalent in patients with EO-CRC that contributes to tumorigenesis under conditions of a HFD by reducing transcriptional induction of the CDX2 gene. Collectively, our study unveils a role for the A98VHNF1A variant, a gene locus that lies at the intersection of the metabolic syndrome and CRC.In conclusion, we show that the HNF1AThe blood samples from patients with EO-CRC were analyzed by the University of Michigan  DNA sequencing core using the Qiagen gene array panel. Next-generation exon sequencing was performed with 158 patients’ blood samples.A98V were created by PCR amplification of human cDNA purchased from Genscript Biotech Corp. HCT116 cells  were purchased from the EMSR Laboratory, University of Arizona Cancer Center, and grown in McCoy’s 5A medium (Thermo Fisher Scientific) supplemented with heat-inactivated 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific). Cells were cultured in atmospheric air enriched with 5% CO2 at 37°C. Cells were transfected with the HNF1A and HNF1AA98V mutant using Lipofectamine 3000 (Invitrogen) and harvested after 48 hours. After collecting the cells from the plate, nuclear and cytoplasmic fractions were prepared using commercial the NE-PER (Thermo Fisher Scientific).Flag-tagged HNF1A and HNF1ACell lysates or fractions were resolved on Invitrogen Novex 4%–20%, Tris-Glycine, 1.0 mm, Mini Protein Gels and transferred to nylon membranes for blotting with antibodies for HNF1A , GAPDH , FLAG , and histone H3 . After treating with HRP-conjugated secondary Ab , the complexes were visualized using Super Signal West Pico chemiluminescent substrate (Thermo Fisher Scientific).Double-strand DNA (5′-CTTGGTTAATAATTCACCAGA-3′) was purchased from Integrated DNA Technologies (IDT) and labeled with biotin following the manufacturer’s instruction (Pierce Biotin 3′ End DNA Labeling Kit). The binding reactions were carried out using the LightShift Chemiluminescent EMSA Kit  and detected using the Chemiluminescent Nucleic Acid Detection Module Kit .A98VHNF1A transgenic mice were generated by the University of Michigan Transgenic Core using Crisper/Cas9 technology and then bred onto a homogenous C57BL/6J genetic background (The Jackson Laboratory). After weaning around 4 weeks of age, mice were fed 3 different diets, including a normal chow , a HFD , and a HSD  or a HSD composed of fructose (Envigo). Blood and organs were harvested, and body weight was measured. Survival was determined using Kaplan-Meier survival curves (GraphPad Prism 8).RNA was extracted from the colonic mucosal scrapings using the Purelink RNA Extraction kit (Invitrogen) and was then submitted for RNA-Seq by Novogene. Fragments per kilobase of transcript sequence per millions base pairs (FPKM) sequenced were analyzed for differential gene expression, principal component, and Gene Ontology (GO) pathway enrichment analysis.Mouse tissues were fixed overnight at 4°C in 4% paraformaldehyde. Tissues were paraffin embedded, cut into 5 μm sections, and placed on frosted glass slides. Immunofluorescence staining was performed as previously described  using KiScrapings of the mouse colonic mucosa were homogenized with a rotor-stator homogenizer in RNA lysis buffer containing 1% β-mercaptoethanol. RNA was isolated using the Purelink RNA Extraction kit (Invitrogen) following manufacturer’s instructions. cDNA was prepared by the iScript Reverse Transcription Supermix for qPCR using 1 μg RNA and diluted by 80 μL nuclease-free water. qPCR was performed using PowerUp SYBR Green Master Mix (Invitrogen) with 20 ng cDNA added to each reaction. Predesigned forward and reverse primer sets were purchased from Integrated DNA Technologies (IDT) and used at a final 500 nM concentration. qPCR was performed using the QuantStudio3 Real-Time PCR System (Applied Biosystems) with the following cycling conditions: 2 minutes at 50°C, 2 minutes at 95°C, denaturing step for 1 second at 95°C, extension and annealing for 1 minute at 60°C, and a dissociation melt curve stage to confirm primer specificity. The results are expressed as fold-increase mRNA expression of the gene of interest normalized to 18S expression by the ΔΔCt method.A98V mutant using Lipofectamine 3000 (Invitrogen) and prepared for ChIP after 48 hours. EZ-Magna ChIP HiSens Kit from MilliporeSigma was applied. The immunoprecipitated purified DNA was used to amplify the Cdx2 gene using SYBR GREEN-based (Thermo Fisher Scientific) RT-PCR analysis. The following primers are used . After 48 hours, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega).The CDX2 -pGL3 basic-luciferase construct  was geneP value of less than 0.05 was considered statistically significant.Results are shown as the mean ± SEM of 3 independent experiments. Shapiro-Wilk test was used to test for normality distribution. If the significance value of the Shapiro-Wilk test was greater than 0.05, the data were considered normal. For comparison among multiple groups, 1-way ANOVA or 2-way ANOVA was used depending on the nature of comparison. When there were more than 1 continuous dependent variables, 1-way multivariate ANOVA (MANOVA) was used to determine whether there were any differences between independent groups. The nonparametric Kruskal-Wallis test followed by the Games-Howell post-hoc test was used for multiple comparisons not distributed normally. Statistical analyses were performed in SPSS v22.0 (IBM SPSS Statistics). A Written informed consent was received prior to participation. Blood samples were collected in accordance with the University of Michigan IRB (HUM00043430). All animal experiments were approved by the University of Arizona IACUC.HS, CAT, EMS, and JLM designed research studies. HS, RAS, JMM, MJV, and SS performed the experiments. HS, SD, JZ, SZ, RP, EMS, and JLM analyzed the data. EK and EMS provided patients’ data. HS, JZ, and JLM wrote the manuscript. JLM supervised the study.


Medicago truncatula and its potential roles in the regulation of response to mineral nutrient deficiency are very limited. Using high-resolution chromosome conformation capture sequencing, we identified the 3D genome structure of M. truncatula in terms of A/B compartments, topologically associated domains (TADs) and chromatin loops. The gene density, expressional level, and active histone modification were higher in A compartments than in B compartments. Moreover, we analysed the 3D chromatin architecture reorganization in response to phosphorus (P) deficiency. The intra-chromosomal cis-interaction proportion was increased by P deficiency, and a total of 748 A/B compartment switch regions were detected. In these regions, density changes in H3K4me3 and H3K27ac modifications were associated with expression of P deficiency-responsive genes involved in root system architecture and hormonal responses. Furthermore, these genes enhanced P uptake and mobilization by increasing root surface area and strengthening signal transduction under P deficiency. These findings advance our understanding of the potential roles of 3D chromatin architecture in responses of plants in general, and in particular in M. truncatula, to P deficiency.Emerging evidence reveals that the three-dimensional (3D) chromatin architecture plays a key regulatory role in various biological processes of plants. However, information on the 3D chromatin architecture of the legume model plant  Medicago truncatulaunder phosphorus deficiency.Reorganization of the 3D chromatin architecture is associated with transcriptional regulation to enhance phosphorus uptake and mobilization in  Chromatin is the main carrier of DNA, which stores the genetic information for living organisms in eukaryotes. Recent developments in spatial genetics have found that the three-dimensional (3D) chromatin architecture plays crucial regulatory roles in diverse biological processes, such as DNA replication and transcription regulation . The difThe chromatin architecture is not always fixed, and reorganization of the 3D chromatin architecture occurs frequently during the diverse biological processes in plants (Medicago truncatula under P deficiency.Phosphorus (P) is one of the essential macronutrients for higher plants. The total P in the soil is abundant. However, the available inorganic phosphate (Pi) to plants is usually low in many types of soils, thus limiting plant growth . About 7M. truncatula ‘Jemalong A17’ were soaked using concentrated sulfuric acid for 7 min, and then rinsed with water. Seeds were sown on wet filter paper to germinate at 26 °C. They were then planted in vermiculite in a greenhouse . All seedlings were pre-cultured by watering with full-strength nutrient solution  for 4 weeks. Thereafter, half of the seedlings were watered using a reduced KH2PO4 concentration (1 µM) for 8 d, as phosphate deficient treatment (PD). Samples were collected for physiological index determination, library construction, and sequencing.Seeds of Samples were dried at 80 °C to constant weight, and then dry weight was recorded . Following this, 100 mg dry samples, 5 ml of nitric acid, and 2 ml of hydrogen peroxide were mixed in digestion tubes, and then samples were digested using a microwave acid digestion system . After diluting and filtering the samples, P concentrations were measured using inductively coupled plasma-atomic emission spectroscopy .Hi-C libraries were constructed using the standard protocol  by mapping reads to the e Mt4.0; ; these r2 function. We calculated the Pearson correlation between patterns of chromosomal interactions of every genome bin, and performed the principle component analysis using a correlation matrix. Positive and negative values of the first principal component were used to separate chromatin regions into A and B compartments, respectively.ICE-normalized interaction matrices at 20 kb resolution were used to detect A/B compartments as described previously using the Cworld-matrix2compartment module  which overlapped with gene regions were defined as proximal ones, while those which did not overlap with genes were defined as distal.We used valid pair reads to call loops using Juicer HICCUPS with default parameters at 5 kb resolution (M. truncatula genome sequence (Mt4.0) by HISAT2 with default parameters  enrichment of differentially expressed genes was implemented using the hyper geometric test (Fisher’s exact test), in which About 2.5 g of leaf samples were crosslinked with 1% formaldehyde. Chromatin immunoprecipitation (ChIP) was performed using previously established methods (M. truncatula genome sequence (Mt4.0) using Bowtie2 software (-N 1 -p 6), and only uniquely mapped reads were used to perform downstream analysis  were watered with a Pi-deficient (1 µM P) solution (PD) for 8 d. Exposure of the seedlings to the Pi-deficient medium significantly reduced dry biomass and foliar P concentrations (P<0.01), while root/shoot ratio was significantly increased (P<0.05) compared with those of CK treatment , respectively, under PD treatment  of six libraries generated 41.29 Gb of data , and theP<0.001  sequencing libraries for H3K4me3 and H3K27ac were constructed using the samples exposed to the same Pi-deficient treatment . Analysin of DNA . Moreoven of DNA , C. We in of DNA . Among tn of DNA . For H3Kn of DNA . As the n of DNA . Moreoven of DNA , H.Chr6g0452171, Chr1g0185401, and Chr1g0149671 was up-regulated by P deficiency. Moreover, the location of these genes in chromosomes changed from B compartments to A compartments by treatment with P deficiency. Enriched H3K27ac or H3K4me3 modifications were related to these switches . It has been reported that 3D chromatin reorganization was associated with histone modification  is an important way to cope with P deficiency. Plants often increase root surface area by producing more lateral roots and root hairs to increase the Pi uptake under Pi-deficient conditions . Auxin sstresses , C, D, w2+ act as signalling molecules in response to numerous abiotic stresses, including response to P deficiency  and a ribosome biogenesis protein (Chr1g0149671) were found to be involved in the response to P deficiency (Abscisic acid (ABA) and Caodelling . The expficiency , F. The M. truncatula, and revealed that transcriptional regulation was associated with 3D chromatin architecture reorganization under P deficiency. Our findings provide a new perspective to explore the mechanisms underlying the regulation of plant gene transcription in response to P deficiency.In conclusion, we constructed the 3D genome architecture of the legume model plant The following supplementary data are available at Fig. S1. Effects of P deficiency on shoot biomass, P concentrations in shoots and root/shoot ratio.Fig. S2. Correlation coefficients of four Hi-C libraries.Fig.S3. Depth in different resolutions.Fig. S4. Pearson correlation of each chromosome.Fig. S5. Correlation coefficients of six transcriptome libraries.Fig. S6. Correlation coefficients of 10 ChIP-seq libraries.Fig. S7. Distribution of histone modification.Fig. S8. Common and specific peaks for ChIP-seq of H3K4me3 and H3K27ac.Fig. S9. Modification level of H3K4me3 and H3K27ac in A and B compartments under PD treatment.M. truncatula.Fig. S10. Western blots using eight commercialized histone antibodies to histone of Table S1. Statistics of Hi-C sequencing.Table S2. Different expression of genes located in CK and PD specific loops.Table S3. Statistics of transcriptome sequencing.Table S4. Up-regulated genes in the B to A compartment switch regions.Table S5. Down-regulated genes in the A to B compartment switch regions.Table S6. Statistics of ChIP sequencing.erac517_suppl_Supplementary_Figures_S1-S10_Tables_S1-S6Click here for additional data file.


