In pH- and temperature-dependent studies, all of the peptide dendrimers were found to be stable in the temperature range up to 40–60 °C and pH values in the range of 6–12. Biological-activity studies showed these peptide dendrimers possessed improved antibacterial activity against different strains of both Gram-positive and Gram-negative strains. Together, these dendrimers also possessed potent selective antiproliferative activity against human cancer cells originating from different organs . The high hemolytic activity of [G10a]2-SHa and [G10a]3-SHa dendrimers, however, limits their use for topical treatment, such as in the case of skin infection. On the contrary, the antibacterial and anticancer activities of Jeff-[G10a]2-SHa, associated with its low hemolytic action, make it potentially suitable for systemic treatment.As the technologies for peptide synthesis and development continue to mature, antimicrobial peptides (AMPs) are being widely studied as significant contributors in medicinal chemistry research. Furthermore, the advancement in the synthesis of dendrimers’ design makes dendrimers wonderful nanostructures with distinguishing properties. This study foregrounds a temporin SHa analog, [G10a]-SHa, and its dendrimers as globular macromolecules possessing anticancer and antibacterial activities. These architectures of temporin SHa, named as [G10a]-SHa, its dendrimeric analogs [G10a] In the past few decades, with the continuous advancement in the technology for peptide development, the potential of the peptides has been thoroughly exploited. Peptide-based therapeutics have played a pivotal role in medical practice and thus has emerged as a promising tool to cure human diseases including tumors and multidrug resistant pathogen-associated infections . It promPelophylax saharicus, was identified as a potent inhibitor of Gram-positive and Gram-negative bacteria 3-SHa as well as a conjugation of dimer with the Jeffamine linker to formulate the conjugate named as Jeff-[G10a]2-SHa   and Helir pylori  was alsoibraries . Herein,0a]2-SHa . The strN-terminus and hydrophobic residues. It is cationic amphipathic and has alpha-helical moiety (net positive charge of +2 at pH 7). [G10a]2-SHa has a length of 27 residues and an amidated C-terminus. This dimer has two free amino groups at the N-terminus and hydrophobic residues. It is cationic amphipathic and has alpha-helical moiety containing D-alanine (net charge of +4 at pH 7). Similarly, [G10a]3-SHa has a length of 41 residues and an amidated C-terminus. This dimer has three free amino groups at the N-terminus and hydrophobic residues. It is cationic amphipathic and alpha-helical with D-Alanine residues (net charge of +6 at pH 7). Conversely, Jeff-[G10a]2-SHa has a length of 26 residues and an amidated C-terminus. This dimer has two free amino groups at the N-terminus and hydrophobic residues. It is cationic amphipathic (net charge of +2 at pH 7) and triple helix as secondary structure with D-Alanine residues. In addition, it has been conjugated to the lipophilic polymer Jeffamine trimer.[G10a]-SHa is D-alanine substituted analog of temporin SHa having antibacterial ,16 and a2O: TFA (60:40:0.08) were used for purification and UPLC to determine the percentage purity of all products.All the reagents and chemicals employed for this experiment were 96–98% pure. Solid support was Fmoc protected Rink amide resin having loading capacity of 0.602 mmol/g. In addition, its mesh size was 200–300. All Fmoc amino acids and coupling reagents were acquired from Novabiochem and rest of the chemicals were purchased from Aldrich. All solvents were of HPLC grade. Chemical shifts are reported in ppm. Recycling preparative HPLC (LC-908) with SP-120-10 column (C-18) and solvent system in the ratio of ACN: H1H-NMR spectrum and NMR table of temporin-SHa and [G10a]-SHa are provided in the supporting information in Temporin SHa and its linear analog [G10a]-SHa were synthesized by using solid-phase peptides synthesis strategy (SPPS) as