A novel salt-tolerant aerobic denitrifier was isolated from a marine recirculating aquaculture system (RAS) and identified as Halomonas alkaliphile HRL-9. Its aerobic denitrification performance in different dissolved oxygen concentrations, temperatures, and C/N ratios was studied. Investigations into nitrogen balance and nitrate reductase genes (napA and narG) were also carried out. The results showed that the optimal conditions for nitrate removal were temperature of 30 °C, a shaking speed of 150 rpm, and a C/N ratio of 10. For nitrate nitrogen (NO3−-N) , the sole nitrogen source of the growth of HRL-9, the maximum NO3−-N removal efficiency reached 98.0% after 24 h and the maximum total nitrogen removal efficiency was 77.3% after 48 h. Nitrogen balance analysis showed that 21.7% of NO3−-N was converted into intracellular nitrogen, 3.3% of NO3−-N was converted into other nitrification products , and 74.5% of NO3−-N might be converted to gaseous products. The identification of functional genes confirmed the existence of the napA gene in strain HRL-9, but no narG gene was found. These results confirm that the aerobic denitrification strain, Halomonas alkaliphile HRL-9, which has excellent aerobic denitrification abilities, can also help us understand the microbiological mechanism and transformation pathway of aerobic denitrification in RASs.Aerobic denitrification microbes have great potential to solve the problem of NO In recent years, the aquaculture industry has developed rapidly and provided abundant aquatic resources for human beings. Nevertheless, this industry also introduced serious environmental problems ,2, such 4+-N) and nitrous nitrogen (NO2−-N) from RASs, which have proven harmful to the farmed organisms [4+-N to nitrate nitrogen (NO3−-N) through NO2−-N [3−-N, to a certain extent, could cause serious harm to cultured organisms [3−-N and NO2−-N could be converted into nitrogen by denitrifiers under anaerobic or anoxic conditions in traditional denitrification theory; four main steps happen in sequence: NO3−-N→NO2−-N→NO→N2O→N2 [Nap), nitrite reductase (NirS or NirK), nitric oxide reductase (Nor), and nitrous oxide reductase (N2OR), correspond to these four steps, respectively [A biofilter system could effectively remove excessive ammonium nitrogen NH+-N and nh NO2−-N . Neverthrganisms ,7,8,9. DO→N2O→N2 ,11. Fourectively . Traditiectively ,14. Neve3−-N under aerobic conditions by aerobic microbes), offers a possible economic solution to nitrate accumulation in RASs. Since the aerobic denitrifier, Thiosphera pantotropha, was first isolated and purified in 1983 [Paracoccus versutus LYM [Paracoccus denitrificans Strain ISTOD1 [Klebsiella pneumonia [Marinobacter strain NNA5 [napA, encoding a periplasmic nitrate reductase, was important for aerobic denitrifiers to remove nitrate under aerobic conditions [narG gene, encoding a membrane-bound reductase, was responsible for bacterial respiration and denitrification under anaerobic conditions [Halomonas campisalis ha3 [Marinobacter sp. F6 [The founding of aerobic denitrifying , which Halomonas alkaliphile HRL-9, was isolated from lab-scale seawater RASs . The bacterial phenotype, the influence of three important factors , a nitrogen balance analysis, and the functional genes responsible for aerobic denitrification were investigated. We aimed to elucidate the influence of important factors on the aerobic denitrification performance, pathways, and functional genes of HRL-9. This study may contribute to understanding the microbiological mechanisms of aerobic denitrification in RASs.In this study, a novel highly salt-tolerant (3%) aerobic denitrifier, 2HPO4 (7.90 g), KH2PO4 (4.50 g), MgSO4 (0.10 g), K2HPO4 (4.78 g), NaCl (30 g), KNO3 (0.72 g), Sodium acetate (3.42 g), and a trace element solution (3.0 mL) [4·7H2O (3.0 g), H3BO3 (1.12 g), CaCl2 (0.6 g), FeSO4·7H2O (0.3 g), CuSO4·5H2O (1.57 g), MgSO4·7H2O (3.0 g), and EDTA (25 g). The agar plates were prepared by DM containing 20 g agar per liter. A Luria–Bertani (LB) medium was used for bacterial enrichment and to extract the genomic DNA of the isolated strain, HRL-9 (per liter): Agar (20 g), Yeast extract (10 g), NaCl (30 g), pH 7.00. All media were autoclaved at 121 °C for 90 min prior to use .The samples were obtained from lab-scale seawater RASs of the Dalian Ocean University . A denitrification medium (DM) was used for bacterial isolation and single-factor affecting experiments. Its components were as follows (per liter): Na(3.0 mL) . The tra−2–10−7 with sterile saline. The samples were uniformly plated on agar plates and cultured at 30 °C for 72 h. The monoclonal colonies were inoculated in DM for 48 h. The purified monoclonal strains were stored at −80 °C with 20% glycerol. A total of 21 strains with aerobic denitrification capacity were obtained from this experiment. The isolates were streaked onto a DM medium and incubated for 48 h at 120 rpm and 30 °C, followed by a test of their denitrification ability. Strain HRL-9 had an excellent removal rate of 93.4% for the nitrate in 24 h and was selected for further analyses.The biological filter samples were taken from the RASs and washed with sterile water. The biofilm was scraped off with a sterilizing blade and gradiently diluted to 10https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree using neighbor-joining method was built by MEGA version 6 .SEM: 2 mL of the bacterial solution was centrifuged at 10,000 rpm for 10 min. The cells were fixed in 3% glutaraldehyde solution for 24 h, and then the morphology of the strain cells was observed by SEM. The genomic DNA of the isolated bacterium HRL-9 was extracted with a TIAamp Bacteria DNA Kit  after cultivation in an LB medium for 24 h. DNA extracts were stored at −20 °C. The amplification of the DNA was performed by polymerase chain reaction (PCR) with 16S universal primers , which were designed by Sangon Biocompany . The PCR was performed under the following conditions: a denaturing step of 90 °C for 5 min, followed by 35 cycles of denaturing for 60 s at 95 °C, annealing for 60 s at 55 °C, and a final extension at 72 °C for 5 min. The 16S rDNA gene was compared with that of other bacteria by way of BLAST . The strain was cultivated at C/N 10 and 30 °C for 48 h. In the temperature experiments, the culture temperature was changed to 20 °C, 30 °C, and 40 °C. The strain was cultivated at 120 rpm and C/N 10 for 48 h. In all single-factor experiments, the strain HRL-9 was inoculated into a 100 mL vial bottle containing 50 mL of a DM medium . The samples were taken at 4 h intervals to measure the growth curve (OD 600), total organic carbon (TOC), NO3−-N, NO2−-N, NH4+-N, and total nitrogen (TN), respectively. All experiments were carried out in triplicate.The denitrification capability of the HRL-9 strain was further determined under different conditions, including DO, C/N, and temperature. In the C/N ratio experiments, different amounts of sodium acetate were added with a constant NO reports ,19 and t3−-N 100 mg·L−1) at optimal conditions . The aerobic denitrification ability was assessed after 48 h. The vial bottle was sealed with sterile breathable sealing membranes. The samples were taken periodically to determine the growth curve (OD600), TOC, NO3−-N, NO2−-N, NH4+-N, and TN. All experiments were carried out in triplicate.According to the single-factor experiment, strain HRL-9 was inoculated into a 100 mL vial bottle containing 50 mL of the DM medium . Samples were sonicated to ensure the final TN contains intracellular TN. The ultrasonication process was as follows: the ultrasonic worked in one cycle consisting of a 4 s pulse and a 6 s pause. Each sample underwent 90 cycles of sonication using a 6 mm ultrasonic probe. The bacterial solution obtained by ultrasonication was centrifuged at 8000 rpm for 10 min. Supernatants were filtered using 0.22 μm membrane filters, and the filtrates were used to detect the contents of the final soluble TN, NOnapA and narG) amplification. The PCR amplification of the napA gene was conducted using primer F: 5′-CAGCTCTATGCCGATCCGAG-3′ and R: 5′-CAGCCCCTCTTCGTTCATGT-3′; The PCR amplification of narG gene was conducted using a primer according to Roussel-Delif et al. [napA and at 50 °C for 30 s for narG, and a final extension at 72 °C for 7 min. The PCR products were electrophoresed on a 2% agarose gel using an electrophoresis apparatus.HRL-9 was inoculated in an LB medium for 24 h. Then, the bacterial suspensions were used for genomic DNA extraction with a bacterial DNA kit . The genomic DNA was used as a template for nitrate reductase gene . The TOC concentration was determined using a TOC-L analyzer . A one-way analysis of variance (ANOVA) was applied to assess the difference of the removal efficiencies under different conditions using SPSS Statistics version 24.0 . Values of p < 0.05 were taken to show statistical significance at a 95.0% confidence level.The concentration of DO was determined by a YSI multi-parameter water quality measuring instrument . The bacterial growth (OD600) was monitored by measuring the optical density (OD) at 600 nm using a spectrophotometer . Concentrations of NHHalomonas alkaliphile HRL-9. A phylogenetic tree was constructed