EE/AA structures of F635A and F872A mutants in complex with nick DNA containing A:C and G:T mismatches demonstrate the importance of DNA end rigidity, as well as uncover a shift in a flexible loop near 5'-end of the nick, which causes an increased barrier to adenylate transfer from LIG1 to the 5'-end of the nick. Furthermore, LIG1EE/AA/8oxoG:A structures of both mutants demonstrated that F635 and F872 play critical roles during steps 1 or 2 of the ligation reaction depending on the position of the active site residue near the DNA ends. Overall, our study contributes towards a better understanding of the substrate discrimination mechanism of LIG1 against mutagenic repair intermediates with mismatched or damaged ends and reveals the importance of conserved ligase active site residues to maintain ligation fidelity.ATP-dependent DNA ligases catalyze phosphodiester bond formation in the conserved three-step chemical reaction of nick sealing. Human DNA ligase I (LIG1) finalizes almost all DNA repair pathways following DNA polymerase-mediated nucleotide insertion. We previously reported that LIG1 discriminates mismatches depending on the architecture of the 3'-terminus at a nick, however the contribution of conserved active site residues to faithful ligation remains unknown. Here, we comprehensively dissect the nick DNA substrate specificity of LIG1 active site mutants carrying Ala(A) and Leu(L) substitutions at Phe(F)635 and Phe(F)F872 residues and show completely abolished ligation of nick DNA substrates with all 12 non-canonical mismatches. LIG1 Although they are highly conserved amongst ATP-dependent DNA ligases from human and other sources, the role of F635 and F872 active site residues for non-canonical substrate discrimination of a human DNA ligase at atomic resolution remains unknown.DNA ligases encoded by the human  domains . The DNA domains -10. Alth domains ,13. The  domains -17. LIG1position -20. There states . The fire states . In thise states . The fire states . Further2+ binding sites that are independent of the catalytic Mg2+, which is required during the initial and final steps of the ligation reaction (EE/AA in the present study) and enables the ligase to better accommodate a nick DNA with oxidative damage (8oxoG) at the 3'-end 346 and Glu(E)592 are the high-fidelity Mgreaction -25. Moree 3'-end . Furthere 3'-end . We repoe 3'-end . Althouge 3'-end -26, it iEE/AA) background of LIG1, and comprehensively characterized the single  and triple  mutants at biochemical and structural levels. We investigated the substrate specificity of these LIG1 mutants in ligation assays in vitro and demonstrated completely abolished nick sealing for all 12 non-canonical mismatches. Additionally, we solved the structures of LIG1EE/AA F635A and F872A mutants with nick DNA complexes containing a cognate A:T, mismatches A:C and G:T, and oxidatively damaged 8oxoG:A ends at the 3'-end. Our mismatch structures for both LIG1EE/AA mutants revealed conformational changes at the DNA ends, as well as a shift in the flexible loop between R736 and R741, which results in the putative formation of additional hydrogen bonds between R738 and the 5'-P of the nick during step 1 of the ligation reaction, which together hinders the ligase for further catalytic steps, in contrast to the LIG1EE/AA/A:T structures which were captured during step 2 when the DNA-AMP intermediate is formed. Furthermore, our LIG1EE/AA F872A/8oxoG:A structure demonstrated a similar DNA end misalignment and the R738 loop shift during the initial step of the ligation reaction and a lack of ligation in vitro, while we captured LIG1 EE/AA F635A/8oxoG:A during step 2 and observed the mutagenic nick sealing of damaged end. Overall, our study contributes to elucidating the mechanism by which LIG1 discriminates against mutagenic repair intermediates with mismatched or damaged ends and the contribution of conserved F635 and F872 residues to faithful ligation during nuclear replication and DNA repair.In the present study, we generated Phe(F) to Ala(A) or Leu(L) mutations at F635 and F872 in the wild-type and low-fidelity (LIG1EE/AA), and triple mutations E346A/E592A/F635A (LIG1EE/AA F635A) and E346A/E592A/F872A (LIG1EE/AA F872A) were prepared using the wild-type LIG1 C-terminal (Δ261) construct as reported previously (We generated Phe(F) to Ala(A) and Leu(L) amino acid substitutions at F635 and F872, residues that reside in Adenylation domain (AdD) and Oligonucleotide-binding domain (OBD) of DNA ligase I (LIG1), respectively, within the catalytic core of the enzyme (Supplementary Scheme 2). Plasmid DNA constructs carrying single mutations F635A, F635L, F872A, F872L, double-mutation E346A/E592A  label at 3'-end were used in DNA ligation assays. Nick DNA substrates containing all 12 mismatches were prepared by annealing oligonucleotides 3'-dA, dT, dG, or dC with template base A, T, G, or C (Supplementary Table 1). Nick DNA substrates containing 8-oxodG were prepared by annealing 3'-8oxodG with template base A or C (Supplementary Table 2). Nick DNA substrates containing A:T, A:C, G:T, and 8oxoG:A were used in the LIG1 X-ray crystallization experiments (Supplementary Table 3).EE/AA, and triple mutants LIG1EE/AA F635A and F872A were purified as described previously (E. coli Rosetta (DE3) pLysS cells and grown in Terrific Broth (TB) media with kanamycin (50 μgml−1) and chloramphenicol (34 μgml−1) at 37 °C. Once the OD600 reached 1.0, cells were induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) and overexpression continued overnight at 20 °C. After centrifugation, cells were lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.0), 500 mM NaCl, 20 mM imidazole, 2 mM β-mercaptoethanol, 5% glycerol, and 1 mM PMSF by sonication at 4 °C. The lysate was pelleted at 31,000 x g for 90 min at 4 °C. The cell lysis solution was clarified and then loaded onto a HisTrap HP column that was previously equilibrated with binding buffer containing 50 mM Tris-HCl (pH 7.0), 500 mM NaCl, 20 mM imidazole, 2 mM β-mercaptoethanol, and 5% glycerol. The column was washed with binding buffer and then eluted with an increasing imidazole gradient 0-500 mM at 4 °C. The collected fractions were then subsequently loaded onto a HiTrap Heparin column that was equilibrated with binding buffer containing 20 mM Tris-HCl (pH 7.0), 50 mM NaCl, 2 mM β-mercaptoethanol, and 5% glycerol. The protein was eluted with a linear gradient of NaCl up to 1 M. LIG1 proteins were further purified by Superdex 200 10/300 column in the buffer containing 50 mM Tris-HCl (pH 7.0), 200 mM NaCl, and 1 mM DTT, and 5% glycerol. LIG1EE/AA F635A and F872A proteins which were used for crystallization experiments were exchanged to crystal storage buffer containing 20 mM Tris-HCl (pH 7.0), 200 mM NaCl, and 1 mM DTT. All proteins purified in this study were concentrated, aliquoted, and stored at −80 °C. Protein quality was evaluated on a 10% SDS-PAGE gel, and protein concentrations were measured using absorbance at 280 nm.Human his-tag LIG1 C-terminal Δ261) wild-type, single mutants F635A, F635L, F872A, and F872L, double mutant LIG11 wild-tyeviously . BrieflyEE/AA, and six active site mutants (Supplementary Scheme 3). For this purpose, we used the nick DNA substrates including all 12 non-canonical 3'-preinserted mismatches as well as 3'-preinserted 8-oxoG:A and 8oxoG:C (Supplementary Tables 1 and 2). The reaction was initiated by the addition of LIG1 (150 nM) to a mixture containing 50 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 100 μgml−1 BSA, 10% glycerol, and the nick DNA substrate (500 nM) in a final volume of 10 μl. The reaction mixture was incubated at 37 °C and stopped at the time points indicated in the figure legends by mixing with an equal volume of loading dye containing 95% formamide, 20 mM ethylenediaminetetraacetic acid, 0.02% bromophenol blue, and 0.02% xylene cyanol. Reaction products were separated by electrophoresis on an 18% Urea-PAGE gel, the gels were scanned with Typhoon PhosphorImager RGB (Amersham), and the data was analyzed using ImageQuant software as described previously . All images were drawn using Graph Pad Prism 9. Thermal shift assays were performed in at least biological duplicates each containing technical triplicates for LIG1 wild-type and all mutants tested in this study.Thermal shift assays were carried out in 96 well plates using the CFX96 RT-PCR detection system (Bio-Rad). Thermal stability experiments were performed in the reaction mixture containing LIG1 (5 μM) in the absence or presence of nick DNA (50 μM) in a total volume of 25 μl. Nick DNA substrate including preinserted 3'-dG:C was used (Supplementary Table 4). After incubation at 4 °C for 1 h, 20X Sypro Orange protein dye (Invitrogen) was added, and the plate was then sealed with an optical seal and centrifuged. The thermal scan ranged from 10-95 °C with a temperature ramp rate of 0.20 °C/min. The fluorescence intensity upon binding of Sypro Orange was measured with excitation/emission of 533/580 nm. Data analysis and report generation were performed by the Maestro instrument software (Biorad). TEE/AA C-terminal (Δ261) F635A and F872A mutants were used for the crystallization experiments. The EE/AA mutation aids in crystallization (−1)/DNA complex solution was prepared in 20 mM Tris-HCl (pH 7.0), 200 mM NaCl, 1 mM DTT, 8 mM EDTA, and 4 mM ATP at 1.5:1 DNA:protein molar ratio and incubated at 37 °C for 3 min. 1 μl of DNA:protein complex was mixed with 1 μl reservoir solution to form the hanging drop (Supplementary Table 5). All crystals grew in 1-2 days. Crystals were harvested and submerged in cryoprotectant solution containing reservoir solution mixed with glycerol to a final concentration of 20% glycerol before being flash cooled in liquid nitrogen for data collection. Crystals were kept at 100 °K during X-ray diffraction data collection using the beamline 22-ID (APS). Diffraction images were indexed and integrated using HKL2000 . All structures were solved through molecular replacement using PHASER with PDB entry 7SUM as the search model  stays adenylated  (The overlay of our current and previously solved structures of LIG1igure 1) . DNA endWe then analyzed the overlay of all our structures near the 5'-end of the nick for both LIG1 mutants . InteresEE/AA F635A and F872A structures . As expected, the 3'-end adopts a canonical Watson-Crick base pair in the LIG1EE/AA/A:T structures . Consistent with what we and others previously demonstrated, the A:C and G:T mismatches undergo wobble base pairing in the LIG1 active site while 8oxoG:A forms a Hoogsteen base pair, where the 8oxoG moiety rotates around the N-glycosidic bond to the syn conformation  . This demonstrates the importance of the A-form transition upstream of the nick on adenylate transfer.We next investigated the base pairing configuration and sugar pucker conformation of the 3'-end in all of our LIG1re 2C-G) , 26. SugEE/AA F635A and F872A that we used to solve the structures of LIG1 active site mutants. In addition to these LIG1 