Magnetic resonance–guided focused ultrasound (MRgFUS) thalamotomy is a safe and effective procedure for drug‐resistant tremor in Parkinson's disease (PD).The aim of this study was to demonstrate that MRgFUS ventralis intermedius thalamotomy in early‐stage tremor‐dominant PD may prevent an increase in dopaminergic medication 6 months after treatment compared with matched PD control subjects on standard medical therapy.We prospectively enrolled patients with early‐stage PD who underwent MRgFUS ventralis intermedius thalamotomy (PD‐FUS) and patients treated with oral dopaminergic therapy (PD‐ODT) with a 1:2 ratio. We collected demographic and clinical data at baseline and 6 and 12 months after thalamotomy.We included 10 patients in the PD‐FUS group and 20 patients in the PD‐ODT group. We found a significant increase in total levodopa equivalent daily dose and levodopa plus monoamine oxidase B inhibitors dose in the PD‐ODT group 6 months after thalamotomy.Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyIn early‐stage tremor‐dominant PD, MRgFUS thalamotomy may be useful to reduce tremor and avoid the need to increase dopaminergic medications. © 2022 The Authors.  The thalamic nucleus ventralis intermedius (VIM) is a widely used target for the surgical treatment of medication‐resistant tremor in patients with essential tremor (ET) and Parkinson's disease (PD).To date, MRgFUS VIM thalamotomy has been mostly focused on ET and proved to be safe and effective.Pharmacological treatment of tremor in PD may be challenging because this symptom may be levodopa resistantWe hypothesized that MRgFUS thalamotomy may be performed at the earliest stages of tremor‐dominant PD aiming to “spare” dopaminergic medications and thus reduce the risk associated with the progressive increase of oral medical therapy over time.In this study, the primary objective was to demonstrate that MRgFUS VIM thalamotomy performed in patients with early‐stage tremor‐dominant PD is associated with a significantly lower dopaminergic therapy daily dosage after 6 months compared with a matched PD control population on standard medical therapy.We included patients diagnosed with early‐stage idiopathic PDData from PD‐FUS were compared with those of a control group of patients with tremor‐dominant PD treated with oral dopaminergic therapy (PD‐ODT), matched by sex, age, and disease duration in a 1:2 ratio. In the PD‐ODT group, we included patients who met inclusion criteria but did not undergo MRgFUS.on medication).All patients were visited by neurologists expert in movement disorders, who assessed motor performance  and Hoehn and Yahr stage in the morning 90 minutes after levodopa intake .Change between baseline and follow‐up visits of MDS‐UPDRS‐III and LEDD were computed and expressed as percentage of change from baseline.AEs were extensively collected for both groups; for PD‐FUS, we also collected AEs associated with thalamotomy and with MRI environment or ultrasonography procedure.MRgFUS VIM thalamotomy screening and procedure were approved by the local Ethics Committee (CE n.59/2020). All patients gave their written informed consent to the use of their anonymized clinical data for research purposes.Details about the statistical analyses are reported in Supporting Information Material A total of 145 patients with tremor‐dominant PD were initially evaluated and referred to a dedicated outpatient clinic for the evaluation for MRgFUS thalamotomy. Eighty‐nine patients were accepted to undergo the full screening evaluations for MRgFUS VIM thalamotomy, of whom 37 underwent the procedure. Of these 37, we included 10 patients with early‐stage PD who fulfilled our a priori defined criteria.Baseline demographic and clinical features were similar between PD‐FUS and PD‐ODT Table .At 6‐month follow‐up, we observed a significant difference in total LEDD between the two groups, because of increased LEDD in the PD‐ODT group versus stable dosage in PD‐FUS Table , Fig. 1AP = 0.001 and P = 0.014) and a mild reduction in LEDD of DA in the PD‐FUS group (P = 0.042)  Fig. .P = 0.002), while in the PD‐ODT group there was only a trend to a statistically significant difference (P = 0.081)  Fig. .P = 0.01), with a significant increase in total LEDD and LEDD of levodopa plus MAOB‐I in the PD‐ODT group after 12 months compared with baseline  . Details are reported in Table ly) Fig. A,B.AEs are detailed in Table MRgFUS thalamotomy is a safe and effective treatment for tremor in patients with PD.Management of dopaminergic therapy has never been specifically investigated by studies reporting data on MRgFUS thalamotomy in PD. In a recent clinical trial, Bond et alWe found a stabilization of LEDD in PD‐FUS after VIM thalamotomy; differences in the study population, such as higher mean disease duration, motor score, and LEDD in the study of Bond et al,In all studies, the MDS‐UPDRS‐III score decreased significantly shortly after the procedure , which was not performed for ethical reasons. However, there are strengths worth mentioning. Our strict selection and matching criteria, as well as the clinical homogeneity of the study population, provided statistically significant results despite the limited sample size. Further studies in larger cohorts with longer follow‐up are needed to confirm whether early MRgFUS may be a cost‐effective therapeutic strategy in early‐stage tremor‐dominant PD, potentially reducing the risk for AEs caused by the progressive increase of dopaminergic medications, such as motor complications and/or ICDs.We acknowledge that our pilot study is limited by the small sample size and a short follow‐up; moreover, given the very limited number of patients who reached 12‐month follow‐up, the results on the long‐term management of dopaminergic medication after MRgFUS thalamotomy should be interpreted with caution. Finally, it would have been interesting to evaluate both groups in a defined In patients with early‐stage tremor‐dominant PD, MRgFUS thalamotomy may be useful to reduce tremor and avoid, in the short term, the need to increase dopaminergic medications. These results may help to understand the correct timing to address patients for MRgFUS thalamotomy, a treatment that, at the time being, should be reserved to patients with proven medication‐refractory tremor.Prospective studies with larger cohorts are needed to confirm these findings and to understand whether this treatment may reduce the incidence of AEs and long‐term motor complications of dopaminergic therapy. Longer follow‐up may additionally provide helpful information on the difference in time to dyskinesias between the two groups.1. Research project: A. Conception, B. Organization, C. Execution;2. Statistical Analysis: A. Design, B. Execution, C. Review and Critique;3. Manuscript Preparation: A. Writing of the first draft, B. Review and Critique.N.G.A.: 1B, 1C, 2A, 2B, 3A, 3B.R.C.: 1A, 1B, 1C, 2A, 2C, 3A, 3B.L.M.R.: 1C, 2C, 3B.S.B.: 1B, 1C, 2C, 3B.G.S.: 1B, 1C, 2C, 3B.A.E.E.: 1B, 2C, 3B.A.N.: 1B, 1C, 2C, 3B.G.M.: 1B, 1C, 2C, 3B.G.T.: 1B, 1C, 2C, 3B.V.L.: 1B, 1C, 2C, 3B.G.D.: 1B, 1C, 2C, 3B.S.R.: 1B, 1C, 2C, 3B.V.G.: 1C, 2B, 3A, 3B.M.G.: 1B, 1C, 2C, 3B.M.S.: 1B, 1C, 2C, 3B.F.G.: 1B, 2C, 3C.S.P.: 1C, 2C, 3B.E.B.: 1B, 1C, 2B, 3B.P.A.: 1B, 1C, 2B, 3B.S.H.M.J.P.: 1B, 1C, 2C, 3B.E.F.M.C.: 1B, 1C, 2C, 3B.M.G.B.: 1B, 1C, 2C, 3B.R.E.: 1A, 1B, 1C, 2A, 2C, 3B.N.G.A., R.C., L.M.R., S.B., G.S., A.E.E., A.N., G.M., G.T., V.L., G.D., S.R., V.G., M.G., M.S., F.G., S.P., E.B., P.A., S.H.M.J.P., E.F.M.C., M.G.B., and R.E. have no financial disclosures to report.R.C. has received speaking honoraria from Zambon; Zambon SAU; Bial Italia Srl; Advisory board fees from Bial; Research support from the Italian Ministry of Health; Editor‐in‐chief of the neuromuscular and movement disorders section of Brain Sciences; Member of the editorial board of Parkinsonism and related disorders, Frontiers in Neuroscience ‐ Neurodegeneration (Associate Editor) and Frontiers in Neurology ‐ Movement Disorders (Review Editor).Appendix S1. Supporting informationClick here for additional data file.Figure S1. flow‐chart with description of the causes of exclusion and inclusion of patients.Click here for additional data file.Figure S2. change in dopaminergic medications and motor outcome at baseline, 6 and 12‐months in PD‐FUS (n = 4) and PD‐ODT‐ (n = 8) patients with 12‐months follow‐up.Click here for additional data file.Table S1. baseline demographics and clinical characteristics and 12‐months of patients with 12‐months follow up .Click here for additional data file.Table S2. characterization of incident Adverse Events observed during the 6‐months follow‐up.Click here for additional data file.


