Polygonum capitatum as an ethnic medicine has been used to treat urinary tract infections, pyelonephritis and urinary calculi. In our previous study, P. capitatum was found to have anti-hyperuricemia effects. Nevertheless, the active constituents of P. capitatum for treating hyperuricemia were still unclear. In this study, an ultra-high-performance liquid chromatography coupled to quadrupole/orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to comprehensively detect the chemical ingredients of P. capitatum and its absorbed constituents in the plasma of hyperuricemia rats for the first time. Xcalibur 3.0 and Compound Discoverer 2.0 software coupled to mzCloud and ChemSpider databases were utilized for qualitative analysis. A total of 114 chemical components including phenolics, flavonoids, tannins, phenylpropanoids, amino acids, amides and others were identified or tentatively characterized based on the exact mass, retention time and structural information. Compared to the previous P. capitatum study, an additional 66 different components were detected. Moreover, 68 related xenobiotics including 16 prototype components and 52 metabolites were found in the plasma of hyperuricemia rats. The metabolic pathways included ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. This work may provide important information for further investigation on the active constituents of P. capitatum and their action mechanisms for anti-hyperuricemia effects. Hyperuricemia (HUA) is one of the most common metabolic conditions characterized by abnormally increased serum urate levels. Long-term HUA is a main etiologic factor for the deposition of monosodium urate crystals (MSU) in joints and soft tissues resulting in gout . MoreovePolygonum capitatum Buch.-Ham. ex D. Don, named Touhualiao in Chinese, was utilized as Miao ethnic medicine in China to treat urinary tract infections, pyelonephritis and urinary calculi − ion through losing ·CH3 and H2O. Moreover, the prominent ion at m/z 139.0390 was assigned as [M − H − CO2]−. Compound 55 was tentatively identified as the reported 4-O-methylgallic acid [50 and 52 gave the [M − H]− ions at m/z 197.0448 and 197.0449 with the same predicted molecular formulae (C9H9O5−), which was C2H4 moiety more massive than that of gallic acid. Notably, the product ions at m/z 169.0133 of compound 50 corresponding to [M − H − C2H4]− indicated the existence of ethyl moiety. The fragment ion at m/z 125.0233 ([M − H − C2H4 − CO2]−) was also observed in the tandem mass spectrometry (MS2) of compound 50. Therefore, compound 50 was tentatively identified as the reported ethyl gallate −) and 166.9976 ([M − H − 2 × CH3·]−) suggesting the presence of two methyl groups in compound 52. Compound 52 was assigned as syringic acid [17 and 35 eluted at 3.20 min and 7.07 min showed additional hexoside groups (162 Da) compared to compounds 52 and 50. The fragmentation pathways of the aglycone ions were similar to those of compounds 52 and 50. Compounds 17 and 35 were tentatively identified as syringic acid-O-hexoside and ethyl gallate-O-hexoside. Compound 13 gave a [M − H]− ion at m/z 329.0858 and exhibited the fragment ions at m/z 167.0341 ([M − H − hexose sugar]−), 152.0105 ([M − H − hexose sugar − CH3·]−), 123.0440 ([M − H − hexose sugar − CO2]−) and 108.0205 ([M − H − hexose sugar-CO2 − CH3·]−) assigned as the moiety of vanillic acid. Based on the above observation, compound 13 was tentatively identified as vanillic acid-O-hexoside. Compound 34 exhibited the quasi-molecular ion at m/z 481.0985 ([M − H]−) which was 152.0127 Da (C7H4O4) more massive than that of compound 13. The [M − H]− ion of compound 34 lost a vanillic acid residue and a dehydrated hexose moiety in succession to form the ions at m/z 313.0564 and 169.0133. Notably, the characteristic fragment ions at m/z 169.0133, 125.0232 and 97.0282 assigned as gallic acid were observed in the MS2 spectrum of compound 34, which primarily implied a galloyl residue connected to the vanillic acid-O-hexoside. Due to the existences of vanillic acid − ions at m/z 615.0987 (err. 1.02 ppm) and 615.0988 (err. 1.23 ppm) with the same predicted molecular formulae (C28H23O16−). Compound 79 gave a similar fragment pattern to that of compound 69, where the [M − H]− ion continuously lost a galloyl unit and a hexose residue to form the ion at m/z 463.0886 and 301.0352. The fragment ions at m/z 151.0027 and 107.0126 were generated from the aglycone ion through the RDA fragmentation reaction. Compound 79 was tentatively identified as the reported quercetin-3-O--β-d-glucopyranoside − and − products of the RDA fragmentation pathway. The observed ions at m/z 463.0881, 316.0223, 164.0104 and 151.0027 were characteristic of myricetin-O-rhamnoside. Myricetin-3-O-rhamnoside-gallate has been identified as a main chemical constituent of Polygonum neofiliforme as homologous plant of P. capitatum − ion and product ions of compound 87 were 13.9797 Da higher than the corresponding ions of compound 79. The observed ions at m/z 327.0357 and 175.0240 of compound 87 suggested a glucuronic acid moiety was connected to the galloyl group. Furthermore, the ions at m/z 301.0353, 151.0027 and 107.0126 were assigned as quercetin. Therefore, compound 87 was tentatively identified as a new compound, quercetin 3-O-galloylglucuronide. Similarly, compounds 102 and 104 were tentatively identified as the reported quercetin-3-O-(6″-O-trans-feruloyl)-β-d-galactopyranoside [O-(4″-O-acetyl)-α-l-rhamnopyranoside [49 eluted at 8.63 min exhibited an [M − H]− ion at m/z 735.1563 and gave the product ions at m/z 583.1091 and 447.0930 through losing a protocatechuoyl moiety and a galloyl group in succession, suggesting the existence of protocatechuoyl and galloyl residues. In addition, the characteristic ions at m/z 447.0930, 301.0348 and 243.0295 in the MS2 spectra implied the presence of quercetin-O-rhamnoside. Although the positions of the protocatechuoyl moiety and the galloyl group were unclear, the finding of quercetin-3-O-(2″-O-protocatechuoyl)-rhamnoside and 3″-O-galloylquercitrin in this plant implied the protocatechuoyl and galloyl moieties were connected to the monosaccharide residue of compound 49. Therefore, compound 49 was tentatively identified as a new compound, quercetin-3-O--rhamnoside.Compound ranoside  and quer0785 C28HO17− and P. capitatum were divided into phenylpropanoid sucrose esters, lignans and chromones derivatives. Compound 111 (tR = 18.36 min) produced an [M − H]− ion at m/z 735.2141 . In the MS2 spectrum, the fragment ions at m/z 559.1661 (C24H31O15−) and 175.0392 (C10H7O3−) were produced via the loss of the dehydrated feruloyl group. The ion at m/z 559.1661 lost an acetylated hexosyl residue to generate the ion at m/z 337.0931, and subsequently discarded another hexosyl residue to form a feruloyl moiety . Compound 111 was tentatively identified as the reported bistoroside B − ion at m/z 705.2040 (C33H37O17−) and gave the product ions at m/z 559.1662 (C24H31O15−), 163.0389 (C9H7O3−) and 145.0284 (C9H5O2−), suggesting the existence of a coumaroyl moiety. The characteristic ions of 6′-acetyl-6(or 3)-feruloylsucrose at m/z 559.1662, 337.0943, 193.0499 and 175.0392 were also observed in the MS2 spectrum of compound 110. Compound 110 was tentatively identified as 6′-acetyl-3(or 6)-feruloyl-6(or 3)-coumaroylsucrose . For compound 77, the fragment ions at m/z 359.1498, 344.1259 and 329.1027 in the MS2 spectrum were generated via losing a hexosyl moiety and subsequent discarding two methyl groups. The RDA fragment ion at m/z 241.0500 was corresponding to the cleavage of 8-8′ bond  to form the aglycone ion at m/z 389.1605 and lost three methyl groups in succession to generate the ions at m/z 374.1370, 359.1136 and 344.0892, respectively, implying the existence of three methoxy groups in the aglycone moiety. Therefore, compound 90 was tentatively identified as 3(or 5′)-methoxyisolariciresinol-9′-O-xylopyranoside.Two isomeric compounds, -8′ bond C. Due toP. capitatum in this study were classified into proanthocyanidins and ellagitannins. Proanthocyanidins were condensed tannins composed of oligomers and polymers of flavan-3-ol moieties linked mainly through 4-8′ bonds. RDA reaction, heterocyclic ring fission (HRF) and quinone methide (QM) cleavage were the main mass fragmentation patterns of proanthocyanidins − ion at m/z 937.0953 (C41H29O26−) and gave the fragment ions at 893.1055 and 785.0838 generated from the loss of a CO2 unit and a galloyl residue, respectively. The product ion at m/z 785.0838 continuously discarded a galloyl residue to form the ion at m/z 615.0617. In addition, the ions at m/z 300.9991and 275.0198 were assigned to the moieties of ellagic acid and urolithin M5. Compound 64 was tentatively identified as davidiin. The proposed mass fragment pathway of compound 64 was shown in 33 (tR = 6.71 min) exhibited an [M − H]− ion at m/z 925.0955 (C40H29O26−). The MS2 spectra for compound 33 revealed the intense product ion at m/z 605.0789 resulting from the neutral loss of a dehydrated HHDP residue (C14H8O9). The characteristic fragment ions at 615.0638 and 309.0245 were generated through losing a C13H9O9 moiety, which implies the existence of the residue region composed of D- and E-rings. Compound 33 was tentatively attributed to phyllanthusiin C. The MS2 fragmentation pathway (Compound  pathway C of phylP. capitatum. The common fragment characteristic of phenolics is a successive or simultaneous loss of H2O and CO groups in their MS2 spectra. Compound 29 at tR 5.58 min gave a deprotonated ion at m/z 247.0246 (C12H7O6−). The MS2 spectrum of compound 29 showed the fragment ions at m/z 219.0293 and 203.0343 resulting from the neutral loss of CO and CO2 unit from quasi-molecular ion, respectively. Furthermore, the ion at m/z 219.0293 went on losing a CO and a CO2 unit in succession to form the ions at 191.0342 and 147.0440. Since brevifolin has previously been found in P. capitatum in the reported literature [29 was tentatively identified as brevifolin. Compound 46 gave a quasi-molecular ion at m/z 275.0197  and the fragment ions at m/z 257.0089 ([M − H − H2O]−), 247.0243 ([M − H − CO]−), 231.0291 ([M − H − CO2]−), 229.0138 ([M − H − H2O − CO]−) and 203.0343 ([M − H − CO − CO2]−). A lactone moiety or a carboxyl group was implied to be present in compound 46. In addition, the ion at m/z 191.0341 was generated through an RDA fragmentation reaction from the ion at m/z 231.0291. Compound 46 was tentatively identified as urolithin M5. Urolithin M5 was an intestinal bacterial metabolite of ellagitannin davidiin from P. capitatum [P. capitatum. Compound 8 (tR = 2.09 min) gave an [M − H]− ion at m/z 243.0507 (C10H11O7−) and exhibited the characteristic ions of gallic acid moiety at m/z 169.0134, 125.0233 and 107.0125. The