mutants, we tested the ligation efficiency of the wild-type and low-fidelity (EE/AA) mutant of LIG1. For this purpose, we used the nick DNA substrates containing all 12 non-canonical mismatches at the 3'-end (Supplementary Scheme 3A).To comprehensively investigate the effect of the active site mutations at F635 and F872 on the mismatch substrate specificity of LIG1, we analyzed the ligation profile of four single mutants F635A, F635L, F872A, F872L as well as the triple-mutants LIG1Consistent with our previous reports , in the In the presence of the single F635A mutation, the ligation efficiency drastically decreased with less than 10% of ligation products for all 12 non-canonical mismatches (EE/AA) that enables LIG1 to seal damaged or mismatched ends more efficiently than the wild-type enzyme can rescue this lack of nick sealing we observed for the single mutants of LIG1 F635A and F872A .We then questioned whether the EE/AA mutation . Overall, these results demonstrate that both F635 and F872 residues play critical roles for mismatch discrimination and do not show any substrate specificity such as a type of mismatch or DNA end architecture . In geneEE/AA F635A and F872A for the nick DNA substrates containing 3'-8oxoG:A and 3'-8oxoG:C to comprehensively understand the effect of the active site mutations on the substrate discrimination against mismatched versus damaged DNA ends (Supplementary Scheme 3B).We next analyzed the ligation efficiency of LIG1 single mutants  and the triple-mutants LIG1EE/AA mutant of LIG1, we observed the mutagenic ligation of the nick DNA substrate with 3'-8oxoG:A, while the formation of the DNA-AMP intermediate during the ligation reaction is accompanied by mutagenic nick sealing of 3'-8oxoG:C   increases the efficiency of 8oxoG:A ligation when F635 is mutated but cannot rescue the lack of ligation for 8oxoG:A when F872A is mutated . This is possibly explained by position of the MgHiFi site which is located upstream of the nick.In line with our previous reports, for the wild-type and LIG1 and 16) ,31. For  and 16) -D. On th mutants . The comm values, which demonstrates similar thermal stability indicating similar global protein folds . LIG1 F872A and F872L mutants exhibit slightly decreased thermal stability, but not drastic enough to demonstrate misfolding or large disturbances in the protein structure. We also tested the thermal stability of the LIG1 wild-type and mutants in the presence of nick DNA. Our results demonstrated an increase in Tm values for all LIG1 variants indicating that all LIG1 variants are able to bind nick DNA and suggesting that DNA binding stabilizes the ligase proteins . We also observed higher thermal stability of both triple mutants suggesting that mutation at the MgHiFi site leads to higher thermal stability upon nick DNA binding .We finally evaluated whether the LIG1 mutations at F635 and F872 impact protein stability and folding by thermal shift protein denaturation assays. For this purpose, we performed the assays in the presence and absence of nick DNA containing 3'-preinserted cognate G:C. Our results demonstrated that all LIG1 proteins tested in this study showed similar T+) and nick DNA substrate binding  or leucine (L), we obtained a drastic ablation of nick sealing efficiency for all possible 12 non-canonical mismatches with only a minor loss of catalytic efficiency for canonical DNA ends. Our findings suggest that these active site residues are both required for efficient non-canonical catalysis and that all mutations at these residues  result in higher fidelity ligation by deterring nick sealing of mutagenic non-canonical ends.EE/AA F635A and F872A mutants in complex with nick DNA duplexes containing a canonical A:T base pair, A:C and G:T mismatches, and 8oxoG:A at the 3'-end revealed further insights into the importance of these active sites for the mismatch discrimination mechanism of LIG1. We observed that the substitution of F to A at both F635 and F872 residues creates a pocket of missing density that the aromatic moiety of phenylalanine would normally occupy, this in turn allows for a greater degree of flexibility at the 3'- and 5'-ends, respectively. This flexibility results in increased sensitivity to the identity of the base-pair at the 3'-terminus which is dependent on the position of a specific point mutation.Structures of LIG1EE/AA F635A/A:C mismatch. Alternatively, it can shift away from the 3'-end, as we observed in the structure of LIG1EE/AA F635A/G:T, which deters mutagenic nick sealing of the G:T mismatch by causing DNA end misalignment. Our results suggests that the direction of the 3'-end shift is likely mismatch dependent. However, this shift was not observed in the structures of LIG1EE/AA F635A for a cognate A:T and damaged 8oxoG:A ends, which suggests that canonical or Hoogsteen base pairing is strong enough to enforce the active site geometry required for ligation even in the presence of missing sidechain density. When the 5'-end is allowed greater flexibility as a result of F872A mutation, the DNA conformation downstream of the nick changes, resulting in a subsequent change upstream of the nick, and therefore misaligns the DNA ends which provide a barrier for catalysis. Unlike F635A mutation, upon F872A mutation, Hoogsteen base pairing is not capable of enforcing the active site geometry required for efficient catalysis, as we observed in the structure of LIG1EE/AA F872A/8oxoG:A. Only a proper canonical base pair is capable of enforcing the active site geometry required for efficient catalysis, as we observed in the structure of LIG1EE/AA F872A/A:T. These results suggest that the 5'-end stability could be more critical for efficient ligation of non-canonical substrates. Our findings suggest that the conserved LIG1 active site phenylalanine residues, F635 and F872, are critical for nick sealing of non-canonical 3'-ends by enforcing rigidity of the 3'- and 5'-ends, respectively, through interactions with the sugar moiety.When the 3'-end is allowed greater flexibility as a result of F635A mutation, the presence of this missing density, in combination with the improperly base paired mismatch, enables the 3'-end to shift into the pocket, as we observed in the structure of LIG1in vitro observation of inefficient non-canonical ligation by all single and triple mutations at F635 and F872. It is not immediately obvious why this shift of the flexible loop only manifests upon F635 or F872 mutation, but it is consistent with previous studies which have also shown that mutations at one side of the nick can influence the ligase active site on the other side of the nick and highlights the sensitivity of the active site of LIG1 to mutation (Furthermore, our LIG1 structures that were captured in step 1 of the ligation reaction revealed a shift in a flexible loop region which results in the positioning of the guanidinium moiety of R738 within hydrogen bonding distance of the 5'-P of the nick. These putative hydrogen bonds provide a barrier to adenylate transfer which, in combination with DNA end misalignment, explains our mutation . Future mutation -56, the


Dioscorea zingiberensis is a perennial herb famous for the production of diosgenin, which is a valuable initial material for the industrial synthesis of steroid drugs. Sterol C26-hydroxylases, such as TfCYP72A616 and PpCYP72A613, play an important role in the diosgenin biosynthesis pathway. In the present study, a novel gene, DzCYP72A12-4, was identified as C26-hydroxylase and was found to be involved in diosgenin biosynthesis, for the first time in D. zingiberensis, using comprehensive methods. Then, the diosgenin heterogenous biosynthesis pathway starting from cholesterol was created in stable transgenic tobacco (Nicotiana tabacum L.) harboring DzCYP90B71(QPZ88854), DzCYP90G6(QPZ88855) and DzCYP72A12-4. Meanwhile, diosgenin was detected in the transgenic tobacco using an ultra-performance liquid chromatography system  tandem MS . Further RT-qPCR analysis showed that DzCYP72A12-4 was highly expressed in both rhizomes and leaves and was upregulated under 15% polyethylene glycol (PEG) treatment, indicating that DzCYP72A12-4 may be related to drought resistance. In addition, the germination rate of the diosgenin-producing tobacco seeds was higher than that of the negative controls under 15% PEG pressure. In addition, the concentration of malonaldehyde (MDA) was lower in the diosgenin-producing tobacco seedlings than those of the control, indicating higher drought adaptability. The results of this study provide valuable information for further research on diosgenin biosynthesis in D. zingiberensis and its functions related to drought adaptability. Dioscorea plants that can meet the requirements for industrial scale extraction. There are about 137 Dioscorea species containing diosgenin, and in more than 30 of them, the content of diosgenin is higher than 1% by dry weight [Dioscorea zingiberensis rhizoma in China [Diosgenin, a well-known steroidal sapogenin, is not only an important and valuable material for the production of many steroid drugs , but it Paris polyphylla, and a high-efficiency cholesterol synthesis system was established promoting the efficiency of diosgenin production in plant chassis [Previous studies have shown that cholesterol is the main precursor of diosgenin biosynthesis, which has the most consistent 27 Carbon skeleton structure with diosgenin b,c 6,7,,10,11,12 chassis .solanaceous plants. In that process, several CYP450s including GAME7/PGA2, GAME8/PGA1, GAME11 and GAME6 sequentially catalyze the hydroxylation and oxidation of cholesterol at the C22, C26 and C16 positions, and finally form the furostanol-type aglycone [Arabidopsis thaliana [Solanum lycopersicum) and DzCYP90B71(D. zingiberensis) were previously reported as cholesterol C22-hydroxylase [PpCYP90G4 in Himalayan paris (Paris polyphylla) and TfCYP90B50 in fenugreek (Trigonella foenum-graecum L.) encoded sterol C16,22-dihydroxylase in the diosgenin biosynthesis pathway coupled with several C26-hydroxylases including PpCCYP94D108, PpCCYP94D109, PpCCYP72A616, TfCYP72A613 and TfCYP82J17 [DzCYP90G6 in D. zingiberensis (monocots) encodes C16-hydroxylase, which is different from its homolog, PpCYP90G4. PGA1 and PGA2 in potato (Solanum tuberosum) belong to the CYP72A subfamily, reported to be C26- and C22-hydroxylase, respectively. Clearly, the CYP450s of the CYP72A subfamily play a crucial role in diosgenin biosynthesis. Recently, a chromosome-scale genome (629 Mb) of D. zingiberensis was generated [D. zingiberensis was performed. A total of 25 CYP72A genes were identified in silico, and nine of them were reported to have high correlation with diosgenin metabolism [It has been reported that furostanol I , protodithaliana , CYP90B2roxylase . Anotherenerated . Based oNicotiana tabacum L.), as a model plant, containing an endogenous cholesterol biosynthesis pathway, can supply sufficient precursors for diosgenin synthesis [DzCYP90B71(22R-oxidation) and DzCYP90G6 (C16-oxidation) in