Coreopsis species have been developed to produce cultivars of various floral colors and sizes and are also used in traditional medicine. To identify and evaluate mutant cultivars of C. rosea and C. verticillata, their phytochemical profiles were systematically characterized using ultra-performance liquid chromatography time-of-flight mass spectrometry, and their anti-diabetic effects were evaluated using the dipeptidyl peptidase (DPP)-IV inhibitor screening assay. Forty compounds were tentatively identified. This study is the first to provide comprehensive chemical information on the anti-diabetic effect of C. rosea and C. verticillata. All 32 methanol extracts of Coreopsis cultivars inhibited DPP-IV activity in a concentration-dependent manner . Thirteen compounds presented as potential markers for distinction among the 32 Coreopsis cultivars via principal component analysis and orthogonal partial least squares discriminant analysis. Therefore, these bio-chemometric models can be useful in distinguishing cultivars as potential dietary supplements for functional plants. Coreopsis species are annual or perennial plants belonging to the Asteraceae (Compositae) family − . At high energy scan, a fragment ion for a quinic acid was observed at m/z 191.0556 [tR 5.21 min) produced a molecular ion at m/z 329.0865 [M – H]− corresponding to C14H17O9− and produced two fragment ions at m/z 167.0338 and 151.0026, assigned to the loss of glucose and [M – H – glucose – O]−, respectively. This peak was tentatively identified as vanillic acid-4-glucoside, which was confirmed by the UNIFI local library and first detected in Coreopsis species. Peak 27 (tR 7.68 min) produced a major ion at m/z 515.1183 [M – H]−  with two stable fragment ions at m/z 353.0869 [M – H – C9H6O3]− and 191.0557 [M – H – 2C9H6O3]− assigned to dicaffeoylquinic acid. Peak 31 (tR 8.04 min) had the same precursor and fragment ions with peak 27. Peaks 27 and 31 were tentatively identified as 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, respectively, by comparing with values in the literature of clearly identified constituents in C. tinctoria − for their glycosides and [M – H – H2O]− for aglycones were produced by the loss of glucose and H2O, respectively. The loss of C8H6O (118 Da) or C8H6O2 (134 Da) were also characteristic fragment ions for flavanones and chalcones −  and yielded fragment ions at m/z 303.0503 [M – H – glucose]− and 285.0397 [M – H – glucose – H2O]− by the loss of glucose and H2O, indicating the presence of a 3′,4′-dihydroxyphenyl group. The fragment ion at m/z 151.0034 [M – H – glucose – H2O – C8H6O2]− showed the presence of a 3-hydroxy group. Peak 3 had the fragment ion at 287.0550 [M – H – glucose – O]−, assuming that the sugar was attached at C-3 in the C ring. Therefore, peaks 1 and 3 were tentatively identified as taxifolin-7-O-glucoside −, corresponding to the molecular formula C21H21O11−. At high energy scan, fragment ions at m/z 287.0554 [M – H – glucose]− and 269.0446 [M – H – glucose – H2O]− were detected by the loss of glucose and H2O and indicated the presence of a 3′,4′-dihydroxyphenyl group for the B ring. Fragment ions at m/z 151.0034 [M – H – glucose – H2O – C8H6O]− suggested no hydroxyl group at C-3. Thus, it was tentatively identified as flavanomarein −  and the fragment ion at m/z 449.1069 [M – H – rhamnose]. Therefore, peak 6 was predicted as isookanin-7-O-rutinoside, which has been described previously − , which is 16 Da less than that of peak 5, and the fragment ion at m/z 135.0449 [M – H – glucose – H2O – C8H6O]− indicated the presence of a 3′,4′-dihydroxyphenyl group and no hydroxyl group at C-3 in the C ring, and consequently confirmed the presence of a hydroxyl group in the A ring. Thus, peak 8 was tentatively identified as butin-7-O-glucoside [tR 6.17 min) produced a molecular ion at m/z 479.0825 [M – H]−, corresponding to the molecular formula C21H19O13−. The fragment ions at m/z 317.0291 [M – H – glucose]− and 166.9963 [M – H – glucose – CH3 – H2O – C8H6O]− represented the presence of two hydroxy groups at C-3′ and C-4′ in the B ring and the absence of a hydroxy group at C-3 in the C ring, and consequently predicted the presence of 5,7-dihydroxy-8-methoxyphenyl group for the A ring. Therefore, peak 9 was tentatively identified as 8-methoxyeriodictyol-7-O-glucoside. Its aglycone, 8-methoxyeriodictyol, has been isolated from several plants [tR 6.23 min) and 11 (tR 6.34 min) produced the same molecular ion at m/z 463.1239 [M – H]−  and the same fragment ion at m/z 165.0188 [M – H – glucose – H2O – C8H6O]−, indicating the presence of a 3′,4′-dihydroxyphenyl group for the B ring and no hydroxyl group at C-3 in the C ring, and consequently predicted the presence of the 7-hydroxy-8-methoxyphenyl group for the A ring. Therefore, peaks 10 and 11 were tentatively identified as coreolanceoline B [tR 6.48 min) produced a molecular ion at m/z 433.1134 [M – H]− , exhibiting the same molecular ions as peak 8; however, an [M – H – glucose – C8H8O]− ion, instead of a [M – H – glucose – H2O]− ion of peak 8, was observed in the fragmentation pattern of peak 12, indicating the presence of one hydroxy group in the B ring and two hydroxy groups in the A ring. Accordingly, peak 12 was tentatively identified as naringenin-7-O-glucoside [tR 6.52 min) produced a molecular ion at m/z 595.1664 [M – H]−  and the fragment ions at m/z 433.1121 [M – H – glucose]− and 271.0604 [M – H – 2glucose]−, indicating the presence of two glucose groups. The fragment ion at m/z 135.0447 [M – H – 2glucose – C8H8O2]− without the loss of H2O resulted in the presence of two hydroxy groups at C-3′ and C-5′ in the B ring. Thus, peak 14 was tentatively identified as 7,3′,5′-trihydroxyflavanone-O-diglucoside. Given that 7,3′,5′-trihydroxyflavanone-7-O-glucoside and its aglycone have been found in Coreopsis species −  and the fragment ion at m/z 287.0552 [M – H – arabinose – glucose] by a loss of the arabinosyl-glucose. The fragment ion at m/z 167.0342 [M – H – arabinosyl-glucose – C8H6O] without the loss of H2O indicated the presence of a hydroxy group in the B ring and three hydroxyl groups in the A ring. Thus, peak 18 was tentatively identified as 4′,5,7,8-tetrahydroxyflavanone-7-O-(6-O-arabinosyl-glucoside), which has not been described previously. Its aglycone, isocarthamidin , has not been reported in Coreopsis species; however, it has been isolated from the Asteraceae plant [tR 7.26 min) produced a molecular ion at m/z 493.0984 [M – H]−  and fragment ions at 331.0447 [M – H – glucose]− and 316.0200 [M – H – glucose – CH3]− by loss of a glucose and a methyl of the methoxy group, respectively. The fragment ion at m/z 164.9830 [M – H – glucose – CH3 – H2O – C8H6O2]− produced a 3′-methoxy-4′-hydroxyphenyl group (or 3′-hydroxy-4′-methoxy phenyl group) for the B ring and a 3-hydroxy group in the C ring. Accordingly, peak 24 was tentatively identified as taxifolin 3′,7-dimethyl ether 3-O-glucoside −  and identical fragment ions with peak 30 [tR 6.51 min) produced a molecular ion at m/z 611.1612 [M – H]−, corresponding to the molecular formula C27H31O16−. At high energy scan, the fragment ions at m/z 449.1080, 287.0551, and 269.0393 were formed by the loss of one glucose, two glucoses, and H2O, respectively, indicating the presence of two glucose groups and a 3,4-dihydroxyphenyl group for the B ring. Hence, peak 13 was tentatively identified as okanin-3′,4′-O-diglucoside, which has been isolated from Bidens pilosa [tR 8.46 min) produced a molecular ion at m/z 611.1398 [M – H]−, corresponding to the molecular formula C30H27O14−. At high energy scan, fragment ions at m/z 449.1109 [M – H – glucose]− and 287.0559 [M – H – 2glucose]− were produced by the loss of two glucoses and 269.0441 [M – H – 2glucose – H2O]− by the loss of H2O, and the fragment ion at m/z 151.0024 [M – H – 2glucose – H2O – C8H6O]− indicated the presence of a 3′,4′-dihydroxyphenyl group for the B ring and the absence of a 3-hydroxy group in the C ring. Therefore, peak 33 was tentatively identified as eriodictyol chalcone-7-O-(glucosyl glucoside) or eriodictyol chalcone-O-diglucoside, which have not been described previously. Eriodictyol chalcone-7-O glucoside, which has one glucose, has been found in Antirrhinum majus [tR 8.73 min) produced a molecular ion at m/z 287.0553 [M – H]− and exhibited the same fragment pathway with that of peak 33, suggesting that it was an aglycone of peak 33, eriodictyol chalcone, which has been identified in Coreopsis species [tR 8.84 min) produced a molecular ion at m/z 477.1396 [M – H]−, corresponding to the molecular formula C23H25O11−. At high energy scan, fragment ions were produced at m/z 315.0864 [M – H –glucose]−, 300.0624 [M – H –glucose – CH3]−, 297.0754 [M – H –glucose – H2O]−, 282.0527 [M – H – glucose – H2O – CH3]−, 163.0747 [M – H – glucose – H2O – C8H6O]−, and 148.00524 [M – H – glucose – H2O – CH3 – C8H6O]−. Therefore, peak 37 was tentatively identified as 4-methoxylanceoletin-4′-O-glucoside, which has been isolated from C. lanceolata −  and fragment ions at m/z 447.0932 [M – H – glucose]− and 285.0392 [M – H – glucose – glucose]− by the loss of two glucoses. Another fragment ion at m/z 151.0033 [M – H – 2glucose – H2O – C8H4O]− indicated the presence of a 3′,4′-dihydroxyphenyl group for the B ring without a 3-hydroxy group in the C ring. Thus, peak 7 was tentatively identified as luteolin-7-O-sophoroside [tR 7.03 min) and 36 (tR 8.80 min) produced molecular ions at m/z 447.0929 [M – H]−  and m/z 285.0398 [M – H]−  that were 162 Da and 324 Da less than that of peak 8, respectively, indicating that the sugar moiety was removed from C-2′′ and C-7 in luteolin-7-O-sophoroside (peak 7). Accordingly, peaks 22 and 36 were tentatively identified as luteolin-7-O-glucoside and luteolin, respectively −  and fragment ions at m/z 447.0932 [M – H – glucose]− and 285.0394 [M – H – 2glucose]− by the loss of two glucoses. The fragment ion at m/z 151.0033 [M – H – 2glucose – H2O – C8H4O2]− suggested the presence of a 3′,4′-dihydroxyphenyl group for the B ring and a 3-hydroxy group in the C ring. As a result, peak 15 was tentatively identified as fisetin-3,7-O-diglucoside, which was first detected in Coreopsis species; however, it has been found in other Sophora species [tR 6.97 min) produced a molecular ion at 463.0885 [M – H]−, corresponding to the molecular formula C21H19O12−. At a high energy scan, the fragment ion at m/z 301.0346 [M – H – glucose]− was observed by the loss of a glucose, and fragment ions at m/z 151.0034 [M – H – glucose – H2O – C8H4O2]− indicated the presence of a 3′,4′-dihydroxyphenyl group by the loss of H2O and the presence of a 3-hydroxy group. Therefore, peak 20 was tentatively identified as quercetin-7-O-glucoside [tR 7.33 min) showed a molecular ion at m/z 461.1085 [M – H]− . At high energy scan, fragment ions at m/z 299.0547 [M – H – glucose]−, 283.0242 [M – H – glucose – O]−, 165.0188 [M – H – glucose – O – H2O – C8H4O]−, and 133.0291 [M – H – glucose – O – C8H6O3]− were observed, indicating the loss of a glucoside at a 3-hydroxy group in the C ring and the presence of 3′,4′-dihydroxyphenyl group for the B ring. Therefore, peak 25 was tentatively identified as 3,3′,4′-trihydroxy-7-methoxyflavone 3-O-glucoside, which has been reported in Aptenia cordifolia [tR 7.58 min) produced a molecular ion at m/z 641.1141 [M – H]−, corresponding to the molecular formula C30H25O16−. At high energy scan, fragment ions were produced at m/z 317.0294 [M – H – caffeoylglucose]−, 301.0342 [M – H – caffeoylglucose – O]−, 285.0381 [M – H – caffeoylglucose – O – O]−, 179.0343 [M – H – C15H9O8 – C6H10O4]−, 161.0224 [M – H – C15H9O8 – C6H10O4 – O]−, 135.0447 [M – H – C15H9O8 – C6H10O4 – CO2]−, and 133.0289 M – H – caffeoylglucose – O – O – C7H3O4]−. This peak was tentatively identified as qurcetagetin-7-O-(6′′-caffeoylglucoside), which was confirmed using the UNIFI local library and was first detected in Coreopsis species. However, this compound has been found in Asteraceae plants, such as Gnaphalium uliginosum and Tagetes maxima [tR 8.74 min) produced a molecular ion at m/z 299.0555 [M – H]− . The fragment ions produced at m/z 284.0319 [M – H – CH3]− by the loss of a methyl of a methoxy group were observed. Moreover, 151.0032 [M – H – CH3 – C8H5O2]− indicated the presence of a hydroxyl group at the B ring and a 3-hydroxy group in the C-ring. Other fragment ions at m/z 151.0032 [M – H – CH3 – C8H6O2]− and 133.0447 [M – H – CH3 – C7H3O4]− indicated the presence of a 4′-hydroxyphenyl group for the B ring with a 3-hydroxy group and two hydroxyl groups in the A ring, respectively. Thus, peak 35 was tentatively identified as kaempferide, by comparison of its mass spectrum with that in the MassBank database −  and fragment ions at m/z 227.0351 [M – H– C2H2O]− and 117.0341 [M – H– C7H4O4]−. Thus, it was tentatively identified as apigenin −  and a fragment ion at m/z 269.0447 [M – H – glucose]− by the loss of a glucose. In addition, fragment ions at m/z 135.0447 [M – H – glucose – H2O – C8H4O]− and 