Eleftheria terra, a betaproteobacterium; odilorhabdins, from Xenorhabdus, are broad-spectrum inhibitors of protein synthesis, and darobactins from Photorhabdus target BamA, the essential chaperone of the outer membrane of Gram-negative bacteria. Xenorhabdus and Photorhabdus are symbionts of the nematode gut microbiome and attractive producers of secondary metabolites. Only small portions of their biosynthetic gene clusters (BGC) are expressed in vitro. To access their silent operons, we first separated extracts from a small library of isolates into fractions, resulting in 200-fold concentrated material, and then screened them for antimicrobial activity. This resulted in a hit with selective activity against Escherichia coli, which we identified as a novel natural product antibiotic, 3′-amino 3′-deoxyguanosine (ADG). Mutants resistant to ADG mapped to gsk and gmk, kinases of guanosine. Biochemical analysis shows that ADG is a prodrug that is converted into an active ADG triphosphate (ADG-TP), a mimic of GTP. ADG incorporates into a growing RNA chain, interrupting transcription, and inhibits cell division, apparently by interfering with the GTPase activity of FtsZ. Gsk of the purine salvage pathway, which is the first kinase in the sequential phosphorylation of ADG, is restricted to E. coli and closely related species, explaining the selectivity of the compound. There are probably numerous targets of ADG-TP among GTP-dependent proteins. The discovery of ADG expands our knowledge of prodrugs, which are rare among natural compounds.With the overmining of actinomycetes for compounds acting against Gram-negative pathogens, recent efforts to discover novel antibiotics have been focused on other groups of bacteria. Teixobactin, the first antibiotic without detectable resistance that binds lipid II, comes from an uncultured  Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii  . This is 46 BGCs , and gam 41 BGCs . Xenorhat larvae , 16, andt larvae . We receorhabdus , 19. DarE. coli that was discovered by a differential screen of Photorhabdus isolates. ADG is an interesting prodrug mimic of guanosine and is produced by a silent operon of Photorhabdus luminescens.In this study, we describe aminodeoxyguanosine (ADG), a novel antibiotic acting against Xenorhabdus and Photorhabdus were previously screened for antimicrobials, but the number of compounds identified is a small fraction of BGCs found in their genomes. To access silent operons, we have been screening concentrated extracts of these bacteria. Darobactin was discovered in such a screen from a 15× concentrated extract of Photorhabdus khanii  separation, producing 48 fractions for each isolate. Following HPLC fractionation, we dried and then solubilized the fractions in water so that the final concentration of each compound in a fraction would be ~200× that of the initial culture supernatant. This resulted in a total of 2,880 concentrated fractions.A collection of 60 10.1128/mbio.00700-22.7TABLE S1E. coli enzymes that require GTP according to Brenda Enzyme Database. Download Table S1, XLSX file, 0.02 MB.(a) ID and library source of the strains screened in this study. (b) Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. luminescens KLE11358 produced a zone of inhibition on an E. coli lawn, while there was no inhibition of S. aureus, and we decided to follow up on this hit. Bioassay-guided isolation of the extract using several HPLC conditions  led to the purification of an active fraction  HRESIMS spectra of (A) pure ADG and (B) authentic synthetic ADG. Download FIG S1, DOCX file, 0.8 MB.(a) HPLC chromatogram and activity of pure ADG that causes a zone of inhibition on Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00700-22.2FIG S21H-NMR spectrum of ADG in D2O (400 MHz). (b) 1H-1H COSY spectrum of ADG in D2O (400 MHz). (c) 13C-NMR spectrum of ADG in DMSO-d6 (100 MHz). (d) 1H-NMR chemical shifts of ADG and 1H-1H COSY correlations . Download FIG S2, DOCX file, 0.9 MB.(a) Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00700-22.3FIG S31H-NMR (400 MHz) in D2O of (A) natural product ADG, (B) synthetic ADG, and (C) 1:1 ratio mixture of natural and synthetic ADG. The left shows the spectra from0 to 10 ppm, the right shows the spectra from 3 to 4.5 ppm. (b) UV spectra of natural (left) and synthetic ADG (right). Download FIG S3, DOCX file, 0.3 MB.(a) Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the X92429.1) was subjected to BLAST search against the P. luminescens genome, reporting a 5-gene putative BGC. These genes were termed adgA-E, with adgB-E being homologous to pur10, pur4, pur7, and pur3 respectively, and adgA was annotated as an MFS transporter gene  under the control of an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter (pNS-ADG). Following confirmation of the desired plasmid construct, we transformed E. coli BAP1 with this plasmid. We also transformed E. coli with the empty PRSF vector as a negative control (E. coli Bap1+pRSFduett-1). Expression of the adgB-E operon was induced by IPTG, and upon LC-MS analysis, a compound with a mass matching ADG and with the same retention time as the ADG standard was identified in the culture supernatant of E. coli BAP1+pNS-ADG . There was little activity against gut bacteria and Gram-positive S. aureus (50) of ADG against mammalian cell lines FaDu and HepG2, and ADG started to show cytotoxicity at 128 μg/mL.ADG showed a MIC of 4 μg/mL against . aureus . These f6E. coli cells on nutrient agar medium containing ADG at 4× MIC. The frequency of resistance was 6.5 × 10−5, which is fairly high for an antibiotic, indicative of a null mutation  into pET Sumo vector downstream of a His6-Sumo ORF and used E. coli BL21(DE3) for expression. Following purification of Gsk and removal of the His6-Sumo tag, we used this enzyme to phosphorylate ADG. Indeed, when treated with ATP and purified with recombinant Gsk, ADG is converted to ADG-MP in vitro, with concomitant production of ADP (E. coli enzymes may further phosphorylate ADG-MP into 5′-diphosphate (ADG-DP) and 5′-triphoshate (ADG-TP) by E. coli guanylate kinase (Gmk) and nucleoside diphosphate kinase (Ndk), respectively (E. coli BL21(DE3) to clone and express Gmk and Ndk and then purified them using the nickel nitrilotriacetic acid (Ni-NTA) affinity procedure. Remarkably, inclusion of the purified Gmk and Ndk in addition to Gsk in an in vitro assay produced ADG-DP and ADG-TP  , right. n of ADP . Despiteectively , right. e of ADG . These d10.1128/mbio.00700-22.5FIG S5FIG S5, DOCX file, 0.2 MB.(a) Kinetics traces of ADG phosphorylation by Gsk compared to guanosine (Guo). Production of ADP is coupled to consumption of NADH and is monitored as the decrease of absorbance at 340 nm. (b) Initial reaction velocity. Error bars are SD from three reaction mixtures set up in separate wells on the same 96-well plate. (c) Identity of AAG, AA(ADG), and AAGA confirmed using mass spectrometry. Download Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the gsk that exhibit full resistance to ADG. We therefore used the E. coli AG1 Gsk overexpression strain from the ASKA library to increase the probability of finding target mutations. We tried to select for resistant mutants by plating E. coli cells carrying additional copies of gsk on medium with ADG at 4× MIC. However, there was no change in the MIC of the colonies following passage to fresh medium, suggesting that the plasmids are not stable. To obtain a stable E. coli strain with two copies of gsk, we introduced a second copy into the neutral fliT gene region . Intriguingly, however, all three resistant mutations mapped to gmk, which encodes guanylate kinase, the second enzyme that adds phosphate to ADG-MP , the membrane stain FM4-64 , and membrane permeabilization stain Sytox green . Cells were incubated at 37°C in a thermostatic chamber and imaged every 15 min under the microscope. The selected time points and phase contrast images were chosen to best represent the elongation of E. coli MG1655 with ADG. Scale bars, 5 μm. This experiment was repeated three times. This image is representative of the three experiments. Download FIG S6, DOCX file, 0.3 MB.Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00700-22.10VIDEO S1E. coli MG1655 cells were spotted onto a 1.5% agarose pad containing ADG (8× MIC), the membrane stain FM4-64 , and membrane permeabilization stain Sytox green . Cells were incubated at 37°C in a thermostatic chamber and imaged every 15 min under the microscope. Phase-contrast images were chosen to best visualize the elongation phenotype of E. coli under ADG treatment. Scale bars, 5 μm. Download Movie S1, MOV file, 5.0 MB.Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Antibiotic discovery is uniquely challenging due to several factors: penetration barrier, especially in Gram-negative bacteria with their additional outer membrane; rapid acquisition of resistance to most compounds; and a very large background of nonspecifically acting compounds in both natural and synthetic collections . In the Xenorhabdus and Photorhabdus, symbionts of entomopathogenic nematode gut microbiomes , that is converted into ADG-TP, a mimic of GTP. In search of compounds acting against Gram-negative bacteria, we focused on robiomes . Severalrobiomes . Howeverrobiomes ; darobacrobiomes , the essnsimycin , 40. Anonsimycin . This isphomonas . When thXenorhabdus and Photorhabdus, the majority of which are silent under laboratory conditions. The standard approach is to engineer the BGC, either in the producing organism or heterologously, for increased production –ADG-MP (Gmk)–ADG-DP (Ndk)–ADG-TP. The product, ADG-TP, is a mimic of GTP. ADG lacks the 3′-OH of guanine, and we determined that it interrupts RNA elongation. Remarkably, this is the same principle that was used by Sanger to interrupt DNA synthesis, which enabled sequencing. Again, human chemists and nature appear to have arrived at a similar solution, in this case how to terminate nucleic acid synthesis.Using this approach, we identified ADG, which acts selectively against ntiviral . The facntiviral , 45. No S. alboniger. Even though this BGC shares homology with puromycin BGC, it codes for a compound that has an entirely unrelated mechanism of action. This finding suggests that even closely related BGCs can diverge to produce secondary metabolites with completely different functions.We identified the BGC of ADG based on homology with the BGC of puromycin in Notably, we identified mutants resistant to ADG in the Gsk and Gmk kinases but not in the RNA polymerase or any other potential target. We also noticed that cells treated with ADG are elongated, suggesting that the compound is interfering with GTP-dependent polymerization of FtsZ. Indeed, the FtsZ septation ring does not form in the presence of ADG. In previous studies, guanosine analogs have been suggested as FtsZ inhibitors and potential antibiotics . For insE. coli to ADG, and this experiment probably emulates natural conditions under which the purine salvage pathway is important. The natural environment of E. coli is the nutrient-poor colon, where Gsk is likely to be expressed. ADG is probably smuggled into the cell and accumulates in the cytoplasm by active transport. We did not identify any resistant mutants in a transporter, which is to be expected, given that there are 3 redundant nucleoside transporters in E. coli: NupC, NupG, and YegT  broth and incubated overnight at 28°C with shaking at 200 rpm, diluted 1:100 in new Falcon tubes with 10 mL LB broth and tryptic soy broth (TSB), and incubated for 8 days at 28°C with shaking at 200 rpm. Following the removal of the cells by centrifuging at 12,000 × g for 10 min, 10-mL culture supernatants were collected, dried, and resuspended in 500 μL MilliQ water to obtain 20 times concentrated sample compared to initial supernatant. These samples were subjected to reverse-phase chromatography, and HPLC elution was fractionated using a C18 semipreparative column . HPLC conditions were the following: solvent A, MilliQ water and 0.1% (vol/vol) formic acid; solvent B, acetonitrile and 0.1% (vol/vol) formic acid. The initial concentration of 2% solvent B was maintained for 5 min, followed by a linear gradient to 50% over 33 min with