O. elatus Nakai is a traditional medicine that has been confirmed to exert effective antioxidant and anti-inflammatory functions, and is used for the treatment of different disorders. However, its potential beneficial effects on drug induced hepatotoxicity and relevant molecular mechanisms remain unclear. This study investigated the protective effect and further elucidated the mechanisms of action of O. elatus on liver protection. O. elatus chlorogenic acids-enriched fraction (OEB), which included chlorogenic acid and isochlorogenic acid A, were identified by HPLC-MS/MS. OEB was administrated orally daily for seven consecutive days, followed by a single intraperitoneal injection of an overdose of APAP after the final OEB administration. The effects of OEB on immune cells in mice liver were analyzed using flow cytometry. APAP metabolite content in serum was detected using HPLC-MS/MS in order to investigate whether OEB affects CYP450 activities. The intestinal content samples were processed for 16 s microbiota sequencing. Results demonstrated that OEB decreased alanine aminotransferase, aspartate aminotransferase contents, affected the metabolism of APAP, and decreased the concentrates of APAP, APAP-CYS and APAP-NAC by inhibiting CYP2E1 and CYP3A11 activity. Furthermore, OEB pretreatment regulated lipid metabolism by affecting the peroxisome proliferator-activated receptors (PPAR) signaling pathway in mice and also increased the abundance of Akkermansia and Parabacteroides. This study indicated that OEB is a potential drug candidate for treating hepatotoxicity because of its ability to affect drug metabolism and regulate lipid metabolism. Drug-induced liver injury (DILI) can be caused by prescription medicines, herbal products or dietary supplements and it is usually a result of mismedication or repeated medication in Europe and the United States . DILI inAcetaminophen (APAP) is a non-steroidal analgesic that is commonly used in clinical practice and does not harm the liver at regular doses. However, APAP overdose is the leading cause of drug-induced liver failure, which makes it an ideal and practical model for investigating DILI . Althoug2O2 and release a number of functional enzymes to destroy dead and dying cells, thereby preparing the environment for wound healing and tissue regeneration. An overdose of APAP causes a large rise in neutrophil infiltration in the liver. When an inflammatory injury occurs, these cells flood into the liver, greatly raising the number of neutrophils . Optimizing the culture conditions for O. elatus ARs in the bioreactor can largely increase its production  and 2, 2-diphenyl-1-picryhydrazyl radical (DPPH) were purchased from Sigma-Aldrich . Chlorogenic acid, isochlorogenic acid A, 4-aminobenzoic acid (PABA), 4-methylpyrazole (4-ME), chlorzoxazone (CHL), nifedipine (NIF), NADPH were purchased from Yuanye biotechnology . 3-(N-Acetyl-L-cystein-S-yl) acetaminophen (APAP-NAC), acetaminophen cysteine (APAP-CYS), 6-OH-chlorzoxazone (6-OH-CHL), dehydro nifedipine (DNIF), ketoconazole (KET) was purchased from Toronto Research Chemicals . Loratadine (LOR) was purchased from Meilunbio . HPLC grade methanol, acetonitrile, and formic acid were purchased from Merck . Deionized water was generated using a Milli-Q system .O. elatus ARs were cultured in an air-lift balloon-type bioreactor using the method of our previous study  were refluxed twice with 5 L 75% EtOH-H2O for 2 h each time. After filtration, the solution was removed under reduced pressure to yield a crude extract (OECE) (185.5 g∼ 37.1%). A portion of OECE (80.0 g) was passed through the HPD-722 macroporous resin column (7 cm × 120 cm) using a successive elution of EtOH-H2O , yielding three fractions: OEA (38.6 g∼ 48.3%), OEB (25.9 g∼ 32.4%), and OEC (5.4 g∼ 6.8%)  . Before m/z 50–2,200.The ESI-MS spectra were recorded on a Bruker amazon SL mass spectrometer . Analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3,000 pump, an Ultimate 3000 DAD, an Ultimate 3,000 column compartment, an Ultimate 3,000 autosampler , and an Alltech (Grace) 2000 ES ELSD  using a Cosmosil Packed C18 column  . Methanol (solvent A) and acidified water  were used as mobile phases with the following gradient: 0 min 10% A-35 min 73% A-45 min 90% A-55 min 100% A-65 min 100% A. The injection volume was 10.0 μl; the column temperature was set at 30°C; and the flow rate was 1.0 ml/min. The mass spectrum data were recorded in negative mode with a scan range of Male C57BL/6 wild-type mice (6–8 weeks) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (China). All the mice used in this study were bred under specific-pathogen-free conditions according to the guidelines for the Care and Use of Laboratory Animals of the South China University of Technology .Experimental design: an APAP-induced mouse model was established using the method referenced to Enzymatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (CHO) and triglycerides (TG) in serum were evaluated by spectrophotometer using diagnostic kits from Shanghai Kehua Bio-engineering Co., Ltd. .Frozen liver samples were homogenized in pre-cooled PBS. The supernatants were collected after the homogenates were centrifuged at 3,000 × g, 4°C for 10 min. Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and glutathione (GSH) levels were measured with a spectrophotometer using the commercially available assay kits following manufacturer instructions . The protein concentrations in the tissue homogenates were measured with Bradford protein assay using bovine serum albumin as the standard.Liver tissues were fixed in 10% formalin and embedded in paraffin for histological assessment. Samples were sectioned at 5 µm and stained with hematoxylin and eosin (H&E). The slides were examined under a light microscope with photo-micrographic attachment.TM below.APAP metabolites were measured according to a previously reported method v/v) with equal gradient elution; the flow rate was 0.2 ml/min; the scan time was 5 min; the injection volume was 10 μl, and the injector temperature was 15°C.Chromatographic conditions: Liquid chromatography was performed on Ultimate 3000 UPLC system . Hypurity C18  column  was used at 40°C; the mobile phase was methanol: 0.1% formic acid water . The ion source was an electrospray ion source with a positive spray voltage of 2,500 V and a capillary temperature of 350°C. Detection of the analyte ions was performed under the selective reaction monitoring (SRM) mode. The transition monitored for the analytes were APAP-NAC, [M + H]g, at 4°C for 20 min. The collected supernatants were placed in ultra-high speed centrifuge tubes, and centrifuged at 100,000 × g, at 4°C for 60 min. Supernatants were discarded and the collected precipitate was washed with potassium pyrophosphate solution and centrifuged again at 100,000 × g, at 4°C for 60 min. The supernatant was discarded, and the microsomes were quantified by resuspension in Tris-HCl buffer.Fresh mice liver samples were rinsed with pre-cooled sucrose solution at 4°C. Two times the volume of liver weight of sucrose solution was added to the liver samples. The solution was manually homogenized on ice, and centrifuged at 16,000 × Hepatic microsomes incubation and sample treatment: Mixed mouse liver microsomes and probe substrates (CHL and NIF), KET and 4-ME were added to the positive control group; methanol was added to the blank group, and OEB solutions  were added to the drug administration group. The system was pre-incubated at 37°C in a metal bath; the reaction was initiated by adding NADPH and incubating for 20 min at 37°C in a water bath; and terminated by adding cold ethyl glacial acetate. After adding 10 μl internal standard LOR solution (2 μg/ml) to the system, it was vortexed for 2 min, rested for 10 min and centrifuged at 3,500 rpm for 10 min. The supernatant was transferred and the organic solvent was evaporated. Before injection, 200 μl of 80% methanol water was added, vortex shaken for 1 min, and centrifuged at 16,000 rpm for 5 min to produce the sample.v/v) with equal gradient elution; the flow rate was 0.3 ml/min; the analysis time was 3.5 min; the injection volume was 10 μl; and the injector temperature was 15°C.Liver CYP450 enzyme activity was measured according to a previously reported method  with sliMass spectrometry conditions: The ion source was an electrospray ion source with a positive spray voltage of 3,500 V, a negative spray voltage of 2,500 V, and a capillary temperature of 350°C. Detection of the analyte ions was performed under SRM mode.-, m/z 184.107→120.20; DNIF, [M + H]+, m/z 345.132→284.107; LOR, [M + H]+, m/z 382.00→266.00, respectively. The concentration ranges included 6-OH-CHL from 0.1 to 6.4 μmol/L, and DNIF from 0.125 to 4 μmol/L. All the samples were measured in the HPLC-MS/MS system to determine the content of the substrate metabolites in the liver microsomes incubated system and to calculate CYP450 enzyme activity.The transitions monitored for the analytes were 6-OH-CHL, [M-H]+ immune cells from APAP group (n = 3) and OEB + APAP group (n = 3) mice were exported for analysis. 3,000 cells were randomly selected from each sample, then merged into one single matrix containing 15 channels. Normalized expression matrix by RunTSNE function in Seurat package (version 3.0.1).Liver cell suspensions were blocked with anti-mouse CD16/32 before surface staining. The cell suspensions were incubated with fluorochrome-conjugated monoclonal antibodies in PBS with 0.2% bovine serum albumin at 4°C for 20 min. The monoclonal antibodies included anti-Gr-1 (FITC and RB6-8C5), anti-CD11c (PerCP-Cy5.5 and N418), anti-KLRG1 (PE and 2F1/KLRG1), anti-NK1.1 (PE-Cy7 and PK136), anti-CD19 (PE-Cy5 and 6D5), anti-CD44 (Alexa647 and IM7), anti-CD45.2 (APC/Cy7 and 104), anti-I-A/I-E (Alexa700 and M5/114.15.2), anti-CD11b (V500 and M1/70), anti-CD3 (BV421 and 17A2), anti-CD4 , anti-CD8a (BV711 and 53–6.7), anti-CD49a , anti-CD62L , and anti-CD69  according the reference with slight modifications . All fluLiver and intestinal samples were collected from the APAP group mice and OEB + APAP group mice, quick-frozen in sterile tubes with liquid nitrogen and stored at −80°C until further use. Liver samples were processed for RNA sequencing by Novegene Bio-Pharm Technology Co., Ltd . The data were analyzed on R language. The intestinal content samples were processed for 16s microbiota sequencing by Majorbio Bio-Pharm Technology Co., Ltd . The data were analyzed on the free online platform of Majorbio Cloud Platform.p < 0.05.All values are expressed as means ± standard error of the mean. Data were analyzed by one-way analysis of variance (ANOVA) using GraphPad Prism 8.0 , and then differences among means were determined using Dunnett’s multiple comparisons test. Statistically significant difference was set at O. elatus ARs is as the flow chart is shown in The extraction process of in vivo, and found that OEB has no effect on liver damage in mice (We then