To address this issue, we developed a series of di- and trinucleotide capping reagents and in vitro transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5′-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, R1ppApG-type structures (where R1 is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (∼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of R2ppUpG-type initiating nucleotides (where R2 is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5′ capping.Many eukaryotic and some bacterial RNAs are modified at the 5′ end by the addition of cap structures. In addition to the classic 7-methylguanosine 5′ cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to  Access nNs-RNA) .in vitro transcription reaction is typically performed by bacteriophage RNA polymerases , using a mixture of four natural nucleoside 5′-triphosphates (NTPs) and a double-stranded dsDNA template (nN’ (N′ is the nucleoside defined by the TSS) structure is added to the transcription mixture, it can serve as an alternative transcription initiator, leading to the corresponding capped RNA (NpnN′-RNA). Two common promoters used for transcription by T7 polymerase are φ 2.5 (...T-1A+1G+2G+3) and φ 6.5 , which trigger initiation with ATP and GTP, respectively. Transcription from templates containing these promoters provides access to purine-initiated uncapped RNAs and RNAs capped with cap structures containing G  or A  as the 5′ terminal nucleotide, thereby producing m7GpppG-capped RNAs or NAD-capped RNAs, respectively , which provided higher RNA capping efficiencies than dinucleotides and enabled more structural variability within the position of the first transcribed nucleotide .1). A linear gradient of 0–50% methanol in buffer A  over 30 min was typically used. All the nucleotides were UV-detected at 254 nm.Analytical RP HPLC was performed on Agilent apparatus using GEMINI LC-18-T HPLC column  column. To this end, the column was loaded with the quenched reaction mixture and washed thoroughly with water until the eluate did not precipitate with AgNO3 solution, in order to remove solvents and unbound reagents. Nucleotides were then eluted with the use of a linear gradient of triethylammonium bicarbonate buffer (TEAB) in deionized water. Collected fractions were analyzed spectrophotometrically at 260 nm and if necessary by RP-HPLC. Fractions containing the desired product were combined. After concentration under reduced pressure with repeated additions of ethanol (96%) followed by precipitation in acetonitrile , compounds were isolated as triethylammonium salts.Isolation of the nucleotides was performed by ion-exchange chromatography on a DEAE Sephadex A-25 (HCO-1) using UV detection at 254 nm. A linear gradient of acetonitrile (from 0 to 50% of ACN for 60 min) in buffer A  was applied except for uridine derivatives which were purified using an isocratic buffer A elution. After repeated freeze-drying of the collected fractions, the products were isolated as ammonium (NH4+) salts.All final products were additionally purified on a semi-preparative RP-HPLC column  were used to determine compound concentrations: 17400 for NAD; 29480 for NADpG; 37000 For FAD, 49080 for FADpG and 19,566 for nucleotides containing U and G nucleobases.Yields were calculated on the basis of UV absorbance of the aqueous solutions of isolated products and corresponding starting materials. Absorbance measurements were performed after diluting an aliquot of the stock solution of the compound in a 0.1 M phosphate buffer at 260 nm (pH 7.0). The following absorption coefficients  spectrometer. NMR spectra were recorded at 25°C with a BRUKER AVANCE III HD spectrometer at 500 MHz (1H NMR) and 202 MHz (31P NMR), or at 400 and 162 MHz, respectively, on a Varian UNITY spectrometer. 1H NMR chemical shifts were calibrated to sodium 3-trimethylsilyl-propionate (TSP) in D2O and for 31P NMR to H3PO4 (20%) in D2O as an external standard. Signal assignments and identification were based on COSY spectra analysis. The raw NMR spectroscopic data were processed by MestReNova v 12.0.2-20,910 Software.The structures and homogeneity of all final compounds were confirmed by RP-HPLC, high-resolution mass spectrometry using electrospray ionization (HRMS-ESI) and H-tetrazole (0.30 M in acetonitrile) and 0.2 M acetonitrile solution of an appropriate TBDMS-protected adenosine or uridine phosphoramidite (step 1) and bis (cyanoethyl)- N,N- diisopropylphosphoramidite (ChemGenes®) (step 2) were recirculated through the column for 15 min. The detritylation step was performed using 3% (v/v) dichloroacetic acid in toluene, the oxidation was performed with 0.05 M iodine in pyridine. The capping step was performed using 20% (v/v) N-methylimidazole in acetonitrile as Cap A and a mixture of 40% (v/v) acetic anhydride and 40% (v/v) pyridine in acetonitrile as Cap B. The 2-cyanoethyl groups were removed after the last cycle by passing 20% (v/v) diethylamine in acetonitrile through the column. The solid support was finally dried with argon, transferred to a falcon tube and the dinucleotide was cleaved from the support at 37°C for 1 h using 5 ml of AMA . The suspension was filtered, evaporated to dryness, freeze-dried from water and then resuspended in 100 μl of DMSO before the addition of 125 μl of triethylammonium trihydrofluoride (TEA·3HF), in order to remove the TBDMS protecting groups. The resulting solution was incubated at 65°C for 2 h and diluted with 10 ml of 0.2 M NaHCO3 to adjust the pH to 7. The crude product was purified by DEAE Sephadex using a linear gradient of TEAB (0–0.9 M) and isolated as a triethylammonium salt. Fractions containing the dinucleotide were combined and evaporated to dryness.The syntheses were performed using an automatic synthesizer ÄKTA Oligopilot plus 10 on a high-loaded polystyrene support  on a 50 μmol scale  using standard phosphoramidite chemistry. In the coupling steps, 5-(benzylthio)-1-A using N-PAC-5′-O-DMT-2′-O-TBDMS-adenosine 3′-O- (ChemGenes®) . This procedure allowed to obtain 3’-(guanyl-5′-yl)-adenosine 5′-monophosphate .The synthesis was performed following the general procedure : Calculated for C20H25N10O14P2- [(M-H)-]: 691.10324; Found: 691.10392.HR-MS A using 5’-O-DMT-2′-O-TBDMS-uridine 3′-O- (ChemGenes®) . This procedure allowed to obtain 3’-(guanyl-5′-yl)-uridine 5′-monophosphate .The synthesis was performed following the general procedure 19H24N7O16P2-([M-H]-): 668.07602; Found: 668.07627.HR-MS: Calculated for Creaction). The resulting precipitate was washed with cold acetone and centrifuged until the supernatant was clear. The resulting powder was then dried under vacuum for few hours, affording the imidazolide (Im) activated nucleotide (ImpNpG or NMP-Im).The desired mono- or dinucleotide was suspended in DMSO (C = 0.1 M), followed by the addition of imidazole, 2,2′-dithiodipyridine, triethylamine, and triphenylphosphine. The reaction was stirred at r.t. Until a complete conversion was determined by RP-HPLC analysis. The reaction was quenched by the addition of a cold solution of sodium perchlorate (3 equiv.) in acetone , imidazole , 2,2′-dithiodipyridine , triethylamine  and triphenylphosphine  in DMSO (0.31 ml). The reaction was stirred at r.t. for 48 h. After precipitation, ImpApG was obtained as a sodium salt .The synthesis was performed following the general procedure B using commercial sodium salt UMP transformed in triethylammonium salt , imidazole  2,2′-dithiodipyridine , triethylamine  and triphenylphosphine  in DMSO (0.9 ml). The reaction was stirred at r.t. for 1 h. After precipitation, UMP-Im was obtained as sodium salt and used directly in further coupling step.The synthesis was performed following the general procedure B using pUpG (0.029 mmol), imidazole  2,2′-dithiodipyridine , triethylamine  and triphenylphosphine  in DMSO (0.3 ml). The reaction was stirred at r.t. for 16 h. After precipitation, ImpUpG was obtained as sodium salt and used directly in further coupling step.The synthesis was performed following the general procedure The nucleotide 5′-phosphorimidazolide (1 equiv.) was dissolved in DMSO or DMF. Then, zinc or magnesium chloride (3–8 equiv.) and the appropriate phosphate nucleophile (1–3 equiv.) were added. The reaction was mixed at r.t and the progress was monitored by RP HPLC. When a complete conversion of the starting material was observed, the reaction was quenched by dilution with a solution of equimolar disodium EDTA in 10 volumes of water and adjusted to pH 7 with sodium bicarbonate. The purification was performed by ion exchange chromatography (DEAE Sephadex) using a linear gradient of TEAB (from 0 to stipulated concentration), followed by a semi-preparative RP HPLC.NADpG (1) was synthesized as described previously .C using ImpApG, , flavine mononucleotide as triethylammonium salt  and zinc chloride  in DMSO (0.75 ml). The reaction was stirred at r.t. for 16 h. After Sephadex (0.9 M TEAB) and RP-HPLC purifications, the expected product FADpG was obtained as ammonium salt .The synthesis was performed following the general coupling procedure 1H NMR : δ1H NMR  8.39 , 8.02 , 7.79 , 7.59 , 7.57 , 5.86 , 5.67 , 4.99–4.89 , 4.78–4.71 , 4.71–4.65 , 4.60 , 4.44–4.40 , 4.39–4.35 , 4.33–4.19 , 4.19–4.03 , 3.94 , 2.36 , 2.28 , 2.00–1.96 . 31P NMR : δ -0.80 , -10.44 , -11.36 . HR-MS : Calculated for C37H44N14O22P3- ([M-H]-): 1129.19729; Found: 1129.19843.C using UMP-Im , N-acetylglucosamine-1-phosphate (NAcGlcp) as triethylammonium salt  and magnesium chloride  in DMF (0.064 ml). The reaction was stirred at r.t. for 2 h. After Sephadex (1 M TEAB) and RP-HPLC purifications, the expected product NAcGlcppU was obtained as ammonium salt .The synthesis was performed following the general procedure 17H26N3O17P2- [(M-H)-]: 606.07429; Found: 606.07455.HRMS : Calculated for CC using ImpUpG (0.015 mmol), glucose-1-phosphate as triethylammonium salt  and magnesium chloride  in DMF (0.2 ml). The reaction was stirred at r.t. for 30 h. After Sephadex (1 M TEAB) and RP-HPLC purifications, the expected product GlcppUpG was obtained as ammonium salt .The synthesis was performed following the general procedure 1H NMR : δ 8.04 , 7.86 , 5.93 , 5.89 , 5.63–5.58 , 4.86–4.81 , 4.70–4.60 , 4.54–4.46 , 4.41–4.30 , 4.24–4.07 , 3.93–3.82 , 3.82–3.73 , 3.53 , 3.45 . 31P NMR : δ -0.70 , -11.35 , -12.76 . HR-MS : Calculated for C25H35N7O24P3- ([M-H]-): 910.09518; Found: 910.09497.C using ImpUpG , N-Acetylglucosamine-1-phosphate as triethylammonium salt  and magnesium chloride  in DMF (0.13 ml). The reaction was stirred at r.t. for 48 h. After Sephadex (1 M TEAB) and RP-HPLC purifications, the expected product NAcGlcppUpG was obtained as ammonium salt .The synthesis was performed following the general procedure 1H NMR : δ 8.01 , 7.86 , 5.91 , 5.88 , 5.51 , 4.83 , 4.66–4.60 , 4.53–4.48 , 4.41–4.30 , 4.22–4.08 , 4.02–3.90 , 3.89–3.76 , 3.54 , 2.06 . 