based on 16S rDNA gene sequences of the isolate and some other phylogenetically related strains  and 150 rpm , the DO concentration decreased to 3.15 mg·L−1 and 4.19 mg·L−1 in 24 h, respectively  at 40 °C  as the sole nitrogen source. The experimental results are shown in 3−-N reached 69.5% at 48 h  was the sole nitrogen source. The NO3−-N concentration decreased to 1.24 mg·L−1 at 48 h, about 21.7% of NO3−-N was converted to intracellular nitrogen, 3.3% of NO3−-N was converted to other nitrification products , and 74.5% of NO3−-N might be converted to gaseous products.The results of the nitrogen balance analysis are shown in napA gene is an essential functional gene for the transformation of nitrate under aerobic conditions [napA gene of 884 bp products was amplified successfully, while the narG gene was not obtained  had significant effects on the aerobic denitrification performance of the strain HRL-9 , the performance of the aerobic denitrification capacity of strain HRL-9 was significantly higher than that under other conditions , which also showed a high DO tolerance limit. Zhou et al. [Pseudomonas stutzeriIt tolerated a high DO level. Therefore, strain HRL-9 has a potential application in the purification process of aquaculture waters, which need to maintain a high DO concentration. Temperature was reported to be an important factor affecting denitrification capacity [P. stutzeri XL-2 [P. stutzeri ZF31 [Halomonas.sp. 5CRT [4+-N concentration on the denitrification rate of HRL-9 were produced under different conditions, but no significant difference was shown. This result is similar to the determination of DNRA phenotype of denitrifying bacteria [At 150 rpm , ammonium nitrogen (NH4+-N), and organic nitrogen)), and 74.5% of NO3−-N might be converted to gaseous products . Single-factor experiment results showed the highest removal rate of nitrate to appear at a C/N ratio of 10, a temperature of 30 °C, and a shaking speed of 150 rpm. A total of 98.0% of NO3−-N was removed at 24 h, and 77.3% of TN was removed at 48 h. A nitrogen balance analysis of strain HRL-9 revealed that approximately 74.5% of the initial nitrogen was removed as gas products, and 21.7% of NO3−-N was converted to intracellular nitrogen. The existence of the napA gene, rather than the narG gene, was responsible for the excellent aerobic denitrification ability of strain HRL-9. This study revealed that strain HRL-9 could efficiently remove nitrogen from salty wastewater. Strain HRL-9 has the potential to solve the accumulation of nitrate in RAS by immobilization and microbial augmentation. Also, the method of feeding carbon source is important. In addition, this study can help us understand the microbiological mechanism of aerobic denitrification in RASs.In conclusion, a novel salt-tolerant aerobic denitrifier,


AbstractAegyptobiabozaiisp. n., is described from Central-Hungary on leaves of the endemic Hungarian statice Limoniumgmeliniisubsp.hungaricum (Plumbaginaceae) based on females, nymphs and larva. The previously described endemic flat mite, Tenuipalpusszarvasensis Bozai, 1970 is redescribed. This species had been treated as a junior synonym of Tenuipalpuscheladzeae Gomelauri, 1960, but our new investigation shows that the two species are not the same.A new tenuipalpid mite species,  Brevipalpus, but pest species are also found in Dolichotetranychus, Raoiella and Tenuipalpus. The family has received considerable attention in some parts of the world, but the majority of the Central European countries have been scarcely investigated. Hungary is no exception, with only 19 recorded species  were collected in a pasture close the border of the village Farmos  from the leaves of an endemic Hungarian plant (Limoniumgmeliniisubsp.hungaricum). The specimens were placed into lactic acid for a week and then slide-mounted in Keifer’s F-medium (in 2014) and Hoyer medium (in 2017). The holotype and some paratypes of the new species are stored in the Hungarian Natural History Museum and other paratypes in the Arachnida collection of the Natural History Museum of Geneva (Switzerland).Specimens of the new species  digital microscope. All measurements and scales are given in micrometers.Taxon classificationAnimaliaProstigmataTenuipalpidaehttp://zoobank.org/CF433BD9-1455-4EE2-84F3-DFF3ABCC858C47°22'30"N, 19°52'08"E, 10 m a.s.l, from the leaves of the Hungarian statice, Limoniumgmeliniisubsp.hungaricum, 2 August 2014, Ripka, G. coll. Paratypes: one female, three deutonymphs, three protonymphs and one larva, locality and date same as for holotype. Other paratypes: four females, Hungary, Farmos, 47°22'30"N, 19°52'08"E, 10 m a.s.l, from the leaves of Limoniumgmeliniisubsp.hungaricum, 2 August 2017, Kontschán, J. and Ripka, G. coll.Holotype: female, Hungary, Pest county, Farmos, PageBreaksmooth and simple; seta f2 present. Length of dorsal setae 6–14. Dorsal opisthosomal pores close to e1. Rostrum extending to middle of tibia I. Genital flap smooth. Intercoxal area between 3a and 4a smooth.. Tarsal claws uncinate. Anterior margin of prodorsal shield with paired projections, prodorsum weakly sculptured, with few irregular lines. Opisthosoma with polygonal reticulation. Propodosomal and opisthosomal setae v2 to h1 240–245; from tip of rostrum 260–266; width 143–147 near setae sc2; distance between setae sc2 120–125; length of legs I–IV (without coxa), leg I 105–110, leg II 80–87, leg III 72–78, leg IV 80–88.. Idiosoma reddish-brown Figure , oval ine1. Prodorsal setae v2 shorter than half distance between their bases. PageBreakLength of dorsal setae: v2 11–12, sc1 12–14, sc2 11–13, c1 8–9, c2 8–9, c3 9–10, d1 9–10, d2 8–9, d3 7–8, e1 8–9, e2 7–8, e3 8–9, f2 7–8, f3 6–8, h1 6–7, h2 7–8.Dorsum Figure : Anterioag, genital and anal plates where longitudinal striations visible. Genital and anal plates smooth. Length of ventral setae, 1a 53–57, 3a 11–12, 4a 8–9, 1b 7–8, 2b 8–9, 3b 8–9, 4b 9–10, 1c 11–12, 2c 10–13, ag 8–9, g1 9–10, g2 7–8. Pseudanal setae, all 6–8. All ventral setae simple and smooth.Venter Figure : Surfacem (4–5).Gnathosoma Figure : Rostruml’) seta present on femora I. Solenidia on tarsi I and II 8–10 long, broad, leaf-like. Tarsal claws uncinate and empodium pad-like.Legs Figures : Setal fv2 to h1 190–200; width 130–140 near setae sc2. (Plumbaginaceae), is an endemic subspecies occurring on salt meadows in Central-Hungary. The mites appeared to prefer leaves of the host plant that were lying close to the surface of the soil. The alkali steppe where the host plant was found is hot and dry in summer, typical habitat for tenuipalpid species, which prefer warm and dry conditions. Up until now, only one species has been reported from Limonium plants: Capeduliamaritima Gerson & Smith Meyer, 1980 was found on the roots of Limoniummeyeri in Israel  close to the town Kecskemét . Anterior margin of prodorsal shield with forked projection; prodorsum smooth medially, with some striae laterally; anterolateral projections carrying setae v2 to h1 305; from tip of rostrum 350; width between setae sc2 190.. Colorization of idiosoma not observable in the holotype. Idiosoma Figure  pentagonPageBreakPageBreaksc2 weakly formed. Opisthosoma smooth anteriorly, with posteromedial reticulation and posterolateral longitudinal striation. Propodosomal setae as follows: v2 short and smooth, sc1 broad and obovate, sc2 long and phylliform. Opisthosomal setae: c1, c3 and d1 broad, long, oblanceolate, d3 short and oblanceolate, e1 short and smooth, h1, f1, f2 and e3 long, oblanceolate, h2 very long and smooth. Rostrum extending to middle of tibia I. Opisthosomal pores between d1 and e1. Length of dorsal setae: v2 9–10, sc1 30–32, sc2 65, c1 45–46, c3 32–34, d1 38–40, d3 12–14, e1 12, e2 45–47, f2 and f3 45–46, h1 36–37, h2 105–110.Dorsum Figure : Anteriog1–g2. Genital and anal plates smooth. Length of ventral setae, 1a 120–122, 3a 26–27, 4a 130–133, 1b 16, 2b 16–17, 3b 18–19, 4bPageBreak13, 1c and 2c 24–25, ag 12, g1–g2 14–16. Pseudanal setae, all 12–13. All ventral setae simple and smooth.Venter Figure : Very feGnathosoma: Rostrum extending to middle of tibia I; palp setal counts as in Figure Legs: Setal formula for leg I–IV (coxae to tarsi): 3-1-4-2-5-9, 2-1-4-2-4-9, 2-2-2-1-3-5, 2-1-1-0-3-5. Shape of the setae on legs illustrated on Figures Tenuipalpusszarvasensis, that the species is very similar to T.cheladzeae, but he mentioned some easy to observe differences  between these two species. Despite these known differences, Tenuipalpuscheladzeae. T.szarvasensis, therefore their opinion was questionable.T.szarvasensis in order to confirm Bozai’s hypothesis that T.szarvasensis differs from Gomelauri’s T.cheladzeae. The differences are presented in Table This year, we studied the types of Bozai’s Tenuipalpus is the largest genus of flat mites, but very few are known from Pinaceae. Apart from the above-mentioned two species, the only other species is T.hondurensis Evans, in Tenuipalpuscupressoides Smith, Meyer & Gerson, 1980 is very similar to T.szarvasensis, but the shape of v2 and e2 and the length of sc1, c1 and d1 are different. In addition, the host plant, Cupressussempervirens of T.cupressoides belongs to the family Cupressaceae and not to Pinaceae (Pinaceae .