designed experiments to determine whether OEB has hepatoprotective activity . H&E sta in mice . These r(p < 0.05).Overdoses of APAP induced liver injury is mediated by toxic APAP adducts first produced by CYPs. Therefore, we investigated whether OEB interferes with the metabolism and detoxification of APAP. The concentrations of APAP and APAP metabolites (APAP-CYS and APAP-NAC) in mice serum were measured using the established HPLC-MS/MS method. Compared to the APAP treatment group, the contents and intensities of APAP, APAP-CYS and APAP-NAC  of OEB pin vitro. The reactions were carried out using CYP2E1 and CYP3A11 substrates (CHL and NIF), CYP2E1 and CYP3A11 inhibitors (4-ME and KET), and OEB at various concentrations . The relative enzyme activities were detected using the HPLC-MS/MS method. As shown in p < 0.001) inhibition of CYP2E1 and CYP3A11 activity at 10 μg/ml. We also found the same phenomenon in vivo, as OEB significantly decreased (***p < 0.001) the expression of CYP2E1 and CYP3A11 , and three clusters of CD8+ T cells  (###p < 0.001). However, the neutrophils percentage of the OEB pretreatment (4.86% ± 2.67%) and OEB-only treatment groups (3.14% ± 1.09%) were both significantly decreased (***p < 0.001) compared to APAP group. (6) significantly increased (###p < 0.001). However, the neutrophils number of the OEB pretreatment (0.91 ± 0.31 × 106) was significantly decreased (***p < 0.001) compared to APAP group (Most study found overdose APAP may impact hepatotoxicity by affecting immune cells . Our fin and 10) . CompareP group. . The neuAP group . These f*p < 0.05) decreased TG (1.27 ± 0.12 mmol/L) and CHO (3.03 ± 0.18 mmol/L) contents in mice serum compared to the APAP group   , Fabp1 (*p < 0.05), Acaa1b (**p < 0.01), and Apoc3 (**p < 0.01) significantly increased. These results indicate OEB offers hepatoprotection by regulating PPAR signaling and lipid metabolism.APAP can affect the lipid metabolism process in AILI, thereby we investigated the CHO and TG levels in serum. Our results found that OEB pretreatment significantly (ctively) . To furtctively) . GSEA syctively) . Furtherctively) . We alsod Apoc3) , We founLachnospiraceae, Oscillibacter and Colidextribacter was lower in the OEB pretreatment group compared with that in the APAP group. However, the percentage of Akkermansia (13.02% ± 5.53%) and Parabacteroides (2.27% ± 1.56%) in OEB + APAP group was significantly higher (*p < 0.05) than APAP group  on the genus level  against AILI was investigated for the first time in the present research. Our data found that OEB decreased ALT, AST, CHO, TG levels and APAP, APAP-CYS, APAP-NAC concentrations in serum. According to the mechanism of APAP metabolism, APAP content was decreased with time dependence in serum in the early stage , which are excreted in urine at regular doses. APAP can also be metabolized by cytochrome P450 enzymes  which are covalently bound to GSH and thus excreted in the early stages of APAP metabolism to toxic metabolites N-Acetyl-p-benzoquinoneimine (NAPQI) . Under rly stage . Except ly stage . Therebyly stage . Furtherin vitro , therebyOverdoses of APAP increase hepatotoxicity by inducing oxidative stress, and hepatocytes undergo apoptosis and necrosis as a result of this rapid onset of oxidative stress. Several studies have shown that natural medicine has antioxidative effect through reducing oxidative stress promotes hepatocyte suppression and hepatic function safety . StudiesImmune cell recruitment and infiltration causes extreme innate immune responses and sterile inflammation, all of which are necessary for the progression of mild liver injury to late liver failure . APAP inp < 0.05) increased TG and CHO contents in mice serum compared to the APAP group (APAP-induced hepatotoxicity activated lipid metabolism in liver. Peroxisome proliferator-activated receptors (PPAR), a member of the nuclear receptor superfamily, control the expression of a battery of genes involved in lipid homeostasis, including those encoding peroxisome and mitochondrial enzymes that carry out fatty acid catabolism . ConsequAP group . TG and AP group  and OEB AP group . These rLachnospiraceae, Oscillibacter and Colidextribacter decreased in the OEB pretreatment group compared with that in the APAP group, and that Akkermansia and Parabacteroides increased on the genus level  and this is associated with an altered gut barrier function leading to an increased plasma LPS levels and eventually triggering low grade inflammation and metabolic disorders (Akkermansia and Parabacteroides.Susceptibility to APAP-induced hepatotoxicity varies a lot from person to person. Age, food intake, genetic variations in APAP-metabolizing enzymes, and concurrent alcohol consumption have all been linked to different clinical outcomes after an APAP overdose. Alterations in gut microbiota have been linked to liver diseases. Diurnal differences in gut microbiota abundance and composition have been linked to APAP-induced hepatotoxicity susceptibility . Howeverus level . Studiesus level . Parabacle acids . Akkermaisorders . In our isorders . OverallIn conclusion, we found the mechanism of hepatoprotective effect of OEB. OEB exerted a potential therapeutic effect against APAP induced hepatotoxicity by inhibiting CYP2E1 and CYP3A11, improving anti-oxidative enzyme activity. Furthermore, OEB decreased APAP hepatotoxicity by regulating lipid metabolism included PPAR signaling. This study indicated that OEB is a potential drug candidate for the hepatoprotection.


Kidney per year , 4, occuper year , 6. As rMachine learning (ML), characterized as the study of algorithms and statistical models that computer systems utilize to learn from previous experience, can assess relationships of multiple variables, create predictions based on characteristics and identify patient groups with comparable patterns . We explParticipants in this retrospective cohort study have been previously described , 5. The 2 per year and (b) eGFR loss > 5.0 mL/min/1.73 m2 per year [Data collected included demographics, clinical laboratory tests, SCD‐related complications, co‐morbid conditions and treatments. At each visit, GFR was estimated using the creatinine‐based CKD Epidemiology Collaboration (CKD‐EPI) formula . Visits per year . Data we0–18), mean age 31.15 (±11.9) years, 133 (56.4%) females, with a mean observation period of 4.9 (±3.1) years were evaluated in the internal cohort. Rapid kidney function decline occurred in 166 (70.3%) patients based on first visit with eGFR decline >3 mL/min/1.73 m2 per year, and in 140 (59.3%) patients based on first visit with eGFR decline >5 mL/min/1.73 m2 per year. Baseline characteristics of the cohorts, stratified by rapid eGFR decline status, are shown in Table 2 per year was observed in 145 (86.3%) patients, while eGFR decline >5 mL/min/1.73 m2 per year was seen in 136 (80.9%) patients.Two hundred and thirty‐six patients , while eGFR decline > 5 mL/min/1.73 m2 at 6 months was predicted with 79% sensitivity, 84% accuracy, and AUC of 0.91 (95% CI: 0.85–0.97). Estimated GFR decline at 12 months was predicted with 53% sensitivity, 70% accuracy, and AUC of 0.67 (95% CI: 0.53–0.81) with the >3 mL/min/1.73 m2 threshold, and with 64% sensitivity, 65% accuracy, and AUC of 0.77 (95% CI: 0.67–0.87) at >5 mL/min/1.73 m2 threshold .Estimated GFR decline >3 mL/min/1.73 m2, in the model predicting decline in 6 months, the AUC was 0.82 (95% CI: 0.72–0.91), with 78% sensitivity, and 78% accuracy. For the >5 mL/min/1.73 m2 threshold, the AUC was 0.87 (95% CI: 0.78–0.95), with 77% sensitivity and 81% accuracy. For the model predicting eGFR decline at 12 months, at the >3 mL/min/1.73 m2 threshold, AUC was 0.60 (95% CI: 0.47–0.72), with 45% sensitivity, and 67% accuracy, and at the >5 mL/min/1.73 m2 threshold, AUC remained unchanged, with 63% sensitivity, and 65% accuracy . Age and eGFR slope 6 months previous were the most strongly predictive of rapid eGFR decline at >5 mL/min/1.73 m2 threshold. Baseline eGFR, age, SBP, serum creatinine and eGFR slope in the previous 12 months predicted rapid eGFR decline for both >3 mL/min/1.73 m2 and >5 mL/min/1.73 m2 thresholds at 12 months.In evaluation of feature importance, age, baseline eGFR, eGFR slope 6 months previously, reticulocyte count and SBP were associated with >3 mL/min/1.73 mRapid eGFR decline at 6 months was predicted with fairly high accuracy using ML models, but less so at 12 months. Despite concerns that the first decline in eGFR might reflect AKI, predictive capacities based on sensitivity analyses were only minimally decreased suggesting that use of the first eGFR decline reflected rapid kidney function decline. Notwithstanding the suitability of ML methods, predictions of distant events may not always be possible as evident by the modest predictive capacities of our models at 12 months. Biomarkers of kidney injury and larger patient populations may be necessary to better predict long‐term outcomes.Our study differs from prior studies evaluating kidney function decline in that we calculated rate of eGFR decline at each visit, while others have calculated decline over the entire observation period , 9. We hDespite limitation by lack of quantification of albuminuria, use of real‐world data, and some eGFR data imputation, this study demonstrates a role for ML models to predict rapid decline in eGFR. With the association of rapid eGFR decline with mortality in SCD, ML may play an important role in identifying patients at high risk for progressive kidney disease as early as 6 months in advance. More studies are required to further evaluate ML models in SCD‐related kidney disease.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Dr. Ataga has received funding from the US FDA (R01‐FD006030), Novartis, and Global Blood Therapeutics, served on advisory boards for Novartis, Global Blood Therapeutics, Novo Nordisk, Editas Medicine, Forma Therapeutics and Agios Pharmaceuticals, and as a consultant for Roche. Dr. Saraf receives research funding support from Novartis, Pfizer, and Global Blood Therapeutics and served on advisory boards for Novartis and Global Blood Therapeutics. The project described was supported in part by the National Institutes of Health through grants R03HL146788 (Santosh L. Saraf) and R01HL153161 (Santosh L. Saraf).Fatma Güntürkün analyzed the data and wrote the manuscript. Daiqing Chen analyzed the data and wrote the manuscript. Oguz Akbilgic analyzed the data and assisted with manuscript preparation. Robert L. Davis analyzed the data and wrote the manuscript. Ibrahim Karabayir analyzed the data and assisted in the manuscript preparation. Maxwell Strome analyzed the data and assisted with manuscript preparation. Yang Dai analyzed the data and assisted with manuscript preparation. Santosh L. Saraf assisted in study design and manuscript preparation. Kenneth I. Ataga designed the study and wrote the manuscript.Supporting InformationClick here for additional data file.