glycerol residue at m/z 91.0388 was found in the spectrum of compound 8. Based on the reported literature [8 was tentatively identified as galloyl-glycerol.Ten phenolics with a small molecular mass (<350 Da) and less than 15 carbons were tentatively identified in apitatum  and was apitatum . The comapitatum  by endopP. capitatum. Compound 6  was tentatively identified as the reported phenylalanine [12 (tR = 2.37 min) gave an [M + H]+ ion at m/z 328.1386 (C15H23O7N+) and lost a fructose moiety to form the characteristic ion of phenylalanine (m/z 166.0859 and 120.0808). Comparing the mass information of compound 12 to those in the reported literature + ion of compound 106 lost a C2H2O2 moiety of hydroxymethylfuran ring and underwent RDA fragment reaction to yield the fragment ion at m/z 180.0804. The product ion at m/z 206.0835 was formed though losing C2H3O2· and CO2 from the [M + H]+ ion. Compound 106 was tentatively assigned as flazin. The proposed mass fragment pathway of flazin was shown for the first time in the study  generated the aglycone ion at m/z 207.1021 (C12H15O3−) through losing hexose sugar, and further lost one molecule of CO2 to form the ion at m/z 163.1118. Compound 41 was tentatively identified as the reported 12-hydroxyjasmonic acid glucoside + ions of probable metabolites from dosed plasma in full-scan mass mode and analyzing the MS2 information of the detected peak. The detected metabolic mechanism of P. capitatum in hyperuricemia rats involved ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. In this study, a total of 52 metabolites of P. capitatum in rat plasma were tentatively identified. Among them, 50 metabolites were revealed for the first time. The details of the characterized metabolites are listed in The procedures for identification of metabolites included speculating probable metabolites according to the biotransformation rules of original compounds, extracting the [M − H]M1–M3, M5–M6, M7–M12, M14, M20 and M24), 3 syringic acid/ethyl gallate-related , 2 dimethylellagic acid-related (M43 and M47), a vanillic acid-related (M17) and a protocatechuic acid-related (M4) metabolites.A total of 22 constituents were identified as phenolic-related metabolites, including 14 gallic acid-related (M18 (tR = 5.67 min) and M19 (tR = 5.94 min) exhibited the same quasi-molecular ion at m/z 373.0775  and the same fragment ion at m/z 197.0448 by the cleavage of a dehydrated glucuronic acid moiety, suggesting the two metabolites were the glucuronidation products. Furthermore, M18 showed the characteristic fragment ions at m/z 197.0448, 169.0144 and 125.0233 of ethyl gallate, while M19 showed the fragment ions at m/z 197.0447, 182.0208, 166.9976 assigned to syringic acid. Therefore, M18 and M19 were identified as ethyl gallate glucuronide and syringic acid glucuronide, respectively. M20 (tR = 6.20 min) and M26 (tR = 7.02 min) exhibited the quasi-molecular ions [M-H]− at m/z 277.0022 which were 79.9573 Da (SO3) more than that of 3,4-O-dimethylgallic acid or syringic acid and yielded the characteristic product ions of the two isomers at m/z 197.0449, 182.0213 and 166.9977. M20 and M26 were preliminarily assigned as 3,4-O-dimethylgallic acid sulfate or syringic acid-4-O-sulfate. To elucidate the exact conjugation site of M20 and M26, their ClogP values were calculated through ChemBioDraw Ultra 20.0 software. Generally, compounds with larger ClogP values tend to form longer retention times in reverse-phase chromatography. The ClogP value of 3,4-O-dimethylgallic acid sulfate (−0.53863) is smaller than that of syringic acid-4-O-sulfate (−0.18863). Thus, M20 and M26 were speculated as 3,4-O-dimethylgallic acid sulfate and syringic acid-4-O-sulfate. M2 and M19 presented [M − H]− ions at m/z 359.0620 (C14H15O11−) and lost a dehydrated glucuronic acid residue, a methyl group and a CO2 unit to form the ions at m/z 183.0292, 168.0068 and 124.0154, implying M2 and M19 were the methylated and glucuronidated products of gallic acid. Considering both 4-O-methylgallic acid and the 3-O-methylgallic acid were the main methylated metabolites of gallic acid − ions at m/z 461.0726 (C21H17O12−) and 364.9972 (C15H9O9S−), respectively. The same fragment ions at m/z 285.0402 of M44 and M49 were corresponding to the loss of a dehydrated glucuronic acid and an SO3 group from their [M − H]− ions, respectively. The ion at m/z 285.0402 furtherly lost a CH2O moiety to form the ion at m/z 255.0293. The fragment pattern of the aglycone ion was consistent with that of kaempferol in P. capitatum extract. According to the reported literature − ion at m/z 571.0396 (C22H19O16S−) and dissociated into the fragment ions at m/z 491.0832 (C22H19O13−) and 315.0511 (C16H11O7−) ascribed to the continuous losses of an SO3 unit and a glucuronic acid residue. The ion at m/z 315.0511 lost a methyl group to yield the product ion at m/z 300.0275 and furtherly was subjected to an RDA fragmentation reaction to form the ion at m/z 148.0155. The cracking path of the ion at m/z 315.0511 was similar to 3-O-methylquercetin − ion at m/z 230.9964 (C8H7O6S−) and an aglycone ion at 151.0393 through loss of an SO3 unit. The [M − H]− ion produced the fragment ions at m/z 187.0063 (C7H7O4S−) and 107.0490 (C7H7O−) by discarding a CO2 group and an SO3 group in succession, which was in high accordance with mass fragment pattern of 3-hydroxyphenylacetic acid sulfate − ion at m/z 287.0233 (C11H11O7S−) and an [aglycone − H]− ion at m/z 207.0504 generated by the loss of an SO3 moiety. The ion at m/z 163.0764 was yielded from the [aglycone − H]− ion through losing a CO2 unit. According to the reported metabolic pathway of (epi)catechin − ion at m/z 242.9966 (C9H7O6S−). In the MS2 spectrum, the fragment ions at m/z 163.0392 ([M − H − SO3]−), 135.0443 ([M − H − SO3 − CO]−) and 119.0491 ([M − H − SO3 − CO2]−) were consistent with the fragment pathway of m-coumaric acid or p-coumaric acid − at m/z 535.1823 (C26H31O12−) and gave the fragment ions at m/z 359.1497 ([M − H − glucuronyl unit]−), 344.1266 ([M − H − glucuronyl unit − CH3·]−) and 329.1036 ([M − H − glucuronyl unit − 2 × CH3·]−) in their MS2 spectra. The ion at m/z 241.0507 was formed from the ion at m/z 329.1036 through an RDA fragmentation pathway. M31, M32, M35 and M36 were preliminarily assigned as glucuronidation of isolariciresinol. The conjugation sites of the four metabolites were speculated by ClogP values. M31 (ClogP = −1.3851) and M32 (ClogP = −1.3851) were tentatively identified as isolariciresinol-4 (or 4′)-O-glucuronide. M35 (ClogP = −0.7404) and M36 (ClogP = −0.7404) were tentatively identified as isolariciresinol-9 (or 9′)-O-glucuronide.A total of 8 phenylpropanoid-related metabolites were detected in rat plasma, mainly from the products of isolariciresinol and 5,7-dihydroxychromone. α-pyrones derivatives [M38) was detected in the BPC of dosed rat plasma. M38 exhibited an [M − H]− ion at m/z 403.0669 (C19H15O10−) and lost a dehydrated glucuronic acid residue to form the ions at m/z 227.0345 ([M − H − glucuronyl unit]−) and 175.0239 (glucuronyl unit), suggesting the M38 was a glucuronic conjugate. The aglycone ion at m/z 227.0345 furtherly lost a CO group and a CO2 unit to generate the fragment ions at m/z 199.0388 and 183.0446, respectively. The ion at m/z 155.0491 was formed through the combined loss of CO and CO2 from the aglycone ion. The fragment pattern of the aglycone ion was consistent with that of urolithin A + ion at m/z 323.1024 (C18H15O4N2+) that was more 14.0161 Da (CH2 moiety) than that of flazin. The MS2 spectrum of M52 in positive ion mode showed the fragment ions at m/z 263.0814, 206.0837 and 180.0806 assigned to flazin as discussed above. Furthermore, M52 detected at m/z 321.0880 ([M − H]−) in negative ion mode yielded the fragment ions at 291.0775 and 277.0612 by losing CH2O and C2H4O, suggesting the existence of a methoxy group in M52. The ion at m/z 277.0612 furtherly lost a CO2 group and a molecule of H2O to form the ion at m/z 233.0719 and 259.0511. M52 was tentatively identified as flazin methyl ether.P. capitatum was collected from Qianxi county, Guizhou province, China and was identified by Qingde Long  as the whole plant of Polygonum capitatum Buch.-Ham. ex D. Don. The voucher specimen of P. capitatum (No.: PC20201103) was deposited in the Herbarium of Guizhou Medical University.The herb of O--β-d-glucopyranoside, quercetin-3-O-β-d-glucopyranoside, cis-N-caffeoyltyramine and 3″-O-galloylquercitrin were obtained in our laboratory. The purities of the reference standards were determined to be more than 98% by HPLC . Urate assay kits were purchased from Nanjing Jiancheng Bioengineering Institute .The reference standards of gallic acid, protocatechuic acid, (+)-catechin, rutin, quercitrin, quercetin and emodin were purchased from Chengdu Chroma-Biotechnology Co., Ltd. . The pure materials of myricitrin, quercetin-3-HPLC-grade methanol and acetonitrile were acquired from Honeywell Burdick & Jackson Company . Formic acid (MS grade) was obtained from Fisher Scientific . Deionized water for HPLC analysis was prepared using a Milli-Q water purification system . All other reagents were of analytical grade.The stock solutions of standards were prepared by weighting appropriate amounts of 13 reference substances individually and dissolving them in methanol at a concentration of 1.0 mg/mL. The final mixed standard solution (200 ng/mL) was obtained by mixing the appropriate volumes of the stock solutions and diluting with 60% methanol before qualitative analysis.P. capitatum (1453 g) was weighed and crushed into power. The obtained powder was immersed in a ten-fold volume of distilled water for 30 min and decocted three times by boiling for 1 h. The decoctions were filtered to remove the herbal residue. The supernatants were merged together and concentrated to yield the extract residue .The dried raw herb of v/v) methanol and ultrasonicated for 30 min at 100 kHz. After centrifuging at 12,000 rpm for 10 min, 10 µL of the supernatant was used for UHPLC-Q-Orbitrap HRMS analysis.10 mg of the obtained extract residue was dissolved in 1 mL of 60%  rats (weighing 200 ± 20 g) were obtained from Changsha Tianqin Biotechnology Company . The rats were housed under a standard 12-h light-dark cycle at 25 ± 2 °C and 60 ± 5% humidity with free access to water and a normal diet for 7 days. All of the experiments were approved by the Animal Care Welfare Committee of Guizhou Medical University  and performed according to the Guide for the Care and Use of Laboratory Animals .P. capitatum extract (5 g/kg/day) was administered orally to the rats in the drug-treated group at 1 h after dosing of the modeling agents for 3 days. The serum urate levels in the control and hyperuricemia rat groups were detected by urate assay kits during the experimental period. The serum urate levels in the hyperuricemia rat group significantly increased compared to those of the control rats (p < 0.05), indicating the successful establishment of the hyperuricemia model.Rats were randomly divided into three groups with six animals per group . Hyperuricemia was induced in the rats according to the described method previously . BrieflyBlood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after the last administration from the retro-orbital plexus into heparinized tubes. Plasma was obtained through centrifugation at 3000 rpm for 10 min. All plasma samples at different time points from each group of rats were combined to produce the pooled sample for eliminating the individual variability. A 500 µL volume of the pooled plasma sample was added with 1.5 mL of acetonitrile and vortexed for 1.0 min to precipitate protein. The sample was centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatant was evaporated to dryness under a gentle flow of nitrogen at room temperature. The residue was redissolved with 200 µL of 60% methanol in water and centrifuged at 12,000 rpm for 10 min. The supernatant was injected into the UHPLC-Q-Orbitrap HRMS system for analysis.