31P NMR : δ -0.78 , -11.59 , -13.09 . HR-MS : Calculated for C27H38N8O24P3- [(M-H)-]: 951.12173; Found: 951.12264.C using ImpUpG , pyrophosphate as triethylammonium salt  and zinc chloride  in DMF (0.3 ml). The reaction was stirred at r.t. for 1 h. After Sephadex (1.2 M TEAB) and RP-HPLC purifications, the expected product pppUpG was obtained as ammonium salt .The synthesis was performed following the general procedure 1H NMR : δ 7.99 , 7.86 , 5.95 , 5.88 , 5.86 , 4.90–4.83 , 4.67–4.62 , 4.50 , 4.40–4.29 , 4.23–4.05 . 31P NMR : δ -0.86 , -11.46 , -22.65 . HR-MS : Calculated for C19H26N7O22P4- [(M-H)-]: 828.00869; Found: 828.00929.NAD-capped RNAs were generated on the template of annealed oligonucleotides, which contained either a T7 A φ 2.5 promoter sequence (CAGTAATACGACTCACTATT) followed by a 35-nt-long sequence (AGG GAAGCGGGCATGCGGCCAGCCATAGCCGATCA), or G φ 6.5 promoter sequence (CAGTAATACGACTCACTATA) followed by a 35-nt-long sequence (GGG GAAGCGGGCATGCGGCCAGCCATAGCCGATCA).in vitro transcription reaction (80 µl) was incubated at 37°C for 4 h and contained RNA polymerase buffer , 10 U/µL T7 polymerase , 1 U/µl RiboLock RNase inhibitor (ThermoFisher Scientific), 0.5 mM CTP/UTP/GTP or CTP/UTP/ATP with 0.125 mM ATP or GTP for 0.1 µM A φ 2.5 or G φ 2.5 DNA template respectively—and 0.375–1.125 mM cap analog of interest (3- to 9-fold excess). Following 4 h incubation, the template was removed by treatment with 1 U/µL DNase I for 30 min at 37 C. The obtained RNAs were purified using RNA Clean & Concentrator-5 (Zymo Research). Transcript homogeneity was analyzed on 15% acrylamide/7 M urea gels/1⨯ TBE and stained with SYBR Gold, whereas the concentration was determined spectrophotometrically.Typical 2 and 50 mM Tris-HCl pH 8.0 for 2 min at 95°C followed by 1 h at 37°C, then DNase I treatment was performed, which allowed to produce 3′-end homogenous 25 nt RNAs. The homogeneity and concentration of the prepared RNAs were analyzed as above. Additionally the transcripts were analyzed on a 1% acryloylphenyl boronic acid in 15% acrylamide/7 M urea gel/1⨯ TBE, with SYBR Gold staining (Icapped and Iuncapped) corresponding to capped and uncapped RNAs according to the equation:To generate homogenous 3′-ends, RNAs were first subjected to HPLC purification using Clarity® 3 µM Oligo-RP phenomenex column at 50 °C, 1 ml/min on Shimadzu apparatus. A linear gradient from 10% to 30% of 200 mM TEAAc pH7/ACN 1/1 in 100 mM TEAAc pH 7.0 was applied. The collection of RNA samples was performed in a way that both capped and uncapped RNA fractions were pooled together . The collected RNAs were freeze dried twice before the DNAzyme treatment. For that purpose, the HPLC purified 35-nt-long transcripts (1 µM) were incubated with 1 µM DNAzyme 10-23  in 50 mstaining . The cap2 and 1 mM DTT at 37 °C for 2 h to release intact FAD. FAD standard curve was prepared under the same reaction conditions using increasing amount of FAD-capped RNA obtained in the presence of 9 equiv. of FADpG. To quantify the released FAD, an FAD assay kit  was used according to the manufacture’s protocol.FAD-CapQ analysis for capping efficiency of FAD-capped RNAs was performed as previously reported.  Briefly,in vitro transcription reaction (20 µL) was incubated at 37°C for 3 h and contained RNA polymerase buffer , 10 U/µl T7 polymerase , 0.0125 U/µl pyrophosphatase (ThermoFisher Scientific), additional 15 mM MgCl2, 1 U/µl RiboLock RNase inhibitor (ThermoFisher Scientific), and either 1) 5 mM CTP/UTP/GTP and 4 mM ATP and 10 mM cap analog of interest (2.5-fold excess of FAD or FADpG) for 40 ng/µl A φ 2.5 DNA template or 2) 5 mM CTP/UTP/ATP with 4 mM GTP and 10 mM cap analog (GlcppUpG or NAcGlcppUpG) for 40 ng/µl of G φ 6.5 DNA template. Following 3 h incubation, the template was removed by treatment with 1 U/µl DNase I for 30 min at 37°C. The obtained RNAs were purified using 500 µg Monarch RNA clean-up kit (New England Biolabs). Transcript homogeneity was analyzed on 15% acrylamide/7 M urea gels/1⨯ TBE and stained with SYBR Gold, whereas the concentration was determined spectrophotometrically.Typical Before analyses and between each series of three technical repetitions the chromatographic system required a washing with 1% phosphoric acid (v/v) in 50% aqueous methanol for 2 h and further 50% aqueous methanol for another 2 h to remove all traces of metal cations unspecifically bound within the system .All analyses were performed on Agilent system  equipped with diode array detector (recording at 260 nm) and mass spectrometer (AB Sciex QTRAP3200) in reversed phase mode . Analytical method was elaborated on the basis of previously published conditions . Flow ra260 = 0.025 (μg/ml)cm−1) was injected per single analysis.A portion of 10 μl of RNA solution : for deconvoluted data [M-15H + 14Na]-1; for signals on spectrogram [M-24H + 14Na]-10, [M-25H + 14Na]-11, [M-26H + 14Na]-12, etc. Qualitative analyses were based on comparison of theoretical data and experimental with accuracy of few daltons  in ordess.org/, . ChemCallc.org/, ). All siFor semi-quantitative analyses Analyst 1.6.3 was used. The most intense signals on the spectrogram of each RNA type were integrated with IntelliQuant mode .Capping efficiency was determined based on the ratio of area corresponding to total capped RNAs versus total capped and uncapped RNAs, which have been identified by means of qualitative analyses. The mean value was calculated from three technical replicates.1–5, respectively) and studied their incorporation into RNA in comparison to their unmodified counterparts, NAD, FAD, GlcUDP and NAcGlcUDP  and φ 6.5 (...A-1G+1G+2G+3) promoters , while the concentration of the initiating non-canonical cap analog was elevated (3-fold relative to the initiating NTP). The resulting RNA was analyzed by denaturing polyacrylamide gel electrophoresis . To ensure more efficient capping with the initiating nucleotides, the concentration of NTP corresponding to the TSS in the promoter sequence  was lower than that of other NTPs , followed by 3′ end trimming. The resulting 25 nt RNAs were analyzed using HRPAGE , which yielded incorporation efficiencies close to 50% .Since the heterogeneity of RNA samples obtained with polymerase T7 arises mostly from the 3′ ends , to enabg HRPAGE . Unfortucis-diol groups, which are present at the RNA 3′-ends and optionally at the 5′-ends if the RNA is 5′ capped (cis-diol function within the RNA) compared to uncapped RNA, allowing an easier separation and assessment of incorporation. Interestingly, analysis of differently modified RNAs in APB gels revealed that a significant improvement in the separation between capped and uncapped RNA was observed only for NAD derivatives, whereas for FAD and nucleotide sugar derivatives, the effect of APB was barely notable . ABP has been used to differentiate the mobility of capped and uncapped RNAs due to interactions with ′ capped . Hence,  notable . We hypo35 was around 11.9 min  and G (φ 6.5), we observed a similar incorporation efficiency but slightly better incorporation of NADpG when the G (φ 6.5) promoter was used, in contrast to FADpG, which showed a better efficiency using the A (φ 2.5) promoter .As a final verification of the capping efficiencies for our novel RNA 5’ end analogs, we performed an LC-MS analysis of 35 nt FAD-RNAs, GlcppU-RNAs, and NAcGlcppU-RNAs obtained by IVT under promoters specified in in vitro transcription, thus promoting the prospect of more efficiently introducing various non-canonical RNA 5′-caps and providing access to molecular tools that could help understand the biological functions of non-canonically capped RNAs. For instance, 3′ biotinylation of non-canonically capped RNAs could yield probes for pull-down assays, to provide information on proteins interacting with the corresponding 5′ caps. Future research may also involve the application of this methodology for non-canonically capped mRNA synthesis in order to assess its biological functions and translational properties. Further studies on longer RNA templates and novel analytical methods would also be useful in the future to determine the full scope and limitations of this capping technology.Herein, we designed and synthesized a set of di- and trinucleotide derivatives of various non-canonical RNA 5′-ends straightforwardly and efficiently. In cases where dinucleotides could not be incorporated into RNA with the usual polymerase/promoter couple, the corresponding trinucleotides were useful and showed good to very good incorporation into RNA by T7 polymerase . For ins


Nelumbo nucifera), an ancient aquatic plant, possesses a unique pharmacological activity that is primarily contributed by benzylisoquinoline alkaloids (BIAs). However, only few genes and enzymes involved in BIA biosynthesis in N. nucifera have been isolated and characterized. In the present study we identified the regiopromiscuity of an O-methyltransferase, designated NnOMT6, isolated from N. nucifera; NnOMT6 was found to catalyze the methylation of monobenzylisoquinoline 6-O/7-O, aporphine skeleton 6-O, phenylpropanoid 3-O, and protoberberine 2-O. We further probed the key residues affecting NnOMT6 activity via molecular docking and molecular dynamics simulation. Verification using site-directed mutagenesis revealed that residues D316, N130, L135, N176A, D269, and E328 were critical for BIA O-methyltransferase activities; furthermore, N323A, a mutant of NnOMT6, demonstrated a substantial increase in catalytic efficiency for BIAs and a broader acceptor scope compared with wild-type NnOMT6. To the best of our knowledge, this is the first study to report the O-methyltransferase activity of an aporphine skeleton without benzyl moiety substitutions in N. nucifera. The study findings provide biocatalysts for the semisynthesis of related medical compounds and give insights into protein engineering to strengthen O-methyltransferase activity in plants.Lotus ( Nelumbo nucifera) and plants belonging to the orders Ranunculales and Magnoliales; they possess important bioactivities and pharmacological values , and the amounts of norcoclaurine, coclaurine, TL and E . The NnOThalictrum tuberosum + (13b) (13b was identified as (S)-tetrahydropalmatine by comparing it with a reference standard (O-methylation of 15 to yield (S)-tetrahydropalmatine (13b), with a conversion rate of 42.4%. These results suggested that N323A exhibited a broader acceptor scope for O-methylation and stronger catalytic activity.To further investigate the catalytic properties of N323A, purified proteins of the mutant N323A were obtained to test activity using compounds bstrates . Mutant y of <2% . Further]+ (13b) . Compounstandard . Further7 and 13  and (S)-scoulerine (86.35 μM) were lower than that of NnOMT6 toward (S)-norcoclaurine. The kcat/Km values of N323A toward asimilobine and (S)-scoulerine were 24.37 ± 0.59 and 20.01 ± 0.99 M−1 s−1, respectively, which were significantly higher than that of NnOMT6 toward (S)-norcoclaurine (16.59 ± 1.48 M−1 s−1). These results indicated that the catalytic efficiency of mutant N323A to BIAs was relatively higher than that of the wild-type NnOMT6.The kinetic parameters of N323A were calculated using 7 