Their excitation spectra consist of two distinct excitation bands with the dominant excitation range from 250 to 450 nm, indicating that they possess strong absorption of near-ultraviolet light. Their emission is located around 693 and 708 nm, respectively, and can be absorbed by the photosensitive pigments Pr and Pfr, proving their great potential for plant growth. Finally, the prepared samples were coated with 365 nm UV chips to fabricate far-red LEDs and W-LEDs with low correlation color temperature (CCT = 4958 K/5275 K) and high color rendering index (Ra = 96.4/96.6). Our results indicate that La2CaSnO6:Mn4+ and La2MgSnO6:Mn4+ red phosphors could be used as candidate materials for W-LED lighting and plant growth.Non-rare earth doped oxide phosphors with far-red emission have become one of the hot spots of current research due to their low price and excellent physicochemical stability as the red component in white light-emitting diodes (W-LEDs) and plant growth. Herein, we report novel Mn As the latest generation of lighting sources, light-emitting diodes (LEDs) have been successfully used in many fields and have contributed to the development of these fields, especially in agriculture, because of their outstanding advantages such as high efficiency, environmental protection, and energy reduction ,2,3,4,5.4+ ion has attracted much attention due to its abundant reserves and low prices. It is well known that Mn4+ ion is a good non-rare earth activator, which usually occupies an octahedral coordination environment in the lattice and has outstanding optical properties, including a broad absorption band covering the UV to visible region and characteristic red luminescence in the wavelength range from red to near-infrared light  octahedra and coordinates with twelve O2−. In the LCSO and LMSO crystal structures, there are three cations available for Mn4+ occupation. Considering the three aspects of ionic radius, valence state and coordination number, among the four ions La3+ , Ca2+ , Mg2+ , and Sn4+ , the Sn4+ site is considered the most suitable Mn4+  doping site.The crystal structures of LCSO and LMSO were obtained by refining the crystallization results in the 2CaSnO6:0.002Mn4+ and La2MgSnO6:0.002Mn4+ at room temperature are shown in 4+-O2− charge transition band (CTB), 4A2g → 4T1g, 4A2g → 2T2g, and 4A2g → 4T2g transitions [−1, 25,943 cm−1, 24,514 cm−1, and 18,768 cm−1 for LCSO:0.002Mn4+, and 28,816 cm−1, 26,381 cm−1, 25,458 cm−1, and 19,342 cm−1 for LMSO:0.002Mn4+. Both exhibit intense red emission under 365 nm UV excitation due to the spin-prohibited 2Eg→4A2g leap of the Mn4+ ions in the octahedral environment, with the peak emission near 693 nm for LCSO: 0.002Mn4+ and around 708 nm for LMSO: 0.002Mn4+, which can be absorbed by the photosensitive pigments Pr and Pfr [2Eg → 4A2g transition, and the Stokes emission [4+ and LMSO:0.002Mn4+ at 7 K. The ZPL of the 2Eg → 4A2g transition was found to occur at 678 nm  and 692 nm , respectively.The excitation and emission spectra of Lansitions ,22. As s and Pfr . These remission ,16. Figu4+ (x = 0.001–0.010) and LMSO:yMn4+ (x = 0.001–0.010) were tested as shown in 4+ (x = 0.001–0.010) and LMSO:yMn4+ (y = 0.001–0.010) have the same trend, and the emission intensity increases with the increase in doping concentration from 0.001 to 0.002 and decreases with the increase from 0.002 to 0.01, and the optimal doping concentration for both was determined as 0.002. When the doping concentration exceeds 0.002, the emission intensity starts to decrease, and the concentration quenching appears and becomes more severe with the increase in the Mn4+ content. The concentration quenching includes two mechanisms: exchange interaction and multistage–multistage interaction. To investigate which mechanism causes the concentration quenching, we calculated the critical distance Rc by the following equation [c is the critical concentration, and N represents the number of sites that the Mn4+ ions can occupy per unit cell. For the LCSO host, V = 282.506 Å3, N = 2, and xc = 0.002; the critical distance Rc is calculated to be 51.29 Å. For the LMSO host, V = 258.650 Å3, N = 2, and xc = 0.002; the critical distance Rc is calculated to be 49.81 Å. Because the exchange interaction is effective only when the distance between activators is shorter than 5 Å, the concentration quenching mechanism of the Mn4+ ions in phosphors is dominated by multipole–multipole interactions. According to Van Uiter’s report, the emission intensity (I) per activator ion follows the equation [4+ emission in LCSO and LMSO.The photoluminescence (PL) spectra of a series of different concentrations of LCSO:xMnequation ,25:(1)Rcequation :(2)Ix=K14+  and LMSO:yMn4+ . All decay curves can be described by fitting the effective decay time τ, which is calculated as [4+ at moment t. As the doping concentration of Mn4+ ions increases, the effective lifetimes were calculated to be 0.289, 0.238, and 0.171 ms for LCSO:xMn4+  and 1.179, 1.011, and 0.635 ms for LMSO:yMn4+ , respectively. The decrease in the effective lifetimes is due to the increase in non-radiative energy transfer with the increase in Mn4+ doping concentration [lated as :(3)τ=∫0∞4+ and LMSO:0.002Mn4+, as shown in 4+ and 35.48% for LMSO:0.002Mn4+ under 365 nm excitation. The IQY of LMSO: 0.002Mn4+ is higher than some previously reported Mn4+-doped samples, as shown in In addition, we tested the internal quantum yields (IQYs) of LCSO:0.002Mn4+ ions in a six-coordinate octahedral environment and its spectral properties, as shown in 4A2g absorb energy and jump to excited states 4T1g, 4T2g, and 2T2g, and then, through a non-radiative energy conversion process, to the lowest excited state 2Eg, and finally return to the ground state; the energy is expressed in the form of red light. In this process, the crystal field strength (Dq) strongly influences the energy level transition of the Mn4+ ions due to the unique 3d3 electron configuration of Mn4+ [4A2g → 4T2g by the following equation [In general, the Tanabe–Sugano energy level diagram can be used to explain the energy level splitting of the Mn of Mn4+ . The cryequation ,34:(4)Dqequation :(5) DqB= follows :(6)x=EA pression :(7)EE 2g4A2g → 4T1g, 4A2g → 4T2g, and 2Eg → 4A2g are 25,943, 18,768, and 14,749 cm−1 for LCSO:0.002Mn4+ and 26,381, 19,342, and 14,451 cm−1 for LMSO:0.002Mn4+, respectively. Substituting the above data into Equation (4)–(7), the values of Dq, B, C, and Dq/B were calculated to be 1876.8 cm−1, 707.37 cm−1, 3160.88 cm−1, and 2.65 for LCSO:0.002Mn4+ and 1934.2 cm−1, 684.67 cm−1, 3107.66 cm−1, and 2.83 for LMSO:0.002Mn4+, respectively. The Dq/B values of both are greater than two and prove that the Mn4+ ions possess strong crystal fields in LCSO and LMSO hosts. In addition, the 2Eg → 4A2g transition of the Mn4+ ions is hardly affected by the crystal field strength as shown by the Tanabe–Sugano energy level diagram. What can affect such a transition is a nephelauxetic effect that depends on the covalent relationship between the Mn4+ ions and the ligand [o is 1160 cm−1 and Co is 4303 cm−1 represent the Racah parameters of Mn4+-free ions. The β values of LCSO and LMSO were calculated as 0.955 and 0.933. On the basis of previous experience with a large number of Mn4+-doped crystals, a rule of thumb was established to predict the emission wavelength of Mn4+ with the following equation [−1, a value close to the typical energy of phonon modes [2Eg energy levels calculated by this empirical formula are in the range 14,318–14,982 cm−1 and 13,960–14,624 cm−1 for LCSO:0.002Mn4+ and LMSO:0.002Mn4+. The experimental values of the 2Eg → 4A2g transition are 14,430 cm−1 for LCSO:0.002Mn4+ and 14,124 cm−1 for LMSO:0.002Mn4+, both of which are within the range.From the excitation spectrum and emission spectrum in e ligand ,36,37,38equation :(9)EE 2gon modes ,32. As s4+ and LMSO:0.002Mn4+ with temperature under 365 nm excitation. The increase in temperature leads to an increase in lattice vibrations, which increases the nonradiative transition and produces a thermal quenching effect with a significant decrease in the intensity of the emission peak [4+ ions in the crystal environment. In addition, the normalized emission intensity curves of the two samples (50%) of the LCSO:0.002Mn4+ and LMSO:0.002Mn4+ samples are about 331 and 397 K, respectively. The LCSO:0.002Mn4+ phosphor exhibits a more serious decay trend between the two. To better understand the thermal quenching properties, the activation energy (Ea) is introduced to represent the energy difference between the cross-relaxation point and the excited state 2Eg, which can be calculated by the Arrhenius formula [0 is the initial emission intensity, I is the emission intensity at temperature T, A is a constant, Ea is the activation energy, and k is the Boltzmann constant equal to 8.626 × 10−5 eV/K. The specific linear relationship is described in a of LCSO:0.002Mn4+ is 0.38 eV, and Ea of LMSO:0.002Mn4+ is 0.40 eV. LCSO matrix structure is less stable than LMSO, resulting in lower activation energy and, thus, more prone to non-radiative processes, which eventually leads to a poor thermal stability. This situation is reflected in the configuration of the Mn4+ ions coordinate diagram (ion peak . The poo samples c indicat formula :(10)lnI0 diagram d, indicaTo demonstrate the potential application of these two red phosphors in plant growth, we prepared two far-red LED models with electroluminescence spectra and luminescence photographs, as shown in 4+ and LMSO:0.002Mn4+ red phosphors can be used as candidates for W-LEDs.In addition, to demonstrate the potential application of these two red phosphors in W-LEDs, three W-LED models were prepared, and their electroluminescence spectra, luminescence photographs, and performance parameters  are shown in 2CaSn1−xO6:xMn4+  and La2MgSn1-yO6:yMn4+  phosphors were synthesized by conventional high-temperature solid-state reaction. The raw materials were La2O3 (A.R.), CaCO3 (A.R.), MgO (A.R.), SnO2 (A.R.), and MnCO3 (5N), which were weighed in the appropriate chemical proportions. After 20 minutes of thorough grinding in an agate mortar, the resulting mixture was transferred to an alumina crucible, warmed up at 4 °C per minute, preheated at 900 °C for 2 hours, and then sintered at 1480 °C for 3 hours in an air atmosphere. Finally, the samples were naturally cooled to room temperature and then reground into homogeneous powder for further