133.0447 [M – H – glucose – C7H4O3]− were also observed, indicating the presence of a 3′,4′-dihydroxyphenyl group for the B ring. Thus, peak 19 was tentatively identified as sulfurein (sulfuretin-6-O-glucoside) −  and identical fragment ions with peak 19 [tR 7.01 min) showed a molecular ion at m/z 447.0929 [M – H]− , which is 16 Da more than that of peak 20, and fragment ions at m/z 285.0397 [M – H – glucose]−, 135.0447 [M – H – glucose – H2O – C8H4O – O]−, and 133.0447 [M – H – glucose – C7H4O3]−, thus, identified as maritimein −, a molecular formula of C26H37O13−. At high energy scan, fragment ions were produced at m/z 233.0650 [M – H – 2glucose]−, 191.0554 [M – H – 2glucose – C3H6]−, and 149.0441 [M – H – caffeoylglucose – C5H8O]−. This peak was tentatively identified as lobetyolinin, which was confirmed by the UNIFI local library and first detected in Coreopsis species; however, it has primarily been found in Lobelia species − m/z 554.2615). Full scan data, MS/MS spectra, accurate mass, and elemental composition were calculated using UNIFI software .A Waters ACQUITY UPLC I-Class system equipped with a binary solvent pump and an autosampler combined with a Xevo G2-XS QTof-MS  was used. Each sample (1 μL) was injected into a ACQUITY UPLC BEH CCoreopsis cultivars was analyzed using a DPP-IV inhibitor screening assay kit  which provides a fluorescence-based method for screening DPP-IV inhibitors. The assay uses the fluorogenic substrate, Gly-Pro-Aminomethylcoumarine (AMC), to measure DPP-IV activity. Cleavage of the peptide bond by DPP releases the free AMC group, resulting in fluorescence that can be analyzed using an excitation wavelength of 350–360 nm and an emission wavelength of 450–465 nm. The tested samples dissolved in DMSO at a concentration of 10 mg/mL were subsequently diluted to a final concentration of 20 to 200 μg/mL using DMSO and were added to a 96-well plate to a final volume of 10 μL and a final concentration of 50 μM. The assay procedure is described in our previous studies [50) was determined using GraphPad Prism software  via dose–response analysis.DPP-IV activity of  studies ,62,63. BtR, range of 0.0–15.0 min), mass-to-charge ratio , and a mass tolerance of 0.04 Da. The resulting data were evaluated using SIMCA 15.0.2  for multivariate statistical analysis. Unsupervised principal component analysis (PCA) was performed using UV (univariate)-scaled and supervised orthogonal partial least-squares discriminant analysis (OPLS-DA) was used to identify and compare different metabolite sizes of the 32 samples. The quality of the OPLS-DA model was evaluated with R2Y value and cumulative Q2 value. The model was further validated with a permutation test (n = 200). Markers for the difference between groups were identified by analyzing the S-plot with pareto scaling, which were generated with covariance (p) and correlation (pcorr) data. These data sets were normalized by dividing with mean value to get a value between 0 and 10 and a heatmap with dendrograms was generated using OriginPro 2021  selecting ward for cluster method. Marker compounds were tentatively identified by comparison to published MS data in literature and databases such as Waters Local Library in UNIFI and Massbank [Data management for the UPLC-QTof-MS analysis was performed using UNIFI software . MS data were processed using UNIFI to obtain a data matrix containing retention times, accurate masses, and normalized peak intensities. Parameters included retention time (Massbank ,29,30,31C. rosea and C. verticillata, were demonstrated for the first time in this study. UPLC-QTof-MS techniques were used to identify several phenolic acids, flavonoids and a polyacetylene in mutant cultivars compared to original cultivars. PCA and OPLS-DA results showed that metabolites discriminate between the mutant and original cultivars and between the two species. In addition, significant changes in metabolite content were observed under different DPP-IV inhibitory activities of cultivars, and chlorogenic acid, butin-7-O-glucoside, sulfuretin-6-O-glucoside, maritimein, 3,5-dicaffeoylquinic acid, coreopsin, luteolin, and butein were abundant in the active extracts. Therefore, the DPP-IV inhibitory cultivars and the metabolites influencing their activities would be favorable for the development of functional foods and the information of the metabolites accumulated differently for each mutant cultivar would be useful as a scientific reference for further studies on plant mutation mechanisms.To the best of our knowledge, a comparative metabolomics approach to identify metabolite composition and DPP-IV inhibitory activities in various cultivars of


H-pyrazole has been determined at 170 K, showing a hydrogen-bonded trimeric molecular assembly that is isostructural to its bromo analogue, 4-bromo-1H-pyrazole.The crystal structure of 4-chloro-1 H-pyrazole, C3H3ClN2, at 170 K has orthorhombic (Pnma) symmetry and is isostructural to its bromo analogue. Data were collected at low temperature since 4-chloro-1H-pyrazole sublimes when subjected to the localized heat produced by X-rays. The structure displays intermolecular N—H⋯N hydrogen bonding and the packing features a trimeric molecular assembly bisected by a mirror plane  running through one chlorine atom, one carbon atom and one N—N bond. The asymmetric unit therefore consists of one and one-half 4-chloro-1H-pyrazole molecules. Thus, the N—H proton is crystallographically disordered over two positions of 50% occupancy each.The structure of 4-chloro-1 As a result of this mirror plane, the NH protons are crystallographically disordered over two positions. As with the bromo analogue , 1.331 (10), and 1.327 (10) Å  and 1.331 (2) Å  with N—N bond lengths of 1.343 (2), 1.344 (2), and 1.349 (2) Å  x, −y + z; (ii) −x + 1, −y + 1, −z] are 2.885 (3), 2.858 (2), and 2.858 (2), respectively, as shown in Table 1H-pyrazole packs in a herringbone arrangement when viewed down the b axis  I. DOI: 10.1107/S2056989021008604/zl5013Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021008604/zl5013Isup3.cmlSupporting information file. DOI: 2103822CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-H, N-phenyl and N-benzyl-2-(4-hexyloxyphenyl)benzimidazoles were prepared and studied by 1H NMR and single-crystal X-ray analysis. The unsubstituted benzimidazole forms intermolecular N—H⋯N bonds while in the crystal structures of the other two compounds, the molecules are assembled only through π–π and C—H⋯π interactions. H-benzimidazole , 2-(4-hexyloxyphenyl)-1-phenyl-1H-benzimidazole  and 1-benzyl-2-(4-hexyloxyphenyl)-1H-benzimidazole  were synthesized and their structures were determined by single-crystal X-ray analysis. The N-substituent at the imidazole moiety slightly affects the interplanar angle between the 4-hexyloxyphenyl ring and the benzimidazole system. The unsubstituted benzimidazole (1) forms intermolecular N—H⋯N bonds while in the crystal structures of 2 and 3, the molecules are assembled only through π–π and C—H⋯π interactions.The title compounds, 2-(4-hexyloxyphenyl)-1 In the structures of 1 and 2, the hydrocarbon chains crystallize in the common trans zigzag conformation, while in the structure of 3 the chain adopts a gauche conformation about the C23–C24 bond.In all three structures, the organic molecules occupy general positions and contain identical benzimidazole and 4-hexyloxyphenyl units and different N-substituents Figs. 1–3 ▸ ▸. 1H NMR spectra of 1–3, a similar set of high-field multiplets assigned to protons of the aliphatic chain was observed. In contrast, the NMR pattern corresponding to the aromatic protons in the substances becomes more complex when going from 1 to 2 and 3. In the aromatic part of 1H NMR spectrum of 1, there are four individual resolved multiplets corresponding to the symmetric benzimidazole part, assuming rapid exchange of the N–H proton on the NMR time scale. Phenyl or benzyl substituents at the nitrogen atom decrease the symmetry of the benzimidazole moiety, which results in the appearance of additional signals that are highly overlapped and make the spectra of 2 and 3 difficult to interpret.In the 1, molecules related by the b glide plane are assembled through N—H⋯N bonds  Å, 126.2 (3)°] and between the H10 atom and the C1–C6centroid . Along the c axis, these relatively dense crystal subunits alternate with less dense regions filled by aliphatic chains held together only via van der Waals interactions  Å, 178.4 (11)°] as well as the H9 atom and the C14–C19centroid . These dimers form the 3-D packing via van der Waals interactions between the alkoxy chains.In the crystal of 2 Fig. 6, there a3  Å, 128.89 (10)°], between the H14 atom and the centroid of the N1–C7 imidazole ring  and between the H3 atom and the centroid of the C9–C14 ring . In addition, each molecule and its symmetry equivalent through the inversion center are linked by C—H⋯π contacts between the H23B atom and the centroid of the C1–C6 ring  and between the H21A atom and the centroid of the N1–C7 imidazole ring . These interactions organize the molecules into thick layers parallel to -4-(2-methylphenyl)-2,4-dihydro--triazole-3-thione is isostructural with compound 2 -1H-benzimidazole (1)in vacuo. Yield 242 mg (82%). Single crystals suitable for X-ray analysis were grown by slow evaporation of the solvent from a solution of the substance in an acetone/water mixture, m.p. = 472–473 KA mixture of 1,2-diaminobenzene , 4-(hexyloxy)benzaldehyde  and sodium metabisulfite  in ethanol (30 mL) was refluxed under Ar for 3 h. The reaction mixture was evaporated to dryness, washed with water and dichloromethane and the white powder was collected and dried 1H NMR : δ 8.19–8.11 , 7.59–7.52 , 7.22–7.13 , 7.12–7.04 , 4.09 , 1.86–1.75 , 1.56–1.44 , 1.43–1.31 , 0.95–0.87 .2-(4-Hexyloxyphenyl)-1-phenyl-1H-benzimidazole (2)N-phenylbenzene-1,2-diamine , 4-(hexyloxy)benzaldehyde  and sodium metabisulfite  in ethanol (15 mL) was refluxed under Ar for 5 h. The solvent was removed in vacuo and the crude product was recrystallized from a water/acetone mixture. Yield 2.083 g (70%). Single crystals suitable for X-ray analysis were collected from the recrystallized product, m.p. = 389–390 K.A mixture of 1H NMR : δ 7.87 , 7.55–7.41 , 7.36–7.28 , 7.28–7.18 , 6.85–6.77 , 3.97–3.89 , 1.82–1.70 , 1.50–1.40 , 1.40–1.27 , 0.96–0.87 .1-Benzyl-2-(4-hexyloxyphenyl)-1H-benzimidazole (3)1  in acetonitrile (30 mL), caesium carbonate  and benzyl bromide  were added. The reaction mixture was stirred at room temperature for 12 h and concentrated in vacuo. The residue was dissolved in a mixture of CH2Cl2 and sat. NaHCO3. The aqueous layer was extracted with CH2Cl2, the organic layers were combined and the solvent was removed in vacuo. Recrystallization from CH2Cl2/EtOH gave the product as a white powder. Yield 188 mg (90%). Single crystals suitable for X-ray analysis were grown by slow evaporation of the solvent from a solution of the substance in a CHCl3/EtOH mixture (3/1 v:v), m.p. = 401–402 K.To a suspension of 1H NMR : δ 7.86 , 7.66–7.58 , 7.39–7.27 , 7.25–7.17 , 7.15–7.10 , 7.00–6.92 , 5.48–5.44 , 4.00 , 1.86–1.74 , 1.49–1.45 , 1.39–1.31 , 0.95–0.86 .1 and 3, hydrogen atoms were placed in calculated positions and refined using a riding model [C—H = 0.94–0.97 Å with Uiso(H) = 1.2–1.5Ueq(C)]. In the structure of 1, the N—H hydrogen atom was located from a difference electron-density map and refined using a riding model [N—H = 0.88 Å with Uiso(H) = 1.2Ueq(N)]. Hydrogen atoms in the structure of 2 were located from difference electron-density maps and were refined freely.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989021004898/mw2176sup1.cifCrystal structure: contains datablock(s) 3, 2, 1. DOI: 10.1107/S2056989021004898/mw21763sup2.hklStructure factors: contains datablock(s) 3. DOI: Click here for additional data file.10.1107/S2056989021004898/mw21763sup7.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021004898/mw21763sup8.cmlSupporting information file. DOI: 10.1107/S2056989021004898/mw21762sup3.hklStructure factors: contains datablock(s) 2. DOI: Click here for additional data file.10.1107/S2056989021004898/mw21762sup6.