Nucleoside/nucleotide analogs that lack the 3′-hydroxy group are widely utilized for HIV therapy. These chain-terminating nucleoside analogs (CTNAs) block DNA synthesis after their incorporation into growing DNA, leading to the antiviral effects. However, they are also considered to be DNA damaging agents, and tyrosyl-DNA phosphodiesterase 1, a DNA repair enzyme, is reportedly able to remove such CTNA-modifications of DNA. Here, we have synthesized phosphoramidite building blocks of representative CTNAs, such as acyclovir, abacavir, carbovir, and lamivudine, and oligonucleotides with the 3′-CTNAs were successfully synthesized on solid supports. Using the chemically synthesized oligonucleotides, we investigated the excision of the 3′-CTNAs in DNA by the human excision repair cross complementing protein 1-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease, which is one of the main components of the nucleotide excision repair pathway. A biochemical analysis demonstrated that the ERCC1-XPF endonuclease cleaved 2–7 nt upstream from the 3′-blocking CTNAs, and that DNA synthesis by the Klenow fragment was resumed after the removal of the CTNAs, suggesting that ERCC1-XPF participates in the repair of the CTNA-induced DNA damage. Nucleoside/nucleotide analogs lacking their 3′-hydroxyl group, such as 3′-azidothymidine (AZT), function as efficient antiviral drugs. Their common therapeutic target is DNA synthesis by DNA polymerases or reverse transcriptases (RTs) . They aret al. reported that the clinical toxicities of CTNAs correlated well with their potential toxicities, which were calculated from the in vitro kinetic rates of CTNA incorporation and CTNA removal by Polγ hept-5-en-3-one, according to the previous reports methanol  hemisulfate and (−)-β-l-2′,3′-dideoxy-3′-thia-cytidine 3TC), were purchased from Sigma-Aldrich Co. , Carbosynth Ltd.  and Wako Pure Chemical Industries, Ltd. , respectively. Carbovir (CBV) was synthesized from optically pure (1R)-(−)-2-azabicyclo[2.2.1]hept-5-en-3-one , according to the previous reports hept-5-en-3-one according to the previous reports [N,N-dimethylformamide dimethyl acetal  was added, and the resulting solution was stirred overnight at room temperature. The solvent was removed in vacuo, and the residue was co-evaporated with toluene three times, and the product was purified with silica gel column chromatography with a stepwise gradient of 0%–6% methanol in chloroform. The fractions containing the product were evaporated to dryness. Yield 475 mg . The product was dissolved in chloroform/methanol solution , and the solution was dropped into hexane/diethyl ether solution  to give a powdery product. TLC  Rf 0.29. 1H-NMR  δ (ppm) 11.2 , 8.59 (s. 1H), 7.72 , 6.11 , 5.86 , 5.51 , 4.71 , 3.47 , 3.15 , 3.03 , 2.88 , 2.65 , 1.64 . FAB-HRMS m/z 303.1573   See .8  was suspended in anhydrous acetonitrile (1.5 mL), and subsequently diisopropylethylamine  and 2-cyanoethyldiisopropylchloro-phosphoramidite  were added. After continuous stirring for 30 min at room temperature, the reaction mixture was diluted with ethyl acetate (40 mL). The organic layer was washed with 2% NaHCO3 (40 mL × 2) and brine (40 mL × 1), and then was dried over Na2SO4. The solvent was evaporated in vacuo. The residue was co-evaporated with toluene once, and the product was purified with silica gel column chromatography with a stepwise gradient of 0%–3% methanol in chloroform containing 0.5% pyridine. The fractions containing the product were evaporated to dryness. The residue was further co-evaporated with acetonitrile to give an oily product. Yield 71 mg . TLC  Rf 0.62. 31P-NMR  δ (ppm) 145.48 and 145.33. FAB-HRMS m/z 503.2650  (See Compound N8P) See .N2-dmf-acyclovir 9 was synthesized according to the previous report [N2-dmf-acyclovir 9  was coevaporated with anhydrous THF three times, and suspended in a mixture of anhydrous THF and DMF . To the suspension, diisopropylethylamine  and 2-cyanoethyldiisopropylchlorophosphoramidite  were added. After continuous stirring for 30 min at room temperature, the reaction mixture turned into a clear solution, and the mixture was diluted with ethyl acetate (40 mL). The organic layer was washed with 2% NaHCO3 (40 mL × 2) and brine (40 mL × 1), and then was dried over Na2SO4. The solvent was evaporated in vacuo. The residue was co-evaporated with toluene once, and the product was purified with silica gel column chromatography with a stepwise gradient of 0%–4% methanol in chloroform containing 0.5% pyridine. The fractions containing the product were evaporated to dryness. The residue was further coevaporated with acetonitrile to give a glassy product. Yield 24 mg . TLC  Rf 0.30. 31P-NMR  δ 145.97 ppm. FAB-HRMS m/z 481.2432  (See s report . N2-dmf-N8P) See .3 (40 mL × 2) and brine (40 mL × 1), and then was dried over Na2SO4. The solvent was concentrated in vacuo. The residue was triturated with diethyl ether, and the precipitate was collected and washed with diethyl ether to give a white powdery product. Yield 112 mg . TLC  Rf 0.50. 1H-NMR  δ (ppm) 11.24 , 8.45 , 8.01 , 7.63 , 7.52 , 7.35 , 6.25 , 5.43 , 5.28 , 3.85 , 3.59 , 3.25 . FAB-HRMS m/z 334.0860   and benzoic anhydride  were suspended in anhydrous ethanol (2.0 mL) in a glass vial, and the vial was tightly sealed with a screw cap. The reaction mixture was heated at 70 °C for 2 h, and then benzoic anhydride  was added to the solution to complete the reaction. After 30 min, the reaction mixture was diluted with chloroform (40 mL). The organic layer was washed with saturated NaHCON3S) See .10  was co-evaporated with anhydrous THF three times, and suspended in anhydrous THF (1.0 mL). To the suspension, diisopropylethylamine  and 2-cyanoethyldiisopropylchlorophosphoramidite  were added. The reaction mixture was stirred for 30 min at room temperature, and then additional diisopropylethylamine  and 2-cyanoethyldiisopropylchlorophosphoramidite  were added to complete the reaction. After 10 min, the reaction mixture was diluted with ethyl acetate (40 mL). The organic layer was washed with 2% NaHCO3 (40 mL × 2) and brine (40 mL × 1), and then was dried over Na2SO4. The solvent was evaporated in vacuo. The residue was co-evaporated with toluene once, and the product was purified with silica gel column chromatography with a stepwise gradient of 40%–80% ethyl acetate in hexane containing 0.1% pyridine. The fractions containing the product were evaporated to dryness. The residue was further coevaporated with acetonitrile to give a glassy product. Yield 54 mg . TLC  Rf 0.80. 31P-NMR  δ (ppm) 147.12, 147.03. FAB-HRMS m/z 534.1938  (See Compound 5PS) See .1–4 were dissolved in anhydrous acetonitrile at a concentration of 0.11 M, and were installed on a 3400 DNA synthesizer . Solutions of nucleoside phosphoramidites for the 5′ → 3′ synthesis and 2′,3′-dideoxycytidine (ddC) phosphoramidite (Glen Research) were also installed. An 18-mer oligonucleotide, d(TCCGTTGAAGCCTGCTTT), was first synthesized in the 5′ → 3′ direction on a 0.2 µmol scale. After the removal of the last 3′-DMT group, the phosphoramidite building blocks of the CTNAs were allowed to react with the exposed 3′-hydroxy group in the presence of tetrazole, on the DNA synthesizer. This process was repeated twice, while omitting the detritylation, capping, and oxidation steps. Subsequently, the supports were treated with an iodine solution to oxidize the newly-generated phosphite triester into the phosphotriester group, to yield the CTNA-attached 19-mer oligonucleotides, d(TCCGTTGAAGCCTGCTTT)X, where X represents the CTNAs and ddC. The synthesized oligonucleotides were treated with 28% ammonia water for 1 h at room temperature. The ammoniac solutions were transferred into glass vials with screw caps, and were heated at 55 °C for 5 h 3TC and ACV) or 24 h (ABC). After evaporation of the solution, the desired oligonucleotides containing the CTNAs were purified by HPLC, with linear gradients of 5%–13% or 6%–14% acetonitrile. The oligonucleotides were characterized by LC-ESI mass spectrometry  containing 10 mM MgCl2, and 2 µL of alkaline phosphatase from Escherichia coli C75  and 23 µL of water were added. The total mixtures (50 µL) were incubated at 37 °C for 2 h. The obtained nucleosides were analyzed by HPLC, and an ODS-3 C18 column  was used at a flow rate of 1.0 mL·min−1 at ambient temperature, with a linear gradient of 2.5%–15% acetonitrile in 0.1 M triethylammonium acetate generated over 20 min. By comparing the peak areas of dC and dG detected at 260 nm, concentrations of the CTNA-containing oligonucleotides were calculated.To quantify the purified oligonucleotides, aliquots of the CTNA-oligonucleotide solutions, in addition to the quantified solution of the oligonucleotide without the CTNA modification, were dried with a SpeedVac centrifugal evaporator, and the residues were treated with S1 nuclease  in 20 µL of 30 mM sodium acetate buffer (pH 4.6) containing 100 mM NaCl and 1 mM ZnCl32P-labeled oligonucleotides were prepared by treating the CTNA-oligonucleotides (100 nmol) with T4 polynucleotide kinase  at 37 °C for 30 min, in 70 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl2, 5 mM DTT and γ-32P adenosine triphosphate . The reaction mixture was heated at 95 °C for 5 min, and was then passed through a MicroSpinTM G-25 column . Aliquots (20 pmol) of the obtained 32P-labeled oligonucleotides were mixed in water with the complementary strands (20 pmol), d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A, G or C, depending on the type of CTNA. The solutions were heated at 80 °C for 2 min, and then were gradually cooled down to 4 °C over 2 h. The hybridized oligonucleotides were used for the enzymatic reaction. After the reaction, an equivalent amount of stop solution, containing 95% formamide, 20 mM EDTA, 0.025% bromophenol blue and 0.025% xylene cyanol, was added to the reaction mixture. The mixtures were heated at 95 °C for 10 min, and then immediately cooled in ice. The reaction products were separated by electrophoresis on a 20% polyacrylamide/7.5 M urea gel. The dried gels were visualized on a GE Healthcare FLA7000 image analyzer. The band intensities in the obtained gel images were quantified by the MultiGauge ver. 3.0 software .The 5′-TM Bradford 1 × Dye Reagent and Quick StartTM Bovine Serum Albumin Standard . The hybridized oligonucleotides (400 fmol) were treated with ERCC1-XPF in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 0.5 mM MnCl2, 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 30 °C for 90 min. The products were analyzed by denaturing PAGE. Time course experiments were performed in the same reaction mixture, except for the enzyme amount (92 fmol), by varying the incubation time .The recombinant human ERCC1-XPF endonuclease was prepared according to the previous report . The con2, 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2, 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity . The total reaction mixtures (15 µL) were incubated at 37 °C for 10 min, and the products were analyzed by denaturing PAGE.The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgClin vivo, cell biological studies will address the involvement of ERCC1-XPF in the repair of the CTNA-induced damage in the future.We successfully synthesized the 3′-CTNA oligonucleotides on a solid support, and used them to investigate the repair of the CTNA-induced DNA damage by ERCC1-XPF endonuclease. The results indicated that ERCC1-XPF can remove the 3′-CTNAs, to expose the re-extendable 3′-OH. Since CTNAs would be metabolized in various ways, depending on their specific characteristics, the repair pathways of the CTNA-induced DNA damage would also be diverse, and our results suggested that structure-specific endonuclease-dependent repair is a possible pathway for the repair of the CTNA-induced DNA damage. Although it is still unclear whether ERCC1-XPF is involved in the repair