measurements.All La2+, green phosphor 2SiO4:Eu2+, red phosphor CaAlSiN3:Eu2+, the as-synthesized La2SnO6:0.002Mn4+, and UV-LED chips (365 nm) were used to fabricate W-LEDs. The luminescence characteristics of the white LED were evaluated using a Starspec SSP6612  .The phase purity of the sintered samples was analyzed using powder X-ray diffraction (XRD)  with graphite monochromatic Cu Kα radiation (λ = 0.15405 nm) operating at 40 kV and 40 mA. Test from 10 to 80° in steps of 0.02° with a counting time of 0.14s per step. XRD Rietveld refinement was performed using the Structural Analysis System (GSAS) program . The X-ray photoelectron spectroscopy (XPS) data were obtained through a Thermo Scientific K-Alpha instrument  with a monochromatized Al Kα line source, and the binding energy was calibrated to the C 1s peak at 284.8 eV. Photoluminescence emission (PL) and excitation (PLE) spectra were measured on a fluorescence spectrophotometer  equipped with a 150 W xenon lamp. The luminescence decay curves and quantum yields (QY) were measured by an Edinburgh Instruments FLS-980 spectrometer  equipped with an Edinburgh Instruments integrating sphere. The temperature-dependent photoluminescence emission (PL) spectra were tested on a phosphor excitation spectrum with thermal quenching analysis system EX-1000 . The commercial blue phosphor BAM:Eu2(Ca/Mg)SnO6:Mn4+ red phosphors were successfully prepared using a conventional high-temperature solid-state reaction. The Rietveld refinements revealed that both La2CaSnO6:Mn4+ and La2MgSnO6:Mn4+ were monoclinic phases and the Mn4+ ions replace the Sn4+ ions. The Tanabe–Sugano energy level diagrams indicate that the Mn4+ ions were in a strong crystal field. In addition, LCSO:0.002Mn4+ and LCSO:0.002Mn4+ quantum yields were 22.90% and 35.48%, respectively. The thermal quenching phenomena of LCSO:0.002Mn4+ and LMSO:0.002Mn4+ were explained by the conformational coordinate diagram of the Mn4+ ions. Finally, the obtained far-red LED devices emitted bright far-red light that matched well with the absorption spectra of plant photosensitive pigments Pr and Pfr. Additionally, the prepared W-LEDs had Ra of 96.4 and 96.6 and CCT of 4958 K and 5275 K, respectively. These results show that LCSO:0.002Mn4+ and LMSO:0.002Mn4+ red phosphors can be used as the red component of W-LEDs as well as in plant growth.In summary, a series of La


Artemis nuclease and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are key components in nonhomologous DNA end joining (NHEJ), the major repair mechanism for double-strand DNA breaks. Artemis activation by DNA-PKcs resolves hairpin DNA ends formed during V(D)J recombination. Artemis deficiency disrupts development of adaptive immunity and leads to radiosensitive T- B- severe combined immunodeficiency (RS-SCID). An activated state of Artemis in complex with DNA-PK was solved by cryo-EM recently, which showed Artemis bound to the DNA. Here, we report that the pre-activated form  of the Artemis:DNA-PKcs complex is stable on an agarose-acrylamide gel system, and suitable for cryo-EM structural analysis. Structures show that the Artemis catalytic domain is dynamically positioned externally to DNA-PKcs prior to ABCDE autophosphorylation and show how both the catalytic and regulatory domains of Artemis interact with the N-HEAT and FAT domains of DNA-PKcs. We define a mutually exclusive binding site for Artemis and XRCC4 on DNA-PKcs and show that an XRCC4 peptide disrupts the Artemis:DNA-PKcs complex. All of the findings are useful in explaining how a hypomorphic L3062R missense mutation of DNA-PKcs could lead to insufficient Artemis activation, hence RS-SCID. Our results provide various target site candidates to design disruptors for Artemis:DNA-PKcs complex formation.  Nonhomologous DNA end joining (NHEJ) is the primary pathway by which DNA double-strand breaks (DSBs) are repaired in vertebrate cells . DividinNHEJ is initiated at DSBs by the Ku70/80 heterodimer binding at the break site, which then recruits core NHEJ factors. DNA-dependent protein kinase catalytic subunit (DNA-PKcs), an important regulator of NHEJ, binds to Ku70/80 bound at dsDNA ends and undergoes autophosphorylation . Even wimammalian target of rapamycin, previously known as FRAP), ATR (ataxia telangiectasia- and Rad3-related protein), ATM (ataxia telangiectasia mutated), and SMG1 , TRRAP (transformation/transcription domain-associated protein) and DNA-PKcs  -related protein kinase (PIKK) subfamily, which includes mTOR (subunit) . Human Dsubunit) . The N-Hsubunit) . Howeverinterest .cis for individual DNA-PKcs molecules (trans for some regions of DNA-PKcs (DNA-PKcs can bind a dsDNA end both in the absence of and in the presence of Ku70/80 and activates itself by autophosphorylation . It has olecules  and in tDNA-PKcs .CPSF-73-Artemis-SNM1-Pso2) subdomains, and an unstructured C-terminal regulatory tail (361–692 aa). The catalytic domain forms the two-metal containing active site (two Zn2+ ions) at the interface of the MβL and β-CASP domains  has weaker binding with D37, H38 and D136. There is also a non-active site Zn2+ (Cys2His2 zinc finger motif) in a peripheral location of the β-CASP domain, which is not present in SNM1A and SNM1B, which are other members of the extended structural family of MβL fold enzymes. The C-terminus is known to interact with other NHEJ components, including DNA-PKcs and DNA ligase IV  fold enzymes, including SNM1A and SNM1B . Artemis domains . One actigase IV , but morigase IV .Artemis, which has intrinsic 5′ to 3′ exonuclease activity , acts aset al. used glutaraldehyde cross-linking to stabilize two forms: a long-range synaptic complex comprising two DNA-PK complexes (each DNA end is bound by Ku70/80 and DNA-PKcs) linked by a Lig IV-XRCC4-XLF-XRCC4-Lig IV scaffold (PDB: 7LT3) and a short-range synaptic complex comprising the two DNA ends bound by Ku70/80, and linked by a Lig IV-XRCC4-XLF-XRCC4-Lig IV scaffold (PDB: 7LSY)  . The Blu  .Although a number of structural and biochemical studies on NHEJ synapsis and function have been reported, structural studies on Artemis:DNA-PKcs and the endonuclease activity have been more difficult. Structural studies with full-length Artemis have been limited because the C-terminal half of Artemis lacks any predicted structure and is likely disordered, promoting full-length Artemis to aggregate. Therefore, reported crystal structures of Artemis are limited to only a small C-terminal peptide (D485–R495) in the complex with DNA ligase IV catalytic domain  ,36 or thB: 6WO0) . Howeverral site . They al2+ instead of Zn2+ at the active site. Also revealed in this structure is a short portion of the C-terminal tail of Artemis (E366–P406) bound to the FAT domain of DNA-PKcs. Using the previous finding that Artemis and DNA-PKcs co-immunoprecipitation is DNA-independent  . It provependent .Since the majority of Artemis appears to exist in complex with DNA-PKcs within cells , it has Endogenous native human DNA-PKcs was purified to near homogeneity from HeLa-S3 cells as follows. The frozen cell pellet was produced by the Cell Culture Company, LLC , using our specifications for cell growth rate and density. The nuclear extract was prepared using a modified protocol based on the method of Dignam . Chromat2, 0.2 mM PMSF, 5 mM DTT and additional protease inhibitors. The resuspended cells were centrifuged at 1,850 ×g for 5 min and the pellets were kept. The supernatant was further centrifuged at 10,000 ×g for 10 min. All the cell pellets were resuspended in NE Buffer A-K10 supplemented with additional protease inhibitors to a final volume of 3× the original packed cell volume. The resuspended cells were incubated on ice for 10 min to swell the cells. The cells were homogenized with a glass Dounce homogenizer (a type B pestle) with 15 strokes. The efficiency and completion of cell lysis (>∼90%) were examined under a microscope by trypan blue staining. The homogenized cells were centrifuged at 3,200 ×g for 15 min and the pellets (i.e. nuclei) were kept. The supernatant was further centrifuged at 25,000 ×g for 20 min and the pellets (i.e. nuclei) were collected. All the nuclei pellets were resuspended in a volume of NE Buffer B-Na20: 10 mM HEPES–NaOH (pH 7.9), 20 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 0.2 mM PMSF, 5 mM DTT and additional protease inhibitors, equal to half of the packed nuclear volume. In a dropwise fashion, while stirring gently, an appropriate volume of NE Buffer C-Na840: 20 mM HEPES–NaOH (pH 7.9), 840 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 0.2 mM PMSF, 5 mM DTT and additional protease inhibitors, was added to the sample to reach ∼420 mM NaCl as the final concentration. The resuspended nuclei were homogenized with the Dounce homogenizer (a type B pestle) with five strokes. The homogenizer was rinsed with NE Buffer D-Na420: 20 mM HEPES-NaOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 0.2 mM PMSF, 5 mM DTT and additional protease inhibitors, to recover as much sample as possible. The nuclei suspension was gently and continuously mixed on an end-over-end rotor for 40 min. The mixed sample was centrifuged at 25,000 ×g for 30 min. At this point, the supernatant  was removed and dialysis was started against DEAE-80 Dialysis Buffer: 50 mM Tris–HCl (pH 7.5), 80 mM KCl, 0.5 mM EDTA, 5% (v/v) glycerol, 0.2 mM PMSF and 5 mM DTT. The nuclear pellets were gently resuspended or rinsed with NE Buffer D-Na420 and mixed on an end-over-end rotor for 30 min. The mixed sample was centrifuged at 25,000 ×g for 30 min. The supernatant  was dialyzed against DEAE-80 Dialysis Buffer: 50 mM Tris–HCl (pH 7.5), 80 mM KCl, 5 mM EDTA, 5% (v/v) glycerol, 0.2 mM PMSF and 5 mM DTT. The dialyzed nuclear extract was centrifuged at 25,000 ×g for 30 min. The supernatant was supplemented with additional protease inhibitors.The frozen cells were thawed in a 37°C water bath while keeping the cells cold. The rest of the procedures were performed at 4°C or on wet ice. The thawed cells were rapidly resuspended in 5× packed cell volume of NE Buffer A-K10: 10 mM HEPES–KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl® 10 DG columns , which were equilibrated with QS-100 Buffer, to desalt. The desalted sample which contained the majority of DNA-PKcs was further purified with the Mono S column. The other desalted sample which contained some portion of DNA-PKcs and Ku70/80 was saved to purify native Ku70/80 later. The sample which contained