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021004898/mw21762sup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021004898/mw21761sup10.cmlSupporting information file. DOI: 10.1107/S2056989021004898/mw21761sup4.hklStructure factors: contains datablock(s) 1. DOI: Click here for additional data file.10.1107/S2056989021004898/mw21761sup5.molSupporting information file. DOI: 2079816, 2079817, 2079815CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces litoralimycin and sulfomycin-type thiopeptides. Bioinformatic analysis revealed a potential biosynthetic gene cluster encoding for the M2T thiopeptides. The strain is Gram-stain-positive, rod shaped, non-motile, spore forming, showing a yellow colony color and forms extensively branched substrate mycelium and aerial hyphae. Inferred from the 16S rRNA gene phylogeny strain M2T affiliates with the genus Streptomonospora. It shows 96.6% 16S rRNA gene sequence similarity to the type species Streptomonospora salina DSM 44593 T and forms a distinct branch with Streptomonospora sediminis DSM 45723 T with 97.0% 16S rRNA gene sequence similarity. Genome-based phylogenetic analysis revealed that M2T is closely related to Streptomonospora alba YIM 90003 T with a digital DNA-DNA hybridisation (dDDH) value of 26.6%. The predominant menaquinones of M2T are MK-10(H6), MK-10(H8), and MK-11(H6) (> 10%). Major cellular fatty acids are iso-C16:0, anteiso C17:0 and C18:0 10-methyl. The polar lipid profile consisted of diphosphatidylglycerol phosphatidyl glycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, three glycolipids, two unknown phospholipids, and two unknown lipids. The genome size of type strain M2T is 5,878,427 bp with 72.1 mol % G + C content. Based on the results obtained from phylogenetic and chemotaxonomic studies, strain M2T (= DSM 106425 T = NCCB 100650 T) is considered to represent a novel species within the genus Streptomonospora for which the name Streptomonospora litoralis sp. nov. is proposed.Strain M2The online version contains supplementary material available at 10.1007/s10482-021-01609-4. Streptomyces Waksman and Henrici 1943  as source to discover new antibiotics  on 20th of July 2017. In our preceding study, we showed that M2T produces the two new thiopeptides litoralimycin 1 (A) and 2 (B) with cytotoxic activity . The plates were incubated at 30 °C and checked for growth after 1–2 weeks. Sub-culturing was performed for purification on GYM medium  according to Wink et al.  were used. DNA amplification was performed by PCR with the following protocol: 5 min at 95 °C; 34 cycles of 30 s at 94 °C, 30 s at 52 °C and 2 min at 72 °C, plus an additional 10 min cycle at 72 °C. The PCR product was analysed by agarose gel electrophoresis. The PCR product was purified with the Nucleo Spin Microbial DNA Mini kit (MACHEREY–NAGEL). The almost full-length 16S rRNA gene fragment (1501 bp) was sequenced by using the 96-capillary system from Applied Biosystems (ABI), 3730xl DNA Analyser    experiments were performed between strain M2T and the three closest type strains Streptomonospora halophila DSM 45075 T, Streptomonospora arabica DSM 45083 T, and Streptomonospora sediminis DSM 45723 T according to the method of Ziemke et al. (Genomic DNA was isolated from a five-day old GYM liquid culture by using Invisorb Spin Plant Mini Kit 250 (Stratec). The 16S rRNA gene sequence was amplified by PCR. For gene amplification the primers F27 (5’AGAGTTTGATCCTGGCTCAG-3’) and R1541  with the IMG genome ID 2,845,838,682. Whole-genome phylogeny was generated using the TYGS server (https://tygs.dsmz.de) (Seemann http://ggdc.dsmz.de)  . Automated ribotyping of the isolates M2T and Streptomonospora halophila DSM 45075 T was accomplished according to Schumann and Pukall .Preparation of samples for matrix-assisted laser-desorption/ionisation time-of-flight spectrometry (MALDI-TOF MS) from approximately eight mg biomass  and recording of spectra were performed according to the protocol of Schumann and Maier  as descrd Pukall , by usinStreptomyces Projects) media ISP2-ISP7  as described by Shirling and Gottlieb . A section of the agar comprising a bacterial lawn was fixed in 5% glutaraldehyde according to the description of Wink et al.  with an Everhart–Thornley SE-detector and an Inlens-SEM detector in a 25:75% ratio utilising the SEM Smart software version 5.05To.To analyse the structure of the mycelium and spores, the cultures were grown on a complex solid growth medium . The suspension of test bacteria (OD600 = 0.01) was prepared in Mueller Hinton Broth medium contained  and suspension of fungi and yeast (OD600 = 0.05) in MYC medium , pH 7.0). 20 µl of crude extract was added to the first row horizontally which contained 280 µl of the microbial test suspension and well mixed, 150 µl withdrawn and transferred to the next row wells. This was repeated serially until the end and 150 µl was discarded from the last row in order to obtain a dilution gradient of 66.6, 33.3, 16.6, 8.3, 4.2, 2.1, 1.0 and 0.52 µg/ml. All plates were incubated on a microplate shaker incubator (SB: Heidolph Titramax 1000) for 24 h at 165 rpm at 30 °C   revealed that M2Tshowed closest relationship to strains belonging to the genus Streptomonospora. The M2T 16S rRNA gene sequence showed highest similarity to the 16S rRNA gene sequences of strains S. halophila DSM 45075 T, S. arabica DSM 45083 T and S. sediminis DSM 45723 T with 98.05, 97.74, and 97.66%, respectively. The phylogenetic tree construction revealed the same three closest type strains to M2T, and their relation is shown in the Maximum-likelihood phylogenetic tree  Genome Server (TYGS). The phylogenomic tree revealed closest relationship between M2T and Streptomonospora alba YIM 90003 T as well as Streptomonospora salina DSM 44593 T . Thus, it can be concluded that strain M2T belongs to the genus Streptomonospora. The Sanger 16S rRNA gene sequence was 100% identical to four 16S rRNA gene sequences in the genome of M2T and one differed with 1 nt. M3 is another strain, which has been isolated from the same sand sample. Since strain M3 had identical physiological and chemotaxonomic characteristics, with the exclusion of a divergent Ribotyper pattern  this study focused on the intensive characterisation of strain M2T and the produced thiopeptides.The comparison of the 16S rRNA gene sequence from strain M2T and DNA from the three closely related type strains. The level of DDH similarity between M2T and Streptomonospora halophila DSM 45075 T was 52.8/45.8%, Streptomonospora arabica DSM 45083 T 44.4/39.2% and Streptomonospora sediminis DSM 45723 T 21.3/38.9%. In silico digital DDH (dDDH) analysis was performed based on the M2T genome sequence. The dDDH analysis identified Streptomonospora alba YIM 90003 T as the most closely related type strain. The dDDH value between the two genomes was 26.64% (24.2–29.1%) (using GBDP distance formula 2). The calculated difference of the G + C content between both strains was 0.26%. Thus, it can be inferred that strain M2T represents a novel species within the genus Streptomonospora.DNA-DNA hybridisation was performed with genomic DNA from strain M2T differs from those of S. halophila DSM 45075 T and other phylogenetically related actinomycetes . However, the resolution of this method is not sufficient for demonstrating strain-specific differences of the two isolates  and a potential prophage/episome with 89,998 bp. The chromosome has a predicted DNA G + C content of 72.1% and includes 5,218 coding sequences, 58 tRNAs, and 15 rRNAs  at pH 4–9 (optimum pH 7–8) and at temperatures between 20–37 °C (optimum 28–30 °C). Predominant diamino acid was meso-diaminopimelic acid. The cell sugars were ribose, galactose, and glucose. The polar lipids of strain M2T were diphosphatidylglycerol (DPG), phosphatidyl glycerol (PG), phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), three glycolipids (GL1-3), two unknown phospholipids  and two unknown lipids . The predominant fatty acids (> 10%) of strain M2T were iso-C16:0 (33.1%), anteiso-C17:0 (24.4%) and 10-methyl C18:0 (13.6%). The distribution of fatty acids detected for M2T was in accordance with those of members of the genus Streptomonospora , MK-10(H6), MK-11(H6), and MK-11(H8) as predominant quinones. Comparative profiles of cellular features for strains M2T, S. halophila DSM 45075 T, S. sediminis DSM 45723 T, S. arabica DSM 45083 T and S. salina DSM 44593 T are listed in Table Strain M2T has no beta-galactosidase activity, which was in contrast to the three analysed Streptomonospora type strains. M2T was hydrolysed gelatin and trypsin as observed for S. sediminis DSM 45723 T and S. arabica DSM 45083 T. Cysteine aryl-amidase activity was found to be negative for strain M2T. Additional comparative data from API ZYM and API Coryne analysis are listed in Table S1.The analysis on enzyme activity showed that M2T showed high activity against Gram-positive bacteria, such as Staphylococcus aureus Newman (0.52 µg/ml), Micrococcus luteus DSM 1790 T (0.52 µg/ml), and Bacillus subtilis DSM 10 T (0.52 µg/ml). No antibacterial activity was observed against tested Gram-negative bacteria, such as Escherichia coli DSM 1116 T and Pseudomonas aeruginosa PA14 DSM 19882 T, except for Legionella pneumophila NCTC 11,192 (12.5 µg/ml). No antibiotic activity was observed against fungal and yeast organisms, such as Candida albicans DSM 1665 T, Pichia anomala DSM 6766 T and Mucor himalis DSM 2656 T in this approach.Minimal inhibition concentration with ratio (1 µg/ml) of crude extracts (1 mg/ml) from M2T . BGC4 shows similarity to other known thiopeptide BGCs encoding radamycin/globimycin in S. globisporus subsp. globisporus, berninamycin A from in S. bernensis  is present in the sulfomycin cluster of S. viridochromogenes but two small open reading frames (ORFs)  are found within BGC4 of M2T . lit11 and lit12 encode the predicted precursor peptides Lit11 (51-aa) and Lit12 (50-aa), whereby Lit11 harbors a N-terminal 17-aa core peptide (CP) sequence consisting of the amino acid sequence pattern SCTTTGCTTSSSSSSSS and Lit12 a 16-aa CP sequence of SCVGCACTCSSTSSSS. The Lit11 CP sequence is 100% identical to the SulA CP sequence described for sulfomycin biosynthesis in S. viridochromogenes, suggesting that lit11 encodes the sulfomycin I thiopeptide precursor in M2T . The Lit12 CP is highly similar to the CP sequence of WP_078651956.1 involved in radamycin biosynthesis in S. globisporus subsp. globisporus with a difference of only two amino acids . Due to the structural similarity of the litoralimycins with radamycin , MK-10 (H2) and MK-11 (H4) were determined in Streptomonospora halophila DSM 45075 T but not in strain M2T. Cysteine arylamidase activity could not be observed for strain M2T, whereas it was positive in all compared three type strains. β-galactosidase activity was detected in all three closest type strains but not in strain M2T. Gelatin hydrolysis activity was determined for strains M2T, S. sediminis DSM 45723 T and S. arabica DSM 45083 T but it was negative for S. halophila DSM 45075 T. Furthermore, A different Ribotyper pattern of strain M2T and closest type strain S. halophila DSM 45075 T could specifically distinguish them from each other. In conclusion, based on genotypic and phenotypic features strain M2T represents a novel species of genus Streptomonospora, for which the name Streptomonospora litoralis sp. nov. is proposed.Cultural, morphological, and chemotaxonomic markers besides phylogenomic tree analysis revealed that strain M2Streptomonospora litoralis . Gram–positive, aerobic actinomycete. The color of substrate mycelium varied depending on the medium; in ISP2 (honey yellow), ISP3 (white), ISP4 (white), ISP5 (light ivory), ISP6 (golden yellow), ISP7 (ivory), Suter synthetic agar media with/o tyrosine (ochre yellow). Aerial mycelium was detected in all media except of ISP4; the color of aerial mycelia is pure white. Diffusible pigments are produced on ISP2, ISP3, ISP5, and ISP7 also on Suter synthetic media with/o tyrosine. Melanin was not produced on ISP6, ISP7 and SSM with/o tyrosine. At maturity on ISP3 medium, aerial mycelium forms spore chains (wrinkled surface) and single non-motile spores (smooth surface). Growth occurs on ISP2 medium with 0–15% NaCl (w/v), at pH 4–9, in 20–40 °C; optimum of growth determined with 7–10% NaCl, pH 7.0–8.0 at 28 °C. Positive for alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine aryl amidase, valine aryl amidase, acidic phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, α-glucosidase, glucosidase. Positive in hydrolyses of pachyman, laminarin, rhamnogalacturunan, pectin, xylose, arabinoxylose, hydroxyethyl-cellulose, barley-β-glucan, and gelatin. No hydrolyses of amylose, pullulan, dextran, galactan, rhamnose, galactorunan, α-cellulose, Avicel-cellulose, chitosan, casein, galactose. Predominant menaquinones > 5% include MK-10(H4), MK-10(H6), MK-10(H8), MK-11(H6), and MK-11(H8). The polar lipids consist of diphosphatidylglycerol (DPG), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), glycolipid (GL), phospholipid (PL). Crude extract from M2T shows high activity against Gram-positive bacteria, such as Staphylococcus aureus Newman (0.52 µg/ml), Micrococcus luteus DSM 1790 T (0.52 µg/ml), and Bacillus subtilis DSM 10 T (0.52 µg/ml). Strain M2T produces cytotoxic thiopeptides litoralimycin A and B, sulfomycin I, methylsulfomycin I and sulfomycin III.T (= DSM 106425 T = NCCB 100650 T) isolated from the sand of Cuxhaven beach. The G + C content of the genomic DNA of M2T is 72.1 mol%. The GenBank accession numbers of the 16S rRNA gene and the genome sequence of M2T are MG551552 and CP036455, respectively. The genome comprises a circular 5,878,427 bp large chromosome and a 89,998 bp large potential prophage/episome. Production of thiopeptides litoralimycin A and B Below is the link to the electronic supplementary material.