and 13 . The Km N. nucifera, only two OMTs involved in the 1-BIA upstream biosynthesis pathway in N. nucifera were identified in vitro. In this study, a novel and regiospecific OMT, NnOMT6, was identified from N. nucifera; it catalyzes the methylation of phenylpropanoid (caffeic acid and chlorogenic acid), 1-benzylisoquinoline ((S)-norcoclaurine, (S)-coclaurine, and N-methylcoclaurine), aporphine (asimilobine and N-methlyasimilobine), and protoberberine (scoulerine) skeletons. NnOMT6 shared a high sequence identity of >70% with three known OMTs  from T. tuberosum and formed a cluster with them in a phylogenetic tree. Therefore, NnOMT6 was determined to be functionally similar to these OMTs and to modify phenylpropanoid and several other BIA skeletons. NnOMT6 can catalyze the 3-O-methylation of caffeic acid with the highest catalytic activity. NnOMT6 showed relatively high methylation activity at position 6 of (S)-norcoclaurine (76.7%) and weak activity at position 7 of (S)-coclaurine (13%) and N-methylcoclaurine (<2%) of the 1-BIA skeleton. Interestingly, the methylation function on aporphine backbone without any substituent at C4′ and C3′ positions from N. nucifera is the first reported, which was considered to result from O-demethylase (ODM) in a previous study [N-methlyasimilobine and at C2 position of the protoberberine alkaloid (S)-scoulerine -norcoclaurine were mutated, resulting in a large decrease in activity for BIA substrates, especially the mutation of residue D316 causing inactivation of NnOMT6 for all substrates-scoulerine substantially increased from <2% to >45%. Moreover, compared with methylation by wild-type NnOMT6, N323A strengthened the 7-O-methylation of (S)-coclaurine by ~2.5 times. It also exhibited two sequential methylations of (S)-norcoclaurine at the C6 and C7 positions. Furthermore, a novel function of N323A was determined—it sequentially catalyzed the methylation of (S)-scoulerine at 2-OH and 9-OH, whereas wild-type NnOMT6 only exhibited weak 2- O-methylation activity for (S)-scoulerine. N323A was found to catalyze (S)-tetrahydrocolumbamine, a new acceptor, at the C2 site, with a conversion rate of 42.4% -norcoclaurine (1), asimilobine (7), and N-methlyasimilobine (8) were purchased from ChemFaces ; (S)-coclaurine (2), lirinidine (9), liensinine (10), and isoliensinine (11) were purchased from Chengdu Push Biotech ; N-methylcoclaurine (3) and (S)-norarmepavine (4) were purchased from BioBioPha ; and armepavine (5), lotusine (6), N-nornuciferine (7a), nuciferine (8a), neferine (12), (S)-scoulerine (13), (S)-tetrahydropalmatine (13b), (S)-tetrahydrocolumbamine (14), jatrorrhizine (15), caffeic acid (16), ferulic acid (17), chlorogenic acid (18), o-coumaric acid (19), and p-coumaric acid (20) were supplied from Shanghai Yuanye Biotech . SAM was from Sigma–Aldrich . BeyoGold™ His-tag Purification Resin was purchased from Beyotime Biotech . Acetonitrile, methanol, and formic acid used in UPLC–MS/MS analysis were obtained from Sigma–Aldrich.Different tissues of the N. nucifera by BLASTP searching. The GenBank accession numbers of the 28 OMT sequences were from the literature and are as follows: Ps6OMT (AAP45315.1), PsSOMT3 (AWJ64118.1), PsN7OMT (ACN88562.1), PsOMT2 (AKO60153.1), Ps4′OMT2 (AAP45314.1), PsOMT1 (AKO60152.1), PsSOMT2 (AWJ64116.1), Ps7OMT (AAQ01668.1), PsOMT6 (AKO60157.1), PsSOMT1 (AFK73709.1), and PsOMT7 (AKO60158.1) from Papaver somniferum; Tf6OMT (AAU20765.1) from Thalictrum flavum; Cc6OMT1 (AXC09385.1) and Cc6OMT2 (AXC09386.1) from Coptis chinensis; Cj6OMT (BAB08004.1), Cj4′OMT (BAB08005.1), CjCoOMT (BAC22084.1), and CjSOMT (BAA06192.1) from Coptis japonica; NnOMT1 (XP_010244054.1) and NnOMT5 (XP_010276063.1) from N. nucifera; Ec4′OMT (BAM37633.1), Ec7OMT (BAE79723.1), and EcSOMT (BBA20643.1) from Eschscholzia californica; Ct7OMT (AXC09387.1) and CtSOMT (AXC09384.1) from Coptis teeta; and Tt6OMT1(AAD29841.1), TtOMT (AAD29845.1), and Tt6OMT2 (AAD29842.1) from Thalictrumtuberosum.A total of 28 OMTs were selected as query sequences for scanning the transcriptome data of In total, 12 fresh lotus tissues, including LS, FS, NR, LBP, RF, LSH, L, P, PT, FSL, O, PS, and SM, were individually ground into powder in liquid nitrogen. Total RNA of 12 lotus tissues was extracted using an RNAprep Pure Kit . RNA purity was verified using a NanoPhotometer spectrophotometer . RNA integrity was assessed using an RNA Nano 6000 Assay Kit on an Agilent Bioanalyzer 2100 system . Thereafter, Illumina paired-end sequencing technology was used.−ΔΔCt method using β-actin as the reference with three replicates. Primer information on NnOMTs for qRT–PCR is given in Total RNA was extracted from different lotus tissues, including TL, ML, F, and E, using an RNA Easy Fast Plant Tissue Kit  and was reverse-transcribed to cDNA using a FastKing RT Kit with gDNase . Quantitative RT–PCR was performed using Taq Pro Universal SYBR qPCR Master Mix  on a Cobas Z 480 real-time PCR system . The expression level of NnOMT genes was evaluated by the 2To extract alkaloids, 100 mg of TL, ML, F, and E fine powder samples were carefully weighed and extracted in 1 ml of 70% methanol and 30% water (v:v) combination, shaken for ~30 seconds at 30-minute intervals six times, and incubated at 4°C overnight. Then, the samples were centrifuged  and the supernatants were filtered with 0.22-μm filters for testing.Determining the content of BIAs and enzyme assay products was performed as described previously . An Agil−1; sheath gas flow, 11 l min−1; collision energy, 30 eV and scan range, m/z 100–1500 Da. The measured masses were modified by internal references (purine and HP-0921) in real time.The ESI source was set to positive ionization mode and the following parameters were set for the QTOF–MS detector: nebulizer, 45 psig; nozzle voltage, 500 V; Vcap, 4000 V; cone voltage, 20 V; sheath gas temp, 350°C; drying gas flow, 8 l min® II One Step Cloning Kit .The abovementioned methods were used to extract the total RNAs from frozen leaves and synthesize first-strand cDNAs. NnOMT genes were amplified from cDNA using KOD-plus-neo polymerase (Toyobo) with primers listed in Alignment of amino acid sequences was performed by ClustalW, and the result was used to construct the phylogenetic tree for NnOMTs and 28 other identified OMTs, using MEGA 7.0 software with 1000 bootstrap replicates.E. coli BL21 (DE3)  for protein expression. The cells were cultured in 300 ml of LB medium (50 μg/ml kanamycin) at 37°C and shaken at 200 rpm until OD600 reached 0.6–0.8. Then, IPTG was added to the cultures to a final concentration of 0.3 mM IPTG and induction was performed at 16°C for 20 hours with shaking (180 rpm). The cell pellets were harvested at 4°C by centrifugation and then resuspended with lysis buffer . Thereafter, the suspended cells were disrupted by sonication for 30 minutes in an ice bath, and centrifuged at 10 000 rpm for 40 minutes at 4°C. The supernatant (crude protein) was subsequently used to perform the initial enzyme assays.Recombinant plasmids with verified sequences were transformed into 1–7, aporphine alkaloids 8–10, and bisbenzylisoquinolines 11–13. The initial reaction was conducted in a 100-μl mixture system comprising 50 mM potassium phosphate (pH 8.0), 200 μM SAM donor, 200 μM acceptor substrate, and 2 mg of crude enzyme. Proteins inactivated by heating at 100°C for 10 minutes were used as negative controls. The mixtures were incubated at 37°C for 12 hours, terminated by the addition of 100 μl methanol, then centrifuged at 12 000 × g for 15 minutes at 4°C before UPLC–MS/MS analysis. The activity of mutants was investigated under the same reaction conditions using 1, 2, 3, 4, 8, 9, and 17 as substrates.The catalytic activity of recombinant NnOMT proteins was initially screened using 13 potential BIA substrates containing monobenzylisoquinolines The crude protein of NnOMT6 and N323A obtained was added to 2 ml of pre-equilibrated BeyoGold™ His-tag Purification Resin  and incubated at 4°C for 4 hours. Thereafter, the lysis buffer and elution buffers containing 250 mM imidazole were used to wash and elute the protein, respectively. The purity of recombinant protein was examined by SDS–PAGE analysis (10% gel) and concentrated using a 10-kDa ultrafiltration tube (Merck Millipore). The concentration of purified enzyme was determined with Bradford Protein Assay kit .1–21 as substrates. One hundred microliters of reaction mixture, including 50 mM potassium phosphate buffer (pH 8.0), 200 μM SAM methyl donor, 100 μM substrates, and 50 μg purified protein, was incubated at 37°C for 12 hours. The reactions were analyzed using UPLC–ESI–QTOF–MS/MS.The catalytic activity of purified NnOMT6 and the mutant N323A was further screened using 1) as substrate. Enzyme assays were performed in 50 mM potassium phosphate buffer (pH 5.0–8.5) and 100 mM Gly–NaOH buffer (pH 9.0–11.0) at 37°C for 4 hours to obtain the optimal pH. The reactions were conducted at 4–60°C for 4 hours (pH 8.0) to optimize temperature. The linear range of product formation was determined for 5–360 minutes.The reaction time, pH and temperature of NnOMT6 were optimized using norcoclaurine (1 (4–500 μM) or 17 (20–500 μM) at a fixed concentration of 200 μM SAM or varying concentrations of SAM (4–500 μM) at a fixed concentration of 200 μM 17. For the kinetic studies of the mutant N323A, reactions were performed with 8 or 14 at a concentration of 4–400 μM in a total volume of 100 μl. The reactions were incubated at 37°C for 30 min and terminated by adding 100 μl methanol. All reactions were conducted in triplicate. The kinetic data were calculated by non-linear regression analysis using a Michaelis–Menten model in GraphPad Prism 8.0 software.The kinetic parameters of NnOMT6 were determined in a 100-μl reaction mixture containing 50 mM potassium phosphate (pH 8.0) and varying concentrations of The structural model of NnOMT6 was constructed using SWISS-MODEL , 39, The2), whereas solvent molecules and hydrogen atoms were free to move. Then, the stage of non-restrained minimization was performed with the steepest descent minimization for 2000 cycles and conjugated gradient minimization for 2000 cycles. Thereafter, the entire system was heated from 0 to 300 K in 100 ps using Langevin dynamics at a constant volume, following which it was equilibrated for 150 ps at a constant pressure of 1 atm. Periodic boundary dynamics simulations were performed for the entire system in an NpT ensemble  at a constant pressure of 1 atm and 300 K during the production step. In the production phase, 100-ns simulation was conducted. The binding free energy of the complex was calculated using the MM/PBSA method.After docking, the complex NnOMT6–SAH–norcoclaurine was optimized using MD simulation performed with AMBER16 . To neutThe mutant plasmids of NnOMT6 were cloned with pET28a-NnOMT6 vector as the template using a Fast MultiSite Mutagenesis System kit . Primer pairs used for PCR amplification are shown in Web_Material_uhac276Click here for additional data file.