a flow rate of 4 mL/min. Fractions were collected every minute. UV detection was monitored at 254 nm. Each fraction was dried down and resuspended in 50 μL of MilliQ water. Overlays were prepared from exponential cultures. After dilution of 1:100 from an overnight culture in cation-adjusted Mueller-Hinton II broth (MHIIB), test pathogens were grown for 2 to 5 h at 37°C with shaking at 220 rpm and diluted to an optical density at 600 nm (OD600) of 0.03 in MHIIB. Cation-adjusted Mueller-Hinton II agar (MHIIA) plates were covered by these cultures, and the excess culture was removed and overlays were left to dry in a biosafety cabinet. Fractions were tested for activity by spotting 6 μL on Mueller-Hinton II agar lawns that were seeded with S. aureus HG003 as a representative of Gram-positive bacteria and E. coli MG1655 as a representative of Gram-negative bacteria for evaluating the activity.P. luminescens KLE11358 was inoculated in a 250-mL Erlenmeyer flask with 100 mL LB broth and incubated at 28°C with shaking at 200 rpm. Following overnight incubation, it was diluted 1:100 into a 2-liter Erlenmeyer flask with 1 liter tryptic soy broth and incubated for 8 days at 28°C with shaking at 200 rpm. Cells were removed by centrifugation at 8,000 × g for 10 min, and XAD16N resin  was added to the culture supernatant at 15% (vol/vol) and incubated overnight under agitation to bind ADG. ADG was eluted from the XAD16N resin using 1 liter of 100% methanol. The eluate was concentrated using a rotary evaporator. The solution was subjected to HPLC fractionation at the preparative scale using a C18 column . HPLC conditions were solvent A, MilliQ water and 0.1% (vol/vol) formic acid; solvent B, acetonitrile and 0.1% (vol/vol) formic acid. The initial concentration of 2% solvent B was maintained for 10 min, followed by a linear gradient to 50% over 43 min with a flow rate of 7 mL/min. Fractions were collected every minute. UV detection was monitored at 254 nm. Active fractions from the previous step were combined and subjected to further fractionation by using C18 semipreparative column . HPLC conditions were solvent A, 50 mM ammonium acetate pH 8; solvent B, acetonitrile. The initial concentration of 2% solvent B was maintained for 3 min, followed by a linear gradient to 40% over 8 min with a flow rate of 5 mL/min. UV detection was monitored at 254 nm. Bioactivity assay led to detection of the active peak. For final purification, active fraction from the previous step was subjected to HPLC using C18 semipreparative column . HPLC conditions were solvent A, MilliQ water and 0.1% (vol/vol) formic acid; solvent B, acetonitrile and 0.1% (vol/vol) formic acid. The initial concentration of 1% solvent B was maintained for 2 min, followed by a linear gradient to 2.2% over 8 min with a flow rate of 5 mL/min. UV detection was monitored at 254 nm. ADG elutes at 5.57 min . The mobile phase was a gradient of 0.1% formic acid in water (A) and acetonitrile (B) at a flow rate of 0.2 mL/min. A linear gradient was initiated with 2% acetonitrile and linearly increased to 95% at 5.1 to 10 min. The flow rate was 0.2 mL/min, and the injection volume was 5 μL. Mass spectra in the m/z range 111 to 3,000 were obtained by positive ion (positive electrospray ionization) modes. The mass spectrometry conditions were the following: gas temperature, 300°C; N2 flow rate, 7 liters/min; nebulizer gas pressure, 35 lb/in2; capillary voltage, 3,500 V; fragmentor potentials, 175 V; Vcap, 3,500 V; skimmer, 65 V; and octopole RFPeak, 750 V. Data acquisition and analysis were conducted using Agilent LC-MS-QTOF MassHunter Data Acquisition Software version 10.1 and Agilent MassHunter Qualitative Analysis Software version 10.0, respectively .LC-MS analysis was conducted on an Agilent HPLC 1260 Infinity II coupled with an Agilent 6530 Q-TOF-LC-MS system . The HPLC column was a reversed-phase ZORBAX RRHT Extend-Cm/z 283.11 [M+H]+ ; 1H-NMR  δ 8.03 , 5.98 , 4.64 , 4.10 , 3.99 , 3.84 , 3.68 ; 1H-NMR  δ 157.4, 154.3, 150.8, 135.0, 116.6, 88.1, 85.1, 75.0, 61.0, 52.6.3′-Amino-3′-deoxyguanosine (ADG): white powder, HRESIMS 1H-NMR spectrum of ADG in D2O showed one singlet aromatic proton (δH 8.03), one doublet anomeric proton , and five signals at δH4.64 to 3.68, suggesting the presence of a sugar unit (δH 5.98) through three methines  to the oxymethylene (δH 3.99 and 3.84) observed in 1H-1H correlation spectroscopy (COSY) spectrum established the existence of a furanose unit (δH 8.03 was observed in the 1H-NMR spectrum. (ii) The UV spectrum showed the characteristic absorption maxima at 255 and 275 nm (18 reversed-phase HPLC column. Considering the similar molecular mass with nucleoside guanosine (283.241 g/mol), together with the evidence described above, the compound was deduced to be the derivative of guanosine. The upfield 1H-NMR signal (δH 3.68) indicated that the methine group at C-3′ in the furanose unit is replaced by the amine group rather than the hygroxy group  . A mixture of two compounds displayed only one set of 1H-NMR signals  . Therefo signals . The syn signals .10.1128/mbio.00700-22.8TABLE S2Table S2, DOCX file, 0.06 MB.Minimal inhibitory concentrations for natural ADG and synthetic ADG. Download Copyright © 2022 Shahsavari et al.2022Shahsavari et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. luminescens WM06 data using Spades  was performed against the producer strain genome, and a small 5-gene BGC candidate consisting genes homologous to pur10, pur4, pur7, and pur3 as well as a gene annotated as an MFS transporter gene were identified  according to manual, and the whole BGC (omitting the MFS transporter gene) was amplified with matching overlap using the primers 5′-GTTAAGTATAAGAAGGAGATATACAATGAAGCTGGTTATAATTGGTTGCG-3′/5′-TGCTCAGCGGTGGCAGCAGCCTATGAATTTAGCGAAGCTATAACCGAATC-3′ and Q5 DNA polymerase  according to the manufacturer’s instructions. Simultaneously, the vector pRSFduett-1 (Invitrogen) was amplified with the primers 5′-GCTGCTGCCACCGCTGAGCA-3′/5′-TGTATATCTCCTTCTTATACTTAACTAATATACTAAGATGGG-3′ using the Q5 DNA polymerase according to the manual, and both fragments were gel purified on 1% Tris-acetate-EDTA (TAE) agarose gels. DNA was recovered from the gels using the large-fragment DNA recovery kit , and both fragments were fused using NEB isothermal assembly mix . E. coli DH5α was transformed with the isothermal assembly reaction using standard methodology and selected on LBKan agar plates. Correct assembly was verified by test restriction using EcoRI/SwaI, and the plasmid pNS-ADG was introduced to E. coli Bap1 using standard electroporation transformation methodology. E. coli Bap1+pNS-ADG and E. coli Bap1+pRSFduett-1, serving as a negative control, were grown in 1 liter LBKan at 37°C and 200 rpm to an OD600 of ~0.5. Expression of the genes was induced by addition of IPTG to 1 mM, and incubation was continued at 30°C and 200 rpm. After 3 days, cells were removed by centrifugation at 8,000 × g for 10 min, and XAD16N resin  was added to the culture supernatants at 15% (vol/vol) and incubated overnight under agitation to bind. Methanol  was used to elute the resin. The eluate was concentrated using a rotary evaporator redissolved in water and subjected to C18 solid-phase extraction. The medium was desalted by application of 6 column volumes (CV) MilliQ water and eluted with 6 CV 80% acetonitrile plus 0.1% formic acid. Elution fractions were evaporated to dryness using a SpeedVac system. The solutions were then subjected to HPLC fractionation using C18 column . HPLC conditions were solvent A, MilliQ water and 0.1% (vol/vol) formic acid; solvent B, acetonitrile and 0.1% (vol/vol) formic acid. The initial concentration of 1% solvent B was maintained for 5 min, followed by a linear gradient to 10% over 37 min with a flow rate of 5 mL/min. Fractions were collected from 5.5 min to 6.5 min based on the elution time of ADG standard (100 μg/mL) under this HPLC condition. Samples were dried, redissolved in water, and subjected to LC-MS analysis.To prove the putative BGC involvement in ADG biosynthesis, the BGC was heterologously expressed in E. coli strains, P. aeruginosa PAO1, A. baumannii ATCC 17978, K. pneumoniae strains, and S. aureus HG003 were diluted 1:100 in MHIIB and incubated at 37°C with aeration at 220 rpm. Exponential cultures with an OD600 of 0.1 to 0.9 were diluted to an OD600 of 0.001 in MHIIB; 98-μL aliquots were transferred into round-bottom 96-well plates containing 2 μL of ADG solutions with 2-fold serial dilution. After overnight incubation at 37°C, the ADG MIC was determined as the minimum concentration at which no growth of strains could be detected by eye. The MIC against intestinal bacteria, Klebsiella variicola KLE 2552, Veillonella ratti KLE 2365, Clostridium bifermentans KLE 2329, Clostridium hylemonae KLE 2503, Escherichia fergusonii KLE 2502, Enterococcus faecalis KLE 2341, and Stenotrophomonas maltophilia KLE 11416 (KLE collection bacteria were isolated from stool under anaerobic conditions and identified by 16S sequencing), was determined under anaerobic conditions . Overnight cultures grown in brain heart infusion (BHI) broth, supplemented with 0.5% yeast extract, 0.1% l-cysteine hydrochloride, and 15 μg/mL hemin (BHI-Ych), were diluted 1:100 in BHI-Ych. The 96-well assay plates were prepared by 2-fold dilution of ADG and included a positive growth control. After 24 h of incubation, the MIC was determined. All MIC assays were performed at least in triplicate. Cytotoxicity assay with FaDu and HepG2 cell lines was conducted as previously mentioned  and subsequently inoculated onto 25-mL MHIIA plates containing 4× MIC ADG at a density of 1.8 × 106 CFU per plate. For E. coligsk++ strain, exponential cultures with a density of 1.5 × 109 CFU per plate were washed in PBS and subsequently inoculated onto 25-mL MHIIA plates containing 16× MIC ADG. After 48 h of incubation the plates were examined for colonies, and the number of colonies was counted. The colonies were restreaked to test for resistance stability. Genome sequencing and variant calling were conducted by the Microbial Genome Sequencing Center . Whole-genome sequencing was performed by paired-end reads (2 × 150 bp) with Illumina NextSeq 550, and E. coli genome information data in NCBI (GenBank accession no. U00096.2) was used for variant calling. GSK-F primer (TTCGCCGCTCAGTTAACCAC) and GSK-R primer (AGGCATCGAGAGCCAAATGC) were used to amplify gsk in resistant mutants and verify the mutations from Illumina whole-genome sequencing. Primers up-gmk-f (CAGTGAATGACAGGCAAATGC) and down-gmk-r (CCTACCTGCATCTGACGAGC) were used to amplify gmk and verify the mutations from Illumina whole-genome sequencing in resistant mutants from gsk++ strain.Three independent colonies were used for each resistance study. For E. coli MG1655 was cultured in MHIIB overnight to stationary phase and the following day was inoculated into fresh MHIIB at 1:100 and grown for 2 h at 37°C. Cells were diluted 10-fold in MHIIB, placed on top of a 1.5% low-melting-point agarose MHIIB pad containing ADG (128 μg/mL) and dyes Sytox Green (0.5 μM) and FM4-64 (10 μg/mL) from Molecular Probes, and observed with a Nikon Ti2-E fluorescence microscope using a 100× oil-immersion lens objective. The fluorescence signals for Sytox Green and FM4-64 were collected sequentially by excitation at 480 nm and 550 nm, respectively, alongside a phase-contrast image. A thermostatic chamber was used to maintain a temperature of 37°C for the duration of the experiment. Images were acquired with NIS-Elements every 15 min at a resolution of 2,048 by 2,048. For FtsZ localization, E. coli MG1655 FtsZ-GFP  was grown as outlined above. After the 2-h outgrowth, 1-mL aliquots were taken and treated with ADG (128 μg/mL), ciprofloxacin (1 μg/mL), cephalexin (8 μg/mL), or no drug for 1 h at 37°C. Cells were spotted onto a 1.5% low-melting-point agarose pad and observed with a Nikon Ti2-E fluorescence microscope using a 100× oil-immersion lens objective. Fluorescence signal from FtsZ-GFP was collected by excitation at 480 nm alongside a phase-contrast image. All images were processed with Fiji software  and gsk-kan-R (5′-GAAGCAGCTCCAGCCTACAACGGTGGTTGCCGGATGTAAAATATG-3′) with the genomic DNA of E. coli MG1655 as the