the majority of DNA-PKcs was injected into the Mono S 5/50 GL column, which was equilibrated with QS-100 Buffer. After washing, DNA-PKcs was eluted in a 0–15% QS-1M Buffer linear gradient. The pooled sample was concentrated down to ∼500 μl using Vivaspin 500 (10 K MWCO) concentrators  and the centrifuged at 16,000 rcf for 10 min. The centrifuged sample was injected into the Superdex 200 Increase 10/300 GL column, which was equilibrated with S200 Buffer11: 25 mM Tris–HCl (pH 7.5), 200 mM NaCl and 10 mM DTT, at a flow rate of 0.75 ml/min.This nuclear extract was passed over the DEAE Sepharose FF column (XK50/20: CV = ∼250 ml), which was equilibrated with DEAE-90 Buffer: 50 mM Tris–HCl (pH 7.5), 90 mM KCl, 0.5 mM EDTA, 5% (v/v) glycerol, 0.2 mM PMSF and 5 mM DTT, using a peristaltic pump. DNA-PKcs was eluted with DEAE Buffer with ∼270 mM KCl. The pooled sample was dialyzed against Heparin-35 Dialysis Buffer: 50 mM Tris–HCl (pH 7.3), 35 mM KCl, 0.5 mM EDTA, 5% (v/v) glycerol, 0.2 mM PMSF and 5 mM DTT, down to ∼113 mM KCl. The dialyzed sample was supplemented with additional protease inhibitors. The post-DEAE sample was passed over the Heparin Sepharose 6 FF column (XK50/20: CV = ∼200 ml), which was equilibrated with Heparin-116.5 Buffer: 50 mM Tris–HCl (pH 7.3), 116.5 mM KCl, 0.5 mM EDTA, 5% (v/v) glycerol, 0.2 mM PMSF and 5 mM DTT. DNA-PKcs was eluted with Heparin Buffer with ∼668 mM KCl. The pooled sample was dialyzed against dsDNA-35 Dialysis Buffer, which is equivalent to the Heparin-35 Dialysis Buffer. The dialyzed sample was supplemented with additional protease inhibitors. The post-Heparin sample was passed over the dsDNA(oligo) Sepharose 4 FF column (XK26/20: CV = ∼40 ml), prepared with a duplex DNA (GW1/2) , 100 mM NaCl and 2 mM DTT to adjust the concentration to ∼1 μM based on the The rest of the peak fractions were diluted with the equal volume of 25 mM Tris–HCl (pH 7.5) and concentrated using Vivaspin 500 (10 K MWCO) concentrators . The concentrated sample was centrifuged at 16,000 rcf for 10 min. The protein concentration was determined by the method of Bradford using Bio-Rad Protein Assay dye reagent  with bovine serum albumin as a standard. The final concentration was adjusted appropriately, and small aliquots were snap frozen in liquid nitrogen and stored at –80°C. This whole procedure from thawing the cell pellet, harvested from 16 L of cell culture, to storing the final protein aliquots was done over 3 days.® 10 DG columns , with QS-100 Buffer#2: 50 mM Tris–HCl (pH 7.5), 100 mM KCl, 0.5 mM EDTA, 5% (v/v) glycerol, 0.2 mM PMSF and 5 mM DTT, to remove Tween-20. The sample was injected onto the Mono S 5/50 GL column, which was equilibrated with QS-100 Buffer#2. After washing, the column was subjected to linear gradients with QS-1M Buffer#2: 50 mM Tris–HCl (pH 7.5), 1 M KCl, 0.5 mM EDTA, 5% (v/v) glycerol, 0.2 mM PMSF and 5 mM DTT. In the region of a 10–20% QS-1 M Buffer#2 linear gradient, Ku70/80 was eluted. The peak fractions were concentrated down to ∼500 μl using Vivaspin 20 and Vivaspin 500 (both 10 K MWCO) concentrators  and the concentrated sample was centrifuged at 16,000 rcf for 10 min. The centrifuged sample was injected into the Superdex 200 Increase 10/300 GL column, which was equilibrated with S200 Buffer11: 25 mM Tris–HCl (pH 7.5), 200 mM NaCl and 10 mM DTT, at a flow rate of 0.75 ml/min. The peak fractions were concentrated using Vivaspin 500 (10 K MWCO) concentrators  and the concentrated sample was centrifuged at 16,000 rcf for 10 min. Once the concentration was determined by Bradford assay, Ku70/80 was aliquoted into a small volume, flash frozen in liquid nitrogen and stored at –80°C.The saved MonoQ fractions, which contained some portion of DNA-PKcs and Ku70/80 above, were injected onto the Mono S 5/50 GL column, which was equilibrated with QS-100 Buffer and the flowthrough, which contained the majority of Ku70/80, was collected. The buffer of the flowthrough was exchanged using Econo-PacTrichoplusia ni and the synthesized gene, with the 3′ flanking sequence for a TEV cleavage site and (8×)His tag, was cloned into pFastBac1 at the BamHI and HindIII sites . The plasmid containing Artemis cDNA + TEV+(8x)His was transformed into DH10Bac competent cells , and the generated bacmid was isolated. The baculovirus was generated by transfecting the generated bacmid into Sf21 cells. The isolated baculovirus was amplified in Sf21 cells and the expression of Artemis was confirmed in both Sf21 and High Five™ cells . The titrated virus was used to infect High Five cells for protein production in a large scale. The infected cells were cultured shaking at ∼130 rpm at 27°C and harvested at 48 h of post-infection, flash-frozen in liquid nitrogen and stored at –80°C.The codon of the human Artemis coding sequence was optimized for ® Superflow Metal Affinity Resin  resin, pre-washed with TALON Binding Buffer, and mixed by rotating end over end for 6 h. After incubation, the resin was washed with TALON Binding Buffer with 800 mM NaCl twice. The washed resin was transferred into an empty Econo-Pac® gravity-flow column , and further washed with TALON Wash Buffer: 50 mM HEPES–NaOH (pH 7.9), 800 mM NaCl, 5% (v/v) glycerol, 50 mM imidazole, 0.005% (v/v) Triton X-100, 1 mM TCEP, 0.2 mM PMSF, 2 μg/ml aprotinin, 1 μg/ml pepstatin A, 10 μM leupeptin, 1 μM bestatin and 10 μM E-64. Artemis was eluted with TALON Elution Buffer: 50 mM HEPES–NaOH (pH 7.9), 500 mM NaCl, 5% (v/v) glycerol, 250 mM imidazole, 0.005% (v/v) Triton X-100, 1 mM TCEP, 0.2 mM PMSF, 2 μg/ml aprotinin, 1 μg/ml pepstatin A, 10 μM leupeptin, 1 μM bestatin and 10 μM E-64. The eluted Artemis was desalted with Econo-Pac® 10DG columns , which were equilibrated with S200 Buffer: 25 mM Tris–HCl (pH 7.5), 200 mM NaCl, 2 mM DTT and 0.005% Triton X-100. The desalted sample was concentrated down to ∼500 μl using Vivaspin 20 and Vivaspin 500 (both 10 K MWCO) concentrators  and the concentrated sample was centrifuged at 16,000 rcf for 10 min. The centrifuged sample was injected into the Superdex 200 Increase 10/300 GL column, which was equilibrated with S200 Buffer without Triton X-100 at a flow rate of 0.75 ml/min. Depending on the height of the peak, the peak fraction was run onto the second round of size exclusion column.The frozen High Five cell pellet harboring recombinant human Artemis-(8×)His was thawed at 37°C, while keeping the cells cold. The rest of the procedures were performed at 4°C. The thawed cells were resuspended in TALON Binding Buffer: 50 mM HEPES–NaOH (pH 7.9), 500 mM NaCl, 5% (v/v) glycerol, 10 mM imidazole, 0.1% (v/v) Triton X-100, 1 mM TCEP, 0.2 mM PMSF, 2 μg/ml aprotinin, 1 μg/ml pepstatin A, 10 μM leupeptin, 1 μM bestatin and 10 μM E-64. The cells were sonicated and centrifuged at 25,000 ×g for 30 min. The supernatant was incubated with TALONFor cryo-EM purposes, a small amount of the Artemis peak fraction was freshly used for complex formation with DNA-PKcs and plunge-freezing. The rest of the peak fractions were supplemented with 0.005% (v/v) Triton X-100, flash frozen in liquid nitrogen and stored at –80°C.TM Protein Ladder  as a marker as indicated.The purity of purified proteins was assessed by Coomassie Brilliant Blue R250 staining or silver staining on 6 or 8% SDS-PAGE using a Spectra™ Multicolor High Range Protein Ladder  or BLUEstain2O and lyophilized. The lyophilized oligo was dissolved in dsDNA Annealing Buffer: 20 mM HEPES–NaOH (pH7.5), 100 mM NaCl and 10 mM MgCl2 and self-annealed. Finally, the quality of dsDNA, as a Y-shape form (GW132H), was assessed by running 20% native PAGE at 4°C. GW132H is configured to be blocked with two short hairpins at one end (creating a Y-shape at this end) and two short overhangs  at the open DNA end.An oligo for Y-shape double hairpins DNA, GW132  with a 1.5 mm space thickness. This procedure describes the making of two gels. First, for each apparatus, ∼2 ml of 20% polyacrylamide gel was prepared from a 30% acrylamide/bis-acrylamide (29:1 (w/w)) stock solution (30%T/3.33%C) and 10× TBE stock solution on the bottom of the gel. About 25 μl of 10% (w/v) ammonium persulfate and 4.5 μl of TEMED were added to make ∼5 ml of 20% polyacrylamide solution. The 2 ml of acrylamide solution was equivalent to ∼1.4 cm of height of the gel from the bottom. For the agarose, SeaKem® Gold Agarose was used . Then, a solution of 1.0% (w/v) agarose (e.g. 0.15 g in 15 ml of 1× TBE) was made in a 125-mL Erlenmeyer flask by heating in a microwave  for 1 min and swirling every 20 s to promote mixing and melting of the agarose. The flask was loosely sealed with aluminum foil and kept in a 45°C water bath to prevent gelation. Then, the acrylamide solution was prepared in another 125-ml Erlenmeyer flask by adding and mixing the following in this order: 8.16 ml of ddH2O, 2.0 ml of 30% acrylamide/bis-acrylamide (75:1 (w/w)) stock solution (30%T/1.32%C), 3.0 ml of 10× TBE, 1.5 ml of 50% (w/v) glycerol, 15 μl of 1 M DTT and 300 μl of 10% (w/v) ammonium persulfate. The mixed solution was degassed for 1 min while swirling, loosely sealed with aluminum foil, and then transferred to the same 45°C water bath for 20 min to reach thermal equilibration. The gel apparatus, which contained the 20% polyacrylamide gel, was half filled with diH2O and placed in a 37°C incubator to pre-warm the glass plates until the 20 min incubation of the acrylamide solution in the water bath ends. After 20 min incubation, the warmed acrylamide solution was poured into the flask containing the molten agarose, along with condensed water on the inside wall of this flask and mixed by gentle swirling. The mixture was quickly poured back to the other flask, which had contained the acrylamide solution, along with condensed water on the inside wall of this flask, mixed by gentle swirling and incubated at 45°C for 1 min. Then, after adding 30 μl of TEMED, the gel mix was rapidly but gently poured into the pre-warmed gel assembly (×2), a short 10-well comb was carefully inserted without air bubbles, and the assembly was immediately transferred to 4°C for 10 min for the agarose to solidify. The cooled gel assembly was taken out after 10 min and kept at room temperature for 1 h to allow full polymerization of the acrylamide. This agarose gelation step prior to polymerization is essential, providing a vital mechanical support to the composite gel since the polymerized acrylamide alone would still be fluid-like at this low concentration (e.g. 2%). Then, after 