described by us earlier . The UPL2 Jeffamine-acid linker s (SPPS) . For this (SPPS) , and itss (SPPS) . The str-MS mass  and 1H-N -10-camphorsulfonic acid was used to calibrate the output of instrument. All dendrimer peptides (1.0 mg/mL) were completely dissolved in SDS (Sodium dodecyl sulphate) in a concentrator of 15 µM and 20 mM SDS. Meanwhile, the wavelength of instrument was set between 185 to 260 nm and bandwidth of 2 nm. Four successive scans were run for each spectrum at the rate of 100 nm/min, while base line was acquired independently under the same condition, and secondary structures of all dendrimers were examined.Far-ultraviolet CD spectra were recorded in a quartz cuvette of 10 mm path length. Temperature was set at 22 °C in CDThe stability of dendrimers at different pH values and temperatures was also determined. For this, a 510 model digital pH meter (Oakton Eutech) with working electrode of glass material and a Ag/AgCl as reference electrode was used for the pH record. Dilute solutions of HCl and NaOH were added to adjust the pH of the solution from 2 to 12. Similarly, stability of monomer [G10a]-SHa and its three dendrimers was checked by heating the peptide dendrimers on different temperature ranges, i.e., room temperature, 35 °C, 40 °C, 50 °C and 60 °C.2-SHa, [G10a]3-SHa, and Jeff-[G10a]2-SHa were evaluated in solution using zetasizer . The dendrimeric solutions of peptides were transferred into transparent cuvette with care to remove bubble formation. The sample was then transferred to the cell holder and study was performed at 90° scattering angle at room temperature. The standard viscosity and refractive index were kept constant and kept at 1.0, 1.33 and 80.4 mPa, respectively.Mean hydrodynamic diameter, surface potential and PDI of dendrimeric peptides aggregates [G10a]Morphology of branched peptides was studied with a digital instrument atomic force microscope  and used to image the dendrimers nanostructures. Mica slide was used for sample drop and it was air dried at ambient temperature. Furthermore, it was mounted on microscope. The whole analysis was performed in non-contact mode. Images collected were optimized and scans were taken at velocity of 1–5 µm/s and 512 × 512-line resolution.Acinetobacter baumannii (DSM 30007); Escherichia coli (ATCC 8739); Enterobacter cloacae (DSM 30054); Helicobacter pylori (ATCC 43504); Klebsiella pneumoniae (DSM 26371); Pseudomonas aeruginosa (ATCC 9027)) and gram-positive bacteria (Bacillus subtilis (ATCC 6633); Enterococcus faecalis (DSM 2570); Enterococcus faecium (DSM 20477); Staphylococcus aureus (ATCC 6538)). In addition, 3 mL of Mueller–Hinton (MH) broth was inoculated on specific agar plates by single colonies of the different bacterial strains. Incubation of tubes was performed at 37 °C for overnight while agitation was performed at 200 rpm. The very next day, 3 mL sterile MH was used to dilute the bacteria solution by 1/100. Moreover, incubation of these bacteria was carried out at 37 °C and 200 rpm to the point where these bacteria reached their log phase growth  at 600nm around 0.6). Bacteria were then diluted in sterile medium to reach bacterial density around 105 bacteria/mL. Then, 100 μL of this bacterial suspension was added per well of 96-well microplates (Greiner BioOne) and exposed to increasing concentrations of peptides . Plates were incubated at 37 °C for 18–24 h in aerobic  or micro-aerobic conditions . At the end of the incubation, OD600nm was measured using microplate reader . The MIC was defined as the lowest concentration of peptides that completely suppressed the visible growth of the bacteria. Experiments were performed in triplicate (n = 3).Antibacterial activity of aforementioned peptides was assessed through determination of the minimal inhibitory concentration (MIC). For this, two-fold serial dilutions of antimicrobial peptides in bacterial liquid media was used, as previously reported by National Committee of Clinical Laboratory Standards NCCLS, 1997) 