The crystal structure of a biologically important pharmacophore containing 1,4-dihydropyridine is reported. 16H21N3O4, the 1,4-dihydropyridine ring adopts a flattened boat conformation, with the imidazole substituent in an axial orientation [dihedral angle between ring planes = 82.9 (6)°]. In the crystal structure, pairs of N—H⋯O and N—H⋯N hydrogen bonds with graph-set notation R22(14) connect the molecules into chains running along the c-axis direction.In the title compound, C The dihedral angle formed between the plane of the 1,4-dihydropyridine (C1–C5/N1) and imidazole (C10–C12/N2–N3) rings is 82.9 (6)°. One of the carbonyl groups (C13=O3) lies on the same side as the methyl group at C16 with the other carbonyl group (C7=O1) on the opposite side. In the crystal, pairs of N—H⋯O and N—H⋯N hydrogen bonds with graph-set notation n Table 1.H-imidazole-2-carbaldehyde (10 mmol) were dissolved in 30 ml of ethanol to give a clear solution. To the mixture, ammonium acetate (10 mmol) was added and the reaction mixture was heated at 365 K for 5 h. After completion of the reaction (monitored by TLC), the mixture was cooled to room temperature to produce a solid product. This solid was recrystallized from ethanol solution to obtain single-crystal of the title compound in 61% yield.Methyl acetoacetate (20 mmol) and 1Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620000346/bt4088sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314620000346/bt4088Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314620000346/bt4088Isup3.cmlSupporting information file. DOI: 1977288CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The refined structure shows that this cubic form is stable at 113 K.The crystal structure of the cubic double-perovskite Sr 2Cr0.84Ni0.09Os1.07O6, grown at high pressure, was solved using intensity data measured at 113 K. The Os site was modelled with a partial Ni occupancy, and the Cr site was modelled with both Os and Ni partial occupancy. The refined structure shows that this cubic form is stable at 113 K.The crystal structure of the cubic double-perovskite Sr In 1998, Sr2FeMoO6, which has the DP structure, was reported as having half-metallic behavior with a high Curie temperature (CT = 420 K)  and Os5+  activate primarily for this state  having 2.2Cr0.84Ni0.09Os1.07O6 has cubic symmetry of space group Fmm, having one Sr, one Os, one Cr, and one O atom on crystallographically independent sites in the asymmetric unit. It corresponds to the fully Cr-containing end-member Sr2CrOsO6 and the low Ni-substituted Sr2Cr0.75Ni0.25OsO6  and Os (B′′) sites show elemental disordering behavior: 96.1 (13)% Os + 3.8 (13)% Ni at the Os site and 85.5 (3)% Cr + 12.1 (3)% Os + 2.4 (3)% Ni at the Cr site. Both the Cr and Os sites form three-dimensional framework structures connected by corner sharing of the coordination octahedra, having Os—O = 1.926 (4) Å (coordination volume CV = 9.5405 Å3) and Cr—O = 1.987 (4) Å (CV = 10.4516 Å3) . From this result, the cubic Sr2Cr0.85Ni0.06Os1.08O6 structure is shown to be stable down to at least 113K.In the structure Fig. 1, the tra3) Fig. 1. The Sr 3.2Cr0.84Ni0.09Os1.07O6 was obtained as a by-product of the synthesis of the polycrystalline Sr2Crx1-NixOsO6 (x = 0.5). The polycrystalline product was synthesized from powders of SrO , CrO2 , NiO , OsO2 . The thoroughly mixed powders (SrO:CrO2:NiO: OsO2:KClO4 = 2:0.5:0.5:1:0.225 mol) were pressed into a pellet and sealed in a Pt capsule. All the processes were carried out in an Ar-filled glove box. A pressure of 6 GPa was continuously applied by a belt-type pressure apparatus , the capsule was heated to 1873 K and held at that temperature for 1 h. The temperature was then quenched to room temperature, following which the pressure was gradually released.A black-colored single crystal of Sr4.SHELXL commands EADP and SUMP, respectively.)Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698902201012X/pk2669sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698902201012X/pk2669Isup2.hklStructure factors: contains datablock(s) I. DOI: 2213752CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Cell Death and Disease (2021) 12:719 10.1038/s41419-021-03977-1, published online 20 July 2021Correction to: The original version of this article contained a mistake. In figure 2E, the “UMUC-3 NC” group of cell cycle was wrong. In figure 2F, the “NC” group should be corrected as “si-NC” in a more appropriate description. In figure 5G “UMUC-3 si-NC 0h” and figure 6G “UMUC-3 NC 0h” was placed with wrong figures. The authors corrected all mistakes with the corrected figs.


The synthesis has been accomplished in high yields. The final structures of both hybrids have been established completely with the help of different spectro-analytical techniques, including NMR, FTIR, HR-MS, and single-crystal X-ray diffraction analyses. In an effort to confirm the experimental findings, versatile quantum mechanical calculations and Hirshfeld Surface analysis have been performed. α-Amylase inhibition assay has been executed to investigate the enzyme inhibitory potential of both hybrids. The low IC50 value (76.92 ± 0.19 μg/mL) of hybrid 2 shows the good α-amylase inhibition potential of the respective compound. Ultimately, the binding affinities and features of the two hybrids are elucidated utilising a molecular docking technique against the α-amylase enzyme.Sulphonamide and 1,3,4-oxadiazole moieties are present as integral structural parts of many drugs and pharmaceuticals. Taking into account the significance of these moieties, we herein present the synthesis, single-crystal X-ray analysis, DFT studies, and  Heterocycles, specially oxadiazoles, have been very important in drug discovery due to their important role in medicines. The use and demand of oxadiazoles motifs in drugs are increasing continuously and can be seen from their increasing number of patents filed, up to increase in 100% in last 10 years from 2000 to 2008, still this number is increasing onProbenecid has been an important drug since the nineteenth century and is used in the treatment of multiple diseases, mainly as a uricosuric agent to fight hyperuricaemia. Probenecid plays a vital role in active brain transportationα-Amylase  is a digestive enzyme found to an ample extent in animals and humans. It is produced by salivary glands and pancreas at a neutral to mildly acidic pH. It behaves as a catalyst in the hydrolysis of complex polysaccharides (starch and glycogen) into shorter chains that ultimately produce glucose, thus facilitating digestion. Excessive α-amylase in blood accelerates starch breakdown resulting high concentration of sugars. Controlling the α-amylase in the body can result in the reduction of hyperglycaemia and obesity. α-Amylase is a good target for developing inhibitors for the treatment of widely spread diabetes and obesityα-amylase and α-glucosidase. However, it is decomposed by bacterial α-amylases in the intestine. Acarbose inhibits excessive α-amylase, thus retarding the hydrolysis of polysaccharides and controlling the blood sugar levels.Acarbose is a commercially available drug prescribed to manage diabetes mellitus type II by inhibiting the salivary and pancreatic α-amylase enzyme. A brief literature survey showed that a series of 5–phenyl)-1,3,4-oxadiazole-2-thiol derivatives had inhibited α-amylase in the range of IC50=40.00–80.00 μg/mL (β-D-glucopyranosyl)-1,3,4-oxadiazoles have been evaluated for their antidiabetic potentialS-benzyl substituted 1,3,4-oxadiazole-2-thiol derivatives have been found to inhibit the α-amylase enzyme in the 100 μM concentration1,3,4-Oxadiazoles have been synthesised in great structural diversity and studied for their inhibition potential against the 00 μg/mL 3. l-2- are recorded on an AV-300 spectrometer (300 MHz) manufactured by Bruker. FT-IR spectra of both hybrids are recorded in ATR  mode by using the Fourier Transform Infra-Red spectrophotometer supplied by Shimadzu.Starting materials, reagents and solvents are procured from Merk and Sigma Aldrich and utilised directly without any further treatment. Melting points of both hybrids are recorded by using the Gallenkamp melting point apparatus (MP-D) and are reported as uncorrected. NMR spectra (2.2.2.2.1.The probenecid (20 mmol) is taken in 30 mL of methanol and 0.5 mL of conc. sulphuric acid is added and stirred at reflux for 12 h. Then concentrated and washed with 150 mL of a saturated aqueous solution of sodium bicarbonate and extracted three times with ethyl acetate (3 × 50 mL). The organic layer is dried over anhydrous sodium sulphate and concentrated to have pure probenecid ester. From probenecid ester, the probenecid hydrazide is synthesised by adopting the following modified procedure from the literatureIn the next step, the probenecid hydrazide (10 mmol) is dissolved in methanol containing potassium hydroxide (30 mmol) at room temperature. After a few minutes of stirring, carbon disulphide (0.06 mol) is dropped into the reaction mixture and further refluxed for 12 h. It is concentrated to less than half, poured into the ice-cold water, and acidified with 1 N HCl to pH = 2. The resultant crude precipitates are then cleaned with warm water and recrystallized from methanol to have pure oxadiazole-2-thiol.S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids 1 and2 in 79% and 83% yields, respectively.To a solution of the corresponding oxadiazole (1 mmol) and bromo-alkyl-substituted phthalimide (1 mmol) in 15 mL acetone, 1 equivalent of potassium phosphate is added and stirred further at room temperature for 4 h. It is concentrated and recrystallized from methanol to obtain pure 2.2.1.1.f): = 0.45 ; melting point; 157–158 °C; 1H-NMR : δ (ppm) 0.87  1.45–1.66  2.23–2.32  3.02–3.19 2) 3.36  3.89  7.70–7.77  7.82–7.88  7.88–7.96  8.07–8.18 ; 13 C NMR : δ (ppm) 11.12, 21.86, 28.42, 29.91, 36.42, 49.81, 76.57, 77.42, 123.35, 126.83, 127.10, 127.63, 131.87, 134.11, 142.93, 164.48, 165.11, 168.34; FT-IR ν(cm−1): 3025 , 2937 , 1708, 1620 (Carbonyl group), 1545 (C = N), 1337, 1151 (SO2 group); High Resolution Mass Spectrum (EI-TOF) [M+] Calcd. for: C25H28N4O5S2: 528.1501; found; 528.1500.Colourless solid; %age yield: 79; Retention factor (R2.2.1.2.f)R: = 0.51 ; melting point; 117–119 °C; 1H-NMR : δ (ppm) 0.88  1.47–1.63  1.91  3.05 − 3.18 2) 3.37  3.75  7.68 − 7.76  7.80 − 7.88  7.90 − 7.96  8.08–8.16 ; 13 C-NMR : δ (ppm) 11.56, 22.30, 26.90, 27.90, 32.37, 37.52, 50.26, 77.43, 77.85, 123.68, 127.32, 127.53, 128.08, 132.38, 134.43, 143.37, 164.84, 168.76; FT-IR (cm−1): 3088 , 2970 , 1703, 1617 (Carbonyl group), 1543(C = N), 1332, 1160(SO2); High Resolution Mass Spectrum (EI-TOF) [M+] Calcd. for: C26H30N4O5S2: 542.1658; found; 542.1656.Colourless solid; %age yield: 83; Retention factor . Multi-scan CrysAlis PRO 1.171.38.46  is used for empirical absorption correction spherical harmonics, implemented in SCALE3 ABSPACK. The structure solution is performed by SHELXT-20141 and 2 have been assigned CCDC numbers 1963535 and 1963536, respectively, and can be obtained free of charge on-demand to CCDC 12 Union Road, Cambridge CB21 EZ, UK. (Fax: (+44) 1223 336–033: data_request@ccdc.cam.ac.uk).2.4.1 and 2). The studied hybrids are first optimised at the B3LYP/6-31G* level of theoryTo verify the experimental results, versatile quantum mechanical calculations are carried out on the two hybrids (2.5.dnorm) surface along with its corresponding two-dimensional (2 D) fingerprint plots is generated to allow the identification of the crucial regions in the crystal packing of the investigated hybrids. The dnorm surfaces are mapped over a fixed colour scale ranging from red (–0.05 au) to blue (+0.75 au). The fingerprint plots are figured out using the translated 1.0 − 2.8 Å range, and reciprocal contacts are considered. As well, the shape index and curvedness properties are mapped with the colour range of −1.0 au (concave) to 1.0 au (convex) and range of −4.0 au (flat) to 0.40 au (singular), respectively. The generated Hirshfeld surfaces and the associated 2 D fingerprint plots are extracted using the CrystalExplorer17 softwareHirshfeld surface (HS) analysis2.6.α-amylase  is 25 000 000, while the genetic algorithm (GA) variable is set to 250. The remaining docking settings are left at their default values. The grid box size is adjusted to 60 Å × 60 Å × 60 Å, which is able to encompass the active site of α-amylase. AutoGrid4.2.6 program is adopted to create grid map files with a spacing value of 0.375 Å. The grid centre is located at the coordinates X = 32.644, Y = 38.464, and Z = −3.166. The root mean square deviation of 1.0 Å is utilised to cluster conformations, and the docking score is adopted to rank them In this work, AutoDock4.2.6 software is employed to perform all molecular docking calculations2.7.