1 h incubation at room temperature, the comb was gently removed with extreme care. The shape of wells was polished with a spatula if necessary. Creating wells with a very flat base was extremely important because of the nature of native gel electrophoresis. Also trimming of the wells must be done carefully because of the fragility of the composite gel and its glutinous nature. But the polishing step must be completed before the gel starts to dry. After the polishing, the gel apparatus was assembled with cold 1× TBE as a running buffer. No air bubbles at the bottom of the gel should be trapped. Usually, the apparatus was kept at 4°C overnight to pre-cool. Then, gel shift assays were carried out at 4°C.An agarose-acrylamide composite native gel shift assay system was further optimized from a method of Suh et al. . The gelThe binding reaction was carried out in a final volume of 10 μl with a buffer containing 20 mM Tris–HCl (pH 7.5), 100 mM NaCl, 2 mM DTT, 5% (v/v) glycerol, 0.3 μM DNA-PKcs, 0.3 μM Ku70/80, 0.3 μM Artemis, 0.36 μM Y-shape duplex DNA GW132H as indicated at 4°C for 30 min. The assembly of each complex was done by incubating them at 4°C for another 30 min. For example, for the assembly of DNA-PK, 0.3 μM DNA-PKcs was added to the complex of Ku70/80:GW132H and incubated at 4°C for another 30 min. Artemis was added to each appropriate complex and incubated at 4°C for another 30 min. During this incubation, the gel was pre-run at 100 V for 30 min. After the complex assembly reactions, the samples were run onto an agarose-acrylamide composite native gel at 4°C at 100 V for 2 h. After the run, the gel was stained with silver nitrate. To visualize free DNA, the gel was overdeveloped.An Artemis peptide (Art: LRHKVPY) and an XRCC4 peptide (X4: APSRKRRQRMQR) were synthesized and analyzed by GenScript USA Inc. . The N-terminus and C-terminus of both peptides were acetylated and amidated, respectively. Various concentrations of peptides  were added into pre-formed Artemis:DNA-PKcs complex, as described above, and incubated at 4°C for another 30 min. Also, to assess whether Artemis binds DNA-PKcs to form a complex in the presence of the XRCC4 peptide, 0.3 μM DNA-PKcs and the 3 mM XRCC4 peptide were mixed and incubated at 4°C for 30 min first, and then, 0.3 μM Artemis was added and incubated at 4°C for another 30 min prior to the gel run. Controls included individual peptides and proteins and the combination of them. The samples were run on an agarose-acrylamide composite native gel at 4°C at 100 V for 2 h. After the run, the gel was stained with silver nitrate.2+ assay, 10 nM Artemis without DNA-PKcs was used to initiate the reaction in the buffer contained 25 mM Tris–HCl (pH 8.0), 10 mM KCl, 10 mM MnCl2, 500 nM ZE16 and 1 mM DTT. For the Artemis:DNA-PKcs with ATP/Mg2+ assay, an equal molar ratio of Artemis and DNA-PKcs was pre-incubated together on ice for 10 min to form a complex. Then, the reaction, which contained 25 mM Tris–HCl (pH 8.0), 10 mM KCl, 100 μM ATP, 10 mM MgCl2, 500 nM ZE16 and 1 mM DTT, was initiated by adding 10 nM Artemis:DNA-PKcs complex and incubating at 37°C for 30 min. The reactions were stopped by the addition of an equal volume of 1:1 40% glycerol:formamide and incubation at 95°C for 5 min. Reaction products were resolved with an 18% denaturing PAGE. A 32 nt marker oligonucleotide (GW35) and an 8 nt marker oligonucleotide (FAM-8 T) were used to aid gel mobility determination.The sequence of the oligos is listed in Cryo-grid preparation was performed using a home-made manual plunger at a typical indoor humidity of 73% and at room temperature. Briefly, UltrAuFoil R 1.2/1.3 grids (Gold 300 mesh)  were glow-discharged using a PELCO easiGlow™ Discharge System  for 40 s on both sides at 0.3 mBar and 20 mA. The Artemis:DNA-PKcs complex was prepared by mixing them at 1.12× molar excess Artemis to a final complex concentration of ∼280 nM and incubating them at 4°C for 30 min. The Artemis:DNA-PKcs:GW132H complex was prepared by incubating DNA-PKcs and 1.12× molar excess GW132H at 4°C for 30 min first, and subsequently adding 1.12× molar excess Artemis to DNA-PKcs to a final complex concentration of ∼280 nM and incubating at 4°C for another 30 min. Then, LMNG was supplemented to both samples at the final concentration of 5.5 nM. The final samples were evenly applied onto both sides of the glow-discharged grids (1 μl and 2 μl). A piece of filter paper (Grade 41 Fast Ashless Whatman Filter Paper)  was applied to one side of the grid to adsorb the sample for ∼3 s. Then, the grid was quickly plunged into liquid ethane, pre-cooled with liquid nitrogen. The plunged-grids were taken to UCI Irvine Materials Research Institute and screened with a JEOL JEM-2100F TEM at 200 kV, equipped with Gatan OneView and K3 cameras. The optimal grid candidates were shipped to the National Cryo-Electron Microscopy Facility (NCEF) of the National Cancer Institute (NCI) for data collection.–/Å2 was fractionated into 40 frames for 3.25 s.At NCEF, movies of the Artemis:DNA-PKcs complex were collected using a Titan Krios operating at 300 kV equipped with a Gatan K3 camera behind a bioquantum imaging filter with an energy filter slit width of 20 eV. For Artemis:DNA-PKcs data, four datasets were collected: one dataset with the super-resolution mode at 0.54 Å/pixel and a defocus range of −1.0 to −2.5 μm and 3 datasets with the counting mode at 1.08 Å/pixel and a defocus range of −0.75 to −3.0 μm. The magnification was 81,000× for all datasets and for each movie, a total exposure dose of 60 e–/Å2 was fractionated into 40 frames for 3.25 s.For Artemis:DNA-PKcs:GW132H complex, two datasets were collected with the counting mode at 1.08 Å/pixel and a defocus range of −0.75 to −2.25 μm. The magnification was 81,000× and for each movie, a total exposure dose of 45 eB-factors to obtain reasonable maps including blurred maps. Also, 3D variability analysis was performed (B-factor at +80 Å2) of the state 2 was used to assist locating the Artemis catalytic part. The head part of DNA-PKcs (FAT + kinase domains) with Artemis tail were segmented out using Segger in Chimera  . Patch m cutoff) . Furthererformed . The blu Chimera  and maskCoot  and the Quality Control Check (https://qc-check.usc.edu) were used to validate the model quality. The refinement statistics were shown in Atomic coordinates of a crystal structure of DNA-PKcs (PDB: 5LUQ) were fit into preliminary maps, from intermediate steps of image processing . Amino aCoot . Then, oCoot . A densiCoot . The resThe crystal structure of the Artemis catalytic domain (PDB: 7AF1) was manually fit into a globular density external to DNA-PKcs of the blurred map. The best orientation was chosen from four possible orientations of the catalytic domain. Flex-EM in CCP-EM v1.5.0 was performed to assess the fit quality for four possibilities for vertical reversal for the catalytic domain of Artemis ,53.The contacts and distances between atoms are calculated with CONTACTS in CCP4 v7.1.018 . RMSD wa2 unmasked GSFSC in cryoSPARC. The decoder network was trained on this dataset with 1.08 Å2 pixel, a 2562 pixel dimension, a z-dimension of 8, encoder space of 1024 × 3, over 25 epochs. Encoder output volumes presented from this work were selected from Umap latent space or PCA traversals as specified.Variation autoencoder methodology incorporated in cryoDRGN was employed to assess structural variability in the data and to provide a greater understanding of the continuous motions of Artemis relative to DNA-PKcs . A conseA full-length Artemis amino acid sequence was used for RoseTTAFold analysis to predict an overall structure of Artemis including the C-terminal tail .A predicted Artemis structure by AlphaFold v2.0 was obtained from the AlphaFold Protein Structure Database .Endogenous DNA-PKcs and Ku70/80 purified from HeLa cells and recombinant human Artemis produced from High Five cells  were visArtemis:DNA-PKcs are believed to reside in a constitutive complex in cells ,39. Our et al.’s state 2 DNA-PKcs (PDB: 6ZFP) is 0.950 Å  and 1.095 Å , respectively  and the largest opening between the FAT and N-HEAT domains, ∼60 and ∼34 Å, respectively . In contectively . All thrThe state 1 structure contains an additional helical density not observed in states 2 and 3 between the helices (Y2184–G2198 and R2228–C2244) in the M-HEAT domain . This reB-factor at +80Å2 .,51.50,51et al. structure with activated Artemis engaging the DNA end , but no detailed assessment on those interactions was discussed , where they placed part of the C-terminal tail of XRCC4 (A267–R278), based on the electrostatic surface (PDB: 7LT3)  in PyMOL, labeling red for negatively charged and blue for positively charged. The surface of contact point 1 of DNA-PKcs is negatively charged Figure  and the B: 7LT3)  (SupplemB: 7LT3) . The map density . However density . Based oWe propose calling this region where the Artemis tail L401–Y407) and XRCC4 tail A267–R278) are proposed to bind, which encompasses the FR2 subdomain of the FAT domain of DNA-PKcs (∼2966–∼3020 aa), the ‘Art-X4 cleft’ Figure . This cl7–R278 ar–Y407 andet al.  was chosen based on Chen et al.  and Arteet al. , lane 4 et al. . While Aet al. , lane 10However, even if these are mutually exclusive sites, because there are quite a number of interaction regions between the C-terminal tail of Artemis and the FAT domain of DNA-PKcs and because there could be potentially other unidentified interaction sites between XRCC4 and DNA-PKcs, such competition assays with peptides may not reflect the complete mechanism. For example, the positively charged XRCC4 peptide still may bind to contact point 3, which is another negatively charged area and in fact more critical for Artemis catalytic domain positioning prior to its transitioning to DNA ends. Therefore, this disruptive effect may be due to the binding of XRCC4 peptide to contact point 3, in addition to contact point 1. More systematic tests such as pull-down experiments will be required to define this precisely.Since XRCC4 is complexed with DNA ligase IV, and DNA ligase IV interacts with Artemis, the interpretation of the data is likely more complicated in a gel shift assay and even in biophysical experiments using those purified proteins in attempts to elucidate the true mechanism. Nevertheless, these competition assays show that the C-terminal portion of XRCC4 (A267–R278) disrupts Artemis:DNA-PKcs complex and that it also prevents the Artemis:DNA-PKcs