® BEH C18 column  eluted with acetonitrile (A) and 0.1% aqueous formic acid (B). The flow rate was set at 0.3 mL/min with an initial mobile phase of 5% (A). The chromatographic elution program was set: 5–5% A at 0–1.0 min, 5–10% A at 1.0–4.0 min, 10–12% A at 4.0–9.0 min, 12–20% A at 9.0–14.0 min, 20–45% A at 14.0–19.0 min, 45–70% A at 19.0–20.0 min, 70–100% A at 20.0–22.0 min, 100–5% A at 22.0–22.1 min, 5–5% A at 22.1–25.0 min. The injection volume was 10 µL.A Dionex Ultimate 3000 UHPLC system  consisted of a quaternary solvent delivery system, a column compartment and a refrigerated auto-sampler. The sample separation was performed on an ACQUITY UPLC2). The parameters were set as follows: ion spray voltage: +3.0 kV and −2.5 kV, capillary temperature: 320 °C, S-lens RF level: 60%. The flow rates of sheath gas and auxiliary gas were set to 35 and 10 arbitrary units, respectively. A full MS/dd-MS2 acquisition program was executed with resolutions of 70,000 and 17,500 FWHM. For the full MS experiments, the scan range was from 80 to 1200 m/z, the automatic gain control (AGC) target was defined as 1e6 and the maximum injection time (IT) was set as auto. For the dd-MS2 experiments, AGC target: 2e5, maximum IT: auto, loop count: 1, the isolation window was 3.0 m/z. The stepped normalized collision energies (NCE) were 20, 40, and 60 eV.A Q-Exactive™ Orbitrap mass spectrometer  equipped with a heated electrospray ionization source (HESI) was used for qualitative analysis. The analysis was carried out both in positive and negative ion modes. The collision and nebulizing gases were ultra-high purity helium (He) and high purity nitrogen  and Compound Discoverer 2.0 software coupled to mzCloud© and ChemSpider© databases. The data processing workflow for the identification of chemical ingredients from P. capitatum and its metabolites was shown in The information of chemical constituents from P. capitatum and its absorbed components in hyperuricemia rats. A total of 114 compounds including phenolic acids, flavonoids, phenylpropanoids, tannins, phenolics, amino acids, amides and others were identified or characterized. At the same time, 68 P. capitatum-related xenobiotics were found in the hyperuricemia rats’ plasma. These exogenous components in hyperuricemia rats might be the potential active constituents of P. capitatum for anti-hyperuricemia and anti-gouty arthritis. The detected metabolic pathway of P. capitatum in hyperuricemia rats included ring fission, hydrolysis, decarboxylation, dehydroxylation, methylation, glucuronidation and sulfation. This study not only supplied a basis for the further investigation of the active components and pharmacokinetics of P. capitatum, but also provided insight into the anti-hyperuricemia mechanism and quality control of P. capitatum.In this study, a sensitive and accurate UHPLC-Q-Orbitrap HRMS method was utilized to systematically analyze the chemical constituents of


Enterovibrio norvegicus DSM 15893 (EnLOX) was cloned and expressed in Escherichia coli BL21 (DE3). EnLOX showed the catalytic activity of 40.34 U mg−1 at 50 °C, pH 8.0. Notably, the enzyme showed superior thermostability, and wide pH range stability. EnLOX remained above 50% of its initial activity after heat treatment below 50 °C for 6 h, and its melting point temperature reached 78.7 °C. More than 70% of its activity was maintained after incubation at pH 5.0–9.5 and 4 °C for 10 h. In addition, EnLOX exhibited high substrate specificity towards linoleic acid, and its kinetic parameters of Vmax, Km, and Kcat values were 12.42 mmol min−1 mg−1, 3.49 μmol L−1, and 16.86 s−1, respectively. LC-MS/MS analysis indicated that EnLOX can be classified as 13-LOX, due to its ability to catalyze C18 polyunsaturated fatty acid to form 13-hydroxy fatty acid. Additionally, EnLOX could improve the farinograph characteristics and rheological properties of wheat dough. These results reveal the potential applications of EnLOX in the food industry.Lipoxygenases have proven to be a potential biocatalyst for various industrial applications. However, low catalytic activity, low thermostability, and narrow range of pH stability largely limit its application. Here, a lipoxygenase (LOX) gene from  Z, 4-Z-pentadiene moieties into hydroperoxides fatty acid with (Z), (E)-diene conjugations , and mK represent the initial rate, the maximum velocity of reaction, the substrate concentration, and the Michaelis constant, respectively.Substrate specificity of EnLOX was determined with different final concentration of PUFAs from 10 to 300 mmol L follows ,30:(1)v0−1 cysteine. The reaction mixtures were extracted according to the method of HUANG et al. [−1. Mass spectrometry was performed using electrospray source in negative ion mode with MS acquisition mode, with a selected mass range of 100–1000 m/z. The lock mass option was enabled using leucine-enkephalin (m/z 556.2771) for recalibration. The ionization parameters were the following: capillary voltage was 2.5 kV, sample cone was 40 V, source temperature was 120 °C, and desolvation gas temperature was 800 °C. Data acquisition and processing were performed using Masslynx 4.1.Product specificity of EnLOX was determined by incubating PUFAs, EnLOX, and Tris-HCl buffer (pH 8.0) at 50 °C for 10 min. Hydroperoxides formed were reduced to their corresponding hydroxides with addition of 10 mmol LG et al. . The proThe effect of pH on EnLOX activity was measured from pH 3.0 to pH 9.5 using citrate sodium citrate buffer (pH 3.0–6.0), phosphate buffer solution (pH 6.0–7.0), Tris-HCl buffer (pH 7.0–9.0), and borax-sodium hydroxide buffer (pH 9.0–9.5) at 50 °C ,33. The The enzyme activity was measured at different temperatures (20–80 °C) to determine the optimum temperature of EnLOX. The maximum activity was set as 100%; the relative activities at different temperatures were plotted to obtain the optimum temperature of EnLOX ,35. Ther−1 of EnLOX, 2 μL of 10X Sypro orange, and 16 μL of 40 mmol L−1 Tris-HCl (pH 8.0) were added to the wells of the 96-well Cycler PCR plate. The plate was heated from 25 to 95 °C with a heating rate of 0.5 °C min−1. The melting point temperature (mT) tested is the results of multiple independent experiments, each in triplicate and reported as mean ± standard deviation (SD).Protein Thermal Shift was performed using Sypro Orange dye from Sigma-Aldrich . The the2, ZnCl2, KCl, MgCl2, FeCl3, FeCl2, MnCl2, NaCl, LiCl, (NH4)2SO4, CuSO4) with a final concentration of 0.1 mmol L−1 and various solvents (phenylmethanesulfonyl fluoride (PMSF), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), IPTG, β-mercaptoethanol (β-ME), and Tween 20) with a final concentration of 1.0 mmol L−1 were added to the reaction system. The residual activity was measured after 4 °C of incubation for 30 min [To investigate the effect of metal ions and solvents on EnLOX activity, different metal ions , 0 (blank control), 5, 10, 20, 30, 40, 50 IU g−1 EnLOX, respectively [Farinographical properties of wheat flour blends were examined using a Brabender farinograph-E according to AACC 54–21 method . Wheat fectively . Mean vaRheological properties were measured by using a Physica MCR301 rheometer with a parallel plate , according to the reported research with some modifications . The wheDuncan’s test and the significance level of p < 0.05 were used [All the tests were performed in triplicate, and data were expressed as mean ± SD. SPSS 21.0 were used to analyze the differences between the variables. Statistical differences in correlation analysis and linear regression analysis were determined by one-way analysis of variance (ANOVA), and ere used ,43.E. norvegicus LOX (WP_017011269). Nevertheless, no characterization experiments of LOX from E. norvegicus have been reported so far. The nucleotide sequences showed significant differences between EnLOX and LOXs from other sources  structure as a template, which revealed that EnLOX was composed of 44.0% α-helix, 8.4% strands, and 47.6% random coil (P. aeruginosa LOX (PDB ID: 4rpe) and Cyanothece LOX (PDB ID: 5med). The results also suggested that EnLOX had the lowest (21.76%) and the highest (37.47%) identity with Gaeumannomyces graminis LOX (PDB ID: 5fx8) and P. aeruginosa LOX, which indicated that it was a novel LOX. Soy flour is used in flour and dough products due to its abundant of soybean LOXs [In order to identify the structural differences with other LOXs, a 3D structural model of EnLOX was predicted. The 3D structure of EnLOX was predicted by Swiss-Model using dom coil a. The prean LOXs . The α-hB. thailandensis (75 kDa) [M. xanthus (80 kDa) [−1, which was higher than that of most other sources of LOXs, such as LOXs from Anabaena (10.4 U mg−1) [B. thailandensis (26.4 U mg−1) [P. aeruginosa (28.5 U mg−1) [Calothrix (73.1 U mg−1) [Rivularia (68.8 U mg−1) [A. aegerita LOX (51.34 U mg−1) [The purified EnLOX showed a single band with a molecular mass of approximately 80 kDa in SDS-PAGE , which w(75 kDa)  and M. x(80 kDa) . The spe U mg−1) , B. thai U mg−1) , and P.  