α-Amylase inhibitory activity of probenecid derived S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids (1 and 2) is achieved using the 3,5-dinitrosalicylic acid (DNS) method [41]. Compound solutions are prepared by dissolving each compound in dimethylsulphoxide (DMSO) to give a concentration of 1 mgmL−1. A measured volume from each compound solution is mixed separately with 200 μL of α-amylase solution  in a test tube and incubated for 10 min at 30 °C. 200 μL of the starch solution (1% in water (w/v)) is added to the tube and incubated for 3 min. 200 μl of DNS reagent (20 mL of 96 mM DNS and 12 g of sodium potassium tartrate tetrahydrate in 8.0 mL of 2 M NaOH) is added to this mixture for reaction termination and the mixture is kept in a boiling water bath for 10 min. After cooling the mixture to ambient temperature, 5 mL of distilled water is added to dilute it, and the absorbance is recorded at 540 nm using a UV-Visible spectrophotometer (Shimadzu UV 1800). A blank reaction mixture is also prepared to remove the background absorbance produced by each compound at various studied concentrations containing all other components except the enzyme solution. Acarbose (250–10 μgmL−1) is used as a potent amylase inhibitor to compare the inhibition potential of compounds (1–3), whereas the control incubation, representing maximum enzyme activity, is carried out by replacing the compound with 2% DMSO. All the tests are performed in triplicate. The α-amylase inhibitory activity is calculated as percent inhibition using the following formula:50 values (μgmL−1) are obtained from the graph plotted for % α-amylase inhibition obtained at five different concentrations (10–200 μgmL−1).3.3.1.S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids (1 and 2) during this work is shown in 1H NMR 13C NMR and HR-MS spectroscopies, and single-crystal X-ray crystallography.The synthetic approach adapted to access probenecid-derived 1H NMR spectra of probenecid derived S-alkylphthalimide-oxadiazole-benzenesulfonamide hybrids (1 and 2), all the eight aromatic protons are found in the range of δ 7.68–8.25 ppm. A multiplet for four protons at δ 3.00–3.22 ppm is assigned to N-(CH2)2 in both compounds. The four protons of the other two methylenes of the n-isopropyl group are observed as a multiplet at δ 1.45–1.66 ppm in 1 and 2. A triplet for six protons of two methyl groups on the n-isopropyl group is found between δ 0.87–0.89 ppm. A triplet for two protons at δ 3.36 and δ 3.37 ppm is assigned to each S-CH2 group in compounds 1 and 2, respectively. The triplets for two protons at δ 3.89 ppm and 3.75 ppm are assigned to the N–CH2 group in compounds 1 and 2, respectively. A multiplet for two protons at δ 2.23–2.32 ppm is assigned to the methylene group in the middle of the n-propyl chain in compound 1 whereas, a doublet of a doublet having coupling constant 4.05 and 1.98 Hz for four protons at δ 1.92 ppm is assigned to two methylene groups in the middle of the n-butyl chain.In 13C NMR spectroscopic studies of compounds 1 and 2, the two carbonyl carbons of phthalimide skeleton are observed at δ 168.34 and 168.76 ppm for compounds 1 and 2, respectively. Similarly, the two carbon atoms present in the oxadiazole part of the hybrid are found at 165.18, 164.55 and 165.34, 164.47 ppm in hybrid 1 and 2, respectively. The carbon atoms in the aromatic rings are found between δ 144–122 ppm. The shift between δ 49.80 and 50 ppm is assigned to N(CH2)2 of the n-isopropyl group in both hybrids. The shift at 36.49 and 37.14 ppm are assigned to S–CH2N and N–CH2 in hybrids 1 and 2, respectively. The chemical shift at δ 29.98 and 31.99 ppm is assigned to S–CH2 in hybrids 1 and 2, respectively. Other carbon atoms of the alkyl chains are observed below δ 30 ppm.In 3.2.1 and 2 are given in 1 (I and molecule II) that are different geometrically with respect to each other. In both molecules, molecule I (C1–C25/N1–N4/O1-O5/S1/S2) and molecule II (C26–C50/N5–N8/O6-O10/S3/S4), one of the propyl groups that is attached to N-atom is disordered over two sets of sites with an occupancy ratio of 0.666 (4): 0.334 (4). The disordered is solved by using EADP, SIMU, DELU, DFIX, and DANG restraints. In molecule I, the isoindoline-1,3-dione ring A (C1–C8/N1/O1/O2), 1,3,4-oxadiazole ring B (C12/C13/N2/N3/O3), and phenyl ring C (C14–C19) are planar with root mean square deviation of 0.0170, 0.0027 and 0.0071 Å, respectively with dihedral angles A/B and B/C is 51.9(6)° and 8.83(8)°, respectively. In molecule II, the isoindoline-1,3-dione ring D (C26–C33/N3/O6/O7), 1,3,4-oxadiazole ring E (C37/C38/N6/N7/O8), and phenyl ring F (C39–C44) are planar with root mean square deviation of 0.0227, 0.0027 and 0.0098 Å, respectively, with dihedral angles D/E and E/F is 54.42(6)° and 11.43(8)°, respectively. In order to further explore the difference between molecules I and II, a molecular overlay plot is formed by using Mercury 4.0  and the maximum deviation (Max. D) between molecules I and II are 1.6359 and 4.7768 Å, respectively. The major difference between molecules is in the orientation of the disordered propyl groups  analysis has been recently apprehended as an informative method to conceive the nature of the chemical systems by indicating the electrophilic and nucleophilic sites on their molecular surfaces1 and 2 that are noticed by the occurrence of deep blue colour regions.Based on the data shown in 3.4.Several studies have addressed frontier molecular orbitals (FMOs) analysis as a consistent tool to elucidate the convenient sites on the molecular surfaces for inter- and intra-interactions. Using FMOs, the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) are generated to assess the stability of the studied hybrids and are illustrated in Subject to data graphed in 3.5.1 and 2. In the context of QTAIM, the bond paths (BPs) and bond critical points (BCPs) are generated and are depicted in Quantum theory of atoms in molecules (QTAIM) protocolAccording to the results presented in 3.6.dnorm along with its associated 2 D fingerprints, shape index, and curvedness, are mapped for the investigated hybrids 1 and 2. dnorm maps and their associated 2 D fingerprint plots, respectively. The generated shape index and curvedness maps are given in Hirshfeld surface (HS) analysis is documented as an authoritative tool to qualitatively assess the nature of intermolecular interactions within crystal structures and thoroughly identify the interactions throughout the surface around the moleculesdnorm maps of both hybrids. Such contacts are also noticed in the 2 D fingerprint plots as a pair of symmetrical spikes at (de + di) ∼ 2.5 Å and 3.0 Å for hybrids 1 and 2, respectively  ∼ 3.5 Å and 2.3 Å for hybrids 1 and 2, respectively. Turning to the proportions of H···C/C···H contacts, prominent red regions are noticed with 13.8% and 9.4% of the total Hirshfeld surface area and characterised by spikes at (de + di) ∼ 3.0 Å and 4.1 Å for hybrids 1 and 2, respectively. The H···S/S···H contacts attributed to weak C–H···S interactions are observed with label 4, as white and red regions in the dnorm maps of hybrids 1 and 2 with 4.8% and 3.5% contributions, respectively. Conspicuously, the π···π stacking interactions show variant contributions to the total Hirshfeld surfaces of hybrids 1 and 2 with values of 2.7% and 3.8%, respectively.The existence of red regions with label 2 in the Hirshfeld surfaces of two hybrids could be ascribed to the occurrence of the reciprocal H···O and O···H contacts that were found in the 2 D fingerprint plots at  is evaluated against α-amylase. Both hybrids (1 and 2) show potent inhibitory potential. The potent inhibitory potential is tested at concentration levels 10, 100, 150, and 200 μg/mL where hybrids 1 and 2 delivered 77.37% and 83.36% inhibition of the α-amylase at 200 μg/mL, respectively (α-amylase activity (IC50 μg/mL). Acarbose, a commercially established α-amylase inhibitor, is used as standard . Both hybrids 1 and 2 have shown IC50 values of 92.23 ± 0.23 and 76.92 ± 0.19 μg/mL, respectively  are accessed in very good yields. The ultimate molecular structures of both compounds are analysed through FT-IR, 1HNMR, 13CNMR and are validated with the help of X-ray diffraction analysis. MEP maps conspicuously announce both the nucleophilic and electrophilic nature of the studied hybrids. The existence of the N···H/H···N, O···H/H···O, C···H/H···C, and S···H/H···S contacts is unequivocally unveiled within the generated Hirshfeld surfaces. α-Amylase inhibition studies have shown that hybrids 1 and 2 have good enzyme inhibition potential against the α-amylase. The synthesised compounds are in silico investigated towards α-amylase with the assistance of the molecular docking technique. The binding affinities reveal that compounds 1 and 2 are potent α-amylase inhibitors.In conclusion, probenecid-derived two


Gαi2 transcripts in HCC tissues is significantly higher than that in the normal liver tissues. Moreover, Gαi2 overexpression in HCC correlates with poor prognosis of the patients. Gαi2 mRNA and protein expression are also elevated in local HCC tissues and different human HCC cells. In patient-derived primary HCC cells and immortalized HepG2 cells, Gαi2 silencing (by targeted shRNA) or knockout  largely suppressed cell proliferation and motility, while inducing cell cycle arrest and caspase-apoptosis activation. Moreover, Gαi2 silencing or KO-induced reactive oxygen species (ROS) production and oxidative injury in primary and HepG2 HCC cells. Whereas different antioxidants ameliorated Gαi2-shRNA-induced anti-HCC cell activity. Using a lentiviral construct, Gαi2 overexpression further augmented proliferation and motility of primary and immortalized HCC cells. Further studies revealed that the binding between the transcription factor early growth response zinc finger transcription factor 1 (EGR1) and Gαi2 DNA promoter was significantly increased in HCC tissues and cells. In vivo, intratumoral injection of Gαi2 shRNA adeno-associated virus significantly hindered HCC xenograft growth in nude mice. Moreover, the growth of Gαi2-KO HCC xenografts in the nude mice was remarkably slow. Gαi2 depletion, oxidative injury, and apoptosis induction were detected in Gαi2-silenced or Gαi2-KO HCC xenografts. Together, overexpressed Gαi2 is required for HCC cell growth in vitro and in vivo, representing as a novel and promising diagnosis marker and therapeutic target of HCC.Hepatocellular carcinoma (HCC) is a global health problem. Its incidence and mortality are increasing. Exploring novel therapeutic targets against HCC is important and urgent. We here explored the expression and potential function of Gαi2  in HCC. The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) database shows that the number of  Liver cancer causes about 800, 000 deaths each year globally , 2. The The multi-kinase inhibitor sorafenib has been utilized as the systemic treatment for unresectable or advanced HCC. Yet, sorafenib could only improve overall survival (OS) of the advanced HCC patients by approximately a few months . LenvatiThe family of Gαi proteins, or G protein inhibitory α subunits, include three members: Gαi1, Gαi2 and Gαi3 , 14. Gαi+ cells suppressed carcinogenesis of colitis-associated cancer ) was inserted into the GV369 lentiviral construct, and the vector transfected to HEK-293 cells along with lentivirus-packing constructs (Genechem). Viruses were then filtered, enriched and added (at MOI = 15) to cultured HCC cells or hepatocytes. After selection by puromycin, two stable cell selections, oe-Gαi2-S1 and oe-Gαi2-S2, were formed. Overexpression of Gαi2 in the stable selections was verified at both mRNA and protein levels. Control cells were infected with empty vector-expressing lentivirus. Overexpression of EGR1 [NM_001964.3] was through the exact same procedure.Cellular functional studies, including nuclear EdU (5-ethynyl-2’-deoxyuridine) staining assaying of cell proliferation, clonogenicity, CCK-8 assaying of cell viability, “Transwell” in vitro cell migration and “Matrigel Transwell” in vitro cell invasion assays, the caspase-3 activity assay, propidium iodide (PI)-flow cytometry assaying of cell cycle progression, nuclear TUNEL  staining of cell apoptosis, Annexin V-PI flow cytometry assaying of cell apoptosis, Histone DNA ELISA, JC-1 (tetraethylbenzimidazolylcarbocyanine iodide) assaying of mitochondrial membrane potential were described in detail in other studies [ studies , 30–33.  