template, and the kanamycin resistance marker was amplified using the primers gsk-kan-F (5′-CATATTTTACATCCGGCAACCACCGTTGTAGGCTGGAGCTGCTTC-3′) and fliT-kan-R (5′-CAGCGTGGGATTACGCATTCTTCGACTCCATTCAAGGGGAACATTAGAAGAATTAGCCATGGTCCATATG-3′) with an in-house-modified plasmid as the template. Those two DNA products were then fused by PCR using the primer pair fliT-gsk-F/fliT-kan-R. Approximately 500 ng of this column-purified DNA product was used to transform electrocompetent cells of E. coli MG1655-pKD46 to perform λ Red recombination. The subsequent steps have been adapted from the Quick and Easy E. coli gene deletion kit . Briefly, 30 μL of an overnight culture of E. coli MG1655-pKD46 was used to inoculate a microtube containing 1.4 mL of LB complemented with ampicillin (100 μg/mL). After 2 h of shaking at 30°C, 0.4% of l-arabinose was added, and the tube was transferred for shaking at 37°C for 1 h. Cells were washed and concentrated with ice-cold 10% glycerol prior to electroporation. The recovery step was performed for 3 h at 37°C with shaking. The selection was performed using resistance to kanamycin (50 μg/mL). Several transformant clones were then restreaked with double selection for resistance to kanamycin (50 μg/mL) and sensitivity to ampicillin (100 μg/mL at 30°C). The neutral fliT site was amplified using the primers fliT-up (5′-CAACGGAAGAACAATGGGAC-3′) and fliT-down (5′-TCATAGGAGTCGTAGCCGAC-3′) and sequenced, and the presence of gsk within this locus was confirmed.To insert a second copy of liT site  through liT site . More prE. coligsk open reading frame  was subcloned into pET Sumo vector (Invitrogen) immediately downstream to the 5′ end of the His6-Sumo ORF (E. coligmk and ndk ORFs were subcloned into pET28b vector between NdeI and XhoI restriction sites (The Sumo ORF . The E. on sites .E. coli BL21(DE3) harboring the corresponding expression plasmid. Expression strains were grown in LB containing 30 μg/mL kanamycin at 37°C to an OD600 of 0.6. The culture was then cooled to 18°C and induced by the addition of 200 μM IPTG. Cells were harvested 16 h postinduction. The cell pellet from 1 liter of expression culture was resuspended in 25 mL lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM KCl, 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 20 μg/mL lysozyme, 10 U Benzonase, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were disrupted through sonication and the lysate was cleared at 12,000 × g for 1 h. Cleared lysate was applied to 1 mL Ni-NTA resin equilibrated with the lysis buffer and allowed to flow through by gravity. The Ni-NTA resin was washed with 5× column volumes of wash buffer 1  and 10× column volumes of wash buffer 2 . Bound protein was eluted with 3× column volumes of elution buffer . Eluates of the Ni-NTA column were dialyzed against 20 mM Tris-HCl, pH 8.0, and 150 mM KCl. His6-Sumo-Gsk then was treated with the Sumo protease, Ulp1 (10 μg per μmol cleavage sites), in the presence of 1 mM TCEP, and His6-Gmk and His6-Ndk with thrombin (10 U per μmol cleavage sites), both at room temperature overnight. Cleavage mixtures were concentrated and Gsk, Gmk, or Ndk was further purified over a Superdex-200 10/300 column run in 20 mM HEPES-Na, pH 7.0, 150 mM KCl (for Gsk) or NaCl (for Ndk and Gmk), and 1 mM TCEP.All three recombinant proteins, namely, His6-Sumo-Gsk, His6-Gmk, and His6-ndk, were expressed in 3 M−1 cm−1 and 15.4 × 103 M−1 cm−1, respectively. For real-time monitoring of the reaction progress, ADP produced from Gsk activity was coupled to the consumption of NADH by the activities of pyruvate kinase (PK) and lactate dehydrogenase (LDH). The overall reaction wasAll nucleotides were quantified using absorbance spectrometry. The wavelength was 251 nm for guanosine and ADG nucleotides and 260 nm for adenosine nucleotides; extinction coefficients were 13.6 × 10A340) every 30 s. ADG and guanosine were each dissolved in water at 100 mM in the presence of equimolar NaOH. ATP was dissolved in water at 100 mM and pH adjusted to 7.4.All reactions were performed at 100-μL scale in 96-well plates at 30°C in a SpectraMax M5 plate reader (Molecular Devices). Absorbance of each reaction was monitored for the absorbance at 340 nm . The reaction was assembled by first mixing all components except ADG or guanosine. This master mix and nucleoside substrates were predeposited in separate wells on the same plate. After preincubating the plate at 30°C for 5 min, the mixture was then transferred to the substrate using a multichannel pipette at t = 0 to initiate the reaction. The initial velocity (in A340/min) was calculated by linear regression of the initial linear segment of each kinetic curve.A 10× pyruvate kinase-lactose dehydrogenase (PK-LDH) mixture was prepared in water that contained phosphoenolpyruvate , NADH , PK , and LDH  . Final r2, and 1 mM TCEP). ADG (1 mM), Gmk (1 μM), Ndk (1 μM), PEP (4 mM), and PK (10 U/mL) were also included as indicated. The reaction mixture was incubated at 37°C for 30 min and then quenched by addition of 100 μL chloroform and vortexing. Following centrifugation at 8,000 × g for 5 min, 2 μL aqueous phase was diluted in 200 μL 5 mM Tris-HCl, pH 8.0, and applied to MonoQ 5/50 running at 1 mL/min with buffer A . Nucleotides were resolved using a linear gradient in which percentage of buffer B  increased from 0% to 40% in 20 mL, starting at V = 2.5 mL.All reactions were performed at 100-μL scale in Eppendorf tubes. Final reaction mixtures contained 1 μM Gsk and 2 mM ATP in reaction buffer  ADG-TP, was lyophilized and redissolved in water for further use. MALDI-TOF [M+H]+m/z calculated for C10H18N6O13P3, 523.011; found, 523.141.In a 15-mL Falcon tube, ADG  was solubilized in 0.5 mL 100 mM NaOH and then diluted in 4 mL reaction buffer. To the solution was added ATP  and PEP . Gsk, Gmk, and Ndk then were added to a final concentration of 3 μM and PK to 10 U/mL. The reaction was expanded to 5 mL using reaction buffer, mixed thoroughly, and incubated at 37°C for 2 h. Analysis using MonoQ 5/50 showed that ADG was completely converted to ADG-TP. ADP was then carefully titrated in to remove excess PEP, which would otherwise be difficult to separate from ADG-TP. Nevertheless, excess ADP would cause Ndk-dependent dephosphorylation of ADG-TP and therefore must be avoided. The reaction was then quenched by addition of 2 mL chloroform and vortexing. Following centrifugation at 8,000 × ntration  at 37°C in vitro transcription assay, and the assay was carried out according to the kit instruction manual. The frozen reagents were thawed, and the transcription reaction mixture consisting of 2 μL of each nucleotide , 2 μL of the reaction buffer, 2 μL of the enzyme mix, 5 μL nuclease-free water, and 1 μL DNA template (the DNA template that was included in the kit was diluted 5-fold and used for this study) were assembled at room temperature. A volume of 18 μL of reaction mixture was moved into 1.8-mL Eppendorf tubes containing 2 μL of ADG-TP solutions with 2-fold serial dilution and one tube containing 2 μL of RNase-free water as a negative control. After mixing thoroughly, the reaction solutions were incubated at 37°C for 4 h. Following incubation, the reaction solutions were mixed with gel loading buffer II at a 1-to-1 volume ratio and loaded on TAE agarose gel containing 0.5 μg/mL ethidium bromide for electrophoresis.MEGAscript T7 transcription kit was purchased to perform the AAGCCTCGCTCCAGTAATACGACTCACTATT-3′ and 5′-AAGACCTCGCTCCAGTAATACGACTCACTATT-3′, respectively. Note that CTP was not added and therefore downstream sequences are not transcribed. DNA oligonucleotides were first annealed in 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 0.1 mM EDTA using a temperature program for 2 min each incubation at 80°C, 75°C, 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, and 40°C (in this order). Transcription was carried out using 6 mM ATP, 2 mM GTP (or ADG-TP), 1 μM double-stranded template, and 1 μM T7-RNA polymerase in the presence of 100 mM Tris-HCl, pH 8.0, 20 mM MgCl2, 2 mM spermidine trihydrochloride, and 20 mM dithiothreitol (DTT). Following incubation at 37°C for 40 min, a 5-μL aliquot was withdrawn from the cloudy suspension, diluted in 0.5 mL cold water, and applied to a MonoQ 5/50 column. Bound nucleotide was eluted using a gradient with the percentage of buffer B increasing from 15% to 36% in 14 mL. To prepare AAG, AAGA, and AA(ADG) for mass spectrometry, 100 μL of the appropriate reaction mix was diluted in 0.9 mL cold water and filtered using an Amicon Ultra 0.5 concentrator with a 10-kDa molecular mass cutoff. The filtrate was applied to a MonoQ 5/50 column and fractionated using a gradient with the percentage of buffer B increasing from 10% to 30% in 20 mL. Volumes of 200 μL from peak fractions containing pppAAG, pppAAGA, and pppAA(ADG) were each treated with 1 μL Quick CIP (NEB) at 37°C for 30 h, and the oligoribonucleotides with 5′-OH were acidified using 10 mM HCl and applied to a Discovery Wide-Pore C18 (Supelco) column  using solvents A (0.1% trifluoroacetic acid [TFA] in water) and B (0.1% TFA in 90:10 acetonitrile-water). All nucleotides were eluted using a gradient with the percentage of solvent B increasing from 0% to 30% in 10 mL. Fractions containing each nucleotide were lyophilized and dissolved in 0.1% formic acid in water for m/z analysis using a Sciex X500B QTOF mass spectrometer. The mass spectrometer was controlled by Sciex OS v.1.6.1 using the following settings: ion source gas 1, 30 lb/in2; ion source gas 2, 30 lb/in2; curtain gas, 35; CAD gas, 7; temperature, 300°C; spray voltage, −4,500 V; declustering potential, −80 V; collision energy, −10 V. Data were acquired from 400 to 2,000 Da with a 0.5-s accumulation time and 4 time bins summed.Templates for the synthesis of AAG and AAAG were fully complementary, double-stranded DNA. Coding strand sequences are '5-E. coli MG1655 from three independent colonies were diluted 1:10,000 into 100 mL MHIIB medium in 250-mL Erlenmeyer flasks and incubated at 37°C with shaking at 220 rpm. After 4 h, 10 mL from each flask was collected (and kept on ice), OD was measured, and ADG was added to the cultures at 1× MIC. After 1 h of incubation, 10 mL from each flask again was collected (and kept on ice) and OD was measured. To normalize the amount of metabolome collected before and after treatment with ADG, OD × volume = 2.5 was used. Cells from each sample were collected using a PVDF membrane and a vacuum filter. The membranes then were moved in a 20-mL glass vial containing 4 mL methanol and vortexed for 1 min to lyse the cells. The membranes then were removed, and 8 mL of chloroform was added, followed by vortexing for 1 min and incubating in an ultrasound bath for 5 min; 4 mL of water containing 0.05 μM internal standards  were added, followed by vortexing for 1 min and incubating in an ultrasound bath for 5 min. The 20-mL vials were placed in 50-mL centrifuge tubes and centrifuged for 10 min at about 3,000 rpm to achieve phase separation. Next, the top layer  was collected and dried using a SpeedVac system. Samples were resuspended in 100 μL of MilliQ water and submitted to the Harvard Center for Mass Spectrometry for measurements. All samples were run on a ThermoFisher IDX using a HILICON column . LC method was solvent A, 20 mM ammonium carbonate, 0.1% ammonium hydroxide, in water; solvent B, acetonitrile 97%, in water. The LC was equilibrated with 100% solvent B, and the initial concentration of 100% solvent B was decreased by a linear gradient to 40% over 19 min followed by 100% solvent A for 5 min with a flow rate of 0.15 mL/min. Peaks of ADG ([M+H]+ = 283.1149 and [M-H]− = 281.1004) and candidate peaks of ADG-MP ([M+H]+ = 363.0813 and [M-H]− = 361.0667), ADG-DP ([M+H]+ = 443.0476 and [M-H]− = 441.033), and ADG-TP ([M+H]+ = 523.0139 and [M-H]− = 520.9994) were integrated for all samples. The ratios of the targeted peak areas divided by the standard peak (IS) were calculated.Overnight cultures of E. coli W0153 strain and ADG at 2× MIC were used in this study; 48 h prior to the assay, E. coli W0153 strain was inoculated onto Trypticase soy agar (TSA) with 5% sheep blood (SB) and incubated at 37°C. One day prior to assay initiation, 3 to 4 colonies from the agar