U mg−1)  but loweandensis .4 U mg−1andensis .4 U mg−1 U mg−1) . In addimaxV, mK, catK, and cat/KmK values of LA were 12.42 mmol min−1 mg−1, 3.49 μmol L−1, 16.86 s−1, and 4.83 L μmol−1 s−1, respectively. The maxV value of LA was the highest among the substrates, indicating that EnLOX had the strongest catalytic rate to LA. This is consistent with the LOXs from basidiomycete fungus [M. xanthus [mK value of LA was the lowest among PUFAs, which exhibited the highest affinity. EnLOX showed a lower mK than most of the LOXs, such as LOXs from Calothrix (6.7 μmol L−1) [Rivularia (7.5 μ mmol L−1) [T. bouteillei (16.5 μmol L−1) [Anabaena (30.3 μmol L−1) [Nicotiana benthamiana LOX (3.9 μmol L−1) [cat/KmK value for LA of EnLOX were significantly higher than soybean LOX (0.17 L μmol−1 s−1) [M. xanthus LOX (0.02 L μmol−1 s−1) [Anabaena (0.54 L μmol−1 s−1) [Pleurotus ostreatus LOX (0.2 L μmol−1 s−1) [Calothrix (13.85 L μmol−1 s−1) [Rivularia (10.79 L μmol−1 s−1) [Kinetic parameters of EnLOX were determined, which showed a discrepancy in the kinetic properties for various PUFAs . The Vma xanthus . The Km mol L−1) , Rivularmol L−1) , T. boutmol L−1) , and Anamol L−1)  and simimol L−1) . The Kcal−1 s−1) , M. xantl−1 s−1) , Anabaenl−1 s−1) , and Plel−1 s−1)  but lowel−1 s−1)  and Rivul−1 s−1) . The res12-C13 double bond, and oxygenation at position C13 to form 13-HFAs [Products of EnLOX catalyzed PUFAs were analyzed by LC-MS/MS . The mai 13-HFAs ,48,49. T1/2) of EnLOX at 50 °C was about 6 h, which was much longer than that of LOXs from soybean (T1/2 = 30 min at 45 °C) [M. xanthus  [P. aeruginosa  [Anabaena  [mT) value of EnLOX was 78.7 °C, much higher than that of human 5-LOX (36.5 °C) [The activity of EnLOX was measured at different temperatures, as shown in , 50 °C) , and Ana, 50 °C) . The thean 5-LOX .5 °C [3636.5 °C) , which r36.5 °C) . These rM. xanthus LOX is only stable in the range of pH 3.0–6.0 [B. thailandensis [Cyanothece [The activity of EnLOX was assayed at different pHs, as shown in  3.0–6.0 . LOXs fr 3.0–6.0 , B. thaiandensis , and Cyaanothece  are only2+, Mn2+, Fe3+, and DMSO enhanced EnLOX activity by 2.37, 2.12, 1.89, and 2.13 times, respectively. The promotion of enzyme activity by metal ions possibly related to the N-terminal PLAT domain, cofactors, and coenzyme of EnLOX [M. xanthus LOX [Cyanobacterium Cyanothece LOX [2+, Li+, and K+ had no effects on the enzyme activity. The activating effect on EnLOX of DMSO may be due to DMSO changing some secondary bonds in the enzyme and influencing the flexibility of the activity site [2+, Mn2+, Fe3+, and DMSO provide new methods for the industrial application of EnLOX to optimize the fermentation conditions.As shown in of EnLOX ,56. Theshece LOX . Zn2+, Lity site . The enzity site . Some soity site . The act−1 EnLOX increased, softening degree decreased. Water absorption, dough development time, and stability time of flours fortified with 20 IU g−1 EnLOX increased by 3.89%, 0.50 min, and 1.14 min, respectively; softening degree was reduced by 39 FU. Fatty acid hydroperoxides generated by EnLOX in the catalytic process can induce protein polymerization [3, had no significant changes apart from the softening degree decreasing significantly. EnLOX exhibited favorable farinographical properties of wheat flour and could be added in flour products to improve its quality.The effects of EnLOX on the farinographical properties of wheat flour are shown in rization , which prization . The indG′) (G″) value (G″/G′) [−1 EnLOX exhibited higher elastic and viscous characteristics than that of the blank group, indicating that EnLOX could enhance the cross-linking of gluten [Anabaena LOX [Rheological characteristics of dough treated with different concentrations of EnLOX were evaluated . Dough sG′) a and vis″) value b; both o (G″/G′) . With thf gluten . EnLOX haena LOX  and soybaena LOX , indicataena LOX .Enterobacter norvegicus was expressed and characterized in this study. EnLOX only showed 37.47% identity with P. aeruginosa LOX, and the enzyme exhibited superior properties; good thermostability at 50 °C; outstanding pH stability at pH 5.0–9.5; and high activity of LA, achieving 40.34 U mg−1. In addition, the application of EnLOX to wheat dough further improved the farinograph and rheological properties. Our research indicated EnLOX is a potential enzyme agent for food industrial applications.To overcome the low enzyme activity, poor thermal stability, and narrow pH range stability of existing LOX enzymes, a novel LOX from


CDK4/6) are key regulatory proteins in the cell division and proliferative cycle in humans. They are overactive in many malignant tumors, particularly in triple-negative breast cancer (TNBC). Inhibition of CDK4/6 targets can have anti-tumor effects. Here, we designed and synthesized a novel derivative of Ribociclib that could affect CDK4/6, named WXJ-202. This study aimed to investigate the effects of compound WXJ-202 on proliferation, apoptosis, and cell cycle arrest in human breast cancer cell lines and their molecular mechanisms. We assayed cell viability with methyl thiazolyl tetrazolium (MTT) assay. Clone formation, migration, and invasion ability were assayed by clone formation assay, wound healing assay, and transwell invasion assay. The effect of compound WXJ-202 on apoptosis and cell cycle was detected by flow cytometry analysis. Western blotting was performed to detect the expression of proteins related to the CDK4/6-Rb-E2F pathway. The anti-cancer effects were studied in vivo transplantation tumor models. WXJ-202 was shown to inhibit cell proliferation, colony formation, migration, and invasion, as well as induce apoptosis and cycle arrest in breast cancer cells. The levels of proteins related to the CDK4/6-Rb-E2F pathway, such as CDK4, CDK6, and p-Rb, were decreased. Finally, studies had shown that compound WXJ-202 exhibited significant anti-tumor activity in transplantation tumor models. In this research, the compound WXJ-202 was shown to have better anti-tumor cell proliferative effects and could be used as a potential candidate against TNBC tumors.Cyclin-dependent kinases 4 and 6 ( Breast cancer is among the most common malignancies in women . Breast CDKs) of the serine/threonine protein kinase family include transcriptional regulators  and cell cycle regulators  , as well as chromosomal instability (changes in chromosome number) to participate in tumor development   . Dysreguelopment . CDKs had growth . In the ein (RB) . This prein (RB) . Overexpt cancer . Inhibitlication . It sele S phase . Thus, tmaciclib . Previou  phenyl) amino)-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo  pyrimidine-6-carboxamide (WXJ-202). We speculated that WXJ-202 and Ribociclib may have similar inhibitory effects against TNBC. Based on the molecular docking results, compound WXJ-202 was found to have a high binding capacity for The following instruments were used: electronic balance , 45–75 μM silica gel powder for column chromatography, silica gel GF254 for thin layer chromatography (TLC), UV lamp (model UV-IIB) for color development, Q Exactive HF LC-MS (Thermo Fisher Scientific) for mass spectrometry, Avance III 500 MHz liquid nuclear magnetic resonance spectrometer . All other reagents were analytically pure and used directly without further processing.CDK4 and CDK6 were downloaded from the PDB data bank (http://www.rcsb.org/) and used for docking studies. The proteins were pretreated with Pymol to remove HOH and metal ions. Ligand structures were constructed using Chem3D. The docking results were visually inspected to determine the binding ability of the analyzed compounds at the active site.All molecular docking research were conducted on a Viglen Genie Intel (R) Core (TM) i5-7300HQ CPU @ 2.50 GHz running Windows 10 1909. AutoDockTools-1.5.6 and Pymol-1.7.0.0.win 32-py2.7 as software for molecular modeling. The X-ray co-crystallized structure of 2 in a constant temperature incubator . 0.25% trypsin was added when the confluence of cells was 70%–80% for digestion, passagework, and culture. For the next study, cells in the logarithmic growth phase and favorable growth state were selected.The human cancer cell lines MDA-MB-231, MCF-7, A498, and Hela cells were purchased from the National Biomedical Laboratory (Beijing) and cultured in DMEM (KGM12800-500) or MEM medium (KGM41500-500) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the cells were incubated at 37°C and 5% CO4 cells per well. After the cells had adhered, the medium in the 96-well plate was removed, and the cell culture medium with various concentrations of the compound WXJ-202 and the positive drug was added to the various groups, respectively. The cells were then treated for 24 h. 10 μL of methyl thiazolyl tetrazolium (MTT) solution was added to each well and continued to be incubated for 4 h. After the supernatant was removed, 100 μL of dimethyl sulfoxide (DMSO) was added to each well for color development. Each group’s absorbance (OD) was measured at 490 nm using a multifunctional microplate detector .Inoculated cells into 96-well culture plate with 1 × 10The following formula was used to determine the inhibition rate based on the OD values obtained from a multipurpose microplate detector . If the inhibition rate was ≤0%, it was recorded as 0%.5 cells/well) were inoculated into 12-well plates and kept overnight at 37°C and 5% CO2. After that, the cells were exposed to different concentrations of WXJ-202, and positive drugs, respectively. Images were taken with an inverted microscope (Gangnam XD-202).The effect of WXJ-202 on MDA-MB-231 cells was determined by morphological changes. MDA-MB-231 cells , cells were counted and inoculated at 10,000 cells per well in a 96-well plate that had been pre-coated with matrix gel . The supernatant was removed after 1 h and 10 μL of MTT solution was added to continue the incubation for another 4 h. The supernatant was discarded and 100 μL of dimethyl sulfoxide (DMSO) was added to each well for color development. Each group’s absorbance (OD) values were then measured at 490 nm using a multifunctional microplate detector  to calculate the adhesion rate.The MDA-MB-231 and MCF-7 cells were plated in 12-well culture plates and cultivated for 24 h at 37°C with 5% CO5 cells/well, respectively. Overnight incubation allowed them to grow as a single cell layer. Scratch over the cell layer in the central portion of each well with a sterile pipette tip to create a scratch. Following that, cells were cleaned of cellular debris using sterile PBS. Cells were incubated with different concentrations of the compound WXJ-202 or positive drug in a complete medium. Images were taken at 0, 24 and 48 h using an inverted optical microscope (Gangnam XD-202). Observed the healing of scratches with time. The percentage of wound closure at 24 and 48 h was estimated from the closed area of migratory cells by using ImageJ software. The formula for calculating the scratch healing rate was as follows.MDA-MB-231 and MCF-7 cells were inoculated into 12-well cell culture plates at a density of 1.5 × 102 in an incubator for 4 h. Different concentrations of compound WXJ-202 and positive drug were added to MDA-MB-231 and MCF-7 cells, respectively, and the negative control concentration was 0 µg/ml. After 24 h, the cells were collected by centrifugation and prepared into 100 μL single cell suspension using serum-free medium containing 0.2% BSA and added dropwise to the upper chamber. Meanwhile, 650 µL of medium containing 10% fetal bovine serum (FBS) was added to the lower chamber as a chemotactic invasion inducer. After 24-48 hours of incubation, cells on the surface of the upper chamber of the Transwell were wiped off with a cotton swab. Cells that invaded the lower chamber of Transwell were fixed in paraformaldehyde, washed with PBS after 15 min, and stained with Giemsa or Crystal violet for 30 min. The staining solution was washed off with deionized water, dried, and placed under an inverted light microscope (Gangnam XD-202) to photograph randomly selected areas. Invasive cells were counted with ImageJ software.The matrix gel  was pre-thawed overnight at 4°C and diluted to 200 μg/mL using DMEM serum-free medium according to the instructions. 