We first demonstrated that long-term increased polyamine  intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation. Polyamines are linear aliphatic hydrocarbons with three or more primary amino groups. The representative polyamines are spermidine (three amino groups) and spermine (four amino groups). Putrescine, a precursor of polyamine, has two amines and is therefore referred to as a diamine, and its biological activities differ from those of polyamines ,2. For ede novo synthesis, cells can take up polyamine from the extracellular space through a polyamine transporter in the cell membrane. For example, polyamines locally administered in the body [In addition to the body  and ingethe body , and arethe body . The majthe body ,13. The the body ,15. And,the body ,16.There is an overwhelming scientific consensus supporting the important role of epigenetic, especially gene methylation, changes in aging-associated pathologies and lifespan alteration ,18. PolyAging is associated with enhanced demethylation of DNA in various organs and tissues in several animals and humans ,21. HoweChanges in polyamine metabolism influencing SAM and dcSAM concentrations and AdoMetDC activity may affect DNMT activities and gene methylation. However, how changes in polyamine metabolism affect substrate concentrations and enzymatic activities involved in gene methylation is not known in detail. In this study, we investigated the effects of decreased polyamine synthesis and of extracellular polyamine supply on substrate concentrations and enzyme activities involved in polyamine metabolism. In our previous studies, spermine concentrations in blood cells of humans and mice fed a high-polyamine diet increased by 1.1- to 1.5-fold, whereas there was no significant increase in spermidine concentration . And, 50Polyamines are contained in all cells in high micromolar to low millimolar quantities. Our previous study showed that spermine concentrations of up to 1 mM for up to 80 h were not toxic . Flow cytometric examination revealed that DFMO treatment (3 mM) for 3 days increased mean fluorescent intensity (MFI) of CD11a. Spermine treatment alone and DFMO plus spermine treatment decreased the MFI of CD11a staining. The percentage of cells negative for ViaProbe was not changed, indicating that these treatments decreased LFA-1 expression without affecting cell viability .HMEpCs does not express LFA-1, therefore we determined the maximum concentration of DFMO that does not decrease cell viability and cell activity. 3--2,5-diphenyltetrazolium bromide (MTT) assay revealed that DFMO concentrations of up to 24 mM were not toxic to HMEpCs .6 cells and 6.45 ± 2.60 µM/1.0 × 106 cells, respectively. When Jurkat cells were cultured with 500 µM spermine, intracellular spermine concentration significantly increased to 10.63 ± 1.65 μM/1.0 × 105 cells (p = 0.008) (5 cells showed significant decrease (p < 0.001)  (indicated as N.S. in 5 cells) (5 cells) (p < 0.001). When spermine and spermindine concentrations were compared between cells cultured with spermine and those cultured with DFMO and spermine, there were no differences in intracellular spermidine (p = 0.365) and spermine (p = 0.184) concentrations.We established optimal concentrations for DFMO of 3.0 mM in Jurkat cells and 24 mM in human mammary epithelial cells (HMEpCs) and for spermine of 500 µM in both cell types. The intracellular spermidine and spermine concentrations in cultured Jurkat cells (controls = cells cultured in unsupplemented culture medium) were 8.27 ± 3.01 µM/1.0 × 10= 0.008) b, whereadecrease a. In Jur< 0.001) a, wherea5 cells) a and spe5 cells) b concent5 cells and 12.93 ± 1.30 µM/1.0 × 105 cells, respectively. When cells were cultured with 500 µM spermine, spermidine significantly decreased to 1.22 ± 0.39 μM/1.0 × 105 cells (p = 0.007) (5 cells showed no significant difference from that in control cells (5 cells (p = 0.002) (5 cells (p = 0.02) (5 cells (p = 0.016) (5 cells), but the change was not statistically significant .The intracellular spermidine and spermine concentrations in control HMEpCs were 13.79 ± 1.41 µM/1.0 × 10= 0.007) c, whereaol cells d. Intrac= 0.002) c and spe = 0.02) d. After = 0.016) c and thanificant d. When ip < 0.001) (p < 0.001) (p < 0.001) (AdoMetDC activity in Jurkat cells (50.81 ± 41.52 pmol/mg protein/min) cultured with DFMO markedly increased in comparison with that in control cells  (1.56 ± 0.77 pmol/mg protein/min) (< 0.001) a. When c< 0.001) a. With a< 0.001) a.p = 0.018) (p = 0.40 vs. control culture), but there was an increase in SAM in cells cultured with DFMO and spermine (2.10 ± 0.19 μM) (p = 0.011 vs. DFMO culture (1.02 ± 0.28 μM) (The SAM concentration in Jurkat cells cultured with DFMO (1.02 ± 0.28 μM) was lower than that in control cells (2.27 ± 0.30 μM) (= 0.018) b. No cha0.28 μM) b.p = 0.001 vs. control culture (0.34 ± 0.12 pM) and significantly decreased in response to spermine addition in the absence (0.043 ± 0.074 pM) (p = 0.031 vs. control culture (0.34 ± 0.12 pM) or presence (dcSAM concentration below the detection limit) (p < 0.001 vs. DFMO culture (7.12 ± 0.74 pM) of DFMO (The dcSAM concentration significantly increased in Jurkat cells cultured with DFMO (7.12 ± 0.74 pM) ( of DFMO c. p = 0.015 vs. control culture) (p = 0.03 vs. control culture (0.015 ± 0.006%) and the presence of DFMO (0.70 ± 0.19%) (p = 0.014 vs. DFMO culture (below the detection limit) (The dcSAM/SAM ratio in Jurkat cells was low in control cells (0.015 ± 0.006%) and markedly increased in cells cultured with DFMO (0.70 ± 0.19%) (culture) d. This rn limit) d.p = 0.59 vs. control (3.17 ± 0.14 pmol/mg protein/min) (p < 0.001 vs. control culture) and presence of DFMO (0.57 ± 0.13 nmol/mg protein/min) (p = 0.002 vs. DFMO culture) (When HMEpCs were cultured with DFMO (AdoMetDC activity 2.99 ± 0.47 pmol/mg protein/min), the increase in AdoMetDC activity observed in Jurkat cells did not occur (ein/min) e. Howeveculture) e.p < 0.001 vs. control culture (1.67 ± 0.040 μM) (p < 0.001 vs. control culture (1.67 ± 0.040 μM) (p < 0.003 vs. DFMO culture (0.71 ± 0.074 μM)  (.040 μM) f, but th.040 μM) g. SAM wa.040 μM) f but inc.074 μM) f. No dif.074 μM) g. p = 0.028 vs. control culture (1.35 ± 0.031%), and its increase was suppressed by adding spermine to cells cultured with DFMO (2.11 ± 0.04%) (p = 0.049 vs. DFMO culture (3.15 ± 0.27%) (The dcSAM/SAM ratio in HMEpCs was increased in cells cultured with DFMO (3.15 ± 0.27%) (± 0.27%) h.Tumor cells such as Jurkat cells grow rapidly, however multiplication of normal cells such as HMEpCs was very slow. Therefore, it was difficult to secure enough HMEpCs to measure protein levels and DNMT activities. Thus Jurkat cells were cultured for examination of protein levels and activities of DNMTs because sufficient enzyme can be obtained from these cells for measurement. p = 0.005 for each DNMT subtype). Addition of spermine significantly activated DNMT 1  (p = 0.069 for each). Addition of spermine to cells cultured with DFMO did not change DNMT 1 activity (0.23 ± 0.24) (p = 0.017) (p = 0.004)  d and tenculture) e and 3B culture) f with no ± 0.24) d, but DN= 0.017) e and DNM= 0.004) f were acIn this study, we have shown that changes in polyamine metabolism affect DNMT activities. Inhibitory effects of DFMO on polyamine concentrations and on activation of AdoMetDC activity varied between Jurkat cells and normal HMEpCs, which may be due to the difference of capability to maintain polyamine homeostasis. Changes in spermine concentration upon spermine supplementation also varied between the two cell types. However, changes in intracellular spermidine concentration were found in both cell types cultured with spermine, which shows that intracellular polyamine metabolism was influenced by the extracellular spermine supply. In Jurkat cells treated with DFMO, spermine supplementation increased both spermine and spermidine concentrations. Since spermidine levels were under detectable levels in cells co-cultured with 3 mM of DFMO, an increase in spermidine concentration by spermine supplementation in DFMO-treated Jurkat cells reflects increased spermine catabolism. Spermine can be converted to spermidine by the enzymatic activities of SSAT/Acetyl CoA and APAO. Spermine supplementation to control Jurkat cells (cultured in unsupplemented culture medium) decreased spermidine concentrations, suggesting ODC inhibition by a negative feedback mechanism. Decreases in spermidine concentrations by spermine supplementation were observed in HMEpCs whether cells were co-cultured with DFMO or not, suggesting that 24 mM of DFMO could not completely inhibit ODC activity. Furthermore, AdoMetDC activity was markedly decreased by spermine in both cells, suggesting that intake of spermine into cells induces a negative feedback mechanism to inhibit polyamine synthesis.As observed in both Jurkat cells and HMEpCs, dcSAM concentration in vivo is generally a few percent of the SAM concentration . The DFMdcSAM is likely to have inhibited activation of DNMT through competition with SAM ,32. Thusde novo methylation [One of the very interesting findings in this study is that changes in polyamine metabolism influenced DNMT activities without affecting their protein levels. The effect of decreased ODC activity was examined previously, and it was reported that protein levels of DNMT 3B in human oral cancer cells decreased when the ODC antizyme-1 gene, which degrades ODC and inhibits its activity, was transfected . The difhylation . A studyhylation , suggestSAM serves as a methyl group donor, therefore increases in SAM concentrations by spermine supplementation indicate increased availability of methyl group. Increased availability of methyl group and increased DNMT activity seem to be the key to maintain DNA methylation status. Supplementation of methyl group by either methionine or SAM affects the DNA methylation status ,40,41, a6 cells/mL and incubated in RPMI-1640 culture medium  containing 10% human inactivated serum  for 72 h before use for subsequent experiments. HMEpCs were adjusted to a cell density of 5.0 × 104cells/mL, incubated