Deficiency in ornithine decarboxylase, a crucial enzyme for polyamine synthesis, results in a severe failure of CD4+ T cells to adopt correct subset specification, underscored by ectopic expression of multiple cytokines and lineage-defining transcription factors across TH cell subsets. Polyamines control TH differentiation by providing substrates for deoxyhypusine synthase, which synthesizes the amino acid hypusine, and mice in which T cells are deficient for hypusine develop severe intestinal inflammatory disease. Polyamine-hypusine deficiency caused widespread epigenetic remodeling driven by alterations in histone acetylation and a re-wired tricarboxylic acid (TCA) cycle. Thus, polyamine metabolism is critical for maintaining the epigenome to focus TH cell subset fidelity.Polyamine synthesis represents one of the most profound metabolic changes during T cell activation, but the biological implications of this are scarcely known. Here, we show that polyamine metabolism is a fundamental process governing the ability of CD4  •+ T cells to differentiate into distinct subsetsPolyamines govern the ability of CD4•Through the synthesis of hypusine, polyamines direct the T cell epigenome•Loss of polyamines or hypusine perturbs the TCA cycle and histone acetylation•H differentiation in polyamine-deficient cellsHAT deletion restores T + T helper cells to develop into specified functional subsets depends upon polyamine-driven metabolic and epigenetic regulation.The ability of CD4 Three major subsets of effector CD4+ TH cells are TH1, TH2, and TH17 cells. These cells differentiate from naive T cells in response to signals from antigen presenting cells during activation and local microenvironmental cues. The functional specialization of TH cells is conferred by the expression of T cell subset-specific transcription factors (TFs) that coordinate genetic programs to direct production of signature cytokines and surface molecules mediating interactions with other cells (H1 cells express the TF T-bet and the cytokine interferon (IFN)-γ and mediate responses to intracellular pathogens. TH2 cells express GATA3 and interleukin (IL)-4 and control helminth infections. TH17 cells synthesize RORγt and IL-17 and limit extracellular bacteria and fungi, particularly at mucosal surfaces , modulates immunity by dampening effector T cell activation and proliferation and expresses the TF Foxp3 , or both . Control mice were absent for cre recombinase. ODC was deleted in T cells from Odc-ΔT mice  littermates , also displayed significantly increased IFN-γ, T-bet, and Foxp3  or Odc−/− T cells (Odc−/− → Rag1−/−)  C, only mickening D.Figure of Odc−/− → Rag1−/− recipient mice relative to WT → Rag1−/− controls  controls E and S2C reduced F. The fr−/− mice E and S2Fin vivo model of TH17 induction in the small intestine. Anti-CD3 antibody treatment drives activation induced cell death in T cells and subsequent engulfment of apoptotic T cells by macrophages leads to IL-6 and TGF-β production, cytokines important for TH17 cell development  increased after CD4+ T cell activation  ablated DHPS in WT and Odc−/− TH17 cells compared to cells with control guides (gCTRL)  D. Dhps dA levels D. As obsout DHPS D. Importing Dhps D.Dhps deletion in WT TH17 cells increased IFN-γ . Control mice were absent for cre recombinase. DOHH deletion in T cells decreased eIF5AH, but not total eIF5A   C. Even uT levels D. Like Oxpansion E and S3Fdivision A and S4BOdc−/− and Dohh−/− T cells strongly supports that the requirement for polyamine metabolism in enforcing TH cell lineage fidelity is due to spermidine acting as a substrate for hypusine synthesis.The robust phenotypic overlap between Dohh-ΔT mice died at ∼10–17 weeks of age . Control mice were absent for cre recombinase. Deletion of DHPS in T cells decreased eIF5AH but not total eIF5A . Various transcriptomic and epigenomic mechanisms underlie this lineage commitment fidelity , finding a dramatically altered transcriptional profile in both Odc−/− and Dohh−/− cells relative to WT cells across all TH cell subsets , Gata3, Rorc, Ifng, and Il17  was comparatively minor compared to that of differentiated Dohh−/− TH subsets . To confirm that expression of factors associated with TH differentiation was not already differentially regulated in naive Dohh−/− T cells, we used Dohh-ΔT-Great mice to assess IFN-γ in naive and effector CD4+ T cells. IFN-γ-YFP expression was restricted solely to CD44-expressing effector CD4+ T cells in the colon, spleen, and lungs of both Dohh-ΔT-great mice and WT littermate controls . Although Tbx21-deletion was efficient  P300 that mediates H3k27 acetylation, a dysregulated mark in Dohh−/− T cells. Treating Dohh−/− T cells with C646 restored IFN-γ and T-bet levels across TH subsets to WT levels  displayed reduced P300 protein compared to T cells with gCTRL  cycle is a central metabolic hub that generates acetyl-CoA, a HAT substrate for histone acetylation. ATP citrate lyase (ACLY) converts the TCA cycle metabolite citrate to acetyl-CoA. We traced carbons from 13C glucose into TCA cycle metabolites in WT and Odc−/− TH cells. Although glucose was metabolized with largely equal efficiency to pyruvate and citrate in WT and Odc−/− TH cells, there was a significant reduction in glucose carbons found in α-ketoglutarate, 2-HG, and succinate—metabolites synthesized after citrate in the TCA cycle  reduced ACLY protein compared to gCTRL cells  . The contribution of these cells to the disease states described here remains to be determined. However, the colitis observed here in Dohh-ΔT and Dhps-ΔT mice is believed to be primarily CD4+ T cell-mediated  , and conrthritis . T-bet+ nfection , and T-bnfection . Becausemediated , and in H lineage fidelity by polyamine metabolism via the epigenome is ultimately driven by TCA cycle regulation. TCA cycle metabolites can impact the epigenome and T cell differentiation .Further information and requests for resources and reagents should be directed and will be fulfilled by the lead contact, Erika L. Pearce .Rag1−/−, CD45.1 SJL, and mice expressing Cre recombinase (CD4Cre) under the control of the CD4 promoter, were all purchased from Jackson Laboratories. Dohhflox/flox and Dhpsflox/flox were a gift from Stefan Balabanov, Zurich. Odcflox/flox mice were purchased from KOMP repository. All mice were bred and maintained under specific pathogen free conditions under protocols approved by the Animal Welfare Committee of the Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany, and The St. Jude Institutional Animal Care and Use Committee, Memphis, USA, in accordance with the Guide for the Care and Use of Animals. Mice used for all experiments were littermates and matched for age and sex . Mice for all strains were typically 6-10 weeks of age. Survival curves in Freiburg and Memphis were plotted using the time post-birth that mice were found with rectal prolapse, at which point mice would be sacrificed.Wild-type C57BL/6, Great, in vitro culture, naive CD4+ T cells were isolated by negative selection using a Stem Cell kit according to the manufacturer’s instructions . Unless otherwise stated, naive CD4+ T cells were isolated from spleen and lymph nodes. Cells were cultured in RPMI 1640 media supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin/streptomycin and 55 μM β-mercaptoethanol in 48 well plates at a seeding concentration of 5x105 cells per well. All activations were done with 5 ug/mL anti-CD3, 2 ug/mL anti-CD28, and 100 U/mL IL-2. TH polarizations were performed as follows: TH0 – 10 ng/mL IL-2, 10 μg/mL anti-IFN-γ, 10 μg/mL anti-IL-4. TH1 – 4 μg/mL anti-IL-4, 10 ng/mL IL-12, 10 ng/mL IL-2. TH2 – 4 μg/mL anti-IFN-γ, 10 ng/mL IL-4, 10 ng/mL IL-2. TH17 – 10 μg/mL anti-IFN-γ, 10 μg/mL anti-IL-4, 5 ng/mL IL-6, 5 ng/mL TGF-β, 10 μg/mL IL-1β. Treg – 4 μg/ mL anti-IFN-γ, 4 μg/mL anti-IL-4, 10 ng/mL TGF-β, 10 ng/mL IL-2. Cells were analyzed on day 4 of polarization. Other blocking antibodies used were anti-IL-2, anti-IL-5 (both BioXcell) and anti-IL-13 (Invivogen). For the 13C arginine tracing under different cytokine conditions in For 2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β- glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin , supplemented with 1 mM PMSF. Samples were frozen and thawed 3 times followed by centrifugation at 20,000 x g for 10 min at 4°C. Cleared protein lysate was denatured with LDS loading buffer for 10 min at 70°C, and loaded on precast 4% to 12% bis-tris protein gels (Life Technologies). Proteins were transferred onto nitrocellulose membranes using the iBLOT 2 system (Life Technologies) following the manufacturer’s protocols. Membranes were blocked with 5% w/v milk and 0.1% Tween-20 in TBS and incubated with the appropriate antibodies in 5% w/v BSA in TBS with 0.1% Tween-20 overnight at 4°C. All primary antibody incubations were followed by incubation with secondary HRP-conjugated antibody (Pierce) in 5% milk and 0.1% Tween-20 in TBS and visualized using SuperSignal West Pico or femto Chemiluminescent Substrate (Pierce) on Biomax MR film (Kodak). Antibodies used: anti-ODC, anti-DHPS, anti-DOHH, anti-β-Actin, anti-KAT3B (P300), α-tubulin (Abcam), anti-GAPDH, anti-KAT2A (GCN5), anti-ACLY , ant-EIF5A (BD Bioscience), anti-hypusine (Millipore).For western blot analysis, cells were washed with ice cold PBS and lysed in 1 x Cell Signaling lysis buffer . 13C glutamine tracing was carried out by culturing cells in glutamine-free RPMI supplemented with 4 mM 13C glutamine (Cambridge Isotope Lab.). 13C glucose tracing was carried out in glucose-free RPMI with 11 mM 13C glucose (Cambridge Isotope Lab.). For 13C proline tracing, cells were cultured in DMEM (that lacks L-proline) with 0.2 mM 13C proline (Cambridge Isotope Lab.). 15N Methionine tracing was performed in methionine-free RPMI supplemented with 0.1 mM 15N methionine (Sigma). 