studies . The sup studies , 34, 35.Tissue or cellular lysates (35 μg per sample) were tested via a commercial TBAR kit , which quantified lipid peroxidation and measured malondialdehyde (MDA) contents colorimetrically at room temperature. TBAR optical density was examined at 545 nm with the reference of 600 nm. TBAR intensity was expressed in nmol per mg of total protein and was always normalized to that of control.Gαi2 promoter luciferase GV238 construct using Lipofectamine 3000 . After 48 h, the firefly luciferase activity was measured by a Glo luciferase reporter assay kit (Genechem).The predicted EGR1-binding site  was sub-Gαi2 DNA promoter sequence [ChIP assay protocols were described early . Brieflysequence  was tests.c.) injected into the flanks of nude mice and pHCC1 xenografts were close to 100 mm3 after three weeks. Mice were then randomized assigned into two groups and ten mice in each group. Mice were subject to intratumoral injection of Gαi2-shRNA AAV (“aav-shGαi2”) or the same amount of scramble control shRNA AAV (“aav-sh-scr”). Tumor volumes, mice body weights, and estimated daily tumor growth were recorded as described. Alternatively, koGαi2 pHCC1 cells or the control Cas9-C pHCC1 cells (at ten million cells per mouse) were s.c. injected into the flanks of nude mice and pHCC1 xenografts measured after seven weeks. Gαi2 IHC staining in xenograft slides were described previously [The nude mice, six-seven weeks old, 18.5-19.5 grams, half male half female, were provided by SLAC animal center . The xenograft model was described previously . The prieviously . The aniP values < 0.05 were considered statistically significant.Data were always with normal distribution and were expressed as means ± standard deviation (SD).One-way analysis of variance (ANOVA) was performed for multiple group comparison, followed by Dunnett post hoc test using SPSS 23.0 . The two-tailed unpaired student t test (Excel 2007) was utilized for the comparison of two groups. Gαi2 expression in HCC. The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) database was first consulted. A total of 374 HCC tissues (“Tumor”) and 50 normal liver tissues  were retrieved. As shown, the number of Gαi2 mRNA transcripts in HCC tissues was significantly higher than that in the normal liver tissues  was significantly higher than that in the matched adjacent normal liver tissues (n = 50)  than that of the Gαi2-low HCC patients  Fig. . FurtherHCC Fig. . These bGαi2 mRNA levels were significantly elevated in the HCC tumor tissues, where its expression was relatively low in the adjacent tissues  and immortalized (HepG2) HCC cells  and matched adjacent normal liver tissues (“N”) were measured. As shown the ues Fig. . Gαi2 prues Fig. . After cues Fig. . IHC stalls Fig. . Moreovells Fig. . These rGαi2 mRNA levels were substantially reduced in shGαi2-s1/2-expressing pHCC1 cells and koGαi2 (sgRNA1) pHCC1 cells . Stable cells were formed after selection using puromycin. Alternatively, the Gαi2 sgRNA1-expressing lenti-CRISPR/dCas9-KO-puro construct was transduced to dCas9-expressing pHCC1 cells, and stable Gαi2 KO cells (“koGαi2”) formed following puro selection and Gαi2 KO verification. The control pHCC1 cells (“Ctrl”) were stably transduced with scramble control shRNA (“sh-scr”) plus lenti-CRISPR/dCas9-puro construct. qRT-PCR assay results showed that lls Fig. , where Gred Fig. . Moreovered Fig. , withoutred Fig. .Fig. 3GαWe next explored the functional consequence of Gαi2 silencing/KO in pHCC1 cells. PI-FACS cell cycle studies revealed that Gαi2 silencing/KO (sgRNA1) resulted in G1-S arrest Fig. . FollowiTwo other CRISPR/dCas-9-Gαi2-KO constructs, with sgRNA2 or sgRNA3 Fig. S, depleteGαi2 mRNA levels were remarkably decreased in shGαi2-s1-expresing HCC cells  pHCC1 cells Fig. . MoreoveGαi2 mRNA levels were remarkably decreased in shGαi2-s1-expressing hepatocytes  mimetic MnTBAP , 41, redGαi2 cDNA sequence was transduced to pHCC1 cells. After treatment with puromycin-containing medium, two stable pHCC1 cell selections, oe-Gαi2-S1 and oe-Gαi2-S2, were formed. As shown, Gαi2 mRNA expression increased over 8-9 folds in oe-Gαi2-expressing pHCC1 cells , were individually transduced to pHCC1 cells. Stable cells were formed following selection. The applied shRNAs resulted in remarkable EGR1 mRNA  were formed following puromycin selection. When compared to the vector control cells (“Vec”), ion Fig.  and the ity Fig.  were sigRNA Fig. , Gαi2 prRNA Fig. , and EGRRNA Fig.  upregulaRNA Fig. . These rGαi2 DNA promoter sequence was significantly increased in HCC tumor tissues of four different primary HCC patients (Patient-1#/-2#/-3#/-4#)  and HepG2 HCC cells, when compared to the low binding between the two in primary human hepatocytes and immortalized HL-7702 cells  Fig. . Where tues Fig. . The binlls Fig. . Thus, ts.c. injected to the flanks of nude mice. pHCC1 xenografts were then formed after three weeks . Thereafter, the nude mice bearing the pHCC1 xenografts were assigned randomly into two groups, with ten mice of each group (n = 10). The first group received intratumoral injection of Gαi2 shRNA (“shGαi2-s1”)-expressing adeno-associated virus (“aav-shGαi2”); Whereas the other group mice were intratumorally injected with scramble control shRNA adeno-associated virus (“aav-sh-scr”). The virus was injected every 48 h for a total of five rounds. As shown, aav-shGαi2 injection remarkably hindered pHCC1 xenograft growth in nude mice  were ice Fig. . The volice Fig. . When caion Fig. . All pHCion Fig. . The bodGαi2 mRNA levels were substantially decreased in aav-shGαi2-expressing pHCC1 xenografts, where Gαi1 and Gαi3 mRNA levels were unchanged  pHCC1 cells and control cells with the lenti-CRISPR/dCas9-KO-puro construct (“Cas9-C”) were ler Fig.  and lighler Fig.  than Casler Fig. . Gαi2 mRfts Fig. , and Gαiged Fig. . TBAR inged Fig. , and SODged Fig. . Increasged Fig.  and nuclged Fig.  in koGαiHCC is currently the third leading cause of cancer-related human death around the world with its incidence rising , 4. RadiOur group and others have identified two other components of Gαi family proteins, Gαi1 and Gαi3, are possible key oncogenic targets for human cancer –19. Gαi1Gαi3 expression is decreased in human HCC tissues and its low expression correlates with poor prognosis in HCC patients [Nguyen et al. reported that Gαi1 protein expression was significantly upregulated in both human alcoholic steatohepatitis (ASH) tissues and non-alcoholic steatohepatitis (NASH) tissues, being more significant in ASH tissues . Interespatients . Zhang epatients .Gαi2 transcripts in HCC tissues is significantly higher than that in the normal liver tissues. Moreover, Gαi2 overexpression in HCC tissues correlates with poor prognosis of the patients. Gαi2 mRNA and protein expression is also elevated in local HCC tissues and different human HCC cells. In primary HCC cells and immortalized HepG2 cells, Gαi2 shRNA or KO largely suppressed cell growth and migration, while inducing cell cycle arrest and caspase-apoptosis activation. Depletion of Gαi2 however failed to significantly inhibit viability and proliferation of normal hepatocytes. In vivo, intratumoral injection of Gαi2 shRNA AAV largely hindered growth of pHCC1 xenografts in nude mice. Moreover, Gαi2-KO pHCC1 xenografts growth was significantly slowed in nude mice. Thus, targeting Gαi2 could be a promising molecularly targeted therapy for HCC.The results of this study implied that Gαi2 could be a promising therapeutic target and novel diagnosis marker of HCC. TCGA-LIHC database shows that the number A fine strategy to inhibit HCC cell growth and to activate apoptosis is through inducing ROS production and oxidative stress. Metabolic activation of PCK1 (phosphoenolpyruvate carboxykinase 1) promoted energy crisis, oxidative stress and apoptosis in HCC cells and inhibited HCC cell growth in vitro and in vivo . Wang etGαi2 could be an important mechanism of Gαi2 overexpression in HCC. In pHCC1 cells, Gαi2 mRNA and protein expression was significantly decreased following EGR1 silencing, but was elevated after ectopic EGR1 overexpression. ChIP assay results showed that the binding between EGR1 and Gαi2 DNA promoter is increased in HCC tissues and cells. Thus, EGR1-induced transcription activation of Gαi2 promoted HCC cell growth in vitro and in vivo.EGR1 could play an important role in tumorigenesis and cancer progression by transcriptional activation its targets. Li et al. showed that EGR1 promoted prostate cancer metastasis by inducing the expression of angiogenic and osteoclastogenic factors . Ma et aOverexpressed Gαi2 is required for HCC cell growth in vitro and in vivo, representing as a novel and promising diagnosis marker and therapeutic target of HCC.Authorship change formFigure S1aj-checklist formAuthor contribution form


N-acylation of lysine residues. However, the importance of many potential acyl modifications remains unclear because antibody-based methods to detect them are unavailable and the in vivo concentrations of their respective acyl-CoAs are poorly characterized. Here, we develop cysteine-triphenylphosphonium (CysTPP), a mass spectrometry probe that uses “native chemical ligation” to sensitively detect the major acyl-CoAs present in vivo through irreversible modification of its amine via a thioester intermediate. Using CysTPP, we show that longer-chain (C13–C22) acyl-CoAs often constitute ∼60% of the acyl-CoA pool in rat tissues. These hydrophobic longer-chain fatty acyl-CoAs have the potential to non-enzymatically modify protein residues.During metabolism, carboxylic acids are often activated by conjugation to the thiol of coenzyme A (CoA). The resulting acyl-CoAs comprise a group of ∼100 thioester-containing metabolites that could modify protein behavior through non-enzymatic   •LC-MS/MS probe to detect the acyl moiety of thioesters using native chemical ligation•in vivoDevelopment of a quantitative and sensitive assay for ∼60 acyl-Co-As •Longer-chain (C13–C22) acyl-Co-As often represent ∼60% of tissue acyl-CoA pools•in vitroThese longer-chain acyl-Co-As can modify protein  Acyl-Co-As can modify protein through non-enzymatic acylation. By using a novel mass spectrometry probe, James et al. demonstrate that longer-chain (C13–C22) acyl-Co-As often constitute ∼60% of the acyl-CoA pool in tissues. S-acylate the thiol of cysteine residues and N-acylate the ε-amino of lysine residues, thereby affecting protein behavior. The importance of N-acylation is implied by the existence of several sirtuins (Sirt1–7), which remove acyl groups from protein lysines and are relevant to the pathology of a wide range of degenerative diseases, including cancer, aging, and diabetes  of a protein lysine deprotonates to become a nucleophile, and this attacks the thioester carbonyl of an acyl-CoA to generate a stable amide-linked modification (Coenzyme A (CoA) is a key cofactor in many branches of metabolism. It contains a thiol that can condense with a carboxylic acid and form a range of activated acyl-CoAs. These reactive acyl-CoAs are metabolic intermediates in the oxidation of carbohydrate and fat in the mitochondrial matrix, as well as providing the building blocks for fatty acid and lipid synthesis in the cytosol . Howeverdiabetes . N-Acetyoteomics , coupledoteomics , suggestoteomics . In addi in vivo . As few fication .N-acylation of lysine residues could explain the benefits of sirtuins in degenerative diseases and aging  probe that uses “native chemical ligation”  cation, which has a fixed charge that greatly enhances detection by MS -cystine A and S1B-cystine B and S2A-cystine .N-acetylated and S-carbamidomethylated (CAM) product (N-acetyl-CysTPP-CAM) at 550 m/z, with its expected fragmentation pattern (m/z for multiple reaction monitoring (MRM) because it contains a molecular memory of the acetyl moiety from acetyl-CoA   led to the predicted  pattern A and S2Betyl-CoA B and S2Bnsition) B. Finallhe amine C.in vivo and we have shown previously that O-acetyl-carnitine does not N-acetylate protein . This is similar to the rate constants for intermolecular S→S acetyl transfer from acetyl-CoA to various thiols, which range from ∼0.05–0.5 M−1 s−1 .2 product . Fragmentation leads to a species at 468 m/z resulting from the neutral loss of −91 Da  for the intermolecular S→S reaction of acetyl-CoA with various thiols  C18 column between 15% and 100% (v/v) acetonitrile  methanol were the most effective at extracting a broad range of acyl-CoAs to a similar extent . Of the standards, only crotonyl-CoA had no detectable signal in extracts. To quantify the remaining 41 species lacking a standard, the standard closest in properties was used  is 24-fold greater than that of the odd-length fatty acyl-CysTPP-CAM peaks (excluding propionyl), consistent with the expected  profile B. Critic profile C. Togeth profile , as wellin vivo, we snap froze rat heart, liver, kidney, and brain tissue in liquid N2 and extracted ∼20 mg of tissue twice with 80% (v/v) methanol. These extracts were reacted with CysTPP, derivatized with IAM, and 63 acyl-CysTPP-CAM transitions were assessed. The acyl-CoA standards allowed direct quantification of all of the non-fatty-acyl species and 85.7%–92.2% of the overall acyl-CoA population in the four tissues  is comparable to a previous direct measurement of acetyl-CoA by LC-MS/MS in mouse liver of 30–40 nmol g tissue−1 , that may damage protein.For the most part, the acyl groups trapped by CysTPP will derive from the acyl-CoA pool and reflect the acyl-CoAs that are measured directly by LC-MS/MS . Howeverydrolase . Furtherydrolase . Thus, mn at 4°C . Anhydrient ions . In contin vivo , succinyl-CoA, palmitoyl-CoA (C16:0), oleoyl-CoA (C18:1), linoleoyl-CoA (C18:2), and docosahexaenoyl-CoA (C22:6). The abundant long-chain acyl-CoAs species identified by CysTPP were consistent with those identified directly by conventional metabolomics . While ain vivo C. We demin vitro . Consistin vitro , 2020, wly ∼0.1% ; consequly ∼0.1%  and 5. Fin vivo and then determine