100 μL was wrapped on the bottom of the upper chamber surface of the Transwell and air-dried at 37°C and 5% CO2 incubator. After the cells were plastered, a culture medium containing different concentrations of compound WXJ-202 or positive control drug was added to each well, while a negative control group was set up. After 12 h of treatment, the medium was replaced with the normal medium, and the culture was continued for another 2 weeks with medium changes every 3 days. After the clones were formed, they were washed with PBS and fixed with 4% paraformaldehyde. The plates were then stained with Giemsa or Crystal violet, dried, and observed for colony formation. Finally, the well plates were inverted, photographed, and counted with ImageJ software.The logarithmic growth phase MDA-MB-231 cells were inoculated into 6-well culture plates and MCF-7 cells were inoculated into 12-well plates, adding 1,000 cells of logarithmic growth stage to each well. The cells were evenly dispersed and incubated at 37°C in a 5% CO5 cells per well and incubated at 37°C with 5% CO2. The following day, different concentrations of compound WXJ-202 and positive drugs were added, respectively. After a 24 h treatment period, cells were collected by trypsinization without EDTA and centrifuged at 1,000 rpm for 10 min. After washing twice with PBS by gentle blowing, incubated with 10 μL Annexin V-FITC solution and 5 μL propidium iodide staining solution for 15 min at room temperature under light-proof conditions. The apoptosis rate of each group of cells was detected on a Guava flow cytometer (Guava easycyte 6HT 2L) and analyzed by Flowjo software.The MDA-MB-231 cells were inoculated into 6-well plates at a density of 5 × 106 cells per well. After the cells adhered, the serum was withdrawn for 24 h to synchronize the cells. A fresh culture medium containing different concentrations of compound WXJ-202 or positive drugs was replaced for 24 h treatment. The cells were digested with 0.25% trypsin and collected by centrifugation at 800 rpm for 5 min. 500 μL of pre-cooled phosphate buffered solution (PBS) was used to resuspend the cells twice, each time at 800 rpm for 5 min. PBS was discarded and the cells were fixed in 70% pre-cooled anhydrous ethanol at 20°C for 12–24 h. After being centrifuged and resuspended for 5 min, the fixed cells underwent three PBS washes. The supernatant was discarded and RNaseA was added for 30 min at 37°C. Finally, 500 μL of propidium iodide (PI) was added and stained for 30 min at room temperature and protected from light, and the cell cycle was measured using a flow cytometer (Guava easycyte 6HT-2 L) and analyzed using Flowjo software.The MDA-MB-231 cells were seeded in 6-well plates at 1 × 10CDK4 , CDK6 , Cyclin D1 , E2F1 , Rb , p-Rb , Caspase-3 , Pro-caspase-3 , β-actin . Incubated overnight at 4°C on a shaker. The next day, the membrane was washed 4 times with 1 × PBST, 5 min/time, and the corresponding secondary antibody diluent  was added, and incubated at room temperature for 45 min. Washed the membrane 4 times with 1 × PBST, 5 min/time. Dropping luminescent liquid to develop on the imaging system. Data were processed using ImageJ software.The MDA-MB-231 cells were inoculated in 6-well plates and treated with different concentrations of compound WXJ-202 or positive drugs after the cells adhered. After culturing in a cell incubator for 24 h, the cells of each group were collected. The cells were lysed with the cell lysis solution. After high-speed centrifugation, the supernatant was extracted and the protein was quantified by the BCA method. An appropriate amount of protein was taken, electrophoresed in 1 × SDS-PAGE buffer, and transferred to the methanol-pretreated PVDF membrane by the “wet transfer method.” The membrane was closed with 5% skimmed milk for 45 min, followed by adding primary antibody dilution : 6 cells/mL. resuspended cells were implanted into the vessels. After 4 days of implantation, a negative control group (DMSO), a positive control group and the experimental group were set up with 6 chicken embryos per group (n = 6). Different concentrations of WXJ-202 and positive drugs were added to the chicken embryo allantoic membrane, respectively. After 3 days of treatment with the drug, the tumors were removed, photographed, weighed, and recorded.The breeding eggs were purchased from the Wufengshan Chicken Farm in Tongling, Anhui, China. All animal experiments followed the standards of the Animal Ethics Committee. Breeding eggs were incubated in a sterile environment at 37°C for 7 days, and then the air chamber was transferred to 1 cm from the umbilical blood vessel. The windows were sealed with sterile plastic wrap for later use. The cells were resuspended using a mixed suspension (1:1) of Matrigel and DMEM and adjusted them to 1.2-1.5 × 10p values under 0.05.GraphPad Prism 9 software was used for the statistical analysis, and the data were expressed as the mean ± S.D of three independent experiments. The t-test was used to compare the data between the two groups. Statistics were considered significant for 2-((3-chloro-4-(pyridin-2-ylmethoxy) phenyl) amino)-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo  pyrimidine-6-carboxamide (WXJ-202).2 , Cs2CO3 , XantPhos  and 1,4-dioxane (25 mL), under the condition of 110°C, after stirring reaction for 5 h, monitoring should be until the raw material point disappears (developing solvent: dichloromethane: methanol = 40:1) under reduced pressure to remove the solvent to give a dark brown oil. Through rapid column chromatography separation , the target product (0.278 g) was obtained as a pale-yellow solid with a yield of 56.6%  δ 8.62 , 8.59 , 8.06 , 7.75 , 7.67 , 7.41 , 7.26–7.20 , 6.94 , 6.42 , 5.29 , 4.73 , 3.15 , 2.65–2.46 , 2.14–2.00 , 1.67 ; Carbon spectrum of compound WXJ-202  δ 164.10, 157.11, 155.41, 152.00, 151.71, 149.04, 148.87, 136.95, 134.70, 131.96, 123.13, 122.66, 121.25, 121.19, 118.24, 114.39, 112.43, 100.91, 71.84, 58.13, 30.15, 24.41; HR-MS m/z: 491.1965[M + H]+.Under nitrogen protection, a 250 mL round bottom flask was charged with 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo pyrimidine-6-carboxamide,3-chloro-4-(2-pyridylmethoxy) aniline , Pd(OAc)of 56.6% . Hydrogeof 56.6% : 1H NMR  WXJ-202 : 13C NMRCDK4 and CDK6 to the compound WXJ-202, the program AutoDockTools-1.5.6 was used to perform a molecular docking study. The position of docking binding of compound WXJ-202 in CDK4 protein kinase , (51.9 ± 1.9%) and (75.8 ± 4.7%), respectively. In comparison to untreated cells [negative control group (42.9 ± 0.5%)], G1-phase cells in the compound WXJ-202 group grew by almost 33% at the 40 μM dose. The G0/G1 phase ratios in the positive control Ribociclib group (53.3 ± 2.3%) and the Imatinib group (56 ± 0.8%) were both lower than that of the compound WXJ-202 group at the same concentration . The aboCDK4, CDK6, Cyclin D1, and E2F1, and the p-Rb/Rb ratio was reduced in a dose-dependent manner , , and , respectively, with significantly enhanced ability to inhibit tumor angiogenesis and stronger effect than the positive drug Ribociclib at the same concentration  (in vivo.To further investigate the ± 2.40%) . Meanwhi± 2.40%) . Our expCDKs are essential regulators of the tumor cell cycle (Cyclin D activates CDK4/6 to form the CDK4/6-Cyclin D complex, which phosphorylates a series of substrates, including Rb (Rb releases the E2F transcription factor, which can activate and transcribe genes required for entry into the S phase (CDK4/6 inhibitors approved by the FDA for the treatment of breast cancer, but patients develop drug resistance with long-term use (The cell cycle is an essential part of cellular life activity, and its proper conduct must be precisely and tightly regulated by regulatory factors at all levels . CDKs arll cycle . Cyclin uding Rb . Phospho S phase . Once th S phase . There i S phase . In thisterm use . WXJ-202CDK4/6 overexpression results in abnormalities in the CDK4/6-Rb-E2F pathway, which is a core cause of the development of breast cancer (CDK4, CDK6, Cyclin D1, and E2F1 expression was inhibited. Meanwhile, the ratio of p-Rb/Rb also decreased with increasing concentration of WXJ-202. These results indicated that WXJ-202, after acting on breast cancer cells, could exert anti-tumor effects by inhibiting the expression of CDK4 and CDK6, thus affecting the formation of CDK4/6-Cyclin D1 complex and inhibiting the phosphorylation of Rb. In addition, the results also showed an increase in the apoptosis-related protein Caspase-3 and a decrease in the expression level of Pro-caspase-3. This suggested that the compound WXJ-202 could exert anti-TNBC effects by interfering with the CDK4/6-Rb-E2F pathway. Uncontrolled Cyclin D1-CDK4/6-mediated Rb phosphorylation in tumor cells leads to sustained cell division and tumor growth (Rb and the blocking effect was superior to that of the positive drug Abemaciclib at the same concentration. Consistent with the above findings, compound WXJ-202 significantly inhibited tumor growth in the chicken embryo chorioallantoic model. These results emphasized that compound WXJ-202 was a potential targeted agent for the treatment of TNBC with excellent and significant anti-tumor cell proliferation activity.t cancer . In our r growth . CompounCDK4/6-Rb-E2F pathway-associated proteins, producing antitumor effects on breast cancer cells in vitro and in vivo. WXJ-202, as a novel Ribociclib derivative, provided the basis for exploring drugs for the treatment of TNBC.In conclusion, our research demonstrated that WXJ-202 induced apoptosis and cycle arrest through affected