tobacco, which could biosynthesize C16,22-dihydrocholesterol form cholesterol to supply the substrate for C26-hydoxylase. Using this platform, potential candidates of CYP450s were studied. Finally, the DzCYP72A12-4 gene was cloned and identified as sterol C26-hydroxylase in D. zingiberensis. The amount of diosgenin in the transgenic tobacco plants was determined using an ultra-performance liquid chromatography system  tandem MS  (UPLC–MS/MS). The column equipped on the UPLC system was the Hypersil GOLD C18 column 690 . Meanwhile, some physiological indexes of the transgenic diosgenin-producing tobacco plants were measured to explore the drought adaptability.Tobacco (ynthesis . In the ynthesis . In the D. zingiberensis have been identified as participating in furostanol hydroxylation so far. A phylogenetic tree was constructed using the neighbor-joining (NJ) method    to confi values)  particip values) .DzCYP94D143 (Clade VI) and DzCYP94N8 (Clade VII) are two characterized C26-hydroxylases in the diosgenin biosynthesis pathway cloned from D. zingiberensis, which means screened sequences in this clade might have similar activity with C26-hydroxylase. Although TfCYP82J17 was in clade V, its identities with the other members were relatively low (<50%), which means no specific homolog of TfCYP82J17 existed in D. zingiberensis.Based on the phylogenetic analysis, the chosen proteins close to C26-hydroxylase could be divided into three groups , consistDzCYP72As (except for DzCYP72A19) as well as three known CYP450s, PpCYP72A616(monocots), GAME7 and TfCYP72A613 (dicots). DzCYP72A sequences were assigned into two clades , which is involved in the inactivation of BRs castasterone (CS) and brassinolide (BL) through the carbon 26 hydroxylation [DzCYP90B71, DzCYP90G6 and PpCYP90G4). In addition, GAMEs reported in the SGAs biosynthesis were scattered into different clades and served as functional references. Obviously, most of the screened sequences were DzCYP72As, which may be related to the later C26-oxidation step  in diosgenin biosynthesis.As we can see from xylation . Clade IDzCYP72A belonging to Clade I (DzCYP1-13/23/24) and Clade II (DzCYP72A18-20), respectively . A 53 amino acid residue deletion occurred in the DzCYP72A12-4 protein sequence  of 9.15. The conserved domain sequence (CDS) analysis of DzCYP72A12-4 revealed that it belonged to the cytochrome P450 (CYP) superfamily and contained a putative transmembrane helix region from position 7 to position 29 and a likely PX domain from position 133 to 244.sequence . AnalysiDzCYP90B71 and DzCYP90G6 were cloned from D. zingiberensis and were co-expressed in tobacco to produce the substrate for C26-hydroxylase. The DzCYP90B71 protein catalyzes cholesterol hydroxylation at the C22 position, producing C22-hydroxycholesterol, which is subsequently hydroxylated by the DzCYP90G6 protein producing 16,22-dihydroxycholesterol. DzCYP94N8 was also cloned as a positive control of C26-hydroxylase and co-transformed into tobacco with DzCYP90B71 and DzCYP90G6 to complete the diosgenin heterogenous biosynthesis pathway.DzCYP90B71/DzCYP90G6/DzCYP72A12-4 (OE3) or DzCYP90B71/DzCYP90G6/DzCYP94N8 . Transgenic tobacco plants harboring DzCYP72A12-4  or DzCYP90B71/DzCYP90G6  were created as background controls. Wild type tobacco was used as the blank control. Genomic DNA PCR was performed for the preliminary screening of positive transgenic plants surviving on MS medium containing 50 ug/mL hygromycin. The expression level of exogenous genes in the selected lines was analyzed by qRT-PCR using the primers listed in The diosgenin heterogenous biosynthesis pathway was reconstituted in stable transgenic tobacco harboring a combination of Ultra-performance liquid chromatography tandem MS was utilized to analyze the metabolic profiling of the tested tobacco leaf extracts, revealing that co-expression of DzCYP90B71, DzCYP90G6 and DzCYP72A12-4 resulted in the heterologous production of diosgenin in tobacco but not in the NC1, NC2 and WT controls . The UPLThe seeds of three OE3 lines and the NC1, NC2 and WT plants were used to test the physiological changes under drought stress. The germination rate and malondialdehyde (MDA) concentration were measured. The germination rate (GR) is a common index of seed vigor. The GRs of all lines were affected by the 15% PEG treatment, but the degree of influence varied with each line A. The seDzCYP72A12-4 gene and drought stress, analysis of the DzCYP72A12-4 expression patterns and levels under drought stress in D. zingiberensis was conducted. The results showed that the transcript level of DzCYP72A12-4 was upregulated at first after the treatments of PEG6000 (15%) reaching a peak level around 4 h; then, it subsequently decreased to a level even lower than the normal control after the 96 h PEG treatment  of berensis , which riosgenin . In addiDzCYP72As were divided into two clades  and Clade II (DzCYP72A18-20), respectively , DzCYP90G6 (C16-oxidation) and DzCYP94N8/DzCYP94D143 (C26-oxidation), in D. zingiberensis were identified and transferred into the cholesterol-producing yeast (Saccharomyces cerevisiae) strain RH6829 to produce diosgenin successfully [DzCYP90B71, DzCYP90G6 and DzCYP94N8. Transgenic tobacco containing DzCYP90B71 and DzCYP90G6 can provide enough substrate for the C26-hydroxylase to biosynthesize diosgenin, and it was exploited as a chassis to test the potential C26-oxidation catalyzing activity of the DzCYP72A candidates. Meanwhile, DzCYP94N8 was used as the positive control. Using this strategy, a novel DzCYP72A12-4 gene encoding C26-hydroxylase was identified from D. zingiberensis. Diosgenin was detected in the transgenic tobacco harboring DzCYP90B71/DzCYP90G6/DzCYP72A12-4 genes and not in the transgenic plants harboring DzCYP90B71/DzCYP90G6 or the transgenic plants harboring DzCYP72A12-4, suggesting that DzCYP72A12-4 encodes a C26-hydroxylase involved in diosgenin biosynthesis.In order to perform high-throughput function characterization of new CYP450s, the tobacco transient expression system was utilized to explore the diosgenin biosynthesis pathway for the first time in Himalayan paris and fenugreek . Then, messfully . Based oDzCYP72A12-4 was upregulated after treatments of PEG6000 (15%) in D. zingiberensis, further proving the relationship between DzCYP72A12-4 and drought stress. However, the specific mechanism needs to be illustrated through further research.Drought resistance can be understood as the ability of plants to sense the water-deficiency signal and initiate coping strategies, which are very complex traits that show up as diverse indicators for assessing improved drought resistance. The germination rate (GR) is considered as a common index of seed vigor. Seed germination usually needs sufficient water. Drought stress can result in decreased vitality and increased harmful substances, such as malondialdehyde (MDA). MDA is the final decomposition product of membrane lipid peroxidation, and the content of MDA can reflect the degree of stress injury suffered by plants. The transgenic tobacco plants (OE3) had a higher germination rate than the controls . Meanwhile, the MDA concentration in 16-day-old OE3 seedlings was lower than that in the controls. The emerging diosgenin could be the main cause of this phenomenon. A previous report showed that diosgenin has the ability to reduce oxidative stress damage . The recD. zingiberensis plants were collected from Ankang of Shaanxi province in China. Fresh rhizomes were transplanted into a greenhouse with strict environmental control (25 ± 2 °C and 16 h light/day). New plants sprouted and were propagated to produce seeds. Samples pending determination were freeze-dried to constant weight at −80 °C and ground into powder with a tissuelyser.Nicotiana benthamiana) was used for the transgenic assays. Mature tobacco seeds were surface sterilized for 60 s with 75% (v/v) ethanol and rinsed with sterile water three times. Sterilized seeds were sown on 1/2 Murashige and Skoog (MS) medium  containing 3% sucrose, and 0.75% agar, adjusted with 1 M KOH to pH 5.8. After two days of vernalizing at 4 °C, the seeds were germinated under the 16 h light/8 h dark photoperiod at 23 °C in a growth chamber. Materials of tissue culture were cultured in other chambers with the same condition.Tobacco . Standard chemicals such as dioscin (98%) and diosgenin (98%) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. .D. zingiberensis genome (NCBI project number: PRJNA716093) against functional characterized CYP450s . The amino acid sequence was analyzed using the online tool ExPASy (https://web.expasy.org/protparam/ accessed on 18 November 2022) [DzCYP72A12-4 proteins were analyzed using the following online tools:http://www.ebi.ac.uk/interpro/search/sequence accessed on 15 November 2022) [InterPro (er 2022) ;http://smart.embl-heidelberg.de accessed on 15 November 2022) [SMART (er 2022) ;https://www.ncbi.nlm.nih.gov/Structure/cdd accessed on 18 November 2022) [NCBI Conserved Domain Database (er 2022) .The basic local alignment search tool (BLAST) was used to search candidate CYP450 genes in the er 2022) . Multipler 2022) . Pairwiser 2022) . The conD. zingiberensis cDNA using the primers shown in ® Master Mix—Assembly (E2611) . Two restriction endonuclease recognition sites were added flanking the cassette simultaneously, facilitating multi-cassette assembly. Single or multiple cassettes were digested by the corresponding restriction endonuclease and inserted into a modified pCAMBIA plasmid via the same restriction endonuclease digested cohesive ends. In summary, four expression vectors were constructed for the plant transformation listed in For stable expression in tobacco, the coding sequences of the selected genes were amplified by PCR from A. tumefaciens strain GV3101 by the electroporation method . The colonies were validated by PCR analysis. A. tumefaciens-mediated transformation for stable expression in tobacco was performed using a leaf-disc infection method. Aseptic seedlings of wild type tobacco cultured for about 30 days were used. The edge of the leaf was cut off, laid on solid MS medium and cultured at 25 °C for 2 days in the dark. After that, the leaves were immersed in the transformed A. tumefaciens prepared in advance for 10 min. The leaves with A. tumefaciens were co-cultivated in the dark at 25 °C continuously for two days. After the co-cultivation, the leaves were transferred to new solid MS medium and cultured at 25 °C in the 16 h light/8 h dark photoperiod. Calluses growing on the edge of the leaves were isolated and cultured on MS medium containing hygromycin (50 mg/L). The clump buds were induced after 20 days of culture. PCR was used to select the positive transgenic plants, which were cultured for further study. Transient expression in tobacco was conducted as previously described [All constructs were verified by sequencing and transformed into escribed , with soD. zingiberensis were used to examine the tissue expression profiles of DzCYP72A12-4. To explore the