whether they can disrupt protein homeostasis, potentially contributing to the many pathologies associated with protein aggregation.Modification of a lysine residue by hydrophobic long-chain acyl-CoAs both neutralizes its charge and greatly enhances its hydrophobicity. Future work will focus on the extent to which low-stoichiometry modification of protein residues by long-chain acyl groups occurs in vivo as they are generated directly by metabolism, acyl-glutathiones, anhydrides, and acyl groups on protein cysteine, residues can form from acyl-CoAs and will generate identical acyl-CysTPP-CAM products. These could contribute to the acyl-CoA signal that is detected if they are present at significant quantities. In this study, the contribution of acyl groups from cysteine residues to the reported acyl-CoA concentration was limited by protein precipitation, but in the future using a modified assay, this population of acyl groups could also be explored with CysTPP.Here, we measure acyl-CoAs in tissues by trapping their acyl moiety with CysTPP. While this allows stable and sensitive detection of acyl-CoAs, there are caveats as CysTPP will also accept acyl groups from other donors if they are present. Although acyl-CoAs will be the main acyl donors to CysTPP in vivo and identifies key modifications that may occur on the surface of proteins. In particular, long-chain acyl-CoAs represent a large proportion of the acyl-CoAs in many of the tissue extracts we assessed here, and their hydrophobic properties are likely to alter the behavior of proteins with which they react.This work develops CysTPP, a highly sensitive LC-MS/MS probe that selectively traps thioester-bound acyl groups using chemistry akin to native chemical ligation. It advances our understanding of the composition of the acyl-CoA pool, demonstrates the broad range of acylating species mpm@mrc-mbu.cam.ac.uk).Michael P. Murphy (2This study generated the following unique reagent: (CysTPP)Richard.Hartley@glasgow.ac.uk) under Materials Transfer Agreements.This reagent is available from MPM or RCH  Act of 1986 and the University of Cambridge Animal Welfare Policy. Wistar rats  were ordered from Charles River Laboratories UK . They were housed under standard laboratory conditions with ad lib food and water and used between 10-12 weeks. Rats were culled by cervical dislocation with accordance to UK Home Office Schedule 1.2 was prepared from L-cystine in three steps (tert-butyl carbamates to give Boc-L-cystine in good yield. Coupling of the free carboxylic acids with (5-aminopentyl)triphenylphosphonium chloride using N,N,Nʹ,Nʹ-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), followed by ion exchange gave (Boc-CysTPP)2 as the bischloride salt. Deprotection with acid gave (CysTPP)2 with its amino groups protonated as the tetrachloride salt, (CysTPP)2•2Cl•2HCl. Full synthetic details are presented below. 1H NMR spectra in DMSO-d6 are referenced to residual protons of partially deuterated solvent at d 2.50 and 13C NMR spectra to the solvent peak at d 39.52 (13C nuclei ortho and meta to phosphorus in the 13C NMR spectrum ((CysTPP)ee steps A. The am d 39.52 . All spespectrum . Raw dattert-butyl dicarbonate  in 1,4-dioxane (50.0 mL). The reaction mixture was allowed to stir at room temperature for 24 h then cooled to 0°C. The solution was acidified to pH 4 with the dropwise addition of concentrated aqueous hydrochloric acid then extracted with ethyl acetate. The combined organic layers were washed with brine, dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give Boc-L-cystine as a white amorphous solid . δH : 1.37 , 2.87 , 3.10 , 4.16 , 7.18 : 28.2 (CH3), 39.4 (CH2), 52.7 (CH), 78.3 (C of tBu), 155.4 (carbamate C=O), 172.4 (carboxylic acid C=O); HRMS : found [M+Na]+ 463.1183. C16H28N2NaO8S2+ requires 463.1179. NMR data assignment assisted by HSQC. Spectral data agree with the literature  in deionized water (100 mL) was added sodium carbonate  followed by the dropwise addition of a solution of di-terature .R)-(tert-butoxycarbonylamino)propanoic acid  in anhydrous N,N-dimethylformamide (20.0 mL) in a flame-dried flask under argon was added N,N,Nʹ,Nʹ-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate  followed by (5-aminopentyl)triphenylphosphonium bromide hydrobromide  and anhydrous N,N-diisopropylethylamine . The reaction mixture was allowed to stir at room temperature under argon for 48 h then partitioned between water and chloroform. The layers were separated, and the aqueous layer was extracted with chloroform. The combined organic layers were washed consecutively with 5% aqueous lithium chloride, 1 M aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and brine then dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue was dissolved in a minimum amount of chloroform and the crude material triturated from hexane. Column chromatography  gave (Boc-CysTPP)2 with mixed counterions as an off-white film. Counterion exchange to give only chloride counterions gave (Boc-CysTPP)2•2Cl as a white hygroscopic amorphous solid . δH : 1.38 , 1.46–1.54 , 1.57–1.66 , 2.92–3.07 , 3.15 , 3.48–3.54 , 4.08–4.23 , 7.73–7.93 ; δC : 20.3 , 21.5 , 27.0 , 28.1 (CH2-4), 28.2 (CH3), 38.1 (CH2N), 40.9 (CH2S), 53.8 (CHN), 78.3 (C of tBu), 118.6 , 130.3 , 133.6 , 134.9 , 155.2 (carbamate C=O), 170.2 (amide C=O); δP : 24.4; HRMS : found [M]2+ 550.2431. C62H78N4O6P2S22+ requires 550.2414. IR (cm−1): 1435 (P-Ph), 1662 (C=O), 1705 (C=O), 2904 (CH), 2972 (CH), 3346 (NH), 3442 (NH); [α]D18 = −105.2° . Data assignment assisted by COSY, DEPT135 and HSQC 2D NMR spectra.To a solution of the disulfide of 3-mercapto-2(R)-(tert-butoxycarbonylamino)propanoylamino]pentyl} triphenylphosphonium chloride  in methanol (10.1 mL) at 0°C was added concentrated hydrochloric acid (4.90 mL) dropwise. The reaction mixture was allowed to stir at room temperature for 16 h then concentrated under reduced pressure. Trituration from diethyl ether gave (CysTPP)2•2Cl•2HCl as a white hygroscopic amorphous solid . δH : 1.41–1.57 , 3.02–3.10 , 3.26 , 3.41 , 3.59–3.57 , 4.09–4.14 , 8.72 , 9.17 : 20.7 , 21.9 , 27.4 , 28.2 (CH2-4), 38.7 (CH2N), 39.3 (CH2S), 51.8 (CHN), 119.1 , 130.7 , 134.1 , 135.3 , 166.9 (C=O); δP : 24.6; HRMS : found [M]2+ 450.1894. C52H62N4O2P2S22+ requires 450.1889. IR (cm−1): 1436 (P-Ph), 1672 (C=O), 2859 (CH), 2924 (CH), 3202 (NH). [α]D21 = −21.5° . Data assignment assisted by COSY, DEPT135 and HSQC 2D NMR.To a solution of the disulfide of {5-[3-mercapto-2. Mitochondria were pelleted from the supernatant by centrifugation . The pellet was resuspended in STE and centrifuged again . Mitochondria were resuspended in STE buffer, placed on ice and used immediately. The protein concentration was subsequently determined using a BCA assay kit with BSA as standard.Crude rat liver mitochondria were isolated by differential centrifugation, with all steps being performed at 4°C using pre-cooled equipment. Rats were culled by stunning followed by cervical dislocation. Tissues were excized and immediately stored in ice-cold STE buffer . The liver was cut into pieces before rinsing thoroughly in ice-cold STE. The tissues pieces were chopped finely with a razor blade, residual blood and connective tissue were removed, before rinsing again with STE. Tissue pieces were homogenized in a glass tube with 5 strokes of a loose-fitting Potter-Elvehjem PTFE pestle followed by 5 strokes with a tight-fitting Potter-Elvehjem PTFE pestle. The homogenate was centrifuged (1000 × On dry ice ∼25 mg of frozen tissue was weighed into a 2 mL tube containing 2.8 mm ceramic beads (Omni). These tubes containing tissue were stored on dry ice before addition of 10 μL of 80% (v/v) methanol/mg of tissue. After a brief period on ice to prevent tubes fracturing, the tissue was disrupted using a Precellys 24 tissue homogenizer  on a 6500 setting for 15s and then immediately placed back on ice for 5 min. Samples were then re-homogenized  and again placed on ice. Each sample was vortexed just prior to removal of 100 μL to a fresh 1.5 mL eppendorf tube. This was kept on ice before centrifugation at 16000 × g for 2 min to pellet protein. 60 μL of the clear supernatant was transferred to a fresh 1.5 mL eppendorf tube and placed on dry ice. Any remaining liquid was removed and discarded before the pellet was resuspended in 20 μL of water to release any remaining acyl-CoA trapped in the pellet. After resuspension of the pellet, 80 μL of methanol was added and the tube centrifuged at 16000 × g for 2 min to repellet protein. 60 μL of this clear supernatant was transferred to a fresh 1.5 mL eppendorf tube on dry ice. The two 60 μL extracts from each sample were then processed separately.2 was prepared as a 50 mM stock solution in DMSO. Acyl-CoA standards were stored at −80°C in 80% (v/v) methanol. Fresh TCEP  was prepared by adding 170 μL of water to 5.6 mg of TCEP-HCl before adding sequentially 8 μL of 5 M NaOH, 20 μL of 1 M NH4HCO3 and finally 2 μL of 5 M NaOH. Sufficient CysTPP master mix (2 mM (CysTPP)2, 20 mM TCEP and 200 mM NH4HCO3 in 20% (v/v) DMSO) was prepared and left on ice for ∼5 min to allow TCEP to reduce (CysTPP)2 to CysTPP. CysTPP master mix (20 μL) was added to each 60 μL sample or standard which was then vortexed, briefly centrifuged and incubated in a shaking water bath at 37°C for 3 h. After addition of 20 μL 25 mM DTT in 80% (v/v) methanol the samples were vortexed, briefly centrifuged and incubated at 37°C for 10 min to remove acyl groups from the thiol of CysTPP. After addition of 100 μL of 100 mM IAM, 100 mM NH4HCO3 in 50% (v/v) DMSO the samples were vortexed, briefly centrifuged and incubated at 37°C for 30 min. The reaction was quenched with 300 μL of 93% (v/v) DMSO containing 0.33% (v/v) formic acid (FA) to give an acid stabilized sample in 80% (v/v) organic solvent . Importantly, ACN is not compatible with early steps in the CysTPP assay as it reacts with CysTPP to generate a false acetyl-CysTPP-CAM signal.(CysTPP)N-acyl-CysTPP-CAMs.Each sample was compared to 22 acyl-CoA standards (0-5 μM of each combined in 60 μL of 80% (v/v) methanol) reacted with CysTPP as above to generate a daily five-point standard curve of their corresponding LC-MS/MS analyses of acyl-CysTPP-CAMs was performed using a Xevo TQ-S triple quadrupole mass spectrometer . Samples in 13% methanol/67% DMSO 0.2% formic acid were analyzed as batches beginning immediately after sample preparation. Samples were kept in glass MS vials at 8°C prior to injection of 2 μL each sample using an autosampler. All acyl-Co-As with standards were analyzed within 7 h of sample preparation. Separations were performed on a I-Class ACQUITY UPLC BEH C18 column  with a UPLC filter  at 30°C using a ACQUITY UPLC I-Class system . The mobile phases were MS solvent A  and B  at a flow rate of 0.2 mL/min with the following gradient: 0–1.5 min, 5% B; 1.5–2 min, 5-15% B; 2-5 min, 15% B; 5-8 min, 15-100% B; 8-9 min, 100% B. The eluate was analyzed by MS for the complete 5 min UPLC gradient. CysTPP products were detected by MRM with electrospray ionization in positive ion mode using a source spray voltage of 3.1 kV and an ion source temperature of 200°C. Cone voltages, collision energies and MS/MS transitions for individual acyl-CysTPP-CAMs are shown in −1) in 100 mM HEPES  was incubated alone or with 2 mM of either octanoyl-CoA or palmitoyl-CoA for 6 h at 37°C. Subsequently each 20 μL sample was incubated with 5 μL of 25 mM DTT, 5% SDS for 15 min at 37°C before thiols were carbamidomethylated with 5 μL of 200 mM IAM for 30 min at 37°C. Protein in each sample was precipitated with 20 volumes of methanol and the pellet trypsinized overnight in 100 μL 25 mM NH4HCO3/10% ACN. Tryptic peptides were injected in either 10% or 45% ACN containing 0.1% TFA. Data was acquired on an Orbitrap QExactive Plus coupled to an RSLC3000 nanoUPLC via an EASY spray source (Thermo Fisher Scientific). Peptides were fractionated using a 75 μm × 50 cm PepMap RSLC C18 column with mobile phases A (0.1% formic acid) and B . Samples were subjected to a gradient rising from 3 to 10% B by 7 mins, to 40% B by 52 mins and to 95% B by 55 min. Data was acquired using a DDA strategy with MS data acquired between 400-1500 m/z at 70,000 fwhm resolution.GDH  with the following parameters: Enzyme: Trypsin (specific), Bos Taurus database (UniProt reference proteome downloaded 05 Aug 2019 containing 23861 proteins) with additional contaminant database (containing 246 common contaminants). Fixed modification at PEAKS DB stage: carbamidomethylation (Cys). Variable modifications at PEAKS DB stage: oxidation (Met), acetylation (Lys), octanoylation  and palmitoylation .Statistical analysis methods used in this study are indicated in the figure legends. Unless otherwise indicated statistical significance was calculated using Prism version 9 for Mac (GraphPad) using a one-way ANOVA followed by a Tukey or Dunnet’s multiple comparison test as indicated. p < 0.05 was considered to indicate statistical significance throughout the study. For animal experiments, efforts were made to achieve this study’s scientific goals with the minimum number of animals.