Myocardial ischemia-reperfusion injury (MIRI) represents a coronary artery disease, accompanied by high morbidity and mortality. Sevoflurane post-conditioning (SPC) is importantly reported in myocardial disease. Accordingly, the current study sought to evaluate the role of Sevo in MI/RI. Firstly, MI/RI models were established and subjected to SPC. Subsequently, pathological injury in the myocardium, myocardial infarction areas, H9c2 cell viability, apoptosis, and levels of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and lactate dehydrogenase (LDH) were all measured. Ubiquitin-specific peptidase (22USP22), lysine-specific demethylase 3A (KDM3A), and Yes1 associated transcriptional regulator (YAP1) were down-regulated in H9c2 cells using cell transfection to verify their roles. The interaction between USP22 and KDM3A and between KDM3A and YAP1 was further validated. USP 22, KDM3A, and YAP1 were found to be down-regulated in MI/RI and SPC protected MI/RI rats, as evidenced by up-regulated expressions of USP22, KDM3A, and YAP1, reduced pathological injury in the myocardium, myocardial infarction areas, apoptosis, and levels of CK-MB, cTnI, and LDH, and enhanced H9c2 cell viability; while the protective effects of Sevo were counteracted by silencing of USP22, KDM3A, and SPC upregulated USP22, which stabilized KDM3A protein levels via deubiquitination, and KDM3A inhibited histone 3 lysine 9 di-methylation (H3K9me2) levels in the YAP1 promoter to encourage YAP1 transcription, to reduce MI/RI.  Sevo post-treatment reduces MIRI and up-regulates USP22 expression.USP22 silencing annuls the inhibitory effect of Sevo on MIRI.USP22 stabilizes KDM3A by deubiquitination, and KDM3A promotes YAP1 transcription.Silencing of KDM3A or YAP1 annuls the inhibitory effect of Sevo on MIRI.Sevo reduces MIRI by promoting USP22 to stabilize KDM3A and enhance YAP1.1.Ischemia-reperfusion injury (I/RI) is attributed to the alterations of normal functioning and structure in the critical period of blood flow restoration after the occurrence of ischemia. More specifically, I/RI is associated with numerous detrimental influences, including tissue non-uniform flow and cell necrosis and swelling despite the beneficial effects from I/RI by reversing ischemia . One sucSevoflurane (Sevo) is well-established for its beneficial effects in improving myocardial function repair by inhibiting inflammatory reactions and reducing pathological injury in the heart . Sevo po2.2.1Guidelines for the Care and Use of Laboratory Animals provisions of administration and usage of laboratory animals  were procured and allowed to acclimatize for a duration of 1 week in a controlled environment, with 12-h light/dark cycles at a temperature of 23°C and humidity of 65%, with ad libitum access to food and water. Subsequently, the rats were intraperitoneally anaesthetized with 1% sodium pentobarbital solution . MI/RI rat modeling was carried out according to a previously published method . Briefly2.33-(LVESD)3]/(LVEDD)3] × 100%, and the value of LVFS% as: [(LVEDD-LVESD)/LVEDD] × 100%.The cardiac function of rats was evaluated in accordance with a previously published method . When th2.4H&E staining was performed according to a previously published method . At 30 m2.5Myocardial infarction area was measured according to a previously published method . At 120 2.6At 120 min after reperfusion, abdominal aorta blood samples were obtained and centrifuged at 4°C at 1800 g for 10 min to harvest the serum . Levels 2.72. Subsequent experimentation was carried out when cells reached 80%–90% confluence. H/R cell models were established according to a previously published method [2 for 15 min at the start of reoxygenation). In accordance with the manufacturer’s instructions of the Lipofectamine 2000 reagent , small interfering (si)-USP22-1, si-USP22-2, si-KDM3A-1, si-KDM3A-2, si-YAP1-1, and si-YAP1-2 plasmids  were transfected into H9c2 cells 6 h prior to H/R.Rat cardiomyocytes H9c2  were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum  and incubated at 37°C and 95% humid air with 5% COd method . Briefly2.8−ΔΔCt method [Total RNA content was obtained from the myocardium or cardiomyocytes using the Trizol reagent , followed by the determination of the concentration and purity of RNA with a NanoDrop spectrophotometer. Subsequently, the obtained RNA was reverse-transcribed into cDNA with the help of reverse transcription kits . The target genes were amplified with 1 μg cDNA each group as a template. qRT-PCR was carried out using an ABI7900 Fast RT PCR System  with SYBR Green Master Mix kits . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was adopted as the internal reference. Primers were shown in t method .Table 1.2.9The protein content was detected using Western blot in accordance with a previously published method . Total p2.102. OD values at a wavelength of 450 nm were assessed using a microplate reader  to evaluate the cell proliferation rate.Cell viability was evaluated at the 0 h, 24 h, 48 h, and 72 h time intervals following the manufacturer’s instructions of the CCK-8 kit  [2.116 cells/mL. Next, 100 μL solution containing 1 × 105 cells were transferred into 5 mL culture tube and cultured with 5 μL Annexin V-FITC and 5 μL propidium iodide at room temperature (25°C) in conditions devoid of light for 15 min with gentle rotation. Additionally, 400 μL 1 × binding buffer solution was supplemented into each tube to analyze the cells using flow cytometry. The fluorescence of FITC and PI was detected with 515 nm and over 560 nm band-pass filters excited at 488 nm.Cardiomyocyte apoptosis was calculated by means of flow cytometry and Annexin V-fluorescein isothiocyanate (FITC) staining . The tre2.12CK-MB and cTnI levels were detected with the help of CK-MB ELISA kits  and cTnI ELISA kits  following the manufacturers’ instructions, respectively ,26. Addi2.13Immunoprecipitation and ubiquitination assays were performed in accordance with previously published literature ,28. For Ubiquitination determination was carried out as the transfected cells were lysed using RIPA buffer containing 0.1% SDS to a final concentration of 0.2% SDS, and then subjected to identical operations as the immunoprecipitation detection. In addition, Western blot analysis was conducted using an anti-Ub antibody .2.14ChIP assay experimentation was conducted with the help of EZ-ChIP kits  . Briefly2.15p < 0.05 was regarded statistically significant.Data analyses were performed using the GraphPad Prism 8.0 software . Measurement data were presented as mean ± standard deviation. One-way or two-way analysis of variance (ANOVA) was carried out for comparison analysis among multiple groups, and Tukey’s multiple comparisons test was adopted for posttest of data. A value of 3.The current study set out to investigate the protective mechanism of sevoflurane post-treatment via regulation of the USP22/KDM3A/YAP1 axis on MI/RI. Herein, we established MI/RI rat models, subjected them to sevoflurane post-treatment, and then observed the changes of myocardial injury and the expression of USP22. Additionally, we utilized lentivirus containing sh-USP22 to infect rats to observe the changes in myocardial injury. Thereafter, we adopted H9c2 cells to establish in vitro models (H/R) through hypoxia reoxygenation, which were post-treated with sevoflurane and transfected si-USP22 to investigate the effects of sevoflurane and USP22 on cardiomyocyte apoptosis and the expression patterns of myocardial injury markers in the H/R models. In addition, the binding relationship between USP22 and KDM3A was verified by means of Co-IP and ubiquitination assay, and the action relationship of KDM3A on YAP1 was detected using a ChIP assay. Lastly, rescue experimentation was carried out by inhibiting KDM3A or YAP1.3.1p < 0.05), disturbed myocardial fiber arrangement, localized degeneration, rupture, cardiomyocyte edema, and inflammatory cell infiltration (p < 0.05), increased myocardial infarction area (p < 0.05), in addition to elevated the levels of CK-MB, cTnI and serum LDH (p < 0.05). On the other hand, the rats in the MI/RI + Sevo group exhibited increased LVEF% and LVFS% (p < 0.05), normal myocardial fiber arrangement, alleviated cardiomyocyte interstitial edema, and quenched inflammatory cell infiltration (p < 0.05), declined myocardial infarction area (p < 0.05), in conjunction with decreased levels of CK-MB, cTnI and serum LDH levels (p < 0.05). Existing evidence also indicates that USP22 is poorly-expressed in MI/RI [p < 0.05). Collectively, our findings indicated that SPC ameliorated rat MI/RI and up-regulated USP22 expression levels in the myocardium.To investigate the protective effects of SPC on MI/RI rats, MI/RI rat models were firstly established and subjected to SPC treatment. The rats in the MI/RI group presented with reduced LVEF% and LVFS% (in MI/RI . To furt3.2p < 0.05). Subsequent findings demonstrated that LVEF% and LVFS% were both reduced (p < 0.05), myocardial fiber arrangement was disturbed, cardiomyocyte interstitial edema was augmented and inflammatory cell infiltration was enhanced (p < 0.05), while myocardial infarction area was increased (p < 0.05) and levels of CK-MB, cTnI and LDH levels in the serum were elevated following USP22 silencing (p < 0.05), indicating that USP22 ablation could reverse the protective effects of SPC on MI/RI rats.To further elucidate whether SPC mitigated rat MI/RI by upregulating USP22 expression, MI/R rats subject to SPC were then injected with LV-sh-USP22 (3.3p < 0.05), suppressed cell viability (p < 0.05), strengthened apoptosis rate (p < 0.05), enhanced cleaved-caspase-3 protein levels (p < 0.05), elevated CK-MB and cTnI levels (p < 0.05) and augmented LDH release (p < 0.05), whereas all these trends were rescued by SPC. Overall, these findings indicated that SPC protected H/R-treated H9c2 cells in vitro and up-regulated USP22 expression.To further explore the mechanism of SPC in MI/R rats, in vitro MI/RI models were established as H9c2 cells were subjected to H/R treatment, which led to down-regulated USP22 expression levels (3.4p < 0.05), followed by combined experimentation with SPC. When USP22 was silenced, H9c2 cell viability was suppressed (p < 0.05), apoptosis rate was augmented (p < 0.05), cleaved-caspase-3 protein levels were enhanced (p < 0.05), CK-MB and cTnI levels were elevated (p < 0.05) and LDH release was strengthened (p < 0.05), highlighting that USP22 knockout could reverse the protective effects of SPC on H/R-treated H9c2 cells.Furthermore, we verified the function of SPC in H/R-treated H9c2 cells via the regulation of USP22 expression in vitro as si-USP22-1 and si-USP22-2 were transfected into H9c2 cells to inhibit USP22 expression (3.5p < 0.05), whereas KDM3A protein levels were poorly-expressed in MI/RI rats and H/R-treated H9c2 cells, and SPC led to elevated KDM3A mRNA levels, while USP22 silencing brought about the opposing trends (p < 0.05). Additionally, KDM3A ubiquitination levels were detected, and it was unraveled that USP22 knockout