effects of drought stress, young leaves were respectively harvested at 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h and 120 h after treatment with 15% PEG 6000 for RNA extraction. The leaves of the normally watered plants were used as the control. The expression pattern of the DzCYP72A12-4 gene under drought stress was analyzed. DzGAPDH was used as an internal control. The qRT-PCR was set up with the following thermal cycler conditions: initial denaturation preheating at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 57 °C for 30 s. Biological and technical replicates were conducted three times to ensure the accuracy of the results. The 2−ΔΔCT method was used for the analysis of the relative gene expression compared to the control [Total plant RNA was isolated using the RNeasy plant mini kit  following the manufacturer’s instruction with modifications. Potentially contaminated DNA was eliminated by treatment with DNase I . The RNA quality and concentration were determined using a NanoDrop 2000 spectrophotometer . First-strand cDNA was synthesized using 1 μg of total RNA and the HiScript III 1st Strand cDNA Synthesis Kit  following the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted following the manufacturer’s instructions of the SYBR Green PCR Master Mix  using the CFX96 Real-Time PCR Detection System . The primers used in this study are listed in D. zingiberensis was performed following the method reported previously with modifications [g for 10 min. The above treatment of each sample was repeated three times. The supernatant was merged each time and filtered by microfiltration (0.22 m) pending determination. The content of diosgenin was determined using the ultra-performance liquid chromatography  tandem MS . A Hypersil GOLD C18 column 690  was equipped on the UPLC system, and the temperature of the chromatography column was set to 40 °C. The flow rate of the mobile phases was 0.3 mL/min.Analysis of diosgenin in transgenic tobaccos and ications ,11,25. Bm/z in positive ion mode. The raw data were processed by XCalibur .To determine the diosgenin content, the UPLC–MS/MS system described above was utilized. The mobile phases were eluent A (water with 5 mM ammonium acetate) and eluent B (methanol with 5 mM ammonium acetate). The gradient program was as follows: from 0 to 3 min, isocratic 65% B; from 3 to 25 min, linear gradient of 65–99% B; from 25 to 33 min, isocratic 99% B; from 33 to 33.5 min, 99–65%; and from 33.5 min to 35 min, isocratic 65% B. The Q Exactive MS was operated in positive target-SIM mode with the following parameters: probe heater temp, 350 °C; ion source, HESI-II; diosgenin precursor ion selection at 415.32 The seeds were sterilized and sown on solid ½MS media. After two days of vernalization at 4 °C, the seeds were further grown at 23 °C under long-day photoperiod conditions of 16 h light/8 h dark. After germination, the seedlings of the same size were transplanted into new MS medium and cultivated for 20 days. All seedlings were grown in the same environment, and tobacco plants of wild type (WT) were used as a control. Then, the 28-day-old transgenic and WT tobacco seedlings were transplanted into new trays filled with sterilized vermiculite and watered with freshly prepared Hoagland solution.The tobacco seed germination test was performed according to the International Rules for Seed Testing. The seeds were laid in petri dishes (9 cm in diameter) underlaid with 2 layers of filter paper. Then, 2 mL 15%PEG-6000 was added to the test groups, while 2 mL distilled water was added to the control group and replenished every day. The growing conditions were controlled as described above. Each petri dish contained 100 seeds, and 3 replicates were set up. The number of germinated seeds was recorded every day, and the germination rate was calculated on the 16th day to determine the physiological indexes.The germination rate (GR) was the number of germinated seeds on day 16. The concentration of malondialdehyde (MDA) in the fresh seedlings was measured after 16 days of growth. The MDA content was determined by the Plant MDA Assay Kit with TBA .t test. Differences were considered statistically significant when p < 0.05 and extremely significant when p < 0.01, marked with * and **, respectively.The qualitative information regarding diosgenin was collected from mass spectra. All experiments were performed for three independent biological repeats, and at least three technical repeats were set each time. The data were analyzed using the Student’s DzCYP72A12-4 gene was screened through comprehensive methods. Diosgenin was detected in the transgenic tobacco plants (OE3) by UPLC–MS/MSv and not in the tobacco plants of the control groups lacking at least one of DzCYP90B71, DzCYP90G6 or DzCYP72A12-4. In addition, the diosgenin-producing tobacco plants showed relatively high drought adaptability. All these findings could strengthen the understanding of the DzCYP72A gene subfamily in diosgenin biosynthesis and lay the foundation for the further exploration of drought resistance in D. zingiberensis.In the present study, a novel DzCYP72A, further study should be conducted to confirm whether other DzCYP72As are involved in diosgenin biosynthesis. In addition, the platform established in this study would be very useful to carry out further identification of DzCYP72As as well as other genes of interest. Meanwhile, since steroid compounds have skeletons of a similar structure, it is worth exploring the structural similarities between key enzymes catalyzing similar substrates. In addition, the relationship between DzCYP72A and drought stress deserves more attention. At present, although the diosgenin biosynthesis from cholesterol has been preliminarily clarified, more research is needed to reveal the full picture of the diosgenin biosynthesis pathway. Specifically, efforts should be made with respect to the following three aspects: (1) finding more key enzymes involved in diosgenin biosynthesis, (2) revealing the possible regulatory mechanism of diosgenin synthesis, and (3) analyzing the interaction effects of different bioactive compounds.Considering the large family of


G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%–100%] with a specificity of 100% (95% CI: 88.78%–100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the  G6PD gene. It affects approximately 500 million people worldwide, mostly among populations living in malaria-endemic (or formerly endemic) areas . Th. ThG6PD https://www.medcalc.org/calc/diagnostic_test.php), according to the following parameters: sensitivity = TP/(TP+FN) ×100; specificity = TN/(TN+FP) ×100. The results were expressed as percentage with 95% confidence interval (CI). The G6PD activity of the population was expressed as median ± interquartile range (IQR) using GraphPad Prism .The calculations of sensitivity and specificity were performed using MedCalc online software  of the studied population, all of which were heterozygotes. G6PD Viangchan was the most common variant in the studied population, accounting for 76.47% (13/17) of the total. Other variants observed by multiplex HRM assays included G6PD Mahidol (2 samples), G6PD Union (1 sample), and G6PD Gaohe (1 sample). Among these 17 G6PD-mutant samples detected by HRM assays, 3 participants (G6PD Viangchan) were identified as G6PD normal by phenotypic test. Ten samples had intermediate G6PD activity between 30% and 80%, and the remaining 4 samples featured G6PD deficiency with enzyme activities of <30% , no mutation was detected by HRM assays . Notablyamplicon . Hence, G6PD mutations . The sequencing results showed that 11 samples having aberrant melting curves in the analysis by G6PD Mahidol primers  and G6PD Aures (c.143T>C), were detected. All G6PD mutations observed in this study are categorized as Class B variants associated with susceptibility to hemolytic toxicity. G6PD Canton was observed in two heterozygous females with average G6PD activity of 8.05 ± 0.70 U/gHb (67.48% of the AMM). G6PD Aures was found in a heterozygous participant with G6PD enzyme activity of 1.55 U/gHb (12.99% of the AMM). It was revealed that individuals carrying G6PD Viangchan also had two other mutations (synonymous or silent mutation c.1311C>T (rs2230037) and intron 11 mutation c.1365-13T>C; rs2071429).Additional nonsynonymous variants that were not included in the HRM assays, namely G6PD gene. The average G6PD activity of c.1311C>T/ c.1365-13T>C was 6.76 ± 1.02 U/gHb for homozygotes and 5.54 ± 1.98 U/gHb for heterozygotes, which were approximately 50% of the normal level  of individuals with enzyme activity <80% of the AMM value had a combination of c.1311C>T and c.1365-13T>C (17 heterozygous and 5 homozygous), in whom no other mutations were observed in the coding exons and intronic flanking regions of the al level . TherefoDNA sequencing also revealed one sample carrying a synonymous mutation c.519C>T (Phe173). Heterozygous c.519C>T mutation was detected in a subject presenting intermediate G6PD activity . The c.519C>T is a rare mutation that has previously been reported in Chinese, Taiwanese, and Thai populations –36. The G6PD gene. However, these methods require additional gel electrophoresis after PCR amplification. Furthermore, PCR-RFLP needs specific restriction enzymes for detecting each variant [G6PD variants within one run, but it also has post-PCR works. Many reagents are required for hybridization and visualization of spots on the membrane. With a manual procedure, RDB-FTH could take up to 4 h and, hence, a rapid hybridization machine is required to reduce hybridization time [G6PD mutation, especially in female heterozygotes, this method is impractical for large-scale screening and routine laboratory testing.Because individuals with deficient as well as intermediate levels of G6PD activity are at risk of developing adverse effects when receiving radical treatment for malaria, the WHO recommends G6PD testing before prescribing 8-aminoquinolines. Individuals with <30% of normal G6PD activity can receive primaquine with a dose adjustment under medical supervision . On the  variant , 40. Revion time , 42. AltG6PD gene by modifying available single-plex analyses [G6PD variants and their zygosity with a run time of 90 min. Six G6PD mutations—G6PD Gaohe, G6PD Chinese-4, G6PD Mahidol, G6PD Viangchan, G6PD Chinese-5, and G6PD Union—were simultaneously detected in two reactions. Cost for detecting 6 mutations in one sample was estimated at approximately 2 USD, excluding PCR instrument and labor. Differences in melting-curve shapes and Tm can identify whether G6PD mutant samples are heterozygous or hemizygous/homozygous . This deletion was previously reported as a sporadic variant in the Chinese population , 60–62.  variant . The c.4 variant –57, 65.  