in serum-free medium , and subcultured according to the manufacturer’s protocol. Cells were collected for use in experiments after sufficient passages resulted in a large enough number of cells, and then incubated for 72 h in the following conditions: 1. control culture ; 2. culture mixed with D,L-alpha-difluoromethylornithine (DFMO) , an irreversible inhibitor of ODC; 3. culture with spermine ; and 4. culture with DFMO and spermine. In Jurkat cells, 3.0 mM DFMO and 500 µM spermine were used in accordance with the protocol of a previous study [Jurkat cells  and human mammary epithelial cells (HMEpCs)  were used in this study. Jurkat cells were adjusted to a cell density of 1.0 × 10us study . HMEpCs 6 Jurkat cells gated in the lymphocyte light scatter region, which were then further analyzed.Jurkat cells cultured in various conditions were fixed in 2% paraformaldehyde for 10 min at 4 °C. To cells suspended in phosphate buffered salts (PBS) containing 0.1% bovine serum albumin (BSA), the following antibodies were added (5 mL per 56105 cells): fluorescein isothiocyanate (FITC)-conjugated anti-human CD11a and FITC-ViaProbe , USA). After incubating for 20 min at 4 °C, cells were washed with 3 times PBS. A FACScan flow cytometer , USA) with CellQuest analysis software was used to identify 1 × 10H-tetrazolium bromide (MTT) . After color changed, the supernatant was removed, and 100 µl of isopropyl alcohol containing 12 M HCl was added to the wells. The absorption at 570 nm and 690 nm was determined with an automated enzyme immunoassay analyzer .HMEpCs cultured in 96-well with various concentrations of DFMO were incubated for 3–4 h in the presence of 0.35 mg/mL 3--2,5-diphenyl-2g, 3 min, 4 °C; HMEpCs: 220 × g, 5 min, 4 °C) and precipitates were suspended in 0.2 M perchlorate in 50 µL per 1.0 × 106 Jurkat cells and 1.0 × 105 HMEpCs. The suspended samples were then homogenized  using an ultrasonic homogenizer , Nishi-ku, Osaka, Japan) and vortexed. The supernatant (50 µL) obtained by centrifugation  was fluorescence-derivatized by reaction with 300 µL of dansyl chloride (10 mg/mL). The dansylated samples were redissolved in acetonitrile (500 µL). The concentrations of polyamines in extracts was determined by reversed-phase high-performance liquid chromatography  using a Capcell pak C18 MG column . 10 µL of the dansylated sample was injected per assay. The HPLC conditions were as follows: column oven at 50 °C; flow rate, 0.9 mL/min; two solvent linear gradient, solvent A consisted of 55% (v/v) 10 mM ammonium phosphate (pH 4.4) + 45% (v/v) acetonitrile, solvent B as 100% acetonitrile; the gradient condition, the solvent B 18% to 100% for 15 min and 100% for further 7 min. The measurement was conducted at an excitation wavelength of 360 nm and an emission wavelength of 500 nm and data were processed using LC Workstation chromatography software .Polyamines were extracted from cells according the previous studies with slight modifications . Cultureg, 4 °C, 3 min) after washing with 1 × PBS(-). Cell pellets obtained were suspended in 25 mM Tris-HCl buffer  containing extraction reagents , homogenized , and centrifuged  to obtain supernatants containing intracellular proteins.Jurkat cells cultured in each condition were centrifuged  after injecting the extraction buffer (300 μL) to obtain supernatants containing intracellular proteins. Protein contents in supernatants were measured by the Bradford method using bovine serum albumin as a standard. HMEpCs attached to a culture dish were washed with 1× PBS(-), collected using a cell scraper, homogenized , and centrifuged  is converted to dcSAM by AdoMetDC. A filter paper was attached inside a 2 mL Eppendorf tube cap; 20 µL of 250 mM phosphate buffer (pH 7.5) and 15 mM DTT, 15 µL 25 mM putrescine dihydrochloride, and a sample (intracellular protein solution) of 80 µL were mixed; and 10 µL of a SAM cocktail of 4.8 mM cold  SAM  and 0.2 mM SAM (carboxyl-14C) (0.1 µCi/reaction)  were added. To adsorb CO2 with alkali, 10% KOH was permeated in the filter paper on the cap. The cap was immediately shut to allow production of CO2 at 37 °C for 30 min and then the tube was cooled on ice for 15 min. To release dissolved CO2, 50 µL 6 M HCl was added to the resulting solution and the cap was immediately shut to allow reaction with CO2 at 37 °C for 15 min. Released 14CO2 was adsorbed on the filter paper. After ice-cooling, the filter paper was put into a vial containing 3.5 mL of scintillation solution and radioactivity was counted three times for 4 min each. The measuring principle for AdoMetDC activity is to determine COv/v) 0.1 M sodium acetate (adjusted to pH 4.50 with acetate) with 10 mM sodium 1-octanesulfonate  and 10% (v/v) methanol, and solvent B consisted of 90% (v/v) 0.2 M sodium acetate (pH 4.50)-acetonitrile (10:3) with 10 mM sodium 1-octanesulfonate and 10% (v/v) methanol; gradient, solvent B 0% to 100% over 50 min; UV detection at 250 nm; data were processed using LC Workstation chromatography software .Intracellular SAM and dcSAM concentrations were determined using reversed-phase HPLC. SAM and dc SAM were extracted from cells by the same method as that used for determination of the intracellular polyamine concentration. Using a Capcell pak C18 SG120 column , 20 µL of sample was injected per assay for reversed-phase HPLC. The reversed-phase HPLC conditions were: column oven at 40 °C; flow rate, 0.5 mL/min; two solvent linear gradient, where solvent A consisted of 90%  . After reacting nuclear protein solutions extracted from cells on plates covered with substances with high affinity for the respective DNMTs (37 °C for 2 h), the concentration of each DNMT was detected by a colorimetric method using a multiplate reader  at 450 nm. Since multiplication of HMEpCs was very slow, it was difficult to secure enough cells to measure protein levels and DNMT activities. Thus, these experiments were performed using Jurkat cells. An EpiQuik Nuclear Extraction Kit I , USA) was used to extract nuclear proteins, using 1 μL of extraction buffer per 1.0 × 10DNMT 1, 3A and 3B activities were determined using an EpiQuik DNA Methyltransferase 1 Activity/Inhibitor Screening Assay Core Kit , a DNMT3A Direct Activity Assay Kit , and an EpiQuik DNA Methyltransferase 3B Activity/Inhibitor Screening Assay Core Kit , USA), respectively. Nuclear protein solutions were adjusted with assay buffer to make the protein concentrations of all DNMTs equivalent. Nuclear protein solutions were incubated with assay buffer on cytosine-rich DNA-covered plates (37 °C for 2 h). DNMT 1 and 3B were detected by a colorimetric method (450 nm) and DNMT 3A was detected by horseradish peroxidase chemiluminescence (1000 ms) using a multiplate reader. Analysis was conducted using Multimode Analysis Software ver. 3.2.0.5 . DNMT activities are reported using indexes that were calculated by dividing measured activity by that of the control culture for each enzyme subtype.t-test was used for homoscedasticity and the Mann Whitney test was used for heteroscedasticity. p < 0.05 was considered to indicate a significant difference in all analyses.Statistical analyses were conducted using EZR software  . For anaDecreases in ODC activity with aging is associated with decreases in DNMT activities and changes in DNA methylation status. Increased polyamine intake elevates blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. The current study addresses the fundamental background of spermine-induced regulation of gene methylation leading to lifespan extension. In cells with decreased ODC activity, dcSAM/SAM ratio increased significantly. When ODC is suppressed, inhibition of AdoMetDC activity by spermine supplementation decreased dcSAM/SAM ratio. The decrease in dcSAM/SAM ratio is associated with an activation of DNMT 3a and 3b.


Escherichia coli has been proved to be one promising platform chassis for the production of various natural products, such as biofuels. Product toxicity is one of the main bottlenecks for achieving maximum production of biofuels. Host strain engineering is an effective approach to alleviate solvent toxicity issue in fermentation.E. coli JM109, and SecB recombinant strain was identified with the highest n-butanol tolerance. The tolerance (T) of E. coli overexpressing SecB, calculated by growth difference in the presence and absence of solvents, was determined to be 9.13% at 1.2% (v/v) butanol, which was 3.2-fold of the control strain. Random mutagenesis of SecB was implemented and homologously overexpressed in E. coli, and mutant SecBT10A was identified from 2800 variants rendering E. coli the highest butanol tolerance. Saturation mutagenesis on T10 site revealed that hydrophobic residues were required for high butanol tolerance of E. coli. Compared with wild-type (WT) SecB, the T of SecBT10A strain was further increased from 9.14 to 14.4% at 1.2% butanol, which was 5.3-fold of control strain. Remarkably, E. coli engineered with SecBT10A could tolerate as high as 1.8% butanol (~ 14.58 g/L). The binding affinity of SecBT10A toward model substrate unfolded maltose binding protein (preMBP) was 11.9-fold of that of WT SecB as determined by isothermal titration calorimetry. Residue T10 locates at the entrance of hydrophobic substrate binding groove of SecB, and might play an important role in recognition and binding of cargo proteins.Thirty chaperones were overexpressed in E. coli. Maximum butanol tolerance of E. coli could reach 1.6% and 1.8% butanol by engineering single gene of SecB or SecBT10A. Hydrophobic interaction is vital for enhanced binding affinity between SecB and cargo proteins, and therefore improved butanol tolerance.SecB chaperone was identified by chaperone mining to be effective in enhancing butanol tolerance of The online version of this article (10.1186/s13068-019-1507-7) contains supplementary material, which is available to authorized users. Escherichia coli has proved to be one of the most important platform microorganism for synthetic biology, considering its diverse genetic engineering tools for pathway and module reconstruction, genomic sequence information for metabolic engineering and rational design, and recently developed computational tools for process optimization [E. coli has been employed in the synthesis of organic solvent-like chemicals including gasoline, diesel, aviation fuel, etc. [Synthetic biology is becoming a promising alternative to synthetic chemistry and has enormous implications for the production of natural products, fine and bulk chemicals, drugs, biofuels, etc. , 2. Eschmization , 4. Due el, etc. .E. coli is one of the main bottlenecks for achieving maximum production of biofuels [E. coli could be severely inhibited [It is widely known that toxicity of bioproducts  to biofuels . Under mnhibited . Penetranhibited . Engineenhibited , 10.P). Organic solvents with logP values between 1 and 5 were found to be particularly toxic due to their similar hydrophobicity to membrane and easy penetration into membrane, especially