plate were restreaked onto another TSA plus 5% SB and incubated overnight at 37°C. On the day of assay, 5 to 7 colonies from the overnight plate were inoculated into TSB and incubated at 37°C with shaking to an OD600 of 0.3 to 0.4. The cultures then were centrifuged, resuspended in fresh TSB or M5T broth (protein synthesis), and adjusted to an OD600 equivalent to 0.3. The impact of ADG was evaluated at 2× MIC. ADG and controls  were diluted to 2 times the final in-well concentration and distributed in triplicate in a volume of 50 μL to wells of a 96-well round-bottomed microtiter plate; 50 μL of bacteria at an OD600 of 0.3 containing the appropriate amount of radiolabeled precursor  were added to the wells containing the compounds and incubated for 30 min at 37°C with shaking. Following the incubation, 100 μL of cold 10% trichloroacetic acid (TCA) was added to the wells of the 96-well plate containing the mixture. The plate was incubated for an additional hour on ice to allow for the radiolabeled precursor-incorporated material to precipitate. Following the incubation, all contents in the 96-well microtiter plate were transferred to a filter plate, and the nonprecipitated radioactive precursor was vacuum filtrated. Each well of the filter plate was washed three times with 200 μL of cold 5% TCA followed by three times with 200 μL of water using vacuum filtration. The filters were transferred to a Microbeta cassette, and 25 μL of scintillation fluid was added to each well. The plate was sealed, and each well was counted on a Perkin Elmer Microbeta scintillation counter.Macromolecular incorporation assay was performed as a service by ImQuest Biosciences. Escherichia coli strain W0153  was acquired from the Yale culture collection  and previously calibrated using explainable artificial intelligence as a bioreporter for known MOAs  on the EpMotion Robotic liquid handler. For the resulting total RNA, RNA integrity number (RIN) values were obtained to check for RNA quality using the 2200 TapeStation . Acceptable values to proceed to ribosomal subtraction were above a RIN of 5. rRNA was subtracted from the total RNA to yield only mRNA for library construction using a bacterial rRNA depletion kit  at half reactions with a total RNA input maximum of 400 ng. The rRNA depleted product was quality controlled using an Agilent Bioanalyzer with the Agilent Pico chip for RNA detection to check for less than 0.5% of rRNA remaining; 2.5 μL of the rRNA-depleted samples, amounting to approximately 2 to 5 ng, is used as the input material to construct each cDNA library for RNA sequencing using the NEBNext ultradirectional RNA library prep kit  at half reactions. The resulting libraries were analyzed using Agilent high-sensitivity DNA chips to ensure library quality. Libraries were quantified and normalized by qPCR and then we sequenced, using the NovaSeq platform, approximately 9 million 75-bp paired-end reads for each library. To identify the primary MOA of ADG, transcriptomes from E. coli W0153 challenged with ADG were evaluated by the CoHEC predictive model (v7.0) and training data from Espinoza-Dupont et al. (https://github.com/jolespin/soothsayer). The predictive model was trained on 41 known antibiotic compounds representing 6 primary MOA containing a mixture of pure compounds and producer-strain extracts. This model was previously used (own MOAs . For thet et al.  using thsly used  to flag


Ibuprofen increased m6A-p53 expression (p < 0.01) by promoting the expression of METTL3 (p < 0.01) and METTL14 (p < 0.05); and increased levels of YTHDF1 (p < 0.001), YTHDF3 (p < 0.001), and YTHDC2 (p < 0.01) that finally reinforced p53 translation (p < 0.01). Therefore, our results present that ibuprofen epigenetically increased p75NTR expression by downregulating promoter methylation and upregulating m6A-RNA-methylation in SGC7901 and MKN45 cells. Our study unveils a novel mechanism for p75NTR regulation by NSAIDs and helps the design of treatment targets.It is acknowledged that nonsteroidal anti-inflammatory drugs (NSAIDs) can participate in various signaling pathways, while information about their epigenetic effects are limited. p75NTR (p75 neurotrophin receptor) can inhibit tumor growth by inducing cell cycle arrest and regulating cell cycle arrest and apoptotic cell death. The expression of p75NTR is influenced by epigenetic roles. We explored the effects of ibuprofen on p75NTR expression and investigated whether promoter methylation and N6-methyladenosine (m6A) RNA methylation regulates this process in human gastric cancer cells (SGC7901 and MKN45). Cell lines were treated with ibuprofen 0, 2.5, 5, 10, 20 µM, and then DNA, RNA, and protein were isolated 24 h later. Expression and promoter methylation of p75NTR were detected by RT-qPCR and Western blot. The levels of m6A-p75NTR were measured by RNA immunoprecipitation. We also used RT-qPCR to determine the levels of m6A-related regulators, METTL3, METTL14, ALKBH5, FTO, YTHDC2, and YTHDF1-3. Ibuprofen attenuated p75NTR promoter methylation (  Ibuprofen epigenetically increased p75NTR expression at the transcriptional and protein levels by increasing m6A-p75NTR methylation in gastric cancer cells.The increased level of m6A-p75NTR was regulated by the increased expression of METTL3 and METTL14, and may occur independently from ALKBH5 and FTO.These results may serve as an alternative pathway for inhibition of gastric cancer induced by ibuprofen.Nonsteroidal anti-inflammatory drugs (NSAIDs) block the cyclooxygenase (Cox) enzyme, thus decreasing the level of inflammatory mediators’ prostaglandins. NSAIDs have also been demonstrated to have anticancer effects, which can inhibit cell growth and metastasis, and lead to apoptosis . Severalp75 neurotrophin receptor (p75NTR), one of the nerve growth factors with low affinity, also has been known as the tumor necrosis factor (TNF) family of receptors . Neural Promoter methylation, within the promoter region of specific genes, plays an important role in gene transcription regulation. mRNA regulation of post transcription is associated with RNA–RNA and RNA–protein interactions . N6-methPreviously, we validated that NSAIDs (such as R-Flurbiprofen) can reverse drug resistance, proliferation and metastasis of gastric cancer cells via activating p75NTR . However6) were inoculated in to 90% confluency in 25 cm3 cell culture flasks (Corning Inc.). Ibuprofen was treated at different concentrations  for 24 h . The membrane was rinsed five times in TTBS  and conjugated with horseradish peroxidase secondary antibody . Rinse the membrane in TTBS five times (10 min at room temperature). Western blots were visualized using the Transparent West ECL Substrate Kit  and images were captured using the Chemidoc XRS+ Molecular Imaging System (Bio-Rad). After detection, the membrane was quenched in hydrogen peroxide , and was treated with housekeep protein and anti-β-actin  to normalize protein expression. Protein expression was measured using Image Lab software version 5.1 (Bio-Rad), and the results were expressed as multiples of the band density (RBD) relative to the control . Centrifugation was performed at 14,000 According to manufacturer’s instructions, genomic DNA was isolated from control and ibuprofen treated gastric cancer cells by Quick-g-DNA MiniPrep kit and DNA Clean and Concentrator™-5 Kit. Using Nanodrop2000 spectrophotometer to determine DNA concentration, and standardization to 4 ng/µl. A260/A280 absorbance ratio of DNA was used as purity assessment. p75NTR promoter methylation was measured by OneStep qMethyl Kit . Cycling conditions were as follows: digestion by methyl-sensitive restriction enzymes , initial denaturation , followed by 45 cycles of denaturation , annealing (60 s), extension , final extension , and a hold at 4°C. The percentage methylation was calculated by formula: Methylation (%) = 100 × 2-ΔCt, where ΔCt = Ct (test) – Ct (reference) .g, 4, 15 min). Nuclear pellet were re-suspended in RNA precipitation buffer as previously described  overnight at 4°C and the antigen–antibody complex was precipitated using protein A beads . Then, the immuno-precipitates were recovered by centrifugation , washed three times in RNA immunoprecipitation buffer, followed by re-suspension in Qiazol Reagent (500 µl). RNA was isolated from both the reference and m6A-precipitated samples, as previously described  and was centrifuged . Differences between the mean values were determined by analysis of variance and a post hoc test. All statistical analyses were performed using SPSS19.0 software  and GraphPad Prism version 5.0 (GraphPad Prism Software Inc.). nificant .The expression of p75NTR is influenced by epigenetic roles. We explored the effects of ibuprofen on p75NTR expression and investigated whether promoter methylation and N6-methyladenosine (m6A) RNA methylation regulate this process in human gastric cancer cells (SGC7901 and MKN45). The aim of this study was to determine the effects of ibuprofen on p75NTR expression and its epigenetic regulation based on transcription and post-transcription through promoter methylation and m6A RNA methylation. The results of this study provide insights into possible mechanisms of ibuprofen-mediated p75NTR regulation at the epigenetic level.p < 0.05), 10 (p < 0.01), 20 (p < 0.001) µM compared with the control. In MKN45 cells, the p75NTR mRNA level was significantly increased by 10 (p < 0.01), 20 (p < 0.001) µM ibuprofen, while the p75NTR protein level was significantly increased by 5 (p < 0.05), 10 (p < 0.01), 20 (p < 0.001) µM ibuprofen, compared with the control.p75NTR, as a tumor suppressor, affects the levels of apoptosis and can halt the cell cycle process of gastric cancer cells . Previoup < 0.001), 5 (p < 0.001), 10 (p < 0.001), 20 (p < 0.001) µM treatment in SGC7901 and MKN45 .Figurep < 0.001), 10 (p < 0.001), 20 (p < 0.001) µM treatment .p < 0.001), METTL14 (p < 0.001), ALKBH5 (p < 0.001), FTO (p < 0.001). Ibuprofen significantly increased the expression of METTL3, METTL14, ALKBH5, FTO in both SGC7901 and MKN45 at 5, 10, 20 µM treatment. This might indicate that ibuprofen could increase m6A-p75NTR levels via regulating the levels of the m6A methyltransferases in gastric cancer cells.As the result of ibuprofen-induced increase in m6A-p75NTR, we assessed the effect of ibuprofen on the mRNA expression of m6A methyltransferases, including METTL3 , p < 0.0 METTL14 , p < 0.0, ALKBH5 , p < 0.001), FTO , p < 0.0p < 0.001), YTHDF1 (p < 0.001), YTHDF2 (p < 0.001), YTHDF1 (p < 0.001) in both SGC7901 and MKN45 at 5, 10, 20 µM treatment compared to the control.m6A can recruit particular readers recognizing m6A altered mRNAs so that it regulates the level of target molecules . Previou, YTHDF1 , p < 0.0, YTHDF2 , p < 0.0, YTHDF1 , p < 0.0Previously, NSAIDs (including ibuprofen) were reported to inhibit invasion and cell proliferation by the induction of p75NTR . HoweverFirstly, our work suggested that ibuprofen significantly increased p75NTR at mRNA and protein level by inducing p75NTR promoter hypo-methylation and increasing m6A-p75NTR expression in both SGC7901 and MKN45 cells at 5, 10, 20 µM. This is in accordance with similar epigenetic studies that the hypo-methylation of tumor suppressor was correlated with the increase in gene expressions ,27. The TP53I11, BRCA2, and CDKN2A [m6A, as the most abundant epi-transcriptomic marker, can recruit particular readers recognizing m6A altered mRNAs so that it regulates the level of target molecules ,28,29. Pd CDKN2A . The knod CDKN2A . It is nd CDKN2A ,37. HoweYTHDC2, YTHDF1-3 specifically identify m6A-altered mRNAs and modify mRNA translation  and degrIn conclusion, this study demonstrated a potential mechanism by which ibuprofen-mediated alterations in p75NTR expression at the epigenetic angle. The results showed that ibuprofen epigenetically increased p75NTR expression at the transcriptional and protein levels by decreasing p75NTR promoter methylation and increasing m6A-p75NTR methylation in gastric cancer cells. This study provides an insight for further studies of ibuprofen and p75NTR in vivo models and for determining the effect of ibuprofen to induce p75NTR mutated proteins by changing m6A levels.Click here for additional data file.