resulted in increased KDM3A ubiquitination levels (p < 0.05). Altogether, these findings suggested that USP22 could modulate KDM3A levels via deubiquitination at the post-transcriptional level.Subsequently, we explored the downstream target gene of USP22. Existing evidence suggests that USP22 is a deubiquitinating enzyme, and is capable of stabilizing protein level via deubiquitination . More imp < 0.05), and si-KDM3A-1, which exhibited better silencing efficiency, was selected to verify whether KDM3A could regulate YAP1 expression. Firstly, H3K9me2 levels were detected in rat myocardium and H9c2 cells, and it was found that H3K9me2 levels were up-regulated in MI/R rat myocardium and H/R-treated H9c2 cells, while being reversed by SPC, and further increased as a result of USP22 or KDM3A silencing (p < 0.05). Next, H3K9me2 enrichment in the YAP1 promoter region was detected using a ChIP assay, which revealed that KDM3A silencing brought about up-regulated H3K9me2 levels in the YAP1 promoter region (p < 0.05). Meanwhile, YAP1 mRNA levels changed in the opposite trend to those of H3K9me2 (p < 0.05). All in all, these findings indicated that KDM3A enhanced YAP1 transcription by down-regulating H3K9me2 level in the YAP1 promoter region.Furthermore, we explored the downstream target genes of KDM3A. KDM3A being a histone demethylase, we speculated whether KDM3A could modulate its downstream genes by histone demethylation in MI/RI. Among the downstream target genes, we focused on the YAP1 gene. Prior evidence suggests that YAP1 expression could be suppressed by H3K9me2 , and YAP3.6p < 0.05), apoptosis rate was strengthened (p < 0.05), cleaved-caspase-3 protein levels were enhanced (p < 0.05), CK-MB and cTnI levels were elevated (p < 0.05) and LDH release was enhanced (p < 0.05), suggesting that KDM3A silencing could reverse the protective effects of SPC on H/R-treated H9c2 cells.To further explore the involvement of KDM3A in the protective mechanism of SPC in H/R-treated H9c2 cells, si-KDM3A-1 was selected to perform combined experimentation with SPC. Following KDM3A silencing, H9c2 cell viability was suppressed (3.7p < 0.05), with si-YAP1-2, the one with better transfection efficiency selected to conduct the combined experiment with SPC. Following YAP1 silencing, H9c2 cell viability was suppressed (p < 0.05), apoptosis rate was strengthened (p < 0.05), cleaved-caspase-3 protein levels were enhanced (p < 0.05), CK-MB and cTnI levels were elevated (p < 0.05) and LDH release was enhanced (p < 0.05), indicating that YAP1 silencing could reverse the protective effects of SPC on H/R-treated H9c2 cells.Lastly, whether YAP1 was implicated in the protective mechanism of SPC in H/R-treated H9c2 cells was verified as si-YAP1-1 and si-YAP1-2 were transfected into H9c2 cells to silence YAP1 expression  of Sevo underscore its potential clinical application in regard to coronary artery disease treatment, and previously published data further suggests that Sevo attenuates myocardial injury and repairs cardiac function, in order to defend against MI/RI . MoreoveMeanwhile, the KDM3A enzyme is known to be capable of eliminating ubiquitination to stabilize protein levels . In addiAdditionally, there is much evidence to suggest that KDM3A can mediate its downstream gene transcription via histone demethylation . For insAltogether, our findings indicated that SPC up-regulated USP22, which stabilized KDM3A protein level via deubiquitination, and KDM3A inhibited H3K9me2 levels in the YAP1 promoter region to encourage YAP1 transcription, leading to reduction of MI/RI. The evidence supported a therapeutic implication for MI/RI alleviation in the form of USP22 upregulation. However, we failed to elucidate whether SPC could directly up-regulate USP22 to target KDM3A. In addition, there are a few limitations to our research, for instance, we solely explored the protective role of SPC in MI/RI; we only researched the effects of SPC on the USP22/KDM3A/YAP1 axis; and we adopted only male rats in the animal experiments; we could not verify the function of the USP22/KDM3A/YAP1 axis in vivo, all of which could influence the overall results. Moreover, screening the dosage of sevoflurane prior to experimentation could be advantageous to our study. We shall experiment further to investigate if Sevo could directly mediate USP22. Additionally, we will continue to explore the role of Sevo pre-conditioning in MI/RI, the regulation of Sevo in other possible axes in MI/RI, and also include female rats for animal experimentation in our future endeavors.5.Sevoflurane post-conditioning can stabilize KDM3A protein levels by up-regulating USP22, whereas KDM3A can protect against MI/RI in rats by reducing H3K9me2 methylation in the YAP1 promoter and promoting YAP1 transcription.


The ribosome, a multicomponent assembly consisting of RNA and proteins, is a pivotal macromolecular machine that translates the genetic code into proteins. The large ribosomal subunit rRNA helix 68 (H68) is a key element in the protein synthesis process, as it coordinates the coupled movements of the actors involved in translocation, including the tRNAs and L1 stalk. Examination of cryo-electron microscopy (cryo-EM) structures of ribosomes incubated for various time durations at physiological temperatures led to the identification of functionally relevant H68 movements. These movements assist the transition of the L1 stalk between its open and closed states. H68 spatial flexibility and its significance to the protein synthesis process were confirmed through its effective targeting with antisense PNA oligomers. Our results suggest that H68 is actively involved in ribosome movements that are central to the elongation process. The ribosome, a macromolecular complex assembly consisting of ribosomal RNA (rRNA) and ribosomal proteins (rProteins), translates genetic information efficiently and accurately into proteins in all living cells. It is composed of large and small subunits, known in prokaryotes as 50S and 30S, respectively, based on their sedimentation coefficients . Apart fIntricate functioning of the ribosome during protein biosynthesis is enabled by the highly integrated flexibility of some of its components . The movin vitro conditions, such as concentration of ions, may lead to the ribosome’s structural variability  at 37°C.It is well-known that disparity of iability , 7. Specomplexes . Fluoresomplexes . Moreoveplexes 1013 that splexes 10 and woulin vitro. Here, we present our structural results, highlighting the conformational variability of H68, as well as our in vitro studies toward its targeting.As, indeed, such studies led to identification of H68 motions, we aimed to prove their significance for protein biosynthesis by targeting the helix with antisense-based peptide nucleic acid (PNA) oligomers. The efficacy of various antisense oligomer sequences to inhibit protein biosynthesis by binding to bacterial rRNA has been already proven by their effective targeting of clinically used antibiotic binding sites . SimilarS. aureus large ribosomal subunit (SA50S) and of the whole ribosome (SA70S) were determined after incubation at physiological temperature for various time durations (5TCU) was used as a baseline reference (no incubation) for comparison with the SA70S ribosome structures determined after incubation for 30 and 50 min. Also, other regions, e.g., rRNA helix 58, appear to undergo similar displacements . Download Copyright © 2022 Cimicata et al.2022Cimicata et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00306-22.2FIG S1FIG S1, PDF file, 16.8 MB.(Ai) Cryo-EM image processing flowchart for SA50S with no incubation. (Aii) Surface (left) and cross-section (right) rendering of the cryo-EM density maps colored according to local resolution. (Aiii) “Gold-standard” Fourier shell correlation (FSC) curves for the final three-dimensional map (black), unmasked map (green), masked map (blue), and phase-randomized masked map (red). (Bi) Cryo-EM image processing flowchart for SA50S with 30 minutes incubation. (Bii) Surface (left) and cross-section (right) rendering of the cryo-EM density maps colored according to local resolution. (Biii) Gold-standard FSC curves for the final three-dimensional map (black), unmasked map (green), masked map (blue), and phase-randomized masked map (red). (Ci) Cryo-EM image processing flowchart for SA50S with 50 minutes incubation. (Cii) Surface (left) and cross-section (right) rendering of the cryo-EM density maps colored according to local resolution. (Ciii) Gold-standard FSC curves for the final three-dimensional map (black), unmasked map (green), masked map (blue), and phase-randomized masked map (red). (Di) Cryo-EM image processing flowchart for SA70S with 30 minutes incubation. (Dii) Surface (left) and cross-section (right) rendering of the cryo-EM density maps colored according to local resolution. (Diii) Gold-standard FSC curves for the final three-dimensional map (black), unmasked map (green), masked map (blue), and phase-randomized masked map (red). (Ei) Cryo-EM image processing flowchart for SA70S with 50 minutes incubation. (Eii) cross-section (right) rendering of the cryo-EM density maps colored according to local resolution. (Eiii) Gold-standard FSC curves for the final three-dimensional map (black), unmasked map (green), masked map (blue), and phase-randomized masked map (red). Download Copyright © 2022 Cimicata et al.2022Cimicata et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00306-22.3FIG S2FIG S2, PDF file, 0.9 MB.Overlay of SA50S_0 (orange) and SA50S_50 (blue) in the SA50S_50 sharpened map (yellow) and unsharpened map (gray). The moveable H58 was not modeled in SA50S, as the resolution of the map was too low to allow an unbiased modeling. Download Copyright © 2022 Cimicata et al.2022Cimicata et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00306-22.4FIG S3FIG S3, PDF file, 0.4 MB.H68 in the EM density maps and the respective fitted structures shown in a vertical images of front and top views. (A) SA50S_30; (B) SA50S_50; (C) SA70S_30; (D) SA70S_0. Download Copyright © 2022 Cimicata et al.2022Cimicata et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Since the density of H68 was visible in all the EM maps corresponding to 50 min incubation time  and D, wSuperposition of both 50S and 70S ribosome structures acquired after 50 min incubation time on the ribosome structures obtained directly without incubation and after 30 min incubation indicated a shift of H68 toward the L1 stalk  and B. D10.1128/mbio.00306-22.5FIG S4FIG S4, PDF file, 0.2 MB.