variant –68. Theroduction , 70. Addoduction .G6PD mutations in other noncoding regions, such as 5’ and 3’ UTRs, and exon 1 and intron 2, in which the designed sequencing primers cannot comprehensively detect. G6PD epigenetic events have been found to downregulate G6PD expression [G6PD expression might also contribute to G6PD deficiency by decreasing transcriptional level or affecting post-translational modification of the G6PD protein [Notably, no mutation was detected among 33 individuals who were considered as intermediate deficiency (30–80% of the AMM). Hematological factors such as white blood cell count, reticulocyte count, and age of red blood cells may affect the phenotypic test results where G6PD activity decreases significantly as erythrocytes age. Additionally, the impaired G6PD activity in these subjects may result from pression . Alterat protein , 74. Bec protein , 76. It  protein .G6PD Mahidol and G6PD Canton, defined as G6PD intermediate according to the 80% threshold, were classified as G6PD normal at the 70% cutoff. The enzyme activities for those with G6PD Mahidol and G6PD Canton were approximately 75% and 72% of the AMM, respectively. This result indicated that an 80% threshold, according to the WHO recommendation, is more reliable for screening G6PD deficiency in heterozygous females of the studied population. It should be noted that the use of an 80% cut-off will exclude malaria patients from effective radical treatment, leaving them vulnerable to relapse, which might result in more serious events than mild hemolysis. Nevertheless, there was no clear consensus that 70% or 80% would be a more appropriate cut-off value of G6PD enzyme activity in females with heterozygous deficiency [To identify those who would be eligible for tafenoquine administration, phenotypic data were analyzed and a normal G6PD was defined as > 70% of the AMM. Two individuals with heterozygous ficiency .G6PD variants by one-step real-time PCR. We show that the HRM assay is a reliable method for detecting G6PD mutations, particularly in females with either normal or deficient G6PD activity, depending on the type of variant and zygosity. In comparison to other genotypic tests, the HRM assay offers potential advantages for rapid, simple, and high-throughput genotypic screening with up to 68 samples that can be analyzed simultaneously. Because the polymorphic frequency of the G6PD gene varies across different populations, our assays may be appropriate for genotyping in areas where the six G6PD variants are widespread but may not be useful in other regions. However, HRM assays can be further developed to detect other G6PD mutations, which can include G6PD variants with normal enzyme activity and rare variants that are at risk for oxidative hemolysis.Our findings suggest that the phenotypic test alone may be insufficient for detecting G6PD deficiency in heterozygous females. Hence, the multiplex HRM assays developed here could be used as a supplementary approach when deciding whether individuals with borderline enzyme activities should be given the drugs. Our assay can be used to identify six G6PD variants common in Thailand. Homozygous and heterozygous females could be distinguished from those with wild-type G6PD by monitoring changes in melting-curve shapes and Tm. Moreover, the assays successfully detected G6PD mutations in heterozygous females with normal G6PD activity. The sequencing results showed a high frequency of intronic variants among all detected variants. This indicated that intronic variants may be more common than previously thought because the molecular characterization of G6PD has mainly focused on the protein-coding exons. We recommend comprehensive molecular analysis to detect genetic variation in the G6PD gene, covering both translated and untranslated regions, because some individuals with impaired enzyme activity have no identified mutations in the exon regions. Finally, the effects of synonymous and intronic variants on G6PD expression should be investigated to understand how these mutations cause enzyme deficiency.In conclusion, the developed multiplex HRM assays correctly identified the genotypes of six missense S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


The P2 MCs show high adsorption capacity for Rhodamine B (642.26 mg/g) and methylene blue (909.25 mg/g), with an extremely low equilibrium adsorption time (~20 min). The P2P MCs exhibited high adsorption capacities of reactive orange K-G (ROKG) and direct yellow 5G (DY5G) which were 404.79 and 451.56 mg/g. Adsorption processes of all dyes onto microcapsules were best described by the Langmuir isotherm model and a pseudo-second-order kinetic model. In addition, the P2P MCs loaded with reactive dyes (P2P–ROKG), could further adsorb rhodamine B (RhB) dye, and P2 MCs that had adsorbed cationic MB dyes could also be used for secondary adsorption treatment of direct dye waste-water, respectively. The present work confirmed that P2P and P2 MCs were expected to become an excellent adsorbent in the water treatment industry.Herein, poly  (PAH)/ poly (styrene sulfonic acid) sodium salt (PSS) microcapsules of (PAH/PSS) Dyes are applied to textile , paintinOnce the dye wastewater is discharged into water, it poses a serious threat to the human and ecological environment because it is toxic, non-biodegradable and even carcinogenic ,10,11. A2PAH and (PAH/PSS)2 microcapsules can selectively remove anionic and cationic dyes based on synergistic effects of porous and hollow structures and electrostatic interactions. The high specific surface area and effective functional groups of (PAH/PSS)2PAH and (PAH/PSS)2 microcapsules’ material promoted the adsorption of anionic and cationic dyes.Many porous materials have been applied to dye adsorption in wastewater . The int2PAH as P2P and (PAH/PSS)2 as P2 microcapsules were constructed by coating PAH and PSS onto the surface of CaCO3 to generate core@shell structured CaCO3@(PAH/PSS)2PAH and CaCO3@(PAH/PSS)2 and then removing the CaCO3 templates. The (PAH/PSS)2PAH (P2P MCs) and (PAH/PSS)2 (P2 MCs) were applied to remove anionic and cationic dyes. The P2 MCs show high adsorption capacity for Rhodamine B and methylene blue, with an extremely low equilibrium adsorption time (~20 min). The P2P MCs exhibited high adsorption capacities for reactive orange K-G (ROKG) and direct yellow 5G (DY5G). The P2P MCs loaded with reactive dyes (P2P–ROKG) could further adsorb RhB, and P2 MCs that had adsorbed cationic MB dyes could also be used for the secondary adsorption treatment of direct dye waste-water, respectively. At the same time, we investigated effects of initial dye concentration, time and solution pH on dye adsorption. In addition, we evaluated adsorption isotherms, adsorption kinetics and possible selective adsorption mechanisms of P2P and P2 microcapsules for four dyes. These studies proved that P2P and P2 microcapsules are promising adsorbents and can be used to treat simulated dye wastewater containing cationic and anionic dyes.We have synthesized polyelectrolyte microcapsules by a layer-by-layer method at room temperature ,37. Holl3 particles with a diameter of 8~12 μm were used as a sacrificial template (2PAH and (PAH/PSS)2 microcapsules through a layer-by-layer (LBL) method. CaCO3 particles were etched by EDTA to yield the hollow space for dye adsorption. 3, P2 and P2P microcapsules are displayed in −1 and the band of PSS was assigned at 1038 cm−1 due to S–O stretching vibration deformation [3 had a big peak in the range of 740–780 °C, but there was no corresponding peak in the corresponding positions of the three microcapsules, which indicates that the CaCO3 template in the microcapsules has been removed, and this result is also consistent with the FTIR and SEM analysis [Mono-disperse PSS doped CaCOtemplate . PAH andormation ,40 , while the surface of P2 microcapsules contain anionic functional groups (–SO3−), so that dyes with opposite electrical properties can be adsorbed through electrostatic interaction [e) of hollow P2P microcapsules to AOCK, DY5K, RhB and MB dyes were 409.80, 451.64, 130.89, and 185.43 mg/g, respectively, which are different from P2 microcapsules , as plotted in The selective adsorption of dyes is of great significance because it not only facilitates the adsorption of dyes, but also facilitates the selective separation of dyes from wastewater. Microcapsules confer selective adsorption properties by interacting with specific types of dyes (anions or cations). The surface of P2P microcapsules contain many cationic functional groups  and P2P(AOKG and DY5G) microcapsules were mixed with the dye solution, respectively, and then digital photos were taken to compare the color change of the dye solution before and after adsorption, as shown in 3+ groups on the surface of the microcapsule wall, which leads to positive charges on the microcapsules. Due to the electronegativity of ROKG and DY5G dyes, hollow P2P microcapsules easily form strong electrostatic interactions with them. However, electrostatic repulsion between adsorbent and cationic dyes results in a lower R of hollow P2P microcapsules to two dyes. Oppositely, as shown in 3−). Similarly, the adsorption of hollow P2 microcapsules for dyes is highly dependent on the electrostatic interaction of –SO3− groups and dye molecules, so it has better selective adsorption capacity for cationic dyes.In As displayed in 3+) of P2P microcapsules and negatively charged anionic dyes (ROKG and DY5G) increases. If the dye concentration was 2500 mg/L, the amount of adsorbent was 2 mg at 25 °C for 5 h, as shown in Under acidic conditions with a pH of 4–7, electrostatic interaction between protonated functional groups , k1 (min−1) and k2 (g·mg−1·min−1) represent the rate constants in quasi first order and quasi second order kinetic models, respectively.The pseudo-first-order and pseudo-second-order kinetic models can be described by Equations (1) and (2) :(1)lnQe2 = 0.9771, which means that the quasi second-order kinetic model is more suitable to describe the adsorption process of P2P and P2 microcapsules than the quasi first-order kinetic model and was very close to eQ. Results can be found in For P2P and P2 microcapsules, pseudo-second-order equation fitted with a pseudo high correlation coefficient value Rik (mg·g−1 min−1/2) is the intraparticle diffusion rate constant and c (mg/g) is a constant involving the thickness and boundary layer.The adsorption kinetics of the three dyes on the microcapsules was simulated by Weber’s model . The modki1) is larger than that of the internal diffusion stage (i1k), indicating that the internal diffusion stage (ki2) is a gradual process, which conforms to Weber’s particle internal diffusion model  of P2P and P2 microcapsules for two anionic dyes ROKG, DY5G and two cationic dyes RhB, MB have been investigated. For researching the adsorption mechanism of P2P and P2 microcapsules on dyes, the Langmuir equation [eC (mg/L) and eQ (mg/g) represent dye concentration at equilibrium and dye adsorption capacity of microcapsules. LK (L/mg) and maxQ, respectively, represent adsorption constant and maximum adsorption capacity in the Langmuir adsorption model. eC is the abscissa and e/QeC is the ordinate. LK and maxQ can be calculated by the straight-line slope and intercept obtained by fitting (FK ((mg/g) (L/mg)1/n) is the adsorption constant in the Freundlich model, and n is the heterogeneity factor [The maximum adsorption capacities (equation  (Formulaequation  (Formula fitting . KF ((mgy factor .2) and equilibrium parameters are listed in 2  of the Freundlich model for dyes  was lower than that of the Langmuir model , which indicates that the Langmuir model was more suitable for describing the adsorption of dye by adsorbent microcapsules. Similarly, the Langmuir model can better fit adsorption data than the Freundlich isotherm model, and the regression coefficient of P2 microcapsules was higher (R2 > 0.99). KL ranges from 0 to 1, indicating that two microcapsules were beneficial for the adsorption of four dyes [Linear fitting results of Langmuir and Freundlich models towards four anionic dyes  are displayed in our dyes .To study the adsorption mechanism of microcapsules for dyes, we measured UV–visible absorption spectra of MB, P2 MCs and P2 MCs–MB, respectively. It can be seen from 3 groups were detected by S 2p spectra of PSS in P2 microcapsules. The S 2p lines were fitted to a spin-split doublet, 2p3/2 and 2p1/2, of energy difference of 1.2 eV. In the P2 microcapsules’ spectrum, the 2p3/2 and 2p1/2 peaks are located at 167.7 and 168.9 eV, respectively [ectively   were purchased from Alfa Aesar (Tianjin) Co., Ltd.  