complex formation. The findings are significant as DNA-PKcs may recruit XRCC4:Ligase IV complex to broken DNA sites.Our previous study shows that C-terminally deleted XRCC4 neither affects DNA ligase IV binding and activity, nor the in vivo function of XRCC4 . Thus, tet al. for the activated state, which shows a similar turn and location of the Artemis tail, the Artemis tail seems to run slightly over these two lysine residues and does not run between them as our density suggests (Contact point 2 occurs between positively charged residues of the FR3 domain (∼3252–∼3309 aa) and Artemis residues D389–P396) Figures . Each ofsuggests  (6). Figures Hydrophobic residues (P396–L401) were assigned to the bridge region between the contact points 1 and 2 as the hydrophobic bridge with no contact with DNA-PKcs Figure –F. As foet al. activated state Artemis:DNA-PKcs:Ku70/80:DNA structure also shows a similar positioning of the Artemis tail relative to DNA-PKcs where L3062 of DNA-PKcs is sandwiched between L372 and L375 of the Artemis tail  and the positively charged residues K368–R379) of Artemis interact with the surface of this region Figures . L3062 ol Figure  (6). How8–R379 ofIn-silico mutagenesis analysis of our model shows that the side chain of L3062R not only would directly interfere with the Artemis tail due to its close proximity but also could interfere with residues of DNA-PKcs, Q3093, Y3090, Q3059 and D3066, potentially altering the overall interaction with the Artemis tail as well as the local FAT domain tertiary structure Figure –E. Moreoet al., 2022), the C-terminal regulatory region of Artemis remains mostly unresolved with ∼280 amino acids still unaccounted for, and this region could interact with other components of the NHEJ complex or with histones on the DNA ends .,73.et als Figure . This ins Figure  20). Im. Imet alet al.  may be supported by two helices (V815–R820 and N827–K832), which can be seen between the N-HEAT and DNA in a DNA-PK structure in a synaptic complex (PDB: 7LT3)  ,36,72. HIn summary, our efforts have been focused on the basal state of the Artemis:DNA-PKcs endonuclease complex because this is the majority state of both Artemis and DNA-PKcs in a cell without DNA damage and even in a cell with DNA damage, since only a small percentage of the 50,000–70,000 molecules of DNA-PKcs and Artemis in each human nucleus become activated. Thus, this is the form that would need to be blocked from activation by an intended inhibitor. We provide structural insight into the basal state of the Artemis:DNA-PKcs interaction and show how dynamic the catalytic domain of Artemis can be. We also describe the Artemis:DNA-PKcs:DNA complex, which is similar to the pre-activated state where DNA-PKcs is bound on DNA, but the phosphate-transfer reaction by the kinase has not yet begun, given the absence of ATP. Our improved agarose-acrylamide composite gel shift assay has the capability of resolving large NHEJ multicomplexes without crosslinking, including Artemis:DNA-PKcs (∼547 kDa) and DNA-PKcs:Ku70/80:DNA (e.g. >∼650 kDa), and the potential for other types of analysis. We utilize this gel system to demonstrate that an XRCC4 peptide, which supposedly binds to the Art-X4 cleft, competes with Artemis and disrupts Artemis:DNA-PKcs complex formation.Recently, the Blundell group reported cryo-EM structures of DNA-PKcs with ATPγS and various inhibitors  including drug candidates undergoing clinical trials, providing insights into the mode of PIKK regulatory domain (PRD) upon ligand binding . TargetiIf one could block the assembly of the Artemis:DNA-PKcs complex, then this would reduce the chance of Artemis conversion to an active form. Inhibition of activation of Artemis in acute lymphoblastic leukemia (ALL) cells would result in chromosomal breaks because the large majority of ALL tumors have ongoing expression of RAG1/2. Even after such pre-B or pre-T cells have exhausted their endogenous V, D and J segments, off-target action by RAG1/2 has been well documented in such cells . Thus, Aet al. : Artemis:DNA-PKcs  and Artemis:DNA-PKcs:DNA . The coordinates for the Artemis:DNA-PKcs has been deposited in the Protein Data Bank (PDB) under the accession code 7TYR.gkac564_Supplemental_FilesClick here for additional data file.


Cunninghamella was employed as an efficient tool to explore active metabolites of DHA in erythrocyte. Microbial transformation products of DHA by Cunninghamella elegans CICC 40250 were detected and analyzed by ultra-performance liquid chromatography (UPLC)-electrospray ionization (ESI)-quadrupole time-of-flight (Q-TOF)-mass spectrometry (MSE), and the main products were isolated and identified. The antimalarial activity of the isolated products was also screened in vitro. Totally, nine products were discovered through UPLC-ESI-QTOF-MSE, and three main products with novel chemical structures were isolated for the first time, which were also detected in red blood cells as the metabolites of DHA. After evaluation, 7β-hydroxydihydroartemisinin (M1) exhibited a good antimalarial activity with an IC50 value of 133 nM against Plasmodium falciparum (Pf.) 3D7. The structure and stereo-configuration of novel compound M1 were validated via X-ray single crystal diffraction. Microbial transformation was firstly employed as the appropriate model for metabolic simulation in erythrocyte of DHA. Three novel metabolites in erythrocyte were obtained for the first time through our microbial model, and one of which was found to show moderate antimalarial activity. This work provided a new research foundation for antimalarial drug discovery.Dihydroartemisinin (DHA) is a sesquiterpene endoperoxide with prominent antimalarial efficacy, which was discovered by Professor Youyou Tu through the reduction of artemisinin in the 1970s. It is always a challenging work for scientists to investigate the metabolites of DHA in the red blood cells due to the complicated matrix background. As a bottleneck, the investigation of metabolites, especially exploring the pharmacodynamic material in the red blood cell, is necessary and significant for metabolism research of antimalarial agent. Recently, microbial transformation provides a green and economical means for mimicking mammal metabolism and synthesis active metabolites, based on which is one efficient route for drug discovery. In this study, a strain from  Dihydroartemisinin (DHA), a famous derivative of artemisinin  through C-10 reduction of lactone carbonyl by sodium borohydride, was discovered by Youyou Tu in 1973 . Owing tin vivo process of DHA, some projects have been conducted through filamentous fungi-mediated strategy, and six derivatives of DHA including 14-hydroxymethyl dihydroartemisinin  CICC 40250, and the products were analyzed and compared with the metabolites of DHA in erythrocytes. As a result, from microbial transformation, a total of nine DHA derivatives (M1–M9) were detected by UPLC-MS and three were isolated, including 7β-hydroxydihydroartemisinin (M1), 1-deoxydihydroartemisinin (M8), and 1-deoxyartemisinin (M9). The three gained products were also identified in erythrocytes. The structure and stereo-configuration of the novel product (M1) were validated via X-ray single crystal diffraction, and reported here for the first time. Moreover, M1 was also proven to exhibit in vitro antimalarial activity against Pf 3D7, with an IC50 value of 133 nM. As well as we know, this is the first attempt to isolate and identify the metabolites of DHA in red blood cells via microbial transformation. The discovery and synthesis of antimalarial products provide the possibility for further drug development by eco-friendly microbial transformation. The structures of DHA and its derivates from C. elegans were shown in Herein, the metabolic study of DHA was performed employing 254 thin layer plates, and silica gel GF254 thin layer preparation plates were purchased from Qingdao Marine Chemical Group Corporation . Silica gel , Silica Flash Column 330 g, and Chormatorex  were used for column chromatography. Water was prepared using a Milli-Q system operating at 18.2 MΩ . All other chemicals used were purchased from Fisher Scientific or Beijing Chemical Works, and were of the highest purity available.DHA was obtained from the Kunming Pharmaceutical Group . The compound had purity ≥ 99%. Methanol, acetonitrile, and formic acid were HPLC grade and purchased from Fisher . Other chemicals in this work were of analytical grade and purchased from Beijing Chemical Works . Silicone filler, silica gel GFE system consisted of a Waters ACQUITY I-class UPLC and Xevo G2-XS Q-TOF Mass Spectrometer . 1H (600 MHz), 13C (150 MHz), and NMR spectra were recorded on a Bruker AV 600 spectrometer  with TMS as an internal reference. X-ray diffraction experiments were performed using a Bruker D8 venture diffractometer with Cu Kα radiation. The UPLC-ESI-Q-TOF-MSC. elegans CICC 40250 (MT1) was obtained from the China Center of Industrial Culture Collection . Culture and biotransformation experiments were conducted in a medium composed of 20 g Sabouraud dextrose broth , 10 g peptone , 15 g sucrose , and 1,000 ml deionized water.Two-stage fermentation was employed in this study . The subE system consisted of a Waters ACQUITY I-class UPLC and Xevo G2-XS Q-TOF mass spectrometer equipped with electrospray ionization (ESI) source. Chromatographic separation was achieved using an ACQUITY UPLC BEH C18 . The mobile phase consisted of solvent A  and solvent B . The gradient program for biosamples included three segments: 5–100% B from 0 to 15 min; followed by 100–5% B from 15 to 17 min; and a postrun of 3 min for column equilibration. The flow rate was 0.4 ml/min, and the temperature was 30°C throughout the analysis.The UPLC-ESI-QTOF-MS2 was used as the drift gas. The low collision energy was set to 6 eV, and the high collision energy was ramped from 12 to 25 eV. MSE analysis was employed for simultaneous acquisition of the exact mass of small molecules at high and low collision energies.The MS was operated in positive ionization mode across a scan range of m/z 50 to 1,000, with a scan time of 0.2 s. Source parameters: source temperature 150°C, cone gas 50 L/h, desolations temperature 450°C, and desolation gas flow 800 L/h. Argon (99.95%) was used for collision-induced dissociation, and NAll data processing was performed using UNIFI 1.9 . Components were identified using the following 3D peak detection features: low-energy limits of 150 and high-energy limits of 20, isotope clustering, and high-to-low energy association within a 0.5 fraction of the chromatographic and drift peak width, with a mass accuracy of ±2 mDa. The maximum number of allowed fragment ions per match was set to 10.Scale-up fermentations were also carried out according to the standard two-stage procedure. Under the same temperature-controlled shaking conditions, each 2000 ml flask contained 500 ml Sabouraud glucose liquid medium, and DHA (6 g) was dissolved in MeOH at a concentration of 25 mg/ml and evenly distributed in all flasks. After incubation for 14 days, mycelia and broth were separated by filtration. The filtrate was extracted with EtOAc  three times, and the extract was evaporated under vacuum to produce a brown residue. The EtOAc extract was fractionated by a Silica Flash Column using a gradient solvent system of petroleum ether-EtOAc to afford 43 fractions (Fr. 1-Fr. 43) according to TLC analysis.M1 (100 mg). Fr. 30 was separated by recrystallization to obtain compound M9 (20 mg). Fr. 40 (1 g) was separated over a CHORMATOREX column and eluted with a gradient solvent system of petroleum ether-acetone to give 14 subfractions. Fr. 40-2 was subjected to thin layer preparation plates to obtain compound M8 (80 mg).Fr. 15 (0.5 g) was subjected to a Chromatorex column and eluted with a gradient solvent system of petroleum ether-EtOAc to afford compound M1, M8, and M9 were diluted in DMSO and examined for antimalarial activity in vitro against the Pf. 3D7 clone. Artemisinin was used as a positive control drug. Inhibition of plasmodium proliferation by different concentrations of transformed products was determined according to a certain formula, and the in vitro antimalarial activity of the drug was evaluated with the IC50.Compounds C. elegans 40250, classified into hydroxylated DHAs (DHA + O), hydroxylated and dehydrogenated DHAs (DHA + O-H2), hydroxylated and dehydrated DHAs (DHA + O-H2-O), deoxygenated DHA (DHA-O), and dehydrated DHA (DHA-H2-O). All identified products were listed in E spectra of DHA biotransformation products were shown in E spectra of its products were shown in The microbial transformation products were identified with UNIFI platform based on chromatographic and mass spectral characteristics provided by reference substances DHA, such as retention time, exact mass, quasi-molecular ions, in-source fragments, characteristic fragments. A total of nine products were found from + was observed at m/z 307.1509. The fragment ions at m/z 267.1590 and 249.1485 were generated by the successive loss of water from m/z 307.1509. The fragment ions at m/z 221.1538 represented the loss of HCOOH from m/z 267.1590. The fragment ions at m/z 203.1432 resulted from the loss of water from m/z 221.1538. Extracted ion chromatograms and mass spectrometry (MSE) spectra of DHA were shown in DHA, the reference substance, was eluted at 7.27 min. Its molecular ion [M + Na]M1-M4) were detected at 3.89, 4.31, 4.97, and 5.14 min. Their molecular ions [M + Na]+ were observed at m/z 323.1457, 323.1461, 323.1455, and 323.1450, respectively. They exhibited the same fragment ions. The fragment ions at m/z 283, 265, and 247 were generated by the successive loss of water from m/z 323. The fragment ions at m/z 219 represented the loss of HCOOH from m/z 265. The fragment ions at m/z 201 resulted from the loss of water from m/z 219. Extracted ion chromatograms and mass spectrometry (MSE) spectra of M1-M4 were shown in Hydroxylated DHAs  was detected at 4.17 min. Its molecular ion [M + Na]+ was observed at m/z 321.1294. The fragment ions at m/z 249 and 231 were generated by the successive loss of water from m/z 267. The fragment ions at m/z 221 represented the loss of HCOOH from m/z 267 or the loss of CO from m/z 249. The fragment ions at m/z 203 resulted from the loss of water from m/z 221. Extracted ion chromatograms and mass spectrometry (MSE) spectra of M5 were shown in Hydroxylated and dehydrogenated DHA  were detected at 3.41 and 4.42 min, respectively. The molecular ions [M + H]+ were observed at m/z 283.1554. Consistent with DHA, these products showed a series of fragment ions resulting from the loss of H2O, CO, or HCOOH, such as m/z 265, 247, 237, 219, and 201. The fragment ions at m/z 265 and 247 were generated by the successive loss of water from m/z 283. The fragment ions at m/z 237 represented the loss of CO from m/z 265. Extracted ion chromatograms and mass spectrometry (MSE) spectra of M6 and M7 were shown in Hydroxylated and dehydrated DHAs  and dehydrated DHA  were detected at 7.16 and 8.65 min, respectively. Their molecular ions [M + Na]+ were observed at m/z 291.1564 and m/z 289.1405, respectively. They showed similar fragment ions except with a 2 Da (a hydrogen atom) mass shift, such as m/z 251, m/z 249, m/z 233, m/z 231, m/z 205, m/z 203, m/z 187, and m/z 185. The fragment ions at m/z 251 and m/z 249 were generated by the successive loss of water from m/z 291 and m/z 289, respectively. The fragment ions at m/z 205 and m/z 203 represented the loss of CO from m/z 233 and m/z 231, respectively. Their ions exhibited the same mechanism resulting from the loss of CO or H2O. Extracted ion chromatograms and mass spectrometry (MSE) spectra of M8 and M9 were shown in Deoxygenated DHA  were evaluated by mass spectral response abundance data. As a result, M1 had the highest content of all compounds, followed by M8 and M9. And the numerical results of response provided the guidance for further isolation. The histogram of the response of all DHA products is shown in Furthermore, as a semiquantitative comparative chromatography analysis, the relative contents of modified dihydroartemisinins (M1 colorless needles (from EtOAc and acetone). ESI-HRMS calcd. for [M + Na]+, C15H24O6Na, 323.1471; found m/z 323.1457. 1H and 13C NMR data are shown in δ 5.39) with C-12 (δ 90.4), C10-OH (δ 4.64) with C-10 (δ 93.7), and H-7 (δ 2.96) with C-7 (δ 72.6), which supported compound M1 as a hydroxylation product. The HMBC spectrum showed correlations of C10-OH (δ 4.64) with C-8a (δ 43.9), C-7 (δ 72.6) and C-8 (δ 31.2), along with the correlations of C-7 (δ 72.6) with H-14 (δ 0.94) and H-15 (δ 1.28). These spectral dates suggested that the hydroxylation occurred at C-7. Moreover, the absolute configuration of the hydroxylated group in the stereogenic carbon center was determined to be β-OH by single-crystal X-ray diffraction analysis (3 was mounted. Data were collected using a Bruker APEX-II CCD diffractometer operating at T = 150.0 K and measured and scans using CuKa radiation. The maximum resolution achieved was = 72.120° (0.81 Å). The final completeness is 98.90% out to 72.120° in. Crystal data: C15H24O6, Mr = 300.34, triclinic, P1, a = 9.6563 (6) Å, b = 9.9540 (6) Å, c = 15.6044 (9) Å, α = 93.832 (3)°, β = 90.860 (3)°, γ = 102.948 (3)°, V = 1,457.78 (15) Å3, Z = 4, Z' = 4, (CuKα) = 0.875, 28521 reflections measured, 10327 unique (Rint = 0.0363), which were used in all calculations. The final wR2 was 0.1013 , and R1 was 0.0383 (I ≥ 2 (I)). Crystallographic data of compound M1 have been deposited to CCDC . Thus, the structure of compound M1 was elucidated as 7β-hydroxydihydroartemisinin.Compound analysis . A singlM8 white powder (EtOAc). ESI-HRMS calcd. for [M + Na]+, C15H24O4Na, 291.1572; found m/z 291.1564. 1H and 13C NMR data are shown in M8 was 1-deoxydihydroartemisinin (Compound emisinin .M9 white powder (EtOAc). ESI-HRMS calcd. for [M + H]+, C15H22O4H, 267.1596; found m/z 267.1589. HR-ESI-MS: [M + H]+ at m/z at 267.1589 . 1H and 13C NMR data are shown in M9 was 1-deoxyartemisinin  spectra of products M1, M8, and M9 and the metabolites of DHA in red blood cells were shown in Based on the analysis results of DHA metabolites in vitro antimalarial activity against Pf. 3D7 with an IC50 (50% inhibition concentration) value of 11 nM. Compound M1 was indicated to exhibit in vitro antimalarial activity against Pf. 3D7 with an IC50 value of 133 nM. Compound M8 and M9 showed no activity against Pf. 3D7.The positive control drug artemisinin exhibited in vitro, microbial transformation can mimic mammal metabolic action according to the similar CYP 450s in microorganisms with the advantage of metabolites accumulation eco-friendly and affordably. Herein, the C. elegans was chosen as an applicable model in vitro for studying DHA metabolism in red blood cells based on the previous research foundation . Artemisinin and its derivatives are the first-line antimalarial agents for saving millions of people’s life. There are four drugs derived from artemisinin, including DHA, artemether, arteether, and artesunate. Given the IC50 value of antimalarial activity against Pf. 3D7 in vitro, the derivatives showed better antimalarial activity than artemisinin, at 4–6 nM approximately, while the value of artemisinin is at 12 nM  is a powerful method for the analysis of compounds, and is increasingly used as a sensitive and efficient tool for metabolite profiling in natural plants or bodies. The numerical results of response, closely related to the conversion rate of the transformation products, were meaningful to guide for isolation work. Herein, UPLC-Q-TOF-MSP. falciparum 3D7 strain due to the cleavage of peroxy linkage (in vitro experiments verify the above conclusion.Artemisinin and its derivatives are excellent antimalarial agents due to the unique peroxide bridge. The mechanism of antimalarial action research indicated that the hallmark of artemisinin activation is the generation of highly reactive carbon-centered radicals via endoperoxide cleavage . And 1-d linkage . FurtherC. elegans.40250 was first employed as a biotransformation tool to gain metabolites that were discovered in erythrocyte. Finally, nine products were detected from microbial system by UPLC-ESI-Q-TOF-MSE, and three intracellular metabolic products were isolated and identified for the first time. Moreover, compound M1 was a novel antimalarial component, with an antimalarial activity IC50 value of 133 nM. In conclusion, the biotransformation of DHA by Cunninghamella proved to be an effective procedure for the investigation of metabolism, especially with the advantage of accumulating metabolites, and one intracellular metabolite of DHA with antimalarial activity was the first time to discover.Microorganisms can mimic mammal metabolism to synthesize metabolites in an environmentally friendly manner. Herein, microbial transformation by