13C putrescine (Cambridge Isotope Lab.) tracing took place in RPMI.With the exception of For harvest, cells were rinsed with cold PBS and metabolites extracted using a mix of cold methanol, acetonitrile and water (50:30:20) kept on dry ice. For polyamine detection, metabolites were extracted in this same mix but with 1.5% hydrochloric acid. Following mixing and centrifugation, the supernatant was collected and dried via centrifugal evaporation. Dried metabolite extracts were resuspended in pyridine and derivatized with methoxyamine (sc-263468 Santa Cruz Bio) for 60 minutes at 37°C and subsequently with N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamid, with 1% tert-butyldimethylchlorosilane (375934 Sigma-Aldrich) for 30 minutes at 80°C. Isotopomer distributions were measured using a DB5-MS GC column in a 7890 GC system (Agilent Technologies) combined with a 5977 MS system (Agilent Technologies). Correction for natural isotope abundance and calculation of fractional contribution was performed as described elsewhere .13C labeled internal standard (ISOtopic Solutions). Polyamines were extracted from CD4+ T cells using a mix of cold methanol, acetonitrile and water (50:30:20) containing 1.5% hydrochloric acid.Metabolites were quantified by LC-MS using HILIC Chromatography on an Acquity UPLC BEH Amide column 1.7 μm, 2.1x100 mm on a 1290 Infinity II UHPLC system (Agilent Technologies) combined with targeted detection in a 6495 MS system (Agilent Technologies). Peak areas were normalized to For tracing of heavy-labeled substrates into polyamines, cells were washed with ice-cold PBS and metabolites were extracted using extraction buffer comprising 50:30:20 methanol:acetonitrile:water (plus 2% acid for polyamine extraction) cooled on dry ice for 30 minutes beforehand. Samples were centrifuged at maximum speed for 10 minutes to remove protein debris and supernatants were dried using a Genevac EZ2 speed vac and then stored at −80 until acquisition. Metabolite analysis was carried out using an Agilent 1290 Infinity II UHPLC in line with a Bruker Impact II QTOF . Details of LC-MS methods are described by in vitro and ex vivo, cells were re-stimulated with 50 ng/mL PMA and 1 μg/mL ionomycin in the presence of brefeldin A (Biolegend) for 5 hours. Intracellular cytokine staining was performed using BD CytoFix/CytoPerm kit (BD Biosciences) and nuclear staining of transcription factors using the FoxP3 Permeabilisation kit (eBioscience). Cells were stained with Live/Dead viability dye (Thermo) prior to antibody staining. Cells were collected on LSR II and Fortessa flow cytometers (BD Biosciences) and analyzed using FlowJo (TreeStar) software. The following antibodies were used: anti-CD4, anti-TCRβ, anti-CD45, anti-IL17A, anti-IL-17B, anti-IFN-γ, anti-T-bet, anti-GATA-3, anti-FoxP3 , anti-RORγt (BD Bioscience), anti-ODC, anti-spermine synthase (both Abcam). For analysis of chromatin marks, cells were gated on Ki-67+ cells and diploid cells with ‘single’ DNA content based on FxCycle  staining in the live cell gate. Primary antibodies were stained for 90 minutes in permeabilization buffer at room temperature, followed by staining with the relevant secondary antibody for 30 minutes. The following antibodies were used: anti-H3k4Me3, anti-H3k9Ac, anti-H3k27Ac, anti-H3k9Me3 , and anti-H3k36Me3 (Abcam).Flow cytometric staining was performed as previously described . To asse5 naive CD4+ T cells (CD45Rbhi CD25- CD44lo CD62L+) from WT or Odc-ΔT mice were adoptively transferred into 12 week old Rag1−/− recipient mice (Jackson) by intravenous injection. For experiments involving the co-transfer of naive CD4+ T cells with or without Tregs, we transferred 4x105 naive CD4+ T cells (CD45Rbhi CD25- CD44lo CD62L+) from CD45.1 SJL mice with or without 1.5x105 Tregs (CD4+ CD25+ sorted by FACS post-CD4+ T cell enrichment) from either Dohh-ΔT mice or their WT littermate controls. Disease score was calculated at the experimental endpoint using the following criteria:4x10Mice were injected intraperitoneally with CD3-specific antibody  three times with 48 hours between each injection. Mice were sacrificed for analysis 4 hours after the third injection.Serum and supernatent cytokine were measured by cytokine bead array using the LEGENDPlex Th cytokine panel according to the manufacturer’s instructions (Biolegend) on a BD Fortessa flow cytometer (BD Biosciences).3 pieces and digested in 1 ug/mL Collagenase A (Sigma) and 0.5 mg/mL Dnase I (Roche) in RPMI at 37°C for 30 minutes on a shaker. Digested organs were then mechanically disrupted through a cell strainer prior to flow cytometry staining. Cell suspensions from the colonic lamina propria were prepared as follows: briefly, colons were isolated, cleaned, cut into small pieces and placed in RPMI/5% FCS supplemented with 5 mM EDTA. Tubes were then placed in 37°C in a shaking incubator to remove intestinal epithelial cells (IECs). This washing process was repeated twice, followed by one incubation with RPMI/5% FCS containing 15 mM HEPES. Digestion of the colon tissue was performed in RPMI/0.5% FCS containing 1 mg/ml type VIII collagenase (Sigma Aldrich) and 40 μg/ml DNase I (Roche) at 37°C for 60 minutes on a shaker. Supernatants were then filtered and a three-layered discontinuous Percoll gradient was used to obtain an enriched leukocyte fraction.For lung, mice were perfused with PBS through the left ventricle. Organs were cut up into 1-3 mmFor the isolation of cells from the small intestine lamina propria – small intestines were isolated, cleaned and cut into 2cm pieces. Tissue was then incubated for 25 minutes in RPMI/3% FCS supplemented with 5mM EDTA and 0.15 mg/ml DTT at 37°C with shaking. After, small intestines were washed 3 times with RPMI containing 2mM EDTA. Tissue was then digested for 30 minutes in RPMI containing 0.1mg/ml Liberase TL (Roche) and 50ug/ml DNase I (Roche) at 37°C with shaking. After that a three-layered discontinuous Percoll gradient was used to enriched for the leukocyte fraction.Samples of the colon and caecum were collected and fixed in buffered 10% of 36% formalin solution for 24 hours and then stored in 70% ethanol prior to processing. Haematoxylin and eosin (H&E) staining was performed on 4–5 mm paraffin-embedded sections.RNA was extracted using the RNeasy Kit (QIAGEN) according to manufacturer instructions and quantified using Qubit 2.0 (Thermo Fisher Scientific) following the manufacturer’s instructions. Libraries were prepared using the TruSeq stranded mRNA kit (Illumina) and sequenced in a HISeq 3000 (Illumina) by the Deep-sequencing Facility at the Max-Planck-Institute for Immunobiology and Epigenetics. Sequenced libraries were processed with deepTools , using S4 T cells treated as described were washed in PBS and then lysed in 10 mM Tris-HCl, pH 7.4,10 mM NaCl, 3 mM MgCl2 and 0.1% Igepal CA-630 . Nuclei were then spun down and then resuspend in 25 μL TD (2x reaction buffer), 2.5 μL TDE1 (Nextera Tn5 Transposase) and 22.5 μL nuclease-free water, incubated for 30 min at 37°C. DNA was purified with the QIAGEN MinElute PCR Purification Kit (Thermo Fisher Scientific). PCR amplification was performed with the NEBNext High-Fidelity 2x PCR Master Mix (New England Labs) using custom Nextera PCR Primers containing barcodes. Adaptors were removed with AMPure XP beads according to manufacturer’s protocol. Libraries were quantified with the Qubit and submitted for sequencing with a HISeq 3000 (Illumina) by the staff at the Deep-sequencing Facility at the Max-Planck-Institute for Immunobiology and Epigenetics. Sequenced samples were trimmed with Trimmomatic 9 and mapped using Bowtie2  adapting a published protocol . Briefly Bowtie2 . Open ch Bowtie2 , while d Bowtie2  with at s on IGV . Bed fil+ T cells were washed in PBS, mixed with the RNP complexes and Electroporation Enhancer in P4 Primary Cell buffer (Lonza) immediately prior to electroporation . Per electroporation, 60 pmol of Cas9 Nuclease and 180 pmol of annealed gRNAs were used. Electroporated cells were recovered in T cell medium for 2 hours prior to activation with αCD3/CD28 and pertinent polarizing cytokines. The following guides were used: Mm.Cas9.Tbx21.1AC and Mm.Cas9.Tbx21.1AD (T-Bet); Mm.Cas9.EP300.1AC and Mm.Cas9.EP300.1AE (P300); Mm.Cas9.KAT2A.1AA and Mm.Cas9.KAT2A.1AD (KAT2A); Mm.Cas9.ACLY.1AE and Mm.Cas9.ACLY.1AF (ACLY); Mm.Cas9.GCGAGGTATTCGGCTCCGCG (Non-specific control guide).All guide RNAs were purchased from IDT. Duplexes of two separate guides per target gene were prepared by annealing  equimolar concentrations of CRISPR gRNA and trRNA, then incubation  with Alt-R S.p. Cas9 Nuclease V3 (IDT). For the delivery of RNP complexes, naive CD4∗ < p0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Data are shown as mean ± s.e.m. Statistics were calculated using GraphPad Prism 6 software.p values were determined using unpaired Student’s t test unless otherwise stated. Differences were considered statistically significant when p < 0.05 (


Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. In unstimulated cells, p50/p65 is retained by the inhibitor IκBα in the cytoplasm that masks the p65-nuclear localization sequence (NLS). Upon activation, p50/p65 is translocated into the nucleus by the adapter importin α3 and the receptor importin β. Here, we describe a bipartite NLS in p50/p65, analogous to nucleoplasmin NLS but exposed in trans. Importin α3 accommodates the p50- and p65-NLSs at the major and minor NLS-binding pockets, respectively. The p50-NLS is the predominant binding determinant, while the p65-NLS induces a conformational change in the Armadillo 7 of importin α3 that stabilizes a helical conformation of the p65-NLS. Neither conformational change was observed for importin α1, which makes fewer bonds with the p50/p65 NLSs, explaining the preference for α3. We propose that importin α3 discriminates between the transcriptionally active p50/p65 heterodimer and p50/p50 and p65/65 homodimers, ensuring fidelity in NF-κB signaling. Nuclear translocation of the p50/p65 heterodimer is essential for NF-κB signaling. Here, the authors identify a bipartite Nuclear Localization Signal in the NF-κB p50/p65 heterodimer that is recognized with high affinity by importin α3. Cytoplasmic cargos targeted for active nuclear import expose a nuclear localization sequence (NLS) recognized by soluble transport receptors. Several pathways for nuclear import have been described, which share similar principles and rely on soluble transport factors of the importin β superfamily (or β-karyopherins)4. These macromolecules promote active, signal-mediated nuclear translocation of cargos by coordinating three activities: high-affinity binding to the NLS-cargo; high-avidity association with the phenylalanine-glycine (FG) repeats lining the inner part of the NPC5; and, in the nucleus, binding to the small GTPase RanGTP, which promotes dissociation from FG-nups and NLS-cargo release.The transport of macromolecules into and out of the cell nucleus is essential for cell physiology and is increasingly linked to the degenerative processes that lead to human diseases. The nuclear pore complex (NPC), the only gateway connecting the cytoplasm and nucleoplasm, functions as a semi-permeable barrier. While smaller molecules (<40 kDa) can diffuse through the NPC passively, most macromolecules in a cell, regardless of their size, shuttle through the NPC via an energy-dependent active transport6 or through specialized adaptor proteins such as snurportin and importin α7. In the classical pathway, importin α binds to importin β through its N-terminal Importin-β binding (IBB) domain9 and to NLS-cargos through its C-terminal 10 stacked Armadillo motifs (Arms) that collectively form an Arm-core11. Importin α possesses two NLS binding pockets located along the concave Arm-core surface: an N-terminal ‘major site’ and C-terminal ‘minor site’ between Arms 2–4 and 6–8, respectively11. There are seven genes for importin α in the human genome, classified into three clades, with importin α1 presumably functioning as the generic adapter for NLS cargos7. Importin α1, and to some extent other isoforms7, is auto-inhibited by the IBB domain that folds back on itself, occupying both NLS sites11. In the presence of importin β and NLS-cargo, the IBB domain is displaced, and the NLS binding sites become accessible for NLS cargo recognition11. A plethora of crystal structures has elucidated the recognition of NLS peptides by importin α12. The classical monopartite NLS, exemplified by the SV40 T-large antigen NLS, contains a short stretch of basic residues (KKKRK) that binds primarily to the major NLS binding site and, to a lesser extent, the minor site10. The bipartite nucleoplasmin NLS (KR-X10-KKKK)12, on the other hand, contains two clusters of basic residues separated