butanol  [E. coli to butanol stress has been shown to lead to oxidative stress, acid stress, heat shock and envelope stress [Clostridium acetobutylicum by 85% through overexpression of groESL [Lactobacillus and Lactococcus strains could grow in the presence of 0.5% (v/v) butanol for 5 h, while the viability of parental strain declined after 1 h [E. coli, where coexpression of groESL and clpB led to dramatic improvements in viability with ethanol, butanol and butane-1,2,4-triol [C. acetobutylicum, E. coli and Zymomonas mobilis [Toxicity effects vary across different organic solvents and are assumed to be closely related to their hydrophobicity   ranking e stress , 13. Mice stress . Inspiree stress , 16, mole stress , TCA cyce stress , knockoue stress , global e stress , adaptive stress , and adde stress . Among tf groESL . Furtherfter 1 h . This st,4-triol . More re mobilis , 27.E. coli [E. coli was carried out to identify functional chaperones related to butanol stress response, enhance viability of host cells under organic solvent challenges, and further understand the mechanisms of chaperones in butanol tolerance.Although overexpression of a variety of chaperones might enhance microbial organic solvent tolerance, studies using genomic libraries or global strategies have often failed to identify them as overexpression targets , 29. OnlE. coli . It is oE. coli JM109 [E. coli [E. coli to high butanol titer in this study. Thirty chaperones responsible for folding/assembly  and localization  were selected from E. coli to evaluate their roles in butanol tolerance. These chaperones were NlpE, HybE, RavA, YcaL, ClpA, ClpX, CbpA, HscC, HslO, IbpA, IbpB, NfuA, PpiD, Skp, SecB, SurA, YcdY, YegD, YrhB, ClpB, HchA, GrpE, HtpG, GroEL, LolA, DjlA, BepA, YajL, DnaK and DnaJ. Although some chaperones were dependent on the co-chaperones (such as DnaK with DnaJ), solo overexpression was implemented to discern the roles of each chaperones, since the co-chaperones exist in host cells. Potential function and accession number of these chaperones are summarized in Additional file E. coli JM109. SDS-PAGE analysis showed that apparent bands were migrated at the appropriate position, indicating that all the chaperones have been successfully expressed  between 0 and 2 h (Eq. T) at 10 h were calculated  and engineered E. coli JM109 without butanol stress was regarded as control, and the screening results are shown in Fig. μ and T of the control (E. coli JM109/pQE80L) were 0.153 h−1 and 16.2%. Among all 30 chaperones tested, 19 recombinant strains displayed increased butanol tolerance, while 11 exhibited decreased tolerance. Apparently, overexpression of DjlA, LolA and PpiD severely inhibited the cell growth, with μ and T lower than 0, especially for PpiD (peptidyl–prolyl cis–trans isomerase), giving μ of − 0.07 h−1 and T of − 5.38%. Chaperones SecB, ClpB and YcdY, achieved the highest butanol tolerance of E. coli, which are protein export chaperone, protein disaggregation chaperone, and putative chaperone, respectively. The μ and T of SecB, ClpB and YcdY were 0.249 h−1 and 27.5%, 0.196 h−1 and 24.0%, 0.220 h−1 and 21.5%. Remarkably, E. coli harboring SecB displayed the highest butanol tolerance; the values of μ and T were 1.6- and 1.7-fold of those of control strain.Butanol tolerance assay was performed under 0.8% (v/v) butanol, where specific growth rate  and 1.16% (amino acid).From the crystal structure of SecB (PDB: 1QYN), holoenzyme of SecB is a homotetramer that assembles as a dimer of dimers , 34. TheE. coli and most of E. coli stopped growing, while 0.8% butanol is more moderate since the growth rates of all the tested variants were similar. In 1.0% butanol, the difference in growth was evident, with ΔOD600 from 0 to 0.5. Consequently, 1.0% butanol was regarded as the appropriate butanol stress for the high-throughput screening of SecB random mutagenesis library. Furthermore, about 2800 E. coli strains harboring SecB mutants were picked up from the random mutagenesis library for screening. Only 48 mutants displayed significant increase in butanol tolerance as summarized in Additional file T10A and SecBE8D. When further increased the butanol concentration to 1.2%, SecBT10A strain displayed 138% and 57% increase in μ and T, while the growth of SecBE8D strain was similar to that of wild-type SecB. As a result, SecBT10A was proved to be effective in enhancing robustness of E. coli under butanol stress. To verify whether the T10A mutation led to the higher expression level of SecB and therefore the increase of μ, quantitative PCR was implemented , valine (4.2), leucine (3.8), methionine (1.9) and alanine (1.8), while mutation into hydrophilic tyrosine (− 1.3) and arginine (− 4.5) led to the significantly decreased growth rate and butanol tolerance , T10L (0.148 h−1 and 12.0%), T10M (0.147 h−1 and 11.5%). Whereas for hydrophilic mutants T10Y and T10R, the lowest μ (0.070 h−1 and 0.088 h−1) and T (4.8% and 4.7%) were noted. Consequently, a hydrophobic pattern was obviously vital for butanol tolerance according to the saturation mutagenesis of T10, except for phenylalanine, a sterically hindered hydrophobic amino acid.To better understand the role of T10, saturation mutagenesis was performed at T10 residue of SecB by whole-plasmid PCR. The  leucine .8, methiE. coli harboring SecB and SecBT10A was also tested against 0.8−2.0% butanol. Without butanol, E. coli strains harboring pQE80L (control), pQE80L–secB and pQE80L–secBT10A displayed similar growth status and OD600 could reach 4.94, 4.72 and 4.71. As shown in Fig. 600 did not increase at 1.4% butanol with tolerance (T) of 0 within 10 h. The μ and T slightly decreased at 1.6% butanol to lower than 0. All above indicated that the maximum butanol-tolerant concentration of E. coli JM109 was 1.4%. SecB strain was able to grow at 1.6% butanol with μ and T of 0.013 h−1 and 0.53%, while no cell growth at 1.8% butanol and with μ and T below than 0. As expected, SecBT10A strain exhibited the highest tolerance among 3 strains, a slightly increased OD600 was observed at 1.8% butanol with μ and T of 0.012 h−1 and 1.21%. At 2.0% butanol, the μ and T of SecBT10A strain were below than 0, while the μ of SecB and control strains was − 0.078 h−1 and − 0.035 h−1, respectively. Consequently, SecB and SecBT10A could enhance the butanol tolerance of E. coli from 1.4% (control) to 1.6−1.8%.Maximum butanol tolerance of T10A, the tolerance against diverse organic solvents with logP values ranging from − 1.0 to 3.2 were determined (Table i-butanol (logP = 0.76), toluene (2.5), and cyclohexane (3.2), were unfavorable for the cell growth, while DMF (− 1.0), acetonitrile (− 0.33), ethanol (− 0.24) and i-propanol (0.39) exhibited better biocompatibility. In the presence of 0.8% octanol, all three strains could not grow at all  method [A) of engineered E. coli toward different solvents could be used to estimate the surface hydrophobicity. For hydrophobic solvents, higher A represents a higher similarity in hydrophobicity of cell surface and solvents, resulting in higher absorbance of solvents and higher inhibition on cell growth, and vice versa. As shown in Table A of E. coli harboring SecB and SecBT10A was 13% and 11% under butanol stress, lower than that of the control (14%), which also explains their improved butanol tolerance. However, for the hydrophilic solvents, such as ethanol, acetone and i-propanol, the A of SecB and SecBT10A strains was higher than those of E. coli JM109. Also, higher A was observed toward solvents with lower logP value. Our results illustrated that the cell surface of SecB and SecBT10A strains was more hydrophilic than the E. coli control, which is conducive to less absorption of hydrophobic solvents.The surface hydrophobicity of the engineered ) method . AdhesioT10A might replenish more proteins to replace the denatured proteins caused by butanol stress. Isothermal titration calorimetry (ITC) experiment was performed to measure the binding affinity between SecB and substrate proteins. The unfolded precursor form of maltose binding protein (preMBP) is one model substrate for SecB, and was selected for ITC experiment. preMBP was prepared by two rounds of PCR, in which the pre-signal was fused to MBP. SecB, SecBT10A and preMBP were purified through nickel affinity chromatography. According to the SDS-PAGE in Additional file T10A to preMBP was optimized to be 6:1. ITC was carried out by titration of SecB or SecBT10A into preMBP at 25 °C. As shown in Fig. T10A toward preMBP increased to 4.99 ± 0.15 from 3.08 ± 0.11 of WT SecB, and the equilibrium dissociation constant (KD) of SecBT10A significantly decreased to 5.2 ± 0.3 μM from 61.7 ± 1.8 μM of WT SecB, indicating 11.9-fold increase of binding affinity toward preMBP. It was apparent that SecBT10A displayed increased heat changes (ΔH) than WT SecB from − 6.18 ± 0.31 to –8.32 ± 0.67 kJ mol−1 during the reaction, however, a considerable decrease in entropy (ΔS) from − 31.2 ± 2.5 to − 27.4 ± 1.6 kJ mol−1 as shown in Fig. T10A was − 17.4 ± 0.9 kJ mol−1, while the ΔG of WT SecB was − 24.8 ± 1.5 kJ mol−1. All above proved that the specific binding of SecB to preMBP was enthalpy-driven and the mutation of threonine at residue 10 into alanine increased the binding affinity toward cargo protein.Since the main function of SecB is protein export, SecB and SecBSerratia proteamaculans (A8GLB9), same alanine appears at residue 10. While in SecB from Vibrio mimicus (D2YLC5) and Pseudomonas fluorescens (WP_060765829.1), it turns out to be glutamine.From the crystal structure Fig. b and seqE. coli, such as butanol [E. coli  after screening. SecB is a component of Sec-dependent pathway; general proteins secretion system consisted of SecA, SecB and SecYEG [E. coli, a vast majority of proteins are translocated across the cytoplasmic membrane by this Sec-dependent pathway. SecB is a highly acidic proteins, and has a long substrate binding groove, which is highly hydrophobic and recognizes the nine amino acid motif enriched in aromatic and basic residues [E. coli with high tolerance against organic solvents was discerned.Molecular chaperones could be classified into foldase , holdase , and disaggregase . Misfolding or aggregation of proteins, resulted by premature termination, miss-folding or stress, is assisted by chaperones to favor proper folding, refolding partially fold proteins, dissolve aggregates, dispose and replenish of irretrievably damaged proteins . HoweverE. coli , GroESL utylicum , Cupriav necator  and Lacts lactis , and Phaoperties  have beed SecYEG . Althougresidues . This spresidues . Besidesresidues  and streresidues , indicatE. coli could improve the butanol tolerance. Under 1.2% butanol, the specific growth rate (μ) and tolerance rate to butanol (T) were 3.6- and 3.2-fold of E. coli JM109. The maximum butanol tolerance increased from to 1.6%. Furthermore, random mutagenesis was performed on SecB, and one mutant SecBT10A was obtained with enhanced effect on butanol