(A) Overlay of SA50S_0 (orange) and SA50S_50 (blue) H69. (B) Overlay of SA70S_0 (orange) and SA70S_50 (blue) H69. Download Copyright © 2022 Cimicata et al.2022Cimicata et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Escherichia coli numbering used throughout), which is a part of the intersubunit bridge B2b observed in the SA50S_50 structure but barely seen in the SA70S_50 structure  is shown in mulberry red. (B) The class with the rearranged H68 obtained from the heterogeneous three-dimensional refinement of the particles used for the three-dimensional reconstruction of SA50_30 is shown in yellow. The rRNA from SA50S_50 (PDB ID 7ASN) is shown in mulberry red. Download FIG S5, PDF file, 0.4 MB.(A) The class with H68 in the original position obtained from the heterogeneous three-dimensional refinement of the particles used for the three-dimensional reconstruction of SA50_30 is shown in blue. The rRNA from SA50S_0  values in the low micromolar range. Remarkably, the slope calculated for all the oligomers is lower than 1, suggesting that only one oligomer binds to each ribosome. In addition, our results show that 12-mer PNA oligomers have a higher inhibition activity than the shorter 10-mer, while transition to the longer 14-mer resulted only in a minor increase of inhibitory activity. The 12-mer oligomers, which appeared optimal in respect to effective inhibition and future penetration through the bacterial cell wall, were therefore selected for the following binding experiments.Following preparation and purification of the PNA oligomers, their capacity to inhibit protein synthesis was tested using an ter gene  to F. As10.1128/mbio.00306-22.8FIG S7S. aureus ribosome. Fitting of the luminescence data for oligonucleotide was performed with GraFit. (B) Inhibition assay using PNA 1862-12 and S. aureus ribosome. Fitting of the luminescence data for oligonucleotides was performed with GraFit. (C) Inhibition assay using PNA 1862-14 and S. aureus ribosome. Fitting of the luminescence data for oligonucleotides was performed with GraFit. (D) Inhibition assay using PNA 1873-10 and S. aureus ribosome. Fitting of the luminescence data for oligonucleotide was performed with GraFit. (E) Inhibition assay using PNA 1873-12 and S. aureus ribosome. Fitting of the luminescence data for oligonucleotide was performed with GraFit. (F) Inhibition assay using PNA 1873-14 and S. aureus ribosome. Fitting of the luminescence data for oligonucleotide was performed with GraFit. (G) Relative inhibition of mismatch sequences compared to the matching sequences. (Gi) PNA oligonucleotides 1862 series (1889 in S. aureus numbering). (Gii) PNA oligonucleotide 1873 series (1900 in S. aureus numbering). Download FIG S7, PDF file, 0.3 MB.(A) Inhibition assay using PNA 1862-10 and Copyright © 2022 Cimicata et al.2022Cimicata et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To evaluate the binding affinity between the various PNA oligonucleotides and SA50S, the fluorescein-labeled 12-mer oligomer of both sets was incubated with SA50S and SA30S, and the mixtures were run on a nondenaturing agarose gel. A mismatched sequence of the oligomer 1862-12 holding 2 mismatches, 1862-12-2, was used as a control. The intense bands in the gel (read at 494 nm)  indicatein vitro transcription/translation assay  conformations, as well as on a third conformation, namely, the intermediate one, positioned between the open and the closed states (5TCU), and in the hybrid P/E state (4V9H)  to provide further insight into the ribosome mechanism of action and for future development of antisense-based antibiotics. Following our findings of H68 movements upon its incubation for 50 min at 37°C, we analyzed H68 interactions with the L1 stalk and tRNA to specifically shed light on its role during translocation. Thus, we superimposed H68 from SA70S_0 and SA70S_50 structures on the L1 stalk both in open  PDB ID   .These overlays show that the 3′ end acceptor arm of the P/E tRNA is involved in a minor groove contact with the SA70S_0 H68 at positions 1850 to 1852 and 1892 to 1893 . HoweverWe suggest that during translocation, the same (or a very similar) movement of the H68 might be triggered by the shift of the tRNA from the hybrid P/E configuration to the classical E-state, as the minor groove interactions are broken when the tRNA is in the exit site . Moreovein vitro conformational changes usually require very long times  of ∼1.5. The bacterial culture was centrifuged three times for 15 min each at 5,000 rpm using a GS-3 rotor at 4°C and the supernatant discarded. The pellet was resuspended and centrifuged at 4,000 rpm for 10 min at 4°C using a GS-3 rotor in a tabletop centrifuge. The supernatants were discarded, and the wet pellets (cells) were weighed. In order to disrupt the cell membranes, the samples were resuspended in 10 mM Tris-acetate buffer, pH 8.0, 14 mM Mg acetate, 1 M KCl, 1 mM dithiothreitol (DTT), and 50 μg/mL lysostaphin and were incubated at 37°C for 1 h and periodically inverted. The lysates were centrifuged for 30 min at 160,00 rpm at 4°C using an SS-34 rotor for removing cell debris. The supernatants were incubated in 670 mM Tris-acetate buffer, pH 8.0, 20 mM Mg acetate, 7 mM DTT, 7 mM Na3-phosphoenolpyruvate, 5.5 mM ATP, 70 mM from each amino acid, and 1.9 mg pyruvate kinase at 37°C for 30 min and dialyzed overnight at 4°C against 10 mM buffer of Tris-acetate, pH 8.0, 14 mM Mg acetate, 60 mM K-acetate, and 1 mM DTT dialysis buffer. The extract was then flash frozen and stored at −80°C.15βMe6, pH 8.0 , and ultracentrifugated twice, each time at 55,000 rpm using a Ti-70 rotor at 4°C for 17 h. The pellet was dissolved in H10M15N150βMe6, pH 8.0, at 4°C. Ribosomal subunits were then separated by zonal ultracentrifugation using a Ti-15 zonal rotor with 8 to 40% sucrose gradient at low Mg2+ (1 mM MgCl2) for 17.5 h at 27,000 rpm. After separation, the concentration of Mg2+ was adjusted to 10 mM, and the ribosomal subunits fractions were concentrated using sequential centrifugations. The samples were kept in H10M15N60K15 , pH 7.6, brought to a final concentration not exceeding 1,000 A260 mL−1, and then flash frozen for storage at −80°C.Cell extract was layered on a 1.1 M sucrose cushion, H10M15N100KN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) and 1-hydroxybenzotriazole , and 8 eq of N,N-diisopropylethylamine (DIEA) (Sigma-Aldrich) in DMF. The reactions vessels  were typically shaken for 3 to 4 h, and then the resin was washed three times with DMF and twice with dichloromethane (DCM) (Macron). Success of couplings, as well as Fmoc removal, was monitored by ninhydrin. In all cases, only one cycle of coupling was required. Simultaneous deprotection of Bhoc and cleavage from the resin were performed using a 95:5 mixture of trifluoroacetic acid (TFA)/m-cresol. After ∼3 h at room temperature, the solution was transferred to cold ether to precipitate the product; this step was repeated three times. After decantation of ether, water was added to the product, and it was lyophilized. The products were then purified using high-pressure liquid chromatography (HPLC) , and their identity and purity were verified by liquid chromatography-mass spectrometry (LC-MS) (Waters). For labeling of the oligonucleotides PNA with 5′-6-carboxyfluorescein (Sigma-Aldrich), the Fmoc-aminoethylethanolamine (AEEA)-OH linker (PolyOrg. Inc.), was first coupled to the resin using the protocol described above. Following the linker Fmoc deprotection, a mixture of 5 eq 5′,6-carboxyfluorescein, 5 eq of HOBt, 5 eq of benzotriazol-1-1-hexafluorophosphate-oxy-Tris-pyrrolidine-phosphonium (PyBOP), and 10 eq of DIEA in DMF was added to the resin after 10 min of preactivation. The tube was then covered with aluminum foil and shaken overnight. A second coupling using the same amounts was performed for an additional ∼3 h. The cleavage from the resin was performed as described above.The PNA oligomers were synthesized manually on Fmoc-Lys(Boc)-NovaSyn TGA resin . The synthesis followed typical PNA Fmoc protocols. In brief, deprotection of Fmoc from Lys as well as the PNA monomers was performed using a solution of 20% piperidine in dimethylformamide (DMF), and couplings were performed with 4 equivalents of Fmoc-PNA monomers (PolyOrg. Inc.), S. aureus ribosomes was tested in a bacterial coupled transcription-translation assay system where the expression of the luciferase gene was measured , 6.5% polyethylene glycol 8000 (PEG 8000), 0.074 mg/mL tyrosine, 208 mM potassium glutamate, 1.8 mM DTT, 1.3 mM ATP, 0.86 mM GTP, 0.86 mM CTP and 0.86 mM UTP, 0.663 mM cyclic AMP (cAMP), 83 mM creatine phosphate, 0.036 mg/mL folinic acid, 28 mM ammonium acetate, and 14.8 mM magnesium acetate was prepared; 30 μL of the reaction mixture was incubated for 50 min at 37°C in the presence of different concentrations of the various oligonucleotides; the reactions were terminated with the addition of 48 μL of water. Luciferin assay reagent  at 5:3 (luciferase/reaction mix) volume ratio was added to the mixture, and photoluminescence was instantly measured; dose-response curves were fitted to the experimental data with GraFit ; 2 μL of 2 μM fluorescein-labeled oligonucleotide (200 nM) dissolved in Milli-Q H2O. We added 3 μL of RNasin to each sample. The sequence of the oligomer used as a positive control is CAGTTCCTAACC. Samples were loaded onto a 1.5% agarose gel and electrophoresed in 1×Tris-borate-EDTA (TBE) buffer. The gel was run for 120 min using 80 V at 4°C.Samples were prepared by mixing 4 μL Milli-Q H−1, defocus range of −0.7 to −1.3 μm, and total dose 40 e/Å2. These data sets were collected at the High-Resolution Imaging Section at the Center for Molecular Microscopy, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA. Micrographs of SA70S_30 and SA70S_50 were recorded on a Falcon 3 (FEI) at a magnification of ×127,000, pixel size, 1.1 Å·Å·px−1, defocus range −0.5 to −1.50 μm, and total dose of 40 e/Å2. These data sets were collected at the Weizmann Institute Electron Microscopy Unit, Israel.Upon incubation at 37°C for different intervals of time , 3 μL of a 1.0-mg/mL ribosome solution was applied on glow-discharged holey carbon grids . The grids were blotted for 3.0 s at 4°C and 100% humidity and vitrified in liquid ethane precooled by liquid nitrogen using a Vitrobot Mark IV (FEI). Cryo-EM micrographs were recorded at liquid nitrogen temperature on a Titan Krios electron microscope (FEI) operating at 300 kV. Micrographs of SA50S_0, SA50S_30, and SA50S_50 were recorded on a Gatan K2 Summit at a magnification of ×29,000; pixel size, 0.859 Å·pxCryo-EM images were subjected to motion correction by using MotionCor2 . ContrasS. aureus ribosome PDB ID 4WCE for SA50S and S. aureus PDB ID 5TCU for SA70S to the calculated density map using Chimera  under accession codes 0243, 11900, 11901, 11902, and 11903 for the 50S and 70S, 0, 30, and 50 min incubation, respectively. The atomic models have been deposited in the Protein Data Bank (PDB) under IDs