The compounds were characterized using physicochemical and spectroscopic methods. The results demonstrate that the free ligand behaves as a tridentate ligand with one oxygen and two nitrogen (ONN) donor atoms in 1:1 metal:ligand ratio. The formation constants of the complexes were found to be (KZn(II) = 2.3 × 106, KCu(II) = 2.9 × 106, and KNi(II) = 3.8 × 105). The thermodynamic parameters indicated that the reactions were spontaneous with exothermic nature of metal-ligand interaction energies. Based on the analyses of the experimental  and DFT results, a distorted tetrahedral, a distorted square pyramidal and square planar geometry for Zn(II), Cu(II) and Ni(II) complexes, respectively, were proposed. The B3LYP calculated IR frequencies and TD-B3LYP calculated absorption spectra were found to be in good agreement with the corresponding experimental results. The powder XRD data confirmed that the Zn(II), Cu(II) and Ni(II) complexes have polycrystalline nature with average crystallite sizes of 27.86, 33.54, 37.40 Å, respectively. In vitro antibacterial activity analyses of the complexes were studied with disk diffusion method, in which the complexes showed better activity than the precursor ligand. Particularly the Cu(II) complex showed higher percent activity index , than both Zn(II)  and Ni(II)  complexes against both E. coli and P. aeruginosa, respectively. Using the DPPH assay, the complexes were further assessed for their antioxidant capacities. All metal complexes showed improved antioxidant activity than the free ligand. Zn(II) and Cu(II) complexes, which had IC50 values of 10.46 and 8.62 μg/ml, respectively, showed the best antioxidant activity. The calculated results of Lipinski’s rule of five also showed that the target complexes have drug-like molecular nature and similarly, the results of binding mode of action of these compounds against E. coli DNA gyrase B and P. aeruginosa LasR.DNA were found to be in good agreement with the in vitro biological activities.Interest is increasingly focused on the use of transition metal complexes as biochemical, medical, analytical, pharmaceutical, agronomic, anticancer, and antibacterial agents. In this study, three complexes of [Zn(H The application of transition metal complexes as biochemical, medicinal, analytical, pharmaceutical, agricultural, antitumor and antimicrobial agents has become center of interest for researchers . Recentl3L= ((E)-2-(((2-((2-hydroxyethyl)amino)quinolin-3-yl)methylene)amino)ethanol, have not been reported. Hence, we hereby report the synthesis of these metal complexes from the corresponding metal salts  properties using disc diffusion and DPPH assay methods, respectively. Furthermore, computational studies had been performed to better understand the properties and activities of the complexes and to correlate with the experimental results.Previous studies show that zinc complexes exhibit antidiabetic , antioxiAcetic anhydride 99.8%, acetic acid glacial 99.5%, aniline 99%, N, N- dimethyl formamide 99%, phosphorus oxychloride 98%, methanol 99.5%, n-Hexane 99%, dichloromethane 98%, Ethyl acetate 99.5%, chloroform 99%, Triethylamine 99%, copper nitrate trihydrate 98%, nickel nitrate hexahydrate 98%, zinc chloride 98%, silver nitrate 99.9%, L-Ascorbic acid 99%, Dimethyl sulphoxide 99% and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were used. All the chemicals and reagents, analytical grade, were purchased from Loba chemie PVT. LtD .3. Chemical shifts (δ) are reported in ppm and the coupling constants (J) are reported in Hz. Fluorescence and UV-visible spectral data were measured using Agilent MY-18490002/PC spectrofluorophotometer and SM-1600 Spectrophotometer, respectively. The absorption spectra of the synthesized compounds were measured using 1.0 × 10–5 M methanolic diluted solution. Elemental composition and morphology were carried out using scanning electron microscopy with energy dispersive X-ray . Mass spectra were recorded with SHIMADZU, LC-MS 8030 . X-ray diffractometer (SHIMADZU model: XRD-7000 X-RAY DIFFRACTOMETER) was used to measure the X-ray diffraction. The patterns of the synthesized metal complexes were performed using powder sample with measurement conditions of X-ray tube target: Cu (λ = 1.5406 Å), voltage: 40.0 kV, current: 30.0 mA, divergence slit: 1.0o, scatter slit: 1.0o, receiving slit: 0.3 mm, scanning drive axis: 2θ, scan range: 5.0–80.0o, scan mode: continuous scan, Scan speed: 3.0 o/min, sampling pitch: 0.02o.The NMR spectra of the ligand were obtained using NMR Bruker Avance 400 spectrometer operating at 400 MHz using DMSO-d6 and CDCl−1 and KBr pellets). Thermogravimetric analyses (TGA) data were recorded using DTG-60H SHIMADZU thermal analyzer under nitrogen-atmosphere (20 ml/min) and heating rate of 10°C/min between 25–800°C. Molar conductance of the complexes were recorded at room temperature in 1 × 10–3 M methanolic solution of the samples using conductometre (AD8000: resolution 0.1 mV (±699.9 mV)/1 mV (±2000 mV), 0.01, 0.1, 1 μS/cm; ppm, 0.01, 0.1 mS/cm; ppt 0.1°C, accuracy at 25°C ± 0.2 mV up to ±699.9 mV, ± 1 mV up to ±2000 mV), ±0.5°C). Melting point analysis was performed using digital auto melting point apparatus .The Chekcell Graphical Powder Indexing (CCP14) program was used to calculate the miller indices, whereas the lattice parameters were determined using the CRYSFIR computer program. FTIR measurements were performed using Perkin-Elmer BX spectrometer  of 383 nm, IR : 3368 ν(O-H), 3275 ν(N-H), 1639 ν(imine C=N). Composition: Calc. for C14H17N3O2; C 64.85; H 6.61; N 16.20; O 12.34%. Found C 64.71; H 6.65; N 16.08 and O 12.56%. 1H NMR : δH 3.65 , 4.72 , 4.92 , 7.19 , 7.55 , 7.72 , 8.21 , 8.5, and 9.55 ; 13C NMR : δC 43.4 (C-14), 60.5 (C-12), 61.2 (C-15), 63.7 (C-11), 117.2 (C-3), 121.9 (C-8), 122.4 (C-4a), 125.7 (C-5), 128.9 (C-6), 131.5 (C-7), 143.0 (C-4), 148.3 (C-8a), 155.4 (C-2), and 163.8 (C-9); DEPT-135 δC 43.4 (C-14 negative), 60.5 (C-12 negative), 61.2 (C-15 negative), 63.7 negative (C-11), 121.9 (C-8), 125.7 (C-5), 128.9 (C-6), 131.5 (C-7), 143.0 (C-4) and 163.8 (C-9). Composition: Calc. for C14H17N3O2; C 64.85; H 6.61; N 16.20; O 12.34%. Found C 64.71; H 6.65; N 16.08 and O 12.56% (The ligand was prepared based on our previously reported procedure  with minO 12.56% .2 (0.13 g), Cu(NO3)2·3H2O (0.232 g), and Ni(NO3)2.6H2O (0.279 g) in methanol  was added dropwise to this solution separately in different flasks  in methanol (10 ml). After 30 min of stirring, a solution (0.96 mmol) of ZnClt flasks . The mixt flasks . Finallyt flasks . The pro1 was found to be light yellow polycrystalline powder with molecular formula [Zn(H2L)Cl], yield 63%, melting point 225–230°C. It is soluble in polar solvents. Compositions calculated for 1 are C 46.82, H 4.49, N 11.70, O 8.91, Cl 9.87 and Zn 18.21%; found: C 46.85, H 4.36, N 11.80, O 8.55, Cl 10.25 and Zn 18.19%. FTIR ): 1657 ν(Imin C=N), 1034 ν(C-O), 524 ν(Zn-O), and 460 ν(Zn-N). UV-Vis : 231 (π→π*), 258 (π→π*), 300 (n→π*) and 380 (n→π*).Complex 2 was found to be deep green polycrystalline powder with molecular formula [Cu(H2L)(H2O)(NO3)], yield 61.5%, melting point 195–200°C. It is soluble in polar solvents. Compositions calculated for 2 are C 41.84, H 4.51, N 13.94, O 23.89 and Cu 15.81%; found: C 41.62, H 4.90, N 13.80, O 24.15, and Cu 15.53%. FTIR ): 1652 ν(Imin C=N), 1059 ν(C-O), 626 ν(Cu-O), and 474 ν(Cu-N). UV-Vis : 235 (π→π*), 267 (n→π*), 317 (n→π*) and 406 (LMCT).Complex 3 was found to be brownish purple polycrystalline powder with molecular formula [Ni(H2L)(NO3)].2H2O, yield 66%, melting point 115–120°C. It is soluble in polar solvents. Compositions calculated for 3 are C 40.52, H 4.86, N 13.50, O 26.99 and Ni 14.14%; found: C 40.86, H 4.95, N 13.35, O 26.90 and Ni 13.94%. FTIR ): 1650 ν(Imin C=N), 1037 ν(C-O), 534 ν(Ni-O), and 462 ν(Ni-N). UV-Vis : 229 (π→-π*), 259 (π→π*), 302 (n→π*) and 401 (LMCT).Complex max 380, 406, and 401 nm, respectively, for complexes 1, 2 and 3, at temperatures of 25, 30, 37 and 40°C. A drop of triethylamine was used to keep the pH of the mixture constant. The metal ion and free ligand mole fractions were changed between 0 and 1 for stoichiometric measurement. From this, the absorbance of the solutions was plotted with these mole fractions, and “n”, the average number of bound ligand, was obtained from the plot, where Xmax was calculated using Zn(II) chloride, Cu(II) nitrate trihydrate, and Ni(II) nitrate hexahydrate standard solutions were pipetted into ten 50 ml volumetric flasks , and aliquots of a similar standard solution of the precursor ligand were added . All absorbance were recorded at λH and ΔS) were derived from the slope and intercept of the lnK vs. 1/T (van’t Hoff) plot, respectively, in order to evaluate the thermodynamic parameters  appropriately (G).Additionally, spectroscopic analysis was used to estimate the complexes’ formation constants . The chapriately . Eqs 2, 1–3) were evaluated using disc diffusion method against two Gram-positive  and two Gram-negative  bacteria by following previously reported media preparation methods (μg/ml) in DMSO. Ciprofloxacin and DMSO were used as a positive and negative control, respectively. The plates were incubated at 37°C for 48 h, and then the bacterial growth data were evaluated by measuring the inhibition zones according to literature , Cu(II) and Ni(II) complexes  was determined from the slope and intercept of the plot of percent radical scavenging activity vs. concentration.The radical scavenging activity study of the ligand and its Zn(II), Cu(II) and Ni(II) complexes were determined using a DPPH assay based on the reported studies . Accordiin silico pharmacokinetic properties  prediction was performed with SwissADME webtool to understand the safety and efficacy of the metal complexes as drug candidates. The Swiss Institute Bioinformatics (SIB) webtool (SwissADME) was used to convert the two dimensional structure into its simplified molecular input line entry system (SMILES) and then to estimate the operties . In lineH3L) and its metal complexes (1–3) were performed using the Gaussian 16 program package (Eg = ELUMO–EHOMO), electronegativity (χ = -½ (EHOMO + ELUMO)), electronic chemical potential (μ = ½ (EHOMO + ELUMO) = −χ), global chemical hardness (η = ½ (ELUMO–EHOMO)), global softness (σ = 1/2η), global electrophilicity index (ω = μ2/2η), nucleophilicity index (Nu = 1/ω), and dipole moment were calculated and analyzed at the same level of theory (Geometry optimizations of the ligand ( package  and the  package  together package  for the  package  was empl package . The pol package  was usedf theory .1–3) were carried out (E. coli DNA gyrase B (PDB ID: 6F86) and P. aeruginosa LasR. (PDB ID: 2UV0). With a grid point spacing of 0.375, the grid box was built using 58, 58, and 40 points that pointed in the x, y, and z directions, respectively. The grid box’s center was 14.527, 56.689, and 5.122. The Scripps Institute website (Using AutoDock 4.2.6 (MGL tools 1.5.7) and a standard methodology, the molecular docking experiments of the free ligand and its metal complexes (ried out  against  website  was util website . Using A website .p < 0.05)  . Groups  < 0.05) .1 – 3 were found to be 5.21, 18.57, and 15.07 Ω−1mol−1cm2, respectively (2L)(Cl)] and this in line with reported studies (vide infra).The molar conductance of complexes ectively , indicatectively . This is studies . Althoug studies . Further3L] ratio (G) and lower negative values of enthalpy change (ΔH) of the chemical reactions. In other cases, the complex formations are entropically favored due to positive values of change in entropy (ΔS) (vide infra).The Job’s plot for mole fraction of the ligand and the metal ions are presented in L] ratio . SimilarL] ratio . The theL] ratio . The negL] ratio . The metopy (ΔS) . Overallopy (ΔS) . This is−1 which was assigned for imine ν(C=N) group (Im) of the ligand , Cu(II) and Ni(II) complexes, respectively  and 460 cm−1 ν(Zn-N) for the Zn(II) complex; 626 cm−1 ν(Cu-O) and 474 cm−1 ν(Cu-N) for the Cu(II) complex; and 534 cm−1 ν(Ni-O), 462 cm−1 ν(Ni-N) for the Ni(II) complex. The DFT calculated frequencies are in close agreement with the corresponding experimental results  groups. Overall, the close agreement between the DFT calculated IR frequencies and the corresponding experimental results  and Cu(II) complexes have weak and broad spectral bands of stretching frequencies in the range 3673–3373 and 3664–3334 cmectively . This co results . The vis results . The preomplexes . Weak O- results  further 10 electron configuration, which prevents it from taking part in the d→d transition (The electronic spectra of the free ligand and the associated Zn(II), Cu(II), and Ni(II) complexes revealed that the ligand absorption bands are primarily caused by π→π* and n→π* transitions in UV-Visible spectral data between 200 and 800 nm. The peak for Cu(II) and Ni(II) complexes are red shifted, whereas very small blue shift was observed for the Zn(II) complex. The free ligand showed absorption peaks at 231, 258 nm (π→π*) and 300, 383 nm (n→π*) . The redansition . This isansition .N-quinoline ring (ligand-based π→π* transitions). The absorption bands above 400 nm could be assigned to the LMCT of Cu(II) and Ni(II) complexes  and the quinoline ring, respectively, inferring the presence of electron transition from metal to the ligand orbital systems for Zn(II) and weak d→d