tolerance. Saturation mutagenesis at T10 revealed that most hydrophobic amino acids displayed similar effect, while hydrophilic amino acids led to decreased specific growth rate and tolerance rate. This T10 site locates at the entrance of hydrophobic substrate binding groove and is not conserved in SecB homologous proteins from different microorganism. The mutation of threonine into alanine was supposed to enhance the hydrophobicity of substrate binding groove and further improve the binding affinity and export efficiency toward cargo proteins. ITC experiment revealed that about 11.9-fold increase in binding affinity (KD) was found in interaction between SecBT10A and preMBP, which was consistent with the result of L42R mutation of SecB [H and decreased ΔS of SecBT10A strain suggested that T10A mutation reduced the mobility of SecB in preMBP binding and increased the proportion of energy supply during the reaction. In the case of mutation threonine into the hydrophobicity phenylalanine, the bulk phenylalanine at the entrance of substrate binding groove might interfere the binding process, which should be responsible for the decreased butanol tolerance of SecBT10F strain. Also, this unconserved T10 is an effective position for the engineering of SecB homology proteins for the secretion of cargo proteins. Finally, the butanol tolerance of E. coli could be further increased to as high as 1.8% butanol by solo SecBT10A. This engineered E. coli strain harboring SecBT10A displayed higher tolerance against i-butanol, n-butanol, and toluene. The surface of engineered E. coli cells is more hydrophilic than that of the E. coli control, which is unfavorable for the absorption of hydrophobic solvents and their penetration across the cell membrane. Recently, chaperone SecB was also evolved to improve the ability in controlling the toxin–antitoxin system of E. coli by directed evolution. Substitution of residues located in substrate binding tunnel was found to be important in modulation the binding of specific substrates without affecting the binding of presecretory proteins [E. coli.Overexpression of SecB in  of SecB . The incproteins . All aboE. coli, and SecB chaperone was proved to be the most efficient, with the highest specific growth rate (μ) and tolerance (T) even at 1.2% (v/v) butanol. Random mutagenesis of SecB revealed that T10A mutation was important for enhancing the binding affinity toward substrate proteins and further improving the butanol tolerance of E. coli. Saturation mutagenesis on T10 indicates a hydrophobic function of this site. With SecBT10A, recombinant E. coli could tolerate as high as 1.8% butanol. In the future, this SecBT10A could be combined with other butanol tolerance prompting components and more SecB homology proteins from different microorganism could be mined for further engineering the organic solvent tolerance of E. coli. Moreover, the potential of the SecB engineered E. coli would be evaluated and optimized in biobutanol synthesis by introducing clostridia coenzyme A-dependent butanol producing pathway constructed in our previous study [Thirty chaperones were evaluated for their functions in butanol tolerance of us study .n-Butanol, methanol, ethanol, i-propanol, i-butanol, toluene, cyclohexane, acetone, tetrahydrofuran, acetonitrile and dimethylformamide (DMF) were analytical grade and purchased from Sigma Aldrich. Plasmid pQE80L carrying T5 promoter and kanamycin-resistant gene was bought from Novagen and utilized for chaperones overexpression.nlpE, hybE, ravA, ycaL, clpA, clpX, cbpA, hscC, hslO, ibpA, ibpB, nfuA, ppiD, skp, secB, surA, ycdY, yegD, yrhB, clpB, hchA, grpE, htpG, groL, lolA, djlA, bepA, yajL, dnaK and dnaJ) were selected and cloned with genomic DNA of E. coli JM109 and primers as listed in Additional file BamHI and HindIII  using Exanse II . The recombinant plasmids were transformed into E. coli JM109 to form the engineered strains. All the chaperones were verified by digestion with BamHI and sequencing.Molecular chaperones were overexpressed to evaluate their role in resistance against organic solvents. Thirty molecular chaperones coding genes  culture was transferred into 30-mL fresh LB medium  and further cultivated at 37 °C and 180 rpm. When the OD600 reached to 0.3, 0.2-mM isopropyl β-d-1-thiogalactopyranoside  was added and incubated at 30 °C and 180 rpm for 6 h. Then, the cells were collected via centrifugation at 8800×g and 4 °C, and disrupted using high-pressure homogenizer . Chaperones were verified by SDS-PAGE.Each colony of engineered 600 reached to 0.3, 0.2-mM IPTG was added to induce the chaperones expression. Furthermore, when the OD600 reached to 0.7–0.8, 1.0% (v/v) n-butanol or other solvents was added into the culture. Samples were withdrawn at 2-h interval and growth status of strains was monitored at 600 nm.Tolerance to organic solvents was determined according to the growth status of each strain. When the ODμ) of E. coli strain was calculated according to Eq. T) to solvents was determined based on the growth of engineered E. coli in the presence and absence of solvent as shown in Eq. t test with a P value < 0.05 as significant difference.Specific growth rate , rTaq polymerase buffer, dNTP, 2 mM MgCl2 and 80 μM MnCl2 for amino acids mutation rate of 1.16%. PCR procedure was set as: pre-denaturation at 95 °C for 5 min, 30 cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 1 min, and further at 72 °C for 10 min. The resultant PCR products was recovered and digested with BamHI and HindШ, and inserted into pQE80L using Exnase II. The recombinant plasmids were transformed into E. coli JM109 and plated on the LB agar plate. Each mono colony was picked up and inoculated into 96-deep-well plate of LB medium and cultivated at 37 °C and 180 rpm overnight to form the random mutagenesis library of SecB.Directed evolution of SecB was implemented to further improve its performance in organic solvent tolerance. Random mutagenesis library of SecB was developed using error-prone PCR with secB-F and secB-R as primers and pET28–600 was monitored at 2-h interval for 12 h. The difference of OD600 at 12 h and 6 h was used for high-throughput butanol tolerance screening.SecB library was transferred into fresh 96-deep-well plate with LB medium and cultivated at 37 °C and 180 rpm for 0.5 h; then 0.2 mM IPTG was added and further cultivated for 1.5 h. Furthermore, 0.8%, 1.0% or 1.2% butanol was supplemented into culture and the ODDpnI to remove parental plasmids and then transformed into E. coli JM109. Then, all the E. coli JM109 strains containing SecB variants were cultivated as above mentioned and were applied for butanol tolerance evaluation.To elucidate the role of SecB in butanol tolerance, saturation mutagenesis at T10 of SecB chaperone was performed using whole-plasmid PCR with primers listed in Additional file ®480 System using 2 µL of diluted cDNA, SYBR® Premix Ex Taq II  and 0.4-µM primers. 16S rRNA gene was used as the housekeeping gene in qPCR. qPCR procedure was set as: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 55 °C for 30 s. 2Ct−ΔΔ method was adopted to evaluate the fold changes in gene expression level.Total RNA for quantitative PCR (qPCR) was isolated from cultures with and without pretreatment of 1% butanol using the Simply P Total RNA Extraction Kit . Reverse transcription PCR step was carried out using Rayscript cDNA Synthesis Kit . Primers for the qPCR are shown in Additional file A), which was a parameter to reflect the surface hydrophobicity of cells. Recombinant E. coli strains harboring pQE80L, pQE80L–secB and pQE80L–secBT10A were induced with 0.2-mM IPTG for 6 h and the cells were harvested at 8800×g and 4 °C. Furthermore, the cell pellets were washed with normal saline and the residual normal saline was thoroughly withdrawn. Then, the cells pellets were re-suspended with PBS buffer  and diluted to OD400 of 0.8–0.9, designated as Abs0. 4.8-mL above-mentioned liquids was intensively mixed with 0.8-mL diverse solvents, and kept standing for 15 min at room temperature for phase separation. The absorbance at 400 nm of water phase was determined as Abs1. A was calculated according to Eq. Microbial adhesion to solvents (MATS) method was employed to determine the adhesion  in fusion with a pre-signal peptide containing 19 residues at T10A and preMBP proteins were purified to homogeneity using nickel affinity chromatography equipped with HisTrap™ FF column . All three proteins could be washed with 300-mM imidazole. Furthermore, preMBP was desalted, concentrated and dissolved in buffer containing 0.5-mM guanidine hydrochloride, 10-mM HEPES and potassium acetate  to avoid spontaneous folding.SecB, SecBT10A and preMBP was studied by isothermal titration calorimetry (ITC) using MicroCal PEAQ-ITC . Buffer of SecB, SecBT10A and preMBP proteins was exchanged with KPB buffer  via a Superdex 75 column. Different molar ratios of SecB and preMBP, SecBT10A and preMBP, were optimized and 120-μM SecB or SecBT10A and 20-μM preMBP was obtained as the best molar ratio and protein loading concentration. Titration experiments were performed at 25 °C with initial injection of 0.4-μL SecB proteins and followed by 18 injections of 2.0-μL SecB proteins  at 120-s interval. Background experiment was performed by titration of SecB into KPB buffer solution. All the experiments were carried out in triplicate.Interaction between SecB or SecBAdditional file 1: Table S1. Function of the molecular chaperones and primers used in this study for the overexpression of chaperones. Table S2. Primers for quantitative PCR and coexpression of SecB and SecA. Table S3. Primers for the saturation mutagenesis on T10 site. Table S4. Primers for the construction of preMBP. Table S5. Sequencing result of the 48 mutants in the random mutagenesis library. Fig. S1. SDS-PAGE analysis of the overexpression of chaperones in Escherichia coli JM109. Fig. S2. Growth curves of Escherichia coli JM109 strains engineered with overexpression of different chaperones. Fig. S3. Growth profiles of E. coli JM109/pQE80L, E. coli JM109/pQE80L-ycdY and E. coli JM109/pQE80L-clpB under different butanol concentrations. Fig. S4. (A) Fold changes of expression level of secB and secA under butanol stress. (B) Growth curves of E. coli JM109 co-overexpressed with SecB and SecA in the presence of different butanol concentrations. Fig. S5. Fold changes of expression level of SecB and SecBT10A. Samples were grown in 1% (v/v) butanol and induced with 0.2 mM IPTG (red column). Fig. S6. Growth curves of recombinant E. coli JM109 harboring saturation mutagenesis variants at T10 of SecB. Fig. S7. Maximum butanol tolerance evaluation of E. coli harboring SecB and SecBT10A. Fig. S8. Growth curves of E. coli JM109 harboring SecB and SecBT10A under diverse organic solvents with different logP values. Fig. S9. SDS-PAGE analysis of the purification of SecB, SecBT10A and preMBP. Fig. S10. Isothermal titration calorimetry analysis of SecB with preMBP and SecBT10A with preMBP.