Direct yellow 5G (DY5G), reactive orange K-G (ROKG), Rhodamine B (RhB) and methylene blue (MB) were bought from Shanghai ANOKY Group Co., Ltd. and their basic data are shown in Ethylenediaminetetraacetic acid (EDTA) was bought from Beijing Puboxin Biotechnology Co., Ltd. Poly (PAH) and poly(styrene sulfonic acid) sodium salt (PSS M3)2·4H2O  solution consisting of PSS  was poured into Na2CO3 (0.025 M) under magnetic stirring. After maintaining for 25~30 min, the CaCO3 templates were washed and collected.In brief, Ca for 20 min under continuous shaking (100–120 rpm/min). Then, the coated particles were incubated in 10 mL PSS solution  using the same procedure. The multilayer structure was formed by alternative assembly of corresponding materials for four or five times ((PAH/PSS)2 as P2 microcapsules or (PAH/PSS)2PAH as P2P microcapsules). The hollow P2 and P2P microcapsules were obtained through etching CaCO3 templates by using 30 mL EDTA solution . The process is shown in CaCO3 particles were analyzed by a HORIBA LA-300 Laser Diffraction Analyzer. The microcapsule samples were freeze-dried for thermogravimetric analysis. The thermogravimetric analyzer STA 409PC (NETZSCH Scientific Instruments Trading Ltd.) was used for detection. X-ray photoelectron spectroscopy (XPS) was analyzed by Thermo Scientific K-Alpha.The P2 or P2P microcapsules after drying, were studied with SEM  under a 20 kV accelerating voltage and the diluted P2 or P2P microcapsules solution was dropped onto a 400-mesh TEM carbon coated copper grid to prepare for TEM  analysis of the sample. FTIR was tested on a Thermo Scientific Nicolet iS20 spectrometer. Particle size of PSS-doped CaCO8) were placed in dye solution  at pH = 4, 5, 6, 7, 8 and 9. Desorption of P2 and P2P microcapsule adsorbed dyes was studied by a methanol acetate mixed solution with a volume fraction of 10%. The adsorption capacity of P2 and P2P microcapsules to dyes under different conditions and the dye removal efficiency in the mixture of acetic acid and methanol (%) were calculated according to the following Equations (6) [C0 (mg/L) and eC (mg/L) are the initial concentration of dye solution and residual dye concentration at adsorption equilibrium. tC (mg/L) represents the concentration of the mixed solution with change of time t after adding adsorbent, and V (L) represents the volume of dye solution. Additionally, m (g) is the mass of microcapsules. The dye removal efficiency  is the percentage of dye desorbed by the desorption agent.The adsorption experiments with P2 or P2P microcapsules were studied under different conditions. A UV-1200 spectrophotometer  was used to test the absorbance of dyes embedded in P2 or P2P microcapsules. P2 and P2P microcapsules (1.0 × 10ions (6)  and (5) ions (6) .(6)Q= and methylene blue (909.25 mg/g), with an extremely low equilibrium adsorption time (~20 min).The P2P MCs exhibited high adsorption capacities of 404.79 and 451.56 mg/g for reactive orange K-G (ROKG) and direct yellow 5G (DY5G), respectively. P2P and P2 MCs’ experimental adsorption data fit the Langmuir isotherm model and pseudo-second-order model well. The excellent adsorption performance of P2P and P2 MCs on four dyes was mainly attributed to mechanisms of electrostatic interaction. The study might offer new ideas for the structure and design of new adsorbents with excellent performance.


Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPGs), are a fatal form of brain cancer. These tumors often carry a driver mutation on histone H3 converting lysine 27 to methionine (H3K27M). DMG-H3K27M are characterized by altered metabolism and resistance to standard of care radiation (RT), but how the H3K27M mediates the metabolic response to radiation and consequent treatment resistance is uncertain.in vitro and in vivo response to de novo and salvage purine synthesis inhibition in combination with RT.We performed metabolomics on irradiated and untreated H3K27M isogenic DMG cell lines and observed an H3K27M-specific enrichment for purine synthesis pathways. We profiled the expression of purine synthesis enzymes in publicly available patient data and in our models, quantified purine synthetic flux using stable isotope tracing, and characterized the de novosynthesis and lower activity of purine salvage due to decreased expression of the purine salvage enzymes. Inhibition of de novo synthesis radiosensitized DMG-H3K27M cells in vitro and in vivo. Irradiated H3K27M cells adaptively upregulate purine salvage enzyme expression and pathway activity. Silencing the rate limiting enzyme in purine salvage, hypoxanthine guanine phosphoribosyl transferase (HGPRT) when combined with radiation markedly suppressed DMG-H3K27M tumor growth in vivo.DMG-H3K27M cells activate purine metabolism in an H3K27M-specific fashion. In the absence of genotoxic treatment, H3K27M-expressing cells have higher relative activity of de novo purine synthesis but adaptively upregulate purine salvage in response to RT. Inhibiting purine salvage may help overcome treatment resistance in DMG-H3K27M tumors.H3K27M expressing cells rely on  Diffuse midline gliomas (DMG) are pediatric high-grade gliomas that arise in midline structures of the brain including the thalamus, cerebellum, and pons–3. In 20de novo from metabolic building blocks or salvage them from pre-formed nucleobases. We find that H3K27M-expressing cells preferentially utilize de novo guanylate synthesis during unperturbed growth, likely due to increased expression of the enzymes utilized in de novo synthesis and decreased expression of those used in salvage. Inhibitors of de novo guanylate synthesis sensitize DMG-H3K27M models to RT in vitro and in vivo but do not cure tumors. Unexpectedly, we find that H3K27M cells upregulate guanylate salvage in response to RT, which likely mediates resistance to inhibition of de novo purine synthesis. Knockdown of the rate limiting enzyme in guanylate salvage greatly increases RT efficacy in orthotopic H3K27M xenografts. These findings indicate that guanylate salvage inhibition may be a promising strategy to overcome RT resistance in DMG-H3K27M.The importance of the H3K27M mutation and its profound effect on metabolism suggest that this mutation may specifically confer RT resistance in DMG-H3K27M tumors. Here, we identify purine metabolism as an H3K27M-specific metabolic vulnerability that governs the RT response. Cells can synthesize the two main classes of purines (adenylates and guanylates) Patient-derived H3K27M-isogenic (H3K27M and H3K27M-KO) cell line pairs (DIPGXIII and BT245) were generous gifts from Dr. Nada Jabado (McGill University) and wereDIPGXIII H3K27M-expressing cells were plated in TSM media containing polybrene transfection reagent and 10X ready-made lentiviral stock containing expression constructs for GFP and firefly luciferase (GFP/LUC) (Lenti-GF1-CMV-VSVG) or ready-made shHPRT1 viral vector stock (Santa Cruz Biotechnology Cat #: sc-40679-V). Cells were centrifuged and washed and expanded in 15cm plates 24hrs later. Successful transduction was confirmed by GFP expression or puromycin selection and loss of HGPRT protein.H3K27M-isogenic cells were lysed in RIPA buffer containing protease  and phosphatase inhibitors  on ice with mechanical disruption at the start of incubation. Lysates were clarified and protein concentration was determined using BCA assay (ThermoFisher). SDS-PAGE was performed using at least 12.5ug protein on 4–20% Tris-Glycine gels. Proteins were semi-dry transferred onto nitrocellulose for 1.5hrs at 24V. Membranes were blocked in non-fat dry milk in TBST before incubation with primary antibody overnight at 4C. Membranes were washed with TBST before incubation with secondary antibody. West Pico Plus (ThermoFisher) ECL reagent and X-Ray film were used to measure expression as previously described. Antibod2SO4 overnight to precipitate histones. Samples were clarified and trichloroacetic acid was added dropwise to the histone-containing supernatant, which were mixed and incubated on ice. Histones were pelleted, washed with ice-cold acetone, and air-dried. Histones were resuspended in ddH2O. Protein content was quantified using a BCA assay.Histone purification was performed as described previously. H3K27M H3K27M-isogenic cells  were plated in 10cm dishes in TSM media and were grown to be small neurospheres (approximately 2 days in culture). Media was changed 2hrs before treatment. Cells were irradiated with 4Gy RT using a Philips RT250  at a dose rate of approximately 2 Gy/minute at the University of Michigan Rogel Comprehensive Cancer Center Experimental Irradiation Shared Resource . Cells were incubated for 2hrs at 37°C. One plate from each group was analyzed by BCA assay quantify protein for standardization. After incubation, all cells were collected in individual 15mL conical tubes and pelleted. Supernatants were removed and metabolites were extracted with ice-cold 80% methanol on dry ice. Pellets were resuspended and incubated for on dry ice. Samples were centrifuged to clarify the metabolite extract and supernatants were transferred to new 15mL tubes. This step was repeated twice to ensure clarity. Metabolite extracts equating to roughly 1000μg of protein were transferred to 1.5mL microcentrifuge tubes before drying in a speed vacuum centrifuge or under a nitrogen blower. Liquid chromatography/mass spectrometery (LC/MS) analyses were performed as previously described. AgilentMetabolite lists were trimmed to exclude metabolites that fell below the noise threshold. Remaining metabolite abundances were median centered. H3K27M isogenic cells were