transition for Ni(II) complex. Since the d-orbitals of Zn(II) are fully occupied, there is no d→d transition in 1  electronic transitions, in line with literature . In addiomplexes  in agreeomplexes . Overallomplexes . Moreovedination . The HOMH3L) and its complexes 1–3 showed emission bands at 526, 608, 471, and 511 nm, respectively (10 electron configuration (Fluorescence study was performed at room temperature. The spectra of the ligand (ectively . Previouguration . In the guration . The comguration . This coguration .1–3) have polycrystalline characteristic peaks (The powder X-ray diffraction (PXRD) patterns of the three synthesized complexes  and 1–3 were found to be 27.86, 33.54 and 37.40 nm, respectively, in good agreement with previous reports for related complexes  tell us that cubic or orthorhombic system  based on the unit cell calculated values. According, the Zn(II) complex showed absence of the forbidden numbers   , indicat 23, 71) . Similar 23, 71)  shows th 23, 71) . Similar 23, 71) , which i 23, 71) .2L)Cl] complex, five characteristic signals corresponding to atoms C, O, N, Cl and Zn were observed which confirmed the formation of pure CHZnNOCl (2L)(H2O)(NO3)] showed four signals, which correspond to C, O, N, and Cu atoms, indicating pure CHCuNO (2L)(NO3)].2H2O complex showed four signals which correspond to C, O, N, and Ni atoms, and indicate the formation of CHNiNO (The compositions of Zn(II), Cu(II) and Ni(II) complexes were analyses from Energy Dispersive X-ray (EDX) analysis, in which the experimental percentage of atoms were found to be very close to the theoretical results . In the CHZnNOCl . Similare CHCuNO . The  (m/z 322.07 (8.75%) (found 322.05) and 259.63 (54.75%) (found 260.14), attributed to [C14H16N3O2Zn]+ and [C14H18N3O2]+ fragments, respectively. Similarly, complex 2 has a parent molecular ion peak at m/z 400.08 (found 401.05) which corresponds to the formula [C14H18CuN4O6] with a molecular weight of 401.86 g/mol (m/z 322 95 (6.70%) (found 322.06), 260.01 (54.75%) (found 260.14) and 226.13 (14.75%) (found 226.13) corresponding to [C14H17CuN3O2]+, [C14H18N3O2]+ and [C14H16N3]+ fragments respectively. Finally, complex 3 exhibited a parent molecular ion peak at m/z 413.09 (found 414.07) attributed to [C14H20N4NiO7] formula with a molecular weight of 415.02 g/mol (m/z 351.09 (10.9%) (found 352.08), 315.09 (30.70%) (found 316.06) and 260.33 (29.9%) (found 260.14) corresponding to [C14H20N3NiO4]+, [C14H16N3NiO2]+ and [C14H18N3O2]+ fragments, respectively. It is important to note that similar observations have been made for other related Ni(II) containing complexes , which indicates a mass loss of 17.80%  corresponding to the loss of chloroethane (C2H5Cl) like moiety. The complex was stable up to 200°C indicating the absence of both lattice and a water ligands in the specified complex (6H4 fragment of the quinoline ring moiety at 360–520°C (DTAmax of 515). The third step of degradation was from mass loss of 29.99%  which corresponds to the loss of C5H5N2O moiety at a temperature range of 535–765°C (DTAmax of 655). The actual weight loss occurred from all these steps are 68.79%, which is in good agreement with the calculated result of 69.53%. Gradual degradation was observed up to 765°C and the residue corresponds to zinc oxide (ZnO), 22.52% , and CHN imine moiety with 7.40%  of the complex.The weight loss of the complexes was measured at temperature ranges 25–800°C. The results are presented in on steps . The fir complex . The sec2 indicates three decomposition steps (3H6N2O and nitrate ion moiety at temperature ranges of 100–225°C (DTAmax of 213). The second step is due to weight loss of 7.26%  at temperature ranges 240–498°C (DTAmax of 245), which correspond to the elimination of C2H4 ethane like moiety. The final step occurs at temperature ranges of 520–640°C (DTAmax of 530) due to the loss of 32.14%  related to quinoline ring C9H6N moiety. The leaving residue of the degradation is CuO, which is 18.91%  of the complex (The TGA diagram of complex on steps . The fir complex . The ove3 showed four degradation steps (max of 125) which correspond to the elimination of two lattice water molecules. The second weight loss of 10.23%  was observed at a temperature ranges of 220–255°C (DTAmax of 229), which is attributed to the loss of C2H4N imine. The third step showed a mass loss of 22.44%  at temperature ranges of 260–361°C (DTAmax of 278) due to loss of (CH3O + NO3) methanol and nitrate ion moiety. The final step of this complex is due to mass loss of 28.74%  at a temperature ranges of 382–610°C (DTAmax of 508), which corresponds to the loss of quinoline ring moiety. The leaving residues of nickel oxide (NiO), representing 29.24%  of the complex. The actual mass loss from all these steps is 70.06%, which is in very good agreement with the calculated result (69.91%).The thermal decomposition of complex on steps . The fir1–3 beyond 765, 640 and 610°C, respectively, showed a straight line, indicating the formation of metal oxides  and Zn(II) complexes arise in three stages, while that of Ni(II) complex occurred in four stages. The thermogram of complexes l oxides . All thel oxides . This isl oxides .in vitro antibacterial activity study of the ligand and complexes 1–3 are presented in μg/ml for the Ni(II) complex against E. coli, and 20.65 ± 0.18 mm at 300 μg/ml for the Cu(II) complex against P. aeruginosa. All the three complexes exhibited good activities against P. aeruginosa  compared to ciprofloxacin with MIZ of 22.98 ± 0.08 mm diameter. These results are in close agreement with previously reported results for related complexes  at both concentrations against E. coli, P. aeruginosa and S. aurous, but has no antibacterial activity against S. pyogenes. Due to the metal’s chelation with the ligand, which encouraged the complexes’ capacity to enter the bacterial strains’ cell membranes, all of the complexes had more antibacterial activity than the precursor ligand  ion is a borderline Lewis acid and easily bind with similar biochemical such as protein and enzyme by “hard soft acid-base (HSAB)” Principle hence copper complexes showed good binding activity with G. negative (Pseudomonas aeruginosa) bacterial strain due to H- bonding interaction with amino acid like arginine having both acid and base end  complex (57 Å2) relative to the ligand’s TPSA value (77.74 Å2) is due to the lipophilicity enhancement of the ligand upon coordination with the Zn(II) ion. This phenomenon was also observed in the boiled egg model  and those with a TPSA 60 Å2 would be well absorbed (>90%) , whereas the number of hydrogen bond acceptors range from 4 to 8 (≤10). The predicted physicochemical properties for drug likeness screening showed that all the synthesized compounds fulfil drug-like molecular nature . Moreovegg model  of the sd (>90%) . From thd (>90%) .−1 (. This inferred that the compounds have no tendency to interact with other drugs fingered by the transporter and hence no drug-drug interactions. The inhibition of CYPs leads to toxicity end points (Skin permeability (logKp) value of the free ligand and its metal complexes were found within the range of −7.10 to −7.53 cms−1 , deducin−1 . Similar−1 . This ind points . The higEg) is correlated with various biological aspects like antibacterial, antioxidant and DNA binding activities  was higher than that of the ligand (3.834 eV) by 0.168 eV, inferring the presence of electron transfer from the HOMO of the metal center to the LUMO of the quinoline part of the ligand  of the ligand showed large enhancement upon coordination to metal ions (vide supra).According to the HSAB principle, soft acids react with soft bases, whereas hard acids react with hard bases . Biologianalysis . Chemicaanalysis . Chemicaanalysis . Therefo1 > H3L , in whictal ions . This geE. coli DNA gyrase B (PDB ID 6F86) (P. aeruginosa LasR (PDB ID: 2UV0)  > Zn(II) > Ni(II) complexes against the E. coli DNA gyrase B   and P. aD: 2UV0)  to underve sites . The resgyrase B . This is results .P. aeruginosa LasR DNA protein interacted with the synthetic metal complexes to acquire further knowledge. The 2D and 3D representation of the interactions for the compounds and ciprofloxacin are presented in in silico docking analysis indicated that the Cu(II) complex interacted with the LasR.DNA residues with a binding energy of −8.2 kcal/mol. This result is comparable with the binding energy of ciprofloxacin . Similar docking activity trends were observed for both E. coli DNA gyrase B  complex showed relatively highest antibacterial activity with mean inhibition zone of 20.65 ± 0.18 mm. Moreover, the Zn(II) and Cu(II) complexes showed better antioxidant activities. The calculated results also showed that none of the target complexed violate Lipinski’s rule of five. The in silico drug likeness and molecular docking results agree very well with the corresponding experimental results. The high antibacterial activity of copper complex against Gram-negative bacteria makes the complexes potential alternative drug for treating diseases caused by Gram-negative bacteria after passing cytotoxicity testing. Overall, there is a chance that the ligand and its metal complexes could be further improved for use as medicinal compounds because they have consequently shown promising antioxidant and antibacterial characteristics.Three new quinoline-based transition metal complexes were synthesized and characterized using PXRD, SEM-EDX, MS,


Polygonatum cyrtonema, one of origins of Polygonata Rhizoma (HuangJing in Chinese), is traditionally steamed repeatedly before being used as herbal medicine in China. However, there has no standard for steaming of HuangJing. Therefore, a comprehensive study for effects of steam on polysaccharides from Polygonatum cyrtonema based on saccharide mapping, a powerful method developed for polysaccharides analysis, and pharmacological activity are still necessary, which is helpful to explore the effect of steam on the physiochemical and biological activities of its polysaccharides and develop steaming standard of Polygonatum cyrtonema.P. cyrtonema polysaccharides (PCP), six polysaccharides named PCP0, PCP1, PCP2, PCP3, PCP4 and PCP5 were extracted from the herb consecutively steamed for 0–5 times, respectively. Their molecular weight distribution, monosaccharide composition and PACE fingerprints were investigated through HPSEC-MALLS-RID, HPAEC-PAD and saccharide mapping based on polysaccharides analysis by using carbohydrate gel electrophoresis (PACE) and HPTLC, respectively. In addition, their antioxidant ability and immunostimulatory activities on RAW 264.7 cells in terms of NO production and phagocytosis were compared.To explore the effect of steam on physiochemical and biological activities of 5-107 Da. They all showed irregularly spherical conformation in aqueous solution based on AFM imaging. Their monosaccharide composition and PACE fingerprints were significantly different after steaming, i.e., galactose increased while glucose and mannose decreased, and β-1,4-Galp appeared while β-1,4-Manp increased, after steaming. Steamed PCP significantly increased scavenging activity against ABTS radicals, while PCP0 had the best immunostimulatory effect on RAW 264.7 in terms of NO production and phagocytosis.Results suggested that molecular weights could be changed during steam, which increased by first steaming and then decreased with further steaming though all polysaccharides’ molecular weights were 10P. cyrtonema. Considering the balance beneficial effects of steaming on antioxidant and immunopotentiation activities of PCP, 2 times of continuous steam is the optimal choice under the given conditions.In summary, steam significantly affected the chemical composition and bioactivities of polysaccharides from The online version contains supplementary material available at 10.1186/s13020-022-00650-3. Polygonatum cyrtonema Hua is one of origins of Polygonata Rhizoma (HuangJing in Chinese), which has a long history of use in China as a tonic herb. It has high medicinal and edible value in medicine and food such as lowering blood sugar  × 100.C (%) = [1-. Cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% FBS and 1% P/S at 37 °C in a humidified atmosphere of 5% CO3 cells/well) were cultured in 96-well microplates overnight, and then treated with LPS (0.4 μg/mL) and a series of concentrations of PCP for 24 h, respectively. An equal volume of culture medium was used as blank control. Subsequently, the original culture medium was discarded and stained with 100 μL of culture medium containing 10% CCK8 for 4 h in dark. The absorbance values were read at 450 nm and the cell viability was calculated as the ratio of absorbance values between the sample and vehicle control group.RAW 264.7 cells (5 × 104 cells/well) were seeded in 96-well microplates overnight, and then cells were treated with a series of concentrations of PCP and LPS (0.4 μg/mL) for 24 h, respectively. An equal volume of culture medium was used as vehicle control. Subsequently, 75 μL of supernatants were collected and mixed with an equal volume of modified Griess reagent at room temperature for 15 min. The absorbance was measured at 540 nm. NO production was expressed as the ratio of absorbance values between sample and LPS treated group.RAW 264.7 cells (5 × 105 cells/well) were cultured in 24-well plates overnight and then incubated with culture