by 10–12 residues, which span both NLS-binding sites. Also, several monopartite NLSs bind selectively to either the major14 or minor NLS site17, and their interaction can be regulated by phosphorylation18. Recent work has revealed that importin α isoforms recognize certain NLS-cargos with a great deal of specificity. For instance, importin α3 selectively imports NF-κB p50/p6520 and RCC121. Likewise, viruses infecting human cells hijack specific isoforms, as shown for HIV-1 INV23, influenza PB27, and Nipah and Hendra virus W protein24 that are specific for importin α3 while influenza NP25 and STAT127 appear to use importin α5. Overall, selective recognition of NLS-cargos by importin α isoforms is likely to play a role in cellular differentiation29 and disease states, including viral infections, cancer7, and developmental defects30.Importin β can import NLS-cargos directly by either binding to their exposed NLSs33. There are five known NF-κB subunits; p65 (RelA), RelB, cRel, and the precursors p105 and p100, which are partially digested to form the p50 and p52 subunits, respectively31. These NF-κB subunits exist as a variety of homodimers and heterodimers, of which p50/p65 represents the most abundant and signaling active species31. Overall, all NF-κB subunits contain an N-terminal Rel homology region (RHR) responsible for DNA binding, dimerization, and nuclear localization  pathway, and specific NF-κB subunits regulate each pathway. In the canonical NF-κB signaling pathway, a diverse subset of stimuli, including inflammatory cytokines and chemokines, activates the p65, p50, and cRel subunits and triggers rapid and transient NF-κB response34. The non-canonical NF-κB pathway, on the other hand, is triggered by a smaller subset of stimuli, which activates RelB and p52 and leads to long-term, persistent NF-κB signaling35. The p50/p65 heterodimer, the most abundant signaling complex in the canonical NF-κB pathway, is expressed in all cell types, while the p65/p65 and p50/p50 homodimers are less abundant36. The p50/p65 cellular abundance reflects the greater structural stability of the NF-κB heterodimer compared to homodimers36. The p50/p65 dimerization interface is stabilized by a network of hydrogen bonds and salt bridges, including an energetically favorable interaction between Asp254 from p50 and the equivalently positioned Asn200 in p6538, which is missing in the p50/p50 and p65/p65 homodimers. Comparing homodimers stability, Tyr267 and Phe307 in p50 are replaced by Phe213 and Val248 in p65, respectively, which reduce both the H-bonding and hydrophobic interactions stabilizing the p65 homodimer40. Thus, the NF-κB p50/p65 is more stable than p50/p50, and the p65/p65 homodimer is the least stable of the three NF-κB dimers.The nuclear factor-kappa B (NF-κB) family consists of essential transcription factors mediating various cellular processes, including immune response, cellular differentiation, and apoptosision Fig. . In cont20 identified a functional NLS responsible for nuclear import at the C-termini of p50 and p65 RHR  which mimics IκB and retains the precursor p105/p50 complex in the cytoplasm43. In the resting state, the NF-κB complex is sequestered in the cytoplasm either by IκBα-mediated retention for the p50/p65 and p65/p65 complexes44 or ARD-mediated retention for the precursor p105/p50 complex46. Upon stimulation, the activated inhibitor of NF-κB kinase complex (NEMO/IKKβ/IKKα)47 promotes IκB phosphorylation, ubiquitination, and the eventual dissociation and proteasomal degradation49, which removes the IκB repression from p50/p65 and p65/p65 complexes. The newly liberated NF-κB p50/p65 complex is then shuttled into the nucleus by the isoforms importin α3 and α4, which are 95% identical in sequence20. The p65/p65 homodimer can also localize to the nucleus by binding to importin α3 and α4 as well as other isoforms, including importin α1 and importin 851. Similarly, for the p105/p50 complex, the activated IKK-mediated signaling leads to ARD cleavage by the 20S proteasome and liberation of the p50/p50 homodimer that exposes the NLSs for importin α3/α4-mediated nuclear import52. Overall, the exact mechanisms of NF-κB homodimers (p50/p50 and p65/p65) nuclear import have not been elucidated. In vitro transfection studies of p65 and p5020 could not fully distinguish between the heterodimeric p50/p65, the most abundant NF-κB species in human cells, and the less abundant homodimeric complexes formed by p50/p50 and p65/p65.The nuclear translocation of NF-κB species is tightly regulated in human cells. Fagerlund et al.RHR Fig. . In the Here, we have investigated how NF-κB complexes interact and discriminate between importin α isoforms. Using hybrid structural methods, we found that the p50-NLS and the p65–NLS bind different regions of importin α3, generating a trans bipartite NLS. Unexpectedly, the two NLSs reveal surprising structural polymorphisms in the context of different NF-κB dimers.42. We assembled and purified three NF-κB dimers . To elucidate the association of either importin α isoform with p50/p65, we added an increasing molar excess of purified p50/p65 to 10 μg of purified ΔIBB-importin α1  though had a slightly decreased number of binding sites at equilibrium (Bmax).To determine how the p65- and p50-NLSs mediate binding to importin α, we generated constructs of p65 and p50 lacking the C-terminal NLS and assembled all possible permutations of dimeric NF-κB complexes  are not only superimposable in the two complexes (RMSD of 1.095)  Fig.  but also. 2 Fig.  versus n α1 Fig. . Accordilex Fig. . Compari1–P5 in a p50-NLS-like conformation, with Glu300, Lys301, Arg302, Lys303, and Arg304, making a total of 11 hydrogen bonds, 101 Van der Waals contacts, and burying 724.2 Å. Unlike the p50-NLS, which inserts Arg361 into the P1 position and is stabilized by Van der Waals interactions, the Glu300 in the p65-NLS orients away from the P1 position  retained and bound to both IκBα and importin α3. In contrast, the p65-NLS residues 294-303 fold into α-helix (referred to as helix 3) in the IκB-bound complex but bind importin α3 in an extended conformation. Unexpectedly, in all complexes with importin α1, the p65-NLS adopts a fully extended conformation at the minor NLS-site, with the P1′–P4′ positions occupied by 301KRKR304 residues  is more energetically favorable than the p65-NLS:importin α1 , which possibly explains the physiological preference for importin α3. Additionally, comparing the p65-NLS in both importin α isoforms, there appears to be a shift in the NLS register at the P2′ position. In the importin α1 structures  observed in the importin α3/p65–NLS complex and are part of the α-helix 4 (res. 306–321) responsible for binding to IκBα42.The mechanism by which different isoforms accommodate the p65-NLS at the minor NLS-binding pockets determines the number and types of contacts made in the two complexes. PISAres Fig. , the P2′ α3 Fig. , the P2′8. DynDom analysis56 of the importin α3 crystal structures in the unliganded state and bound to p50-NLS and p65-/p50-NLSs revealed two structural changes in the peripheral arches in response to the p65-NLS binding. At the N-terminus, a hinge movement57 was identified within Arm 4 (res. 198-201) in response to p50-NLS and p50-/p65-NLS binding compared to the apo structure   and even more from the apo α324 (RMSD 2.80 Å between residues 72 and 486). Meanwhile, importin α1 residues 75–497 are perfectly superimposable (RMSD 0.26–0.39 Å) in all complexes of importin α1 bound to either p65-, p50-, and both NLSs  of 44.21 ± 0.28 Å  calculated from the SAXS data indicates a maximum diameter Dmax ~ 137 Å  p65 Fig.  bound to8 Å Fig. , and a v7 Å Fig. . We calcmer Fig. . The dirRg of 48.56 + 0.22 Å  calculated from the SAXS data indicates a maximum diameter Dmax ~ 157 Å   calculated from the SAXS data indicates a maximum diameter Dmax ~ 151 Å, which is slightly larger than the p50/p65:∆IBB-importin α3 complex   and helix 4 (res. 306–315) in complex with IκB67. While our data suggests the p65-NLS is not a major binding determinant for importin α3 in the p50/p65 heterodimer, it is a functional NLS for the p65/p65 homodimer  independently regulated to ensure sustained gene expression76. The p65/p65 is the least stable NF-κB dimer, and while its concentration is likely low, this homodimer can also be inhibited and retained in the cytoplasm by IκBα44 2 2 Fig. , that isIn conclusion, this paper describes the mechanisms of NF-κB dimer recognition by the isoforms α3. Our work paves the way to identifying inhibitors that target the p50-NLS:importin α3 association, reducing the accumulation of NF-κB in the nucleus and dampening aberrant NF-κB signaling.41. ∆NLS-p65 (residues 1–290) was generated by introducing a stop codon at position D291 of p65 using the QuikChange site-directed mutagenesis kit (Stratagene). A synthetic gene encoding human p50 (residues 1–366), p50 lacking the DNA-binding domain ΔDBD-p50, residues 243–366), and p65 lacking the DNA-binding domain ΔDBD-p65, residues 191–316) were purchased from Genewiz and cloned between BamHI and XhoI restriction sites of expression vectors pGEX-6P-1 (GE Life Sciences). Plasmids encoding ∆IBB-importin α1 (residues 70–529) and ∆IBB-importin α3 (residues 68–487 and 63–487) were cloned into vectors pET-30a and pGEX-6P or pET-15b, respectively, as previously described21. Untagged IκBα (residues 67–287) was cloned in the pET-11a67. Peptides encompassing the p65-NLS (294-DRHRIEEKRKRTYETFKSIMKK-315) and p50-NLS (355-DKEEVQRKRQKLMP-368) were synthesized from GenScript .The plasmids encoding human p65 (residues 1–306) and ∆NLS-p50 (residues 41–351) cloned in the pLM1 vector were a generous gift of Dr. Stephen HarrisonE. coli strain in the presence of ampicillin or kanamycin. Plasmids encoding GST-ΔDBD-NF-κB and ΔIBB-Importin α3 were co-transformed in BL21 DE3 E. coli strain and expressed in the presence of ampicillin and kanamycin. Bacterial cultures were grown in LB medium at 37 °C until A600 = ~0.6 AU and all p65 induced at 16 °C with 0.25 mM IPTG for 12–16 h, all p50 induced at 18 °C at 0.25 mM IPTG for 12–16 h, and all importin α1 and α3 at 28 °C with 0.5 mM IPTG for 4 h. Cell pellets expressing all untagged p65 and p50 constructs  were sonicated in lysis buffer 1  glycerol) followed by 0.2% polyethyleneimine precipitation for 1 h at 4 °C and 55% ammonium sulfate precipitation for 2 h at 4 °C. The p65, ∆NLS-p65, and ∆NLS-p50 ammonium sulfate pellets were re-suspended in Re-Suspension Buffer  glycerol), loaded onto 5 mL HiTrapTM SP HP Column , washed with 5 CV of Buffer A , and the proteins were eluted using a linear gradient from 0% to 100% Buffer B . Cell pellets expressing GST-p50, GST-ΔDBD-p50, GST-ΔDBD-p65 and co-expressed GST-ΔDBD-NF-κB and ΔIBB-Importin α3 complexes were sonicated in Lysis Buffer 2  TWEEN 20, 1 mM EDTA), followed by 0.2% polyethyleneimine precipitation for 1 hour at 4 °C. GST-p50, GST-ΔDBD-p50, GST-ΔDBD-p65, and GST-ΔDBD-NF-κB:ΔIBB-Importin α3 complexes were purified by affinity chromatography on Glutathione Resin (GenScript) and incubated overnight with 100 U of PreScission Protease in PPase Cleavage Buffer  TWEEN 20). Untagged p50, ΔDBD-p50, ΔDBD-p65, and ΔDBD-NF-κB:ΔIBB-Importin α3 complexes were recovered off the beads. All GST-tagged7 and His-tagged ∆IBB-Importin