Notably, compound c4 was found to be most active among the tested calycanthaceous analogues and might be a novel potential leading compound for further development as an antifungal agent. The results could pave the way for further design and structural modification of calycanthaceous alkaloids as antimicrobial agents.A total of 29 novel tetrahydropyrroloindol-based calycanthaceous alkaloid derivatives were synthesized from indole-3-acetonitrile in good yields. The synthesized compounds were evaluated against nine strains of bacteria and a wide range of plant pathogen fungi. Bioassay results revealed that majority of the compounds displayed similar or higher in vitro antimicrobial activities than the positive control. The biological activities also indicated that substituents at R Chimonanthus praecox [As a part of conventional agriculture, agrochemicals play an important role in the protection of vegetable and cereal crops. However, the unrestricted usage of highly toxic agrochemicals over the past several decades had caused negative effects on environment and poisoned non-targeted species. Therefore, to reduce the negative impacts of agrochemicals, new compounds with high efficacy against target species are desired. Natural-product-based libraries provide a rich source for new agrochemical discovery ,2,3,4. C praecox , and hav praecox ,11,12,13 praecox ,23,24,25To the best of our knowledge, the antimicrobial activities of the synthetic derivatives are reported herein for the first time.1–8 were synthesized according to the corresponding references and their spectral data were consistent with literature values [1H-NMR, and 13C-NMR spectroscopy and ESI-MS.The synthetic route is outlined in e values ,27,28. TB. cereus, S. aureus, and S. epidermidis).Antibacterial results are shown in B. cereus, S. aureus, and S. epidermidis. Compounds a3, a7–a12, a16, a20, b1–b4 and c1–c4 showed higher degrees of activity against B. cereus than gentamicin, with b3 and a12 being the most effective, with MIC values of 7.81 µg·mL−1 and 15.63 µg·mL−1, respectively. Compound a4 showed comparable control efficacy against S. epidermidis to that of gentamicin. Noticeably, compound c4 revealed potent activity against B. cereus, S. aureus, and S. epidermidis, with MIC values of 62.5 µg·mL−1, 31.25 µg·mL−1, and 31.25 µg·mL−1, respectively.E. coli, S. typhimurium, and S. flexneri. 28 compounds showed high degrees of activity against E. coli, P. aeruginosa, and R. solanacearum. Compounds a1, a8, a11, a19, b1–b3, and c3–c4 exhibited high degrees of activity against more than two kinds of Gram-negative bacteria than that of gentamicin and streptomycin. Noticeably, compound c4 showed a broad spectrum and remarkably high activity against E. coli, S. typhimurium, S. flexneri, Escherichia sp., P. aeruginosa, and R. solanacearum, with MIC values of 31.25 µg·mL−1, 62.5 µg·mL−1, 31.25 µg·mL−1, 16.5 µg·mL−1, 62.5 µg·mL−1, and 1.96 µg·mL−1, respectively.As for inhibitory effects of the synthesized compounds against gram-negative bacteria and gram-positive bacteria, 23 compounds exhibited high degrees of activity against P. capsici, V. dahliae, F. oxysperium sp. vasinfectum, C. orbiculare, P. citrinum, Cytospora juglandis, A. sflavu, A. solani, C. lunaia, F. oxysporum, and A. niger.Inhibitory effects of calycanthaceous alkaloid analogues against a wide range of plant pathogen fungi are listed in P. capsici, V. dahliae, F. oxysperium sp. vasinfectum, and C. orbiculare. Nineteen compounds showed high degrees of activity against P. citrinum, A. sflavu, and A. solani. Nine of the synthesized compounds manifested high degrees of activity against C. lunaia. 27 compounds illustrated high degrees of activity against F. oxysporum. Twenty eight compounds manifested high degrees of activity against A. niger. Compounds a11, b2, b3, and c4 manifested higher degrees of activity against V. dahliae than amphotericin B and carbendazim, with c4 being the most effective with a MIC value of 62.5 µg·mL−1. Compounds a3, a4, a7–a8, a18, b2–b3, and c1–c2 displayed better degrees of activity against C. lunaia than that of carbendazim, with b3 and a12 being the most effective with MIC values of 62.5 µg·mL−1. Compounds a4, a7, a8, a12, a15, c1, c2, and c4 illustrated higher degrees of activity against F. oxysporum than that of amphotericin B, with a4 being the most effective with MIC value of 15.63 µg·mL−1.It is observed that this series of compounds generally exhibits more effective antimicrobial activity than the positive control. Twenty three compounds exhibited high degrees of activity against a4, a8, c1, and c2 indicated higher degrees of activity against C. lunaia and F. oxysporum than that of amphotericin B and carbendazim. Compound a7 displayed higher degrees of activity against Cytospora juglandis, C. lunaia and F. oxysporum than amphotericin B and carbendazim. Compound a8 illustrated higher degrees of activity against, C. lunaia and F. oxysporum than amphotericin B and carbendazim. Compound b2 manifested higher degrees of activity against V. dahliae, C. lunaia and Cytospora juglandis than amphotericin B and carbendazim. Compound b3 illustrated higher degrees of activity against V. dahliae, and C .lunaia than amphotericin B and carbendazim. Especially, compound c4 displayed higher degrees of activity against C. orbiculare, and C. lunaia than amphotericin B and carbendazim.Compounds 254 . Melting points were measured on an Electrothermal digital apparatus  and were uncorrected. The 1H-NMR (500 MHz), and 13C−NMR (125 MHz) were obtained on an AM-500 FT-NMR spectrometer  with CDCl3 as the solvent and TMS as the internal standard. MS were recorded under ESI conditions using a LCQ Fleet instrument . Optical rotation was measured by an Autopol II polarimeter . Yields were not optimized. The title compounds were synthesized under a nitrogen atmosphere.All reagents and solvents were reagent grade or purified according to standard methods before use. Analytical thin-layer chromatography (TLC) was performed with silica gel plates using silica gel 60 GFDMSO:VHCl = 1:5) dropwise at 0 °C. The resulting mixture was allowed to warm to room temperature. Then the mixture was stirred for 1 h. The solvents were removed to obtain the white solid. The solid was crystallized from acetone to provide the desired product 2 .To a stirred solution of indole-3-acetonitrile  in DMSO (30 mL) was added hydrogen chloride  in THF (10 mL) dropwise at 0 °C. To the resulting mixture was added benzyl chloride  in THF (5 mL) dropwise at 0 °C. Then the reaction mixture was quenched with ammonium chloride (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography on silica (petroleum ether–ethyl acetate = 4:1) gel afforded compound 3 .To a solution of NaH  in THF (20 mL) was added the compound 3  in THF (20 mL) was added LiAlH4  at 0 °C. The resulting mixture was refluxed for 2 h, then, it was allowed to reach room temperature and stirred for another 1 h, quenched with H2O (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography (petroleum ether–ethyl acetate = 2:1) on silica gel afforded compound 4 .To compound 4 in pyridine (10 mL) was added the corresponding desired reagent at 0 °C. The resulting mixture was refluxed for 2 h. The resulting mixture was allowed to warm to room temperature and then stirred for a 1 h. Then, the reaction mixture was quenched with methanol (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography on silica gel afforded the compounds a1–a21 in yields from 80% to 96%.  on silica gel afforded the compound 5 .To a solution of NaH  in THF (20 mL) was added indole-3-acetonitrile  in DMSO (30 mL) dropwise at 0 °C. The resulting mixture was allowed to reach room temperature for a further 30 min. Then, the resulting mixture was added the compound RBr in THF (5 mL) dropwise at 0 °C. The reaction mixture was quenched with ammonium chloride (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na5  in DMSO (30 mL) was added hydrogen chloride  dropwise at 0 °C. The resulting mixture was allowed to warm to room temperature and then stirred for 1 h. Then, the solvent was removed. Purification by flash chromatography (petroleum ether–ethyl acetate = 2.5:1) on silica gel afforded the compound 6 .To a stirred solution of 6  in THF (10 mL) dropwise at 0 °C. The resulting mixture was added the compound RX in THF (5 mL) dropwise at 0 °C. The reaction mixture was quenched with ammonium chloride (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography (petroleum ether–ethyl acetate = 3:1) on silica gel afforded the compound 7 .To a solution of NaH  in THF (20 mL) was added compound 7  in THF (20 mL) was added LiAlH4  at 0 °C. The resulting mixture was refluxed for 2 h, then allowed to warm to room temperature for a further 1 h. Then, the reaction mixture was quenched with H2O (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography (petroleum ether–ethyl acetate = 1.5:1) on silica gel afforded compound 8 .To compound 8 in pyridine (10 mL) was added the corresponding desired reagents at 0 °C. The resulting mixture was heated to reflux for 2 h. The resulting mixture was allowed to warm to rt. Then, the mixture was stirred for 1 h. The reaction mixture was quenched with methanol (1 mL), and extracted three times with ethyl acetate. The organic extracts were combined, washed with brine, dried over Na2SO4, and concentrated. Purification by flash chromatography on silica gel afforded the compounds b1–c4 in yields, from 91% to 94%). , to a concentration of 1.02 mg/mL, 100 μL of the solutions were added to the first well and serially diluted from first well by taking 100 μL into second. This two-fold dilution was continued down the plate and 100 μL from the 8th column of the plated discarded. The 9th column of the plate was reserved for negative control wells (without inocula) and the 10th column, for the positive growth control wells . The antibacterial concentrations were 256, 128, 64, 32, 16, 8, 4 and 2 μg/mL, respectively. The antibacterial test plates were incubated aerobically at 37 °C for 24 h, the antifungal test plates were incubated aerobically at 28 °C for 48 h. The MICs, MBC and MFC were examined. MBC and MFC were determined by plating 10 μL from each negative well and from the positive growth control on LB Agar and Sabouraud Dextrose Agar. MBC and MFC were defined as the lowest concentration yielding negative subcultures or only on colony. All tests were performed in triplicate and repeated if the results differed.4 and R5 significantly affect the activities of title calycanthaceous alkaloid analogues. Notably, compound c4 displayed the broadest and most effective activities among the tested calycanthaceous analogues and might be a novel potential leading compound for further development of antifungal agents. These results will pave the way for further design, structural modification, and development of calycanthaceous alkaloids as antimicrobial agents.Twenty nine novel tetrahydropyrroloindole-based calycanthaceous alkaloid derivatives were synthesized using indole-3-acetonitrile as the starting material via acylation at the N3 position, and their antimicrobial activity against nine strains of bacteria and a wide range of plant pathogen fungi were evaluated. According to the bioassay results, most of the title compounds showed moderate to excellent activities against the selected bacteria and a wide variety of plant pathogen fungi and were more effective than the positive controls. The compounds with substituted aliphatic groups at positions R4 and R5, showed higher antimicrobial activities than with the aromatic substituents. This suggested that substituents at R