medium, LPS (0.4 μg/mL) and a series of concentrations of PCP for 18 h, respectively. Then the cells were treated with FITC-dextran (0.1 mg/mL in culture medium) and incubated at 37 °C for an additional 1 h in dark. After incubation, the cells were collected with cold PBS after washed for three times. BD Accuri™ C6 Cytometer  was used for analysis. The percentage of phagocytosis was expressed as ratio of phagocytic rate between the treatment and control cells.FITC-dextran was used for phagocytic assay. RAW 264.7 cells  was milky white, after the first steam the color turned reddish brown with a lighter color in the center (PC1). Then, after the second time of steam treatment, slice PC2 changed to dark brown. Slice PC3 changed into black and began to appear luster, the color of PC4 and PC5 almost no further change compared with PC3. The color of the polysaccharide powders was also changed accordingly, similar to the raw materials , dispersibility index (DPI) and radius of gyrations (Rg) of polysaccharides without standards . Figure yrtonema , 34.Fig.After complete acid hydrolysis, compositional monosaccharides of PCP were determined by HPAEC-PAD  is 0.2–0.4 for branched polymers with a compact helical chain conformation, 0.3 for spheres, 0.5–0.6 for flexible polymers in good solvents, and 0.6–1.0 for semi-flexible chains [v index of PCP0-PCP5 was concentrated between 0 and 0.3  has also become a powerful tool to directly characterize the structure and properties of polymers . The pla50 values of PCP1-PCP5 were 4.89, 1.81, 1.79, 2.21 and 3.04 mg/mL, respectively. Change of steamed PCP in antioxidant capacity may attribute to Maillard reaction of polysaccharides during steam processing [50 showed that antioxidant activity of both PCP2 (1.81 mg/mL) and PCP3 (1.79 mg/mL) reached to the most potent, and then antioxidation decreased with increase of steaming times. Anyway, steaming significantly enhances antioxidant capacity of PCP, which is beneficial to its efficacy in delaying aging [P. cyrtonema should be further comprehensively investigated though 2–3 times steaming seemed have good antioxidant activity. Actually, the optimum steaming times could be different based on various steaming conditions and investigated biological activities [ABTS scavenge ability of PCP was shown in Fig. ocessing , which wocessing , IC50 shng aging , 40, 41 tivities , 18.Fig.Macrophages play an indispensable role in the innate and adaptive immunity of human body . StudiesP. cyrtonema in promoting TNF-α, IL-6 production and NO release at 2 h and 4 h, respectively, of steam treatment. The difference might attribute to different steaming conditions.Flow cytometry was used to determine the fluorescence intensity in cells after RAW 264.7 cells devoured FITC-dextran. The results showed that LPS (0.4 μg/mL) and PCP could promote the phagocytic activity of macrophages in a dose-dependent manner Fig. D. With tP. cyrtonema, one of the origins of HuangJing, a well-known tonic herb. In brief, steaming could significantly increase the molecular weights, UV absorption and antioxidant activity of polysaccharides from P. cyrtonema. Polysaccharides with glycosidic linkages such as β-1,4-Galp and β-1,4-manp obviously increased after steaming, but fructan could be completely degraded. In addition, steaming could significantly decrease the immunopotentiation activity of polysaccharides from P. cyrtonema in certain range. Considering the balance beneficial effects of steaming on antioxidant and immunopotentiation activities of PCP, 2 times of continuous steam is the optimal choice under investigated conditions. However, further study is still necessary to well understand the effect of steaming on HuangJing.Steaming treatment significantly influenced physicochemical properties and bioactivities of polysaccharides from Additional file 1: Table S1. HPAEC-PAD monosaccharide analysis liquid phase conditions. Table S2 Molar ratios of compositional monosaccharides for completely acid/pectinase hydrolysates of PCP. Figure S1. Ion chromatograms of monosaccharide composition by (A) complete acid hydrolysis and (B) pectinase hydrolysis. 1–10 in descending order: Fucose, Arabinose, Rhamnose, Galactose, Glucose, Xylose, Mannose, Fructose, Galacturonic Acid, Glucuronic Acid. Figure S2. Percentage of compositional monosaccharides molar ratio of (A) supernatant alcohol solution, (B) complete acid hydrolysate and (C) pectinase hydrolysate of PCP0-PCP5. Figure S3. The HPSEC-MALLS-RID chromatograms of PCP0-PCP5 after treat with pectinase (PE), β-1,4-Galactanase (GA) and β-1,4-Mannanase (MA). Figure S4. HPAEC chromatograms of PCP0-PCP5 after treat with pectinase, β-1,4-Galactanase, β-1,4-Mannanase and TFA. Figure S5. The rms conformation plot and conformation plot slope of PCP0-PCP5.


Enterovirus (EV) infections are being increasingly seen in younger infants, often being more severe than in older children. The risk factors of EV infection in infants have been inadequately investigated till date.We conducted a retrospective study on hospitalized children with laboratory-confirmed EV infection (50 infants aged 0–3 months and 65 older than 3 months) at a tertiary care center in China. Prevalence, clinical characteristics, and genetic features of the virus were analyzed, and independent predictors for severe infection were assessed.p < 0.001), with higher morbidity of pneumonia, meningitis, and sepsis (p < 0.01). EV-B types were detected more frequently in infants aged 0–3 months than in older children . Echovirus 11 was the most identified EV-B, and it recombined with E6 in P2 and P3 regions. Risk factors for severe EV infection included EV-B types infection, age less than 3 months, elevated alanine aminotransferase level, abnormal platelet count, and abnormal cerebrospinal fluid characteristics.Clinical findings showed that severe infection was more common in infants aged 0–3 months than in older children (78.0% vs. 35.4%, Our data indicated that EV-B types mainly cause severe infection in infants aged 0–3 months. Therefore, knowledge about EV-B types could have implications in designing effective intervention and prevention strategies for young infants with severe EV infection.The online version contains supplementary material available at 10.1186/s12985-023-01965-9. Picornaviridae, with genomes approximately 7500 nucleotides in length. Currently, more than 100 EV types, assigned to four species, have been found to infect humans, namely enterovirus A (EV-A), EV-B, EV-C, and EV-D, based on genetic divergence , CVA4 [14 (12.2%)], CVA16 [6 (5.2%)], CVA10 [2 (1.7%)], and CVA5 [1 (0.9%)], were identified in 66 (57.4%) cases. EV-B, accounting for the remaining 49 (43.6%) cases, included E11 [30 (26.1%)], E18 [6 (5.2%)], CVA9 [3 (2.6%)], E25 [2 (1.7%)], E9 [2 (1.7%)], CVB3 [2 (1.7%)], CVB2 [2 (1.7%)], E3 [1 (0.9%)], and E6 [1 (0.9%)]. The median age of EV-B infections was 21 days (IQR 12–27), which was significantly less than the 16.5 months (IQR 13.0–26.0) observed for EV-A infections  of 65 vs. 6 (12.0%) of 50, p < 0.001], and only 5 (7.7%) were infected with EV-B. E11 was the predominant serotype identified in 29 (58.0%) of the 50 infants, with a noticeably higher proportion than that in older children [1 (1.5%) of 65] (p < 0.001). Additionally, E18 was detected in five (10.00%) infants, whereas only one such case (1.5%) was found in older children. However, CVA6 was the most commonly observed serotype in older children [42 (64.6%) of 65], followed by CVA4 [10 (15.4%)] and CVA16 [6 (9.2%)].The species of EV infecting at different ages were significantly different Fig. b. EV-B wp < 0.01). Although the clinical features of infants aged 0–3 months were not typical, the median days of hospital stay were significantly longer than those of older children .Clinical case files of the 115 patients, with symptoms registered by the pediatrician upon hospital admission, were carefully analyzed, according to age and EV species  vs. 8 (12.3%), p < 0.001], and sepsis  than older children. Hepatitis occurred in six (5.2%) infants and two (1.7%) children, and all the cases were associated with EV-B infections.The disease spectrum of patients with EV infection varied in this study. HFMD [59 (51.3%) of 115], pneumonia [44 (38.3%)], meningitis [36 (31.3%)], sepsis [19 (16.5%)], and gastrointestinal dysfunction [23 (20.0%)] were the main disorders frequently observed in 115 patients. Of the 59 patients who fulfilled the definition of HFMD, 55 (93.2%) cases were caused by EV-A, and four (6.8%) were caused by EV-B. Majority [58 (98.3%) of 59] of HFMD cases were children older than 3 months, and only one was an infant. However, the infants aged 0–3 months had significantly higher prevalence rates of pneumonia .Blood test results, according to age and EV serotypes, are presented in Table p < 0.001). In addition, the positive rate of EV in CSF samples from young infants was 76.7% (23 of 30), which was significantly higher than the 20.0% (5 of 25) in the children group (p < 0.001).CSF parameters according to EV type and age are shown in Table In this study, among the 115 patients with EV infections, 62 53.9%) developed severe disease. Risk factors were analyzed between severe and non-severe EV infections by multiple logistic regression analysis using the forward stepwise logistic regression model. As shown in Table .9% develTo understand the molecular epidemiology of EV-B types better, a portion of the VP1 gene from all 49 viruses isolated from EV-B infections was amplified and selected for phylogenetic analysis Fig. . The phyTo help determine the evolution of predominant EV type in infants aged 0–3 months during the study year, we randomly performed full-length genome sequencing of E11 from five infants. Phylogenetic analysis revealed that all five E11 strains that circulated in Guangzhou city during 2019 clustered monophyletically with the E11 strains (MN597937 and MN597943) isolated from sewage samples in 2018 in Guangzhou city.Based on the heatmap results of evolutionary divergence between E11 sequence and other closely related EV sequences, we found that the nucleotide sequences of 5ʹ UTR, VP1–VP4, 2A, and 2B regions of E11 strains displayed higher sequence identity with the strains  isolated in Guangzhou city in 2018, whereas 2C and 3A–3D genes showed minimum evolutionary divergence with E6 strains from Jiangsu province (MK791151) and Zhejiang province (MN145871) of China in 2018 (Fig. Similarity plot and bootscanning were conducted between E11 and E6 strains to investigate the recombination phenomenon. Simplot analyses Fig. b showed This study showed that seasonal variations in EV infections with respect to age were evident. Most infants aged 0–3 months had a seasonal pattern of infections. They presented during summer, with a peak from April to July, which was apparently different from the epidemic features in older children, and consistent with previous studies , 11, 18.EV infection in neonates can present with clinical symptoms, ranging from asymptomatic, non-specific febrile illness to severe, life-threatening disease, and it is difficult to distinguish from non-EV infection based on the clinical signs only. In this study, fever was the most common symptom in both infants aged 0–3 months and older children. However, the other clinical symptoms, including rashes, tachycardia, coughing, vomiting, startle, and convulsions, frequently observed in children with HFMD, were not typical for infants, as suggested in earlier retrospective studies , 21. RasSevere illnesses due to EV are commonly seen in neonates; mortality is exceptionally high when meningitis, myocarditis, and hepatitis occur , 13, 23.Pneumonia is the most common comorbidity in lower respiratory tract-EV-B infection, and can be rapidly progressive, leading to severe pneumonia . In thisUnderstanding the risk factors and monitoring the parameters associated with severe infection may lead to effective prophylaxis and prompt aggressive treatment to reduce morbidity and mortality. Infection with EV-B and younger age are considered the major risk factors for developing severe infections . A retro. had previously reported that an abnormal platelet count could be an independent predictor of severe EV infection [Xiao-Qing Lv et alnfection . Our resnfection , and eleGenomic recombination is a major driving force in the evolution, diversification, and shaping of genetic architecture of EVs , and timIn summary, EV affects infants aged 0–3 months differently and more severely than in older children. Clinical manifestations in infants with EV mainly included meningitis, sepsis, pneumonia, and even death. EV-B types were the most common in neonatal EV infection, and recombination events were observed in the P2 and P3 regions of predominant type E11 with E6 from China. In addition, we identified independent predictors of severe EV infection, including EV-B infection, age less than 3 months, elevated ALT level, abnormal platelet count, and abnormal CSF characteristics. Taken together, EV-B infections should be routinely considered in neonates with meningitis, sepsis, and pneumonia, with or without a rash, particularly during EV season.Additional file 1: Table S1. Primers used for complete genome amplification of echovirus 11Additional file 2: Table S2. Clinical characteristics of children with EV infection according to EV species, Guangzhou, China, 2019