α1 and α321 were purified as previously described. IκBα was expressed and purified as previously described67. All purified samples were concentrated with a 10- or 30 kDa Millipore PES concentrator and injected on a Superdex 200 16/60 column  pre-equilibrated with Gel Filtration Buffer 1  glycerol). Sample purity was confirmed by SDS–PAGE (13.5%) analysis. Complexes eluted from beads were further purified on a Superdex 200 16/60 column  pre-equilibrated with Gel Filtration Buffer 1. Co-purified ΔDBD-NF-κB:ΔIBB-Importin α3 complexes were further purified on a Superdex75 10/300 column .Expression plasmids for all human p65, p50 (and relative mutants), ∆IBB-importin α1, ∆IBB-importin α3 were transformed and expressed in BL21 DE3 g at 4 °C for 30 min to remove precipitation41. Homodimeric NF-κB  complexes were concentrated with a 30 kDa Millipore PES concentrator and injected on a Superdex 200 16/60 column  pre-equilibrated with GF Buffer 1. All NF-κB trimeric complexes were assembled by incubating pre-formed NF-κB hetero/homodimers in a 1.5-molar excess of ∆IBB-importin α3 or IκBα, followed by incubation at 4 °C for 1 h. Complexes were concentrated with a 30 kDa Millipore PES concentrator and injected on a Superose 12 10/30 GL column  pre-equilibrated with GF Buffer 1 followed by SDS–PAGE analysis. Trimeric complexes of NF-κB p50 or p65 dimerization domains bound to importin α3 ΔDBD-p50/ΔDBD-p50:α3 and ΔDBD-p65/ΔDBD-p65:α3, Supplementary Fig. Heterodimeric p50/p65 complexes  were assembled by incubating in a 1:1.5 p50:p65 molar ratio, respectively, together at 25 °C for 30 min, then at 37 °C for 1 h, followed by centrifugation at 12,000 × 1, NF-κB hetero/homodimers  were coated on ELISA plates  and incubated at 4 °C overnight. The plates were blocked with PBS containing 5% skim milk for 2 h at room temperature and were washed three times with PBS containing 0.05% Tween-20 (PBST). The His-importin α samples were diluted in PBST (from 1000 to 0 nM) and 100 μL added into the wells, and the plates were incubated at room temperature for 1 h. The plates were washed three times with PBST and incubated with anti-6xHis-4HRP-conjugated rabbit polyclonal antibody (1:3500) (Abcam ab1187) for 1 h at 37 °C. After washing with PBST three times, the reaction was initiated by the addition of 100 μL of tetramethylbenzidine (TMB) substrate (1-Step Ultra TMB-ELISA (Ref 34028), Thermo Scientific). The colorimetric reaction was stopped by adding 100 μL of 2 M H2SO4, and absorbance was read at 450 nm wavelength using a Tecan microplate reader. All experiments were carried out in duplicates. One-site specific binding analysis using least-squares fit was performed using GraphPad Prism 9 for Windows, GraphPad Software, LLC (version 9.2.0). Native gel electrophoresis was performed on a 1.5% agarose gel, as previously described82. In the p50/p65:α titration assay in Fig. For microtiter binding assay−1 and crystallized using the vapor diffusion hanging drop method by mixing 2 µl of purified protein with 0.5 µl containing a 2.5-fold molar excess of NLS peptide. The best crystals for ∆IBB-importin α3 bound to p50-NLS were obtained using 0.2 M potassium thiocyanate, 20% PEG3350, 0.1 M BIS–TRIS pH 6.5. ∆IBB-importin α3 bound to both the p50- and p65-NLS peptides was crystallized in the presence of 0.2 M ammonium sulfate, 0.1 M BIS–TRIS pH 6.5, 25% PEG 3350. For ∆IBB-importin α1, all complexes with the p50-NLS and p65-NLS alone or p50-NLS + p65-NLS peptides were crystallized in the presence of 0.5-0.65 M sodium citrate, 0.1 M HEPES pH 6.0, 10 mM β-Mercaptoethanol. All crystals were harvested in nylon cryo-loops, cryo-protected with 27% ethylene glycol, and flash-frozen in liquid nitrogen. Complete diffraction data were collected at beamlines 9-2 and 12-1 at the Stanford Synchrotron Radiation Lightsource (SSRL) on a Dectris Pilatus 6 M detector (Table 83 (version 2.8). The p50-NLS was built in an unbiased Fo–Fc electron density map using Coot84 (version 0.9.4), and the model was subjected to several rounds of real-space refinement using phenix.real_space_refine85 (version 1.18.2-3874) in combination with reciprocal-space and TLS refinement using phenix.refine86 (version 1.18.2-3874). This model was then used as a search template to solve the structure of ∆IBB-importin α3 bound to both the p65- and p50-NLSs, also using PHASER83. The p65-NLS and p50-NLS were modeled in unbiased Fo-Fc densities readily visible at the minor and major NLS-binding pockets, respectively, using Coot84. The complete model of ∆IBB-importin α3/p50-NLS/p65-NLS was refined as described above. The final models of ∆IBB-importin α3 bound to just the p50-NLS or both the p65- and p50-NLSs have a Rwork/Rfree of 19.9/22.0 and 19.6/23.7, at 2.10 and 2.85 Å resolution, respectively  equilibrated in SEC–SAXS Buffer . SAXS data were recorded on an EIGER 4 M detector  in vacuo at 2 s per frame with a fixed camera length of 1.709 m and 10.03 keV (1.237 Å) energy allowing the collection of the angular range q between 0.008 and 0.54 Å−1. Primary reduction of the SAXS data was performed using RAW88 (versions 1.6.4 and 2.0.3), and ATSAS software89 (version 2.6.0). To minimize the effects of damaged material accumulating on the X-ray sample window and to help compensate for any baseline drift, the buffer profile was constructed by averaging the frames before the sample peak frames. The Guinier plots of the subtracted profiles were linear to the lowest measured q value. GNOM90 was used to calculate P(r) plots from the scattering data. Ab initio model calculations to generate an average electron density from solution scattering data were done using DENSS91, as implemented in RAW. The DENSS densities have a Fourier shell correlation (FSC) of 52.2 Å (p50/p65/∆IBB-importin α3), 64.7 Å (p50/p50/∆IBB-importin α3) and 50.7 Å (p65/p65/∆IBB-importin α3), respectively. Docking of PDB models inside the SAXS density was done manually and improved by rigid-body refinement using Chimera92. Theoretical solution scattering curves were calculated using the FoXS web server93. SEC–SAXS data collection and analysis statistics are in Supplementary Table SEC–SAXS analysis was performed at ID7A1 station at MacCHESS, which is equipped with an AKTA Pure FPLC system . The trimeric p50/p65:∆IBB-importin α3, p65/p65:∆IBB-importin α3 and p50/p50:∆IBB-importin α3 complexes were pre-assembled and concentrated at 4.5, 6.6, and 6.29 mg ml92. Binding interfaces were analyzed using PISA55 and PDBsum94. Secondary structure superimpositions were done in Coot82 and analyzed using SuperPose 1.0 server59. DynDom56 was used to identify importin α3 domain movements upon cargo-binding.All structural illustrations were generated using PyMoL  and ChimeraFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Xanthium strumarium and Pueraria montana, we found that 1) X. strumarium reactivated HIV-1 latently infected cells in J-Lat 8.4, J-Lat 9.2, U1, and ACH-2 cells in vitro; 2) 27 nuclear regulatory proteins were associated with HIV-1 5′-LTR using deoxyribonucleic acid affinity pull-down and LC-MS/MS analyses; and 3) among them, silencing of XRCC6 reactivated HIV-1 5′-LTR transcriptional activity. We found that X. strumarium inhibits the 5′-LTR associated XRCC6 nuclear regulatory proteins, increases its viral 5′-LTR promoter transcriptional activity, and reactivates HIV-1 latently infected cells in vitro. These findings may contribute to understanding the 5′-LTR activity and the host cell nuclear regulatory protein machinery for reactivating HIV-1 and for future investigations to eradicate and cure HIV-1 infection.Chinese herbal medicines (CHMs) are widely used in Asian countries. They show multiple pharmacological activities, including antiviral activities. The 5′-long terminal repeat (LTR) region of HIV-1, required for viral transcription, is a potential drug target for HIV-1 reactivation and intrinsic cell death induction of infected or latently infected cells. Modulation of HIV-1 reactivation requires interactions between host cell proteins and viral 5′-LTR elements. By evaluation of two CHMs-  Howeverof HAART .- and 3′- long terminal repeat (LTR) regions  Greuter (X. strumarium) (Family Asteraceae), also called Cocklebur or Xanthii Fructus, is a CHM. X. strumarium has multiple pharmacological activities including the antiviral activity (Pueraria montana var. lobata (Willd.) Sanjappa and Pradeep (P. montana) (Family Fabaceae) has antiviral activity against the human respiratory syncytial virus (HRSV)  have been widely used in HIV-1 infected patients . Xanthiuactivity . As a trs (HRSV) . Further surface . HoweverX. strumarium and P. montana, we found that X. strumarium reactivated HIV-1 latently infected cells in vitro. HIV-1 5′-LTR DNA affinity pull-down coupled with LC-MS/MS analyses identified 27 nuclear regulatory proteins in X. strumarium-treated cells. Characterization of the HIV-1 5′-LTR promoter activity by silencing these newly identified nuclear regulatory proteins was also examined. To our knowledge, this is the first study to describe X. strumarium exerting a HIV-1 reactivation activity via a mechanism that modulating the composition of nuclear regulatory proteins in the 5′-LTR region.Evaluating the two CHMs- nef coding sequence and a frameshift mutation in the env gene (J-Lat cell lines (clones 8.4 and 9.2) were clonal Jurkat cells with a latent HIV-1 provirus, in which the enhanced green fluorescent protein (EGFP) replaces the env gene . J-Lat cl-glutamine (Gibco).ACH-2 (T cell-derived) and U1 (promonocyte-derived) cell lines were cells with full-length proviruses, without EGFP replacing coding sequence -1,3-benzene disulfonate (WST-1) assay  as described previously . After tA high-performance LC system  coupled with a quadrupole-time-of-flight MS (Q-TOF MS)  was used with the full scan, DDA and multiple reaction monitoring (MRM) function .X. strumarium  was used with a flow rate of 0.2 ml/min and the mobile phases of solvent A (0.1% formic acid) and solvent B (100% acetonitrile). Three LC gradient methods, A and B, were used to produce suitable separation conditions for different compounds. In method A, solvent B was maintained at 5% for 1 min and then increased to 99% for 18 min. After maintaining this for 2.5 min, solvent B was decreased to 5% and held at this concentration for 3 min at a flow rate of 0.2 ml/min. Method A was used to analyze the marker compounds of chlorogenic acid and 1,3-dicaffeoylquinic acid in rumarium . In meth montana .The mass spectrometer was operated in negative ion mode for chlorogenic acid and 1,3-dicaffeoylquinic acid analysis in the m/z range 50–1,000 at 2 Hz. The capillary voltage of the ion source was set at -2500 V for the negative mode, and the endplate offset was 500 V. The nebulizer gas flow was 1 bar, and the drying gas flow rate was 6 L/min. The drying temperature was set to 200°C. For daidzein, genistein, and ononin, the mass spectrometer was operated in positive ion mode using an m/z range of 50–800 at 2 Hz. The capillary voltage of the ion source was set at +4500 V, and the endplate offset was 500 V. The nebulizer gas flow was 1 bar, and the drying gas flow rate was 8 L/min. The drying temperature was set to 200°C.X. strumarium and P. montana (1 or 2 mg/ml) for 24 and 48 h, respectively, at 37°C, followed by Western blot