analyzed in pairs. All metabolites whose abundance changed by a Log2 FC of |0.15| following RT in either H3K27M or H3K27M-KO cells were selected and combined into a single list. These lists were ordered by the absolute value of difference in post-RT Log2FC between K27M and KO cells. The top 25 metabolites were selected for pathway enrichment analysis using MetaboAnalyst 5.0 where the top 10 most significantly represented pathways were selected. Statist15N-glutamine ). One replicate plate from each group was set aside and given unlabeled TSM media to act as unlabeled control samples. Untreated and irradiated cells (4Gy) were incubated at 37°C for 3hrs after RT at which point metabolites were extracted.H3K27M isogenic cells (> 4.0×10^6 cells/replicate) were plated in 10cm dishes in TSM and grown into small neurospheres. Approximately 30min before RT, cells were pelleted and washed and replated with TSM media with stable isotope tracer molecules replacing the naturally occurring metabolites  or 4mM LC/MS Analysis was performed on an Agilent system consisting of an Infinity Lab II UPLC with a 10-port valve coupled with a 6545 QToF mass spectrometer (Agilent Technologies) using a JetStream ESI source in negative mode. Source parameters: Gas Temp: 250 °C, Gas Flow: 13 L/min, Nebulizer: 35 psi, Sheath Gas Temp: 325 °C, Sheath Gas Flow: 12 L/min, Capillary: 3500 V, Nozzle Voltage: 1500 V.Chromatographic separation was performed on an Agilent ZORBAX RRHD Extend 80Å C18, 2.1 × 150 mm, 1.8 μm column with an Agilent ZORBAX SB-C8, 2.1 mm × 30 mm, 3.5 μm guard column. The column temperature was 35 °C. Mobile phase A consisted of 97:3 water/methanol and mobile phase B was 100% methanol; both A and B contained tributylamine and glacial acetic acid at concentrations of 10mM and 15mM, respectively. The column was backflushed with mobile phase C  between injections for column cleaning.The LC gradient was as follows: 0–2.5 min, 0% B; 2.5–7.5 min, linear ramp to 20% B min, 7.5–13 min, linear ramp to 45% B; 13–21 min linear ramp to 99% B and held at 99% B until 25 min. At 25 minutes, the 10-port valve was switched to reverse flow (back-flush) through the column, and the solvent composition changed to 95% C and held there for 3 min. From 28 to 28.5 min, the flow rate was ramped to 0.8 mL/min, held until 32.5 min, then reduced to 0.6mL/min. From 32.5 to 33.25 the solvent was ramped from 99–0% C while flow was simultaneously ramped down from 0.6–0.4mL/min and held until 39.9 min., at which point flow rate was returned to starting conditions at 0.25mL/min. The 10-port valve was returned to restore forward flow through the column at 40 min. An isocratic pump was used to introduce reference mass solution through the reference nebulizer for dynamic mass correction. Total run time was 30 min. The injection volume was 5μL. Data was analyzed using MassHunter ProFinder 8.0 software (Agilent Technologies).H3F3A or HIST1H3B) and the Z-score for each gene of interest was extracted and sorted based on mutational status. Statistical analyses were performed using a two-tailed t-test in GraphPad Prism 9.0.mRNA-seq Z-score expression and patient survival data from Mackay et al. 2017) was obtained using PedCBioPortal 017 was o, 31. DatH3K27M cells (3×10^5–4×10^5) were cultured in 6-well dishes for 48hrs until they formed small spheres which were then treated with mycophenolic acid (MPA) for 6hrs prior to irradiation in the previously described apparatus. Approximately 18hrs later, spheres were dissociated and replated at low density (500 cells/well) in 96-well plates. Plates were incubated for 5–10 days in a BioSpa incubator (Agilent Technologies) and imaged every 24hrs in a Cytation 5 plate reader with the 4x objective. Gen5 software (Agilent Technologies) was used to analyze the data. A 2D area threshold of 2600um^2 was used to pick spheres, approximating the 50-colony threshold used in standard clonogenic survival assays. End-point sphere numbers were used to calculate the surviving fraction of cells. The Dmid of the 0μM MPA control was divided by the Dmid of the MPA-treated groups to calculate the Enhancement Ratio (ER) to display changes in RT efficacy.in vivo experiments.Rag1-KO C57BL/6 mice were anesthetized and provided carprofen analgesic before removing the scalp fur and sterilizing the incision site. An incision was made along the midline of the scalp and a small hole in the skull was made using an electric burr hole drill or handheld drill bit at coordinates 2–3mm lateral, 0.5–1mm rostral or caudal (depending on the injection rig used) from the bregma. Approximately 1.5×10^5–2.0×10^5 cells (~ 5.0×10^4 cells/μL) in a volume of 3μL were implanted in the cortex at depth of approximately 2.5mm using 10μL Hamilton syringes. After injections, wounds were sutured and were given triple antibiotic ointment. Atipamezole was given reverse the anesthetic in heated cages. Mice were given diet gel supplement and monitored for 10 days, including a second dose of carprofen the day following the surgery. For some experiments, a PhD Ultra multi-syringe pump (Harvard Apparatus) was utilized to facilitate simultaneous implantation of up to 8 mice at once. A minimum of 6 mice were used per treatment group in subsequent Mice were administered sterile-filtered 30mg/mL luciferin solution (Syd Laboratories) via IP injection. Ten minutes post-injection, BLI signal was measured using an IVIS Spectrum imaging station and Luminescent Flux Values were obtained for each tumor. BLI flux values were used to randomize mice into four treatment groups (10 mice/group) based on BLI flux average signal. BLI was repeated 1–2x weekly during efficacy studies. Treatment groups included: Vehicle Control  alone, RT alone, and MMF + RT. MMF (150mg/kg) was administered by oral gavage for 11 consecutive days starting between 13–18 days post-implantation. Mice were sedated using 2.5% isoflurane and RT was administered in 2Gy fractions on days 2, 4–5, 8–9, and 11 of MMF treatment using an Orthovoltage irradiator. Mice were euthanized upon development of neurological symptoms and survival data was analyzed using the Kaplan-Meier method using GraphPad Prism 9.0.Altered cellular metabolism is a hallmark of cancer. To begiSome of these metabolic alterations might be caused specifically by the H3K27M mutation while otWe hypothesized that the H3K27M mutation influences the metabolic response to RT, thereby conferring RT resistance–35. UsinTo better understand which metabolic changes were H3K27M-specific, we calculated the differences in fold-change (FC) following RT between H3K27M and H3K27M-KO cell lines for each metabolite –G, H. Otde novo or the salvage synthesis pathways (de novo synthesis (DNS) using 15N-glutamine as it is critical for the formation of newly synthesized purine rings . Purine salvage was measured using 2D-Hpx that is converted into the common purine precursor, IMP, which can then be converted into GMP or AMP . Here, we observed that DIPGXIII H3K27M-expressing cells have a significantly higher ratio of 15N-glutamine:2D-Hpx labeling of GMP than do H3K27M-KO cells, indicating that H3K27M cells prefer DNS to create GMP  and lower 2D-Hpx labeling  and of GMP in H3K27M cells than in H3K27M-KO cells. There was no H3K27M-specific preference for DNS-derived AMP  and guanylate salvage (HGPRT). Using publicly available RNAseq data from patients bearing pediatric high-grade gliomas (pHGG). We also did not observe a difference in the expression of GMPS, the gene-encoding the enzyme downstream of IMPDH1/2 .Given the H3K27M-specific differences in GMP synthesis, we assessed the expression of the rate limiting enzymes in both me HGPRT . At the me HGPRT  and decrme HGPRT  in H3K27de novo guanylate synthesis that is facilitated by increased IMPDH protein expression  radiosensitizes adult GBM brain tumors and is being evaluated in a Phase 0/1 clinical trial (NCT04477200) in human GBM patients, the pro-drug of MPA, has efficacy in intracranial adult GBM models. RT alone increased mouse survival over vehicle control (31.5d), but not in a statistically significant manner. Combination MMF + RT significantly extended survival over vehicle controls , but did not cure tumors . These findings suggest that H3K27M cells do not increase DNS following RT.We reasoned that DMG-H3K27M tumors might upregulate purine metabolism in response to RT and limit the efficacy of MMF owing RT , S8. We KO cells , S4E-F. o change . We alsoincreased hypoxanthine salvage into GMP post-RT  (HPRT1 expression (We then wanted to determine if HGPRT inhibition could increase RT efficacy in DMG-H3K27M xenograft tumors. No blood-brain barrier (BBB)-penetrant HGPRT inhibitors currently exist, so we utilized a pooled shRNA to knockdown shHPRT1) . These cshHPRT1) . Mouse wshHPRT1) . Median shHPRT1)  and 6E. shHPRT1) –F. Lastlpression (2). Togein vitro and in vivo but did not cure tumors. Intriguingly, H3K27M cells upregulated guanylate salvage in response to RT, and DMG-H3K27M xenografts had abundant intratumoral guanine. This suggested that RT-induced guanylate salvage might be an adaptive mechanism of RT resistance. Consistent with this model, inhibition of guanylate salvage in DMG-H3K27M tumors overcame RT resistance and led to complete responses in several mice. Together, our findings suggest that DMG-H3K27M tumors rely on both de novo and salvage guanylate synthesis, with salvage preferentially contributing to radiation resistance.In this study, we have defined new metabolic strategies to overcome treatment resistance in devastating DMG-H3K27M tumors. Using steady-state and stable isotope tracing metabolomics in patient-derived H3K27M-isogenic cell lines, we found that H3K27M cells preferentially rely on the DNS of guanylates. This reliance is likely due to low HGPRT and high IMPDH1 expression in H3K27M cells. Inhibiting DNS of guanylates potentiated the effects of RT on H3K27M models de novo pyrimidine synthesis and methionine consumptionPRT is an HGPRT inhibitor that is effectively used in acute lymphoblastic leukemia (ALL) patients to maintain remission and has been shown to induce apoptosis in renal cancer cells, 42. How/HG(X)PRT. These AThese studies present lingering questions that still need to be answered. While we know that RT induces HGPRT expression, we do not know the molecular mechanism behind this upregulation. The rapid change in HGPRT expression (< 3hrs) suggests an epigenetic mechanism that could be related to the underlying H3K27M mutation. Additionally, we do not know if the ratio of DNS to salvage changes further as time progresses. We also do not know the source of the abundant guanine in our xenograft model, which could be due to high turn-over from malignant cells or secretion from non-cancerous cells such as atrocytes, 44. Lasde novo purine synthesis inhibition with RT, an approach that we are utilizing clinically for adult patients with GBM(Together, our studies suggest that multiple routes of purine synthesis are important for DMG growth. Combining  with GBM, may the