N-oxide is presented, which gives a two-dimensional supramolecular crystal structure.The molecular structure of pyridine-4-carboxamidoxime  N-oxides led to the crystallization of pyridine-4-carboxamidoxime N-oxide. Herein we report the first crystal structure of the title compound, C6H7N3O2 [systematic name: (Z)-4-(N′-hydroxycarbamimidoyl)pyridine N-oxide]. The hydroxycarbamimidoyl group is essentially coplanar with the aromatic ring, r.m.s.d. = 0.112 Å. The compound crystallizes in hydrogen-bonding layers built from the formation of strong O—H⋯O hydrogen bonds between the oxime oxygen atom and the oxygen atom of the N-oxide, and the formation of N—H⋯O hydrogen bonds between one amine nitrogen atom and the N-oxide oxygen atom. These combined build R34(24) ring motifs in the crystal. The crystal structure has no π–π interactions.Our work in the area of synthesis of metal–organic frameworks (MOFs) based on organic  We are particularly interested in their uses in coordination polymers and as potential catalysts. The utility of these aromatic N-oxides to facilitate organic oxotransfer reactions has been well documented over the years ° with respect to the pyridine ring plane. N1—O1 has a distance of 1.3226 (18) Å and is consistent with normal N-oxide distances. The crystal structure contains a strong intermolecular hydrogen bond between O2⋯O1i which forms a chain running parallel to the b axis; the O2⋯O1i separation is 2.6747 (19) Å. Another hydrogen bond is formed between N3⋯O1ii which links neighboring chains together; the N3⋯O1ii separation is 2.899 (2) Å [symmetry codes: (i) x, y + 1, z; (ii) x, −y + z + Herein we report the first crystal structure of pyridine-4-carboxamidoxime de Fig. 1, which cR(24) synthons, generating sheets of hydrogen-bonding molecules parallel to the (100) face of the unit cell  was weighed and dissolved in a 25 ml beaker in enough methanol to form a solution that allowed to slowly evaporate at room temperature. The clear crystals were analyzed on a Rigaku Xtal Miniflex.An amount of 0.025 g of pyridine-4-carboxamidoxime Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314620013358/bh4056sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314620013358/bh4056Isup2.hklStructure factors: contains datablock(s) I. DOI: 2035503CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The growth-suppressive properties of 1α,25-dihydroxyvitamin D3 and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1α,25-dihydroxyvitamin D3 and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indicated suppression of p70-S6 kinase levels by 1α,25-dihydroxyvitamin D3 and tacalcitol in T98G cells, whereas the levels of PLCγ, a target for phospholipid signaling, was slightly increased.The active hormonal form of vitamin D, 1α,25-dihydroxyvitamin D3 and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol , indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1α,25-dihydroxyvitamin D3 and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumorigenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.Activation of STAT3, an important regulator of malignancy, was suppressed by 1α,25-dihydroxyvitamin D •Vitamin D and its analog tacalcitol affect signaling in glioblastoma cell lines.•Suppression of the oncogenic STAT-3 is stronger by tacalcitol than by vitamin D.•Tacalcitol and vitamin D inhibit anchorage-independent growth in T98G and U251 cells.•Vitamin D analogs may be useful to combat oncogenicity and invasion of glioblastoma. In addition to its well-known roles in the maintenance of calcium levels and bone health, the vitamin D hormone affects a number of additional systems and processes, including e. g. neurotransmission, immune function and cellular growth and survival . The inacitriol) . 1α,25-dunctions . 1α,25-Dpathways . Alternapathways . 1α,25-Diability . The grof cancer . Many stnd colon . Furthernd colon ,9. Unforcalcemia . Therefocalcemia . Vitamincalcemia ,14. Howe3 and the vitamin D analog tacalcitol   A on the 3 on signaling pathways and anchorage-independent growth in human glioblastoma cells T98G and U251. The findings of the present study indicate that the cellular response to these two molecules, despite their close resemblance in structure, is partly different. In addition, we report significant suppression of anchorage-independent growth of T98G and U251 cells by tacalcitol and 1α,25-dihydroxyvitamin D3, supporting a functional role for these compounds in decreasing tumorigenicity of glioblastoma.In the present study we explored the effects of tacalcitol and 1α,25-dihydroxyvitamin D22.13  and tacalcitol  were stored as stock solutions in – 20 °C, dissolved in toluene and pure ethanol 1:1. The toluene was removed prior to experimentation and the compounds were added to the cell cultures in ethanol. In all experiments, the final ethanol concentration was kept below 0.1% (v/v).1α,25-Dihydroxyvitamin D2.2Human T98G and U251 glioblastoma cells were cultured in DMEM + GlutaMAX  and RPMI 1640 + Glutamax  respectively, supplemented with 10% (v/v) fetal bovine serum .2.32 one day prior to treatment. Cells were treated with ethanol, 1 μM 1α,25-dihydroxyvitamin D3 or 1 μM tacalcitol for 24 h. Cells were then rinsed with ice-cold Dulbecco's phosphate-buffered saline  and lysed with a buffer containing 20 mM Tris HCl pH 8, 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM EDTA and 0.1% SDS, supplemented with a protease and phosphatase inhibitor cocktail . After centrifugation at 12 000 g for 15 min, the supernatant was collected as whole cell lysate and 1 × NuPAGE™ LDS Sample Buffer, 50 mM dithiothreitol (DTT) was added. Samples were denatured at 95 °C for 5 min before loading on SDS-polyacrylamide gels for electrophoresis. Chameleon Duo Pre-Stained Protein Ladder (LI-COR Biosciences) was used for size reference. After electrophoresis, protein was transferred to PVDF-FL membranes. Membranes were blocked for 2 h at room temperature in Intercept blocking buffer  and incubated at 4 °C over-night with primary antibodies. The following primary antibodies were used: rabbit anti-pY783-PLCγ1 , mouse anti-PLCγ1 , mouse anti-pThr389-p70-S6 Kinase , rabbit anti-p70-S6 Kinase , rabbit anti-pY705-STAT3 , mouse anti-STAT3 , rabbit anti-pS473-AKT , mouse anti-AKT , rabbit anti-pT202/Y204-ERK1/2 , rabbit anti-ERK1/2 , mouse anti-β-Actin , mouse anti-pT180/182-p38 , rabbit anti-p38  and rabbit anti-β-Actin . The membranes were washed 3 × 10 min in 0.05% Tween-20 in TBS both before and after incubation for 1 h with secondary antibodies . The membranes were scanned and protein expression levels were analyzed using an Odyssey Scanner and ImageStudio v5.2.5 software (both from LI-COR Biosciences). β-Actin was used as loading control.For Western blot experiments, the cells were seeded in 6-well plates at a density of 25 000 cells/cm2.4T98G cells were incubated with ethanol or 1 μM tacalcitol for 24 h followed by RNA isolation using RNeasy mini kit (Qiagen). cDNAs were synthesized with qPCRBIO cDNA Synthesis Kit (PCR Biosystems) and used for real-time PCR amplification with qPCRBIO SyGreen Mix (PCR Biosystems) on a CFX Connect Real-Time PCR System (Bio-Rad). β-Actin, TBP (TATA-binding protein) and 18S were used as control (housekeeping) genes. Forward and reverse primers were as follows: 5′-TCTGCCGGAGAAACAGTTGG-3′ (forward) and 5′-AGGTACCGTGTGTCAAGCTG-3′ (reverse) for STAT3; 5′-TCTACAATGAGCTGCGTGTG-3′ (forward) and 5′-AGCCTGGATAGCAACGTACA-3′ (reverse) for β-actin; 5′-TGCACAGGAGCCGCCAAGAGTGA-3' (forward) and 5′-CACATCACAGCTCCCCACCA-3' (reverse) for TBP; 5′-AGTCCTGCCCTTTGTACACA-3' (forward) and 5′GATCCGAGGGCCTCACTAAAC3' (reverse) for 18S.−ΔΔCT method.Each PCR was done in triplicate and the experiment was repeated four times. Data were analyzed using the 22.52) and treated for 24 h with ethanol, 1α,25-dihydroxyvitamin D3 or tacalcitol at the indicated concentrations. Preparation of nuclear cell lysates and colorimetric STAT3 detection was carried out according to the manufacturer's protocol. A BCA kit  was used to measure the protein concentration. The absorbance, proportional to activation of STAT3, was measured at OD 450 nm using a FLUO STAR Omega microplate reader (BMG LABTECH).Activation of STAT3 in T98G and U251 cells was measured by a colorimetric ELISA-based STAT3 assay kit . Cells were cultured in T175 flasks (25 000 cells/cm2.62) in medium containing 0.3% agarose  and 3% (for T98G) or 10% (for U251) FBS. Cell suspensions were treated with ethanol, 1α,25-dihydroxyvitamin D3 (0.2–5 μM) or tacalcitol (0.2–5 μM) and placed on a 0.6% agarose layer in a 48 well plate. Each treatment condition was tested in triplicate. Cells were cultured for three weeks with addition of new ethanol, 1α,25-dihydroxyvitamin D3 or tacalcitol in fresh media every 72 h, followed by counting the number of formed T98G (≥70 μm) or U251 (≥40 μm) colonies.Cells were trypsinized and re-suspended  for Western blotting.For quantification using STAT3 Transcription Factor Assay, data are expressed as average values relative to the control ± SD. The values of experimental samples were divided by the value of control sample for each experiment and one sample Student's t-test was performed to test whether the average differs significantly from the control.P-values *<0.05, **<0.01, ***<0.001 and ****<0.0001 were considered significant.33.13 . The results of these experiments confirmed that tacalcitol has a marked effect on STAT3 and dose-dependently suppresses STAT3 activation in T98G cells  and/or by hydroxyderivatives other than 1α,25-dihydroxyvitamin Desponses . Our stuTo summarize, the current results contribute to our understanding of effects by structurally similar vitamin D-related compounds in glioblastoma and underlines the potential for vitamin D analogs in future cancer therapy.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.