analysis  for 48 h  for 48 h  for 48 h  Chang-Gung University). The pNL4-3 plasmid was used as the DNA template for the in vitro synthesized 621-bp-long DNA fragment of the HIV-1 5′-LTR (GenBank AF324493.2: nt 1–621) by polymerase chain reaction (PCR). The following primers were used: forward primer, 5′-/desBioTEG/TGGAAGGGCTAATTTGGT-3′ and reverse primer: 5′-/Cy3/CCACACTGACTAAAAGGGTCTG-3′.The pNL4-3 plasmid contained a HIV-1 5′- LTR fragment  upstream of the X. strumarium (2 mg/ml) for 24 h. The treated cells were harvested, washed, and resuspended in ice-cold PBS. Cells were used as negative controls. Thereafter, the treated cells and cell-only controls were lysed and extracted using a nuclear extraction kit .J-Lat 9.2 cells were treated with The J-Lat 9.2 cell nuclear extracts were pre-incubated with magnetic beads. Biotin-tagged HIV-1 5′-LTR DNA fragments were then incubated with J-Lat 9.2 cell nuclear extracts for 4 h at 4°C . Biotin-The purified proteins were identified using a nanoflow UPLC system  coupled to a quadrupole time-of-flight (Q-TOF) mass spectrometer . The peptides were separated using an RP C18 capillary column (25 cm × 75 µm id) at a 300 nL/min flow rate, and eluted with a linear ACN gradient from 10 to 50% ACN in 0.1% formic acid for 90 min. The eluted peptides from the capillary column were sprayed into the MS using a captive spray ion source . Data acquisition from Q-TOF was performed using Data Analysis software . Proteins were identified by the nanoLC-MS/MS spectra  by searcThe TZM-bl cells were transfected with pcDNA HIV-1 viral tat-flag gene plasmid and siRNAs including siHMGA1, siNCL, siRBBP4, siYBX1, siIFI16, siNOLC1, siXRCC5, siXRCC6, siTOP2B, and siSRSF1 for 24 h or 48 h, respectively . siNC waHMGA1, forward: 5′-GAAAAGGACGGCACTGAGAA-3′and reverse: 5′-CTCTTAGGTGTTGGCACTTCG-3′; NCL, forward: 5′-CCACTTGTCCGCTTCACA -3′and reverse: 5′-TCTTGGGGTCACCTTGATTT-3′; SRSF1, forward: 5′-GGCGGTCTGAAAACAGAGTG-3′and reverse: 5′-TTTAAATCCTGCCAACTTCCA-3′; IFI16, forward: 5′-ACTCCTCAGATGCCACCAAC-3′ and reverse: 5′-TCATTTTGGAGATTGTGTCTTCAC-3′; NOLC1, forward: 5′-ATAAGTTCGCCAAAGCGACA-3′ and reverse: 5′-CTAAGAGGGAAGAGGCATTGG-3′; RBBP4, forward: 5′-CGGATGAACAAAACCATCTTG-3′ and reverse: 5′-CTGAACCAAAACCTCCAAATTC-3′; TOP2B, forward: 5′-AGCCTGGAAAGAAGCACAAG-3′ and reverse: 5′-TCGGAGGAACTATCATCATGC-3′; XRCC5, forward: 5′- GAGCCCACTTCAGCGTCT-3′ and reverse: 5′-CAGCAGGATTCACACTTCCA-3′; XRCC6, forward: 5′-CCTTTTGACATGAGCATCCA-3′ and reverse: 5′-AATTTTTGTCTTTCTCGGTACCAT-3′; YBX1, forward: 5′-GGAGGGTGCTGACAACCA-3′ and reverse: 5′-GCTGTCTTTGGCGAGGAG-3′; firefly luciferase, forward: 5′-AGGTCTTCCCGACGATGA-3′ and reverse: 5′-GTCTTTCCGTGCTCCAAAAC-3′; GFP, forward: 5′-GAAGCGCGATCACATGGT-3′ and reverse: 5′-CCATGCCGAGAGTGATCC-3′.Cellular RNA was isolated using a QIAamp® RNA Mini Kit according to the manufacturer’s instructions . RNA was eluted in 60 μL buffer, and real-time TaqMan RT-PCR assays were used to determine siRNA knockdown efficiency. The primers used for quantitative PCR (qPCR) amplification were as follows: Reverse transcription was performed in a 10 μL reaction mixture consisting of 2 μL RNA template, 1 μL RT primer mix, 1 μL dNTP mix (10 mm each), and 6 μL RNA/DNAse-free water at 65°C for 5 min. Next, a reaction mixture of 4 μL 5 × MMLV buffer, 0.8 μL MMLV enzyme, and 5.2 μL RNA/DNAse-free water was added to each RNA sample. Reverse transcription reactions were performed at 42°C for 60 min cDNA was amplified by PCR in a 20 μL reaction mixture containing 5 μL cDNA, 10 μL, 2 × Mastermix, 1 μL primer/probe mix, and 4 μL RNA/DNAse-free water. Real-time TaqMan RT-PCR conditions were 95°C for 10 min, 50 cycles of 95°C for 10 s, and 60°C for 60 s. RNA levels were detected using a 7900HT Fast Real-Time PCR System .p < 0.05. A statistical package (SPSS 19.0) was used for all analyses.A Student’s t-test was used to compare the differences between the two groups . StatistX. strumarium and P. montana, their corresponding marker compounds  control, allowing the detection of HIV-1 reactivation. In this study, to evaluate the HIV-1 reactivation activity for X. strumarium and P. montana, J-Lat cells were used -treated cells served as the positive controls. The activation of EGFP protein expression under HIV-1 5′-LTR control was detected using SDS-PAGE and Western immunoblotting against anti-EGFP antibodies (J-Lat cells included J-Lat 8.4 and 9.2 cells . These Jere used . Water etibodies .X. strumarium (Lanes 4 and 5) resulted in significant EGFP protein expressions in the cell lysates at 24 and 48 h when compared with the cells only (Lane 1) (P. montana (Lanes 2 and 3) resulted in very low EGFP protein expression when compared with the cells only (Lane 1) (X. strumarium-treated cells (Lanes 4 and 5). These results suggested that X. strumarium reactivated HIV-1 latently infected cells via inducing EGFP protein expressions under the HIV-1 5′-LTR control.As shown, for J-Lat 8.4 cells, cells treated with 1 or 2 mg/ml (Lane 1) . TNF-alp(Lane 1) . Tubulin(Lane 1)  and is u(Lane 1) . SignifiX. strumarium on HIV-1 reactivation activity, HIV-1 p24 virus capsid proteins in the cell culture supernatants were detected in J-Lat 9.2, U1, and ACH-2 cells using enzyme-linked immunosorbent assay (ELISA) analysis (X. strumarium (2 mg/ml) resulted in significantly increased HIV-1 p24 virus capsid protein expressions (900 pg/ml) in the cell culture supernatants at 48 h when compared with the un-treated cell controls (100 pg/ml)  .X. strumarium  resulted in significant increased HIV-1 p24 virus capsid protein expressions ) in the cell culture supernatants at 48 h when compared with the un-treated cell controls (1765 pg/ml)  also resulted in increased HIV-1 p24 virus capsid protein expressions ) at 48 h when compared with the un-treated cell controls (950 pg/ml) (X. strumarium reactivated J-Lat 9.2 cells and another two kinds of HIV-1 latently infected cells by inducing HIV-1 p24 virus capsid protein expression under the HIV-1 5′-LTR control.The U1 cells (promonocyte-derived cell line) and ACH-2 cells (T cell-derived cell line) were the cells with the full-length HIV-1 proviruses to detect the reactivation of HIV-1 gene products . U1 cell5 pg/ml) . ACH-2 c0 pg/ml) . These rX. strumarium exhibited HIV-1 reactivation activity, which was controlled by HIV-1 5′-LTR. HIV-1 5′- LTR-associated nuclear regulatory proteins may be involved in HIV-1 reactivation. To identify nuclear regulatory proteins affected by X. strumarium, J-Lat 9.2 cells were treated with or without X. strumarium. The cell nuclear extracts then interacted with the biotin-labeled HIV-1 5′- LTR DNA fragments . There were no significant differences in HMGA1, SRSF1, and RBBP4 mRNA expression between siRNA-treated and siNC-treated cells . There were no significant differences in HMGA1, SRSF1, NOLC1, and RBBP4 mRNA expression between siRNA-treated and siNC-treated cells . Cells transfected with siXRCC6 and siXRCC5 + siXRCC6, respectively, resulted in increased mRNA levels of the firefly luciferase gene when compared with the siNC transfected cells at 48 h after transfection .To investigate the effect of sfection . As showX. strumarium and the 2 nuclear regulatory proteins- XRCC5 and XRCC6, J-Lat 8.4 cells were treated with X. strumarium (2 mg/ml) for 48 h  reactivated the GFP mRNA expressions to 35,000% when compared with the untreated cells (J-Lat 8.4 cells only: 100%). As shown in XRCC5 mRNA expression between the untreated and treated cells. As shown in XRCC6 mRNA expression was reduced to 10% in the X. strumarium (2 mg/ml)-treated cells when compared with the untreated cells (J-Lat 8.4 cells only: 100%). These results suggest that X. strumarium may reduce XRCC6 mRNA expression. Silencing of 5′-LTR associated XRCC6 nuclear regulatory protein may reactivate latent HIV-1 infected cells in vitro.To investigate the relationship between for 48 h . The mRNX. strumarium reactivated three HIV-1 latent cell models. Our deoxyribonucleic acid (DNA) affinity pull-down assay identified 27 nuclear regulatory proteins were associated with HIV-1 5′-LTR region in X. strumarium treated cells. In addition, silencing of the XRCC6 nuclear regulatory protein reactivated HIV-1 5′-LTR promoter activity.Chinese herbal medicines (CHMs) exhibit multiple pharmacological properties. Chinese herbal medicines (CHMs) have been widely used in Taiwan, including in HIV-1 infected patients . This stX. strumarium reactivated the HIV-1 5′-LTR driven transcription of the GFP reporter gene in J-Lat 8.4 and J-Lat 9.2 cells. In addition, X. strumarium exhibited the activation activity of the HIV-1 5′-LTR-driven protein expressions in J-Lat 9.2, U1, and ACH-2 cells by inducing HIV-1 viral p24 protein expressions. No similar studies have reported that X. strumarium can reactivate HIV-1 latently infected cells. Cary et al. showed that Euphorbia kansui, a CHM, can effectively activate CD4+ T cells and reactivate HIV-1 from latency, particularly when combined with HDACi (SAHA) or BETi (JQ1)  inducer prostratin and stimulates the latent HIV reservoir (X. strumarium reactivated HIV-1 latently infected cells in vitro. Further studies in isolating active compounds from X. strumarium should be performed.We first found that Ti (JQ1) . EK-16A,eservoir . A combieservoir . This stX. strumarium-treated nuclear extracts. We also found that X. strumarium may reduce XRCC6 expressions in the mRNA level (Our results showed that deoxyribonucleic acid (DNA) affinity pull-down assay identified 27 nuclear regulatory proteins associated with the HIV-1 5′-LTR region from the NA level . The reaNA level . These HNA level .XRCC6). Ku is a dimeric protein complex that binds to DNA double-strand break ends and is required for the non-homologous end joining (NHEJ) pathway of DNA repair. The Ku heterodimer acts as a transcription repressor and regulates transcription from the HIV-1 promoter  by increasing the affinity of the catalytic subunit PRKDC to DNA by 100-fold. The XRCC5 and XRCC6 dimers are involved in stabilizing the broken DNA ends and bringing them together. The XRCC5 and XRCC6 dimers, together with APEX1, act as negative regulators of transcription.Our study showed that the reactivation activity of the HIV-1 5′-LTR region was silenced by X-ray repair cross-complementing 6 (promoter . Ku70 (XX. strumarium may modulate the composition of these 5′-LTR associated nuclear regulatory proteins, affect 5′-LTR promoter activity, and reactivate HIV-1 latently infected cells in vitro. We identified 27 nuclear regulatory proteins associated with the HIV-1 5′-LTR region in X. strumarium-treated cells. Silencing of XRCC6 reactivates HIV-1 5′-LTR transcriptional activity. A limitation of this study was the lack of the use of primary human cells and isolation of active compounds from X. strumarium. However, with the use of current cell lines, we observed that X. strumarium reactivated HIV-1 in vitro. Future studies on primary human cells and isolation of active compounds from X. strumarium will be conducted. These findings may contribute to understanding the viral 5′-LTR promoter activity and the mechanism by which drug-induced host cell nuclear regulatory proteins reactivate HIV-1 and for future investigations to eradicate and cure HIV-1 infection.To our knowledge, this study is the first to demonstrate that