3-SHa < Jeff-[G10a]2-SHa < [G10a]-SHa < [G10a]2-SHa  technique was used to measure the average hydrodynamic diameter of about 149.5 nm of [G10a]−55.0 mV , which i changes . The polwas 0.34 , showingcroscopy B.3-SHa  technique illustrated the average hydrodynamic diameter of about 46.13 nm of [G10a]3-SHa A, a rangmV range  and hencmposites . The morcroscopy B.2-SHa  technique depicts the average hydrodynamic diameter of about 174.1 nm of Jeff-[G10a]2-SHa A. The ze−41.7 mV . It showmposites . The morcroscopy B. As inv2-SHa and [G10a]3-SHa showed considerable stability up to 60 °C, while the peptide conjugated with polymer Jeffamine Jeff-[G10a]2-SHa was stable at temperatures RT, 35 °C and 40 °C.Stability is an important parameter for the practical application of peptides. The change in particle size and its environment, including concentration, pH of the medium and temperature, also affect the stability of peptides. The goal of the present study is to assess the stability of dendrimeric peptides as a function of temperature and pH. The stability of monomer [G10a]-SHa and its three dendrimers was checked by heating the peptide dendrimers at different temperature ranges, i.e., room temperature, 35 °C, 40 °C, 50 °C and 60 °C, and their absorbance spectrum was recorded as shown in 2-SHa and [G10a]3-SHa were found to be significantly stable at pH values up to 10 and unstable at high pH values of 12 and 14. However, comparatively, Jeff-[G10a]2-SHa, a polymer-conjugated peptide dendrimer, showed considerably good stability at higher pH values but was not very stable at low pH ranges of pH 2 and pH 4. This may be due to the effect of the lipophilic polymer on the properties of peptides and the deprotonation of an OH functional group in a basic medium, which enhanced the stability of this conjugated peptide and reversed the trend as compared to other non-conjugated dendrimers, as shown in the Similarly, the pH of dendrimers was adjusted by the addition of dilute HCl and NaOH solutions and a study of dendrimeric peptides was also completed at different pH ranges . The pH H. pylori -SHa was not effective against E. cloacae, P. aeruginosa, and E. faecium (MIC > 100 µM). When comparing the activity of the dendrimers with [G10a]-SHa, it appeared that, depending on the strain tested, the dendrimers can have lower, similar or higher activity compared to [G10a]-SHa. For example, on A. baumannii, dendrimer [G10a]3-SHa was found to be four times more active than [G10a]-SHa, [G10a]2-SHa or Jeff-[G10a]2-SHa (MIC of 3.12 µM versus 12.5 µM). Similarly, on E. coli, [G10a]3-SHa was found to be four times more active than [G10a]-SHa (MIC of 25 µM versus 100 µM), whereas [G10a]2-SHa and Jeff-[G10a]2-SHa were found to be inactive (MIC > 100 µM). On H. pylori, [G10a]2-SHa, [G10a]3-SHa and Jeff-[G10a]2-SHa were all found to be less active than [G10a]-SHa . On K. pneumoniae, although [G10a]-SHa gave a weak activity (MIC of 100 µM), none of the dendrimers were found to be active (MIC > 100 µM). Regarding Gram-positive bacteria, although the dendrimers gave the same MIC value as [G10a]-SHa on B. subtilis , differences were found using other strains. On E. faecalis, dendrimer [G10a]2-SHa was found to be twice as active than [G10a]-SHa or the other dendrimers (MIC of 12.5 µM versus 25 µM). On E. faecium, [G10a]-SHa was found to be inactive, but the dendrimers were active with MIC ranging from 6.25 to 12.5 µM. Finally, on S. aureus, [G10a]2-SHa was found to be active like [G10a]-SHa (MIC of 3.12 µM), but [G10a]3-SHa and Jeff-[G10a]2-SHa were less active (MIC of 6.25 µM).Temporin SHa has been shown to possess antibacterial activity. Similarly, its derivative [G10a]-SHa was tested and found to be active against . pylori  and meths (MRSA) . The ant50 on the proliferation of human normal or cancer cells , except on the breast cancer MCF-7 cells (IC50 of 20.36 ± 5.64 µM), as previously described 2-SHa and [G10a]3-SHa exhibited IC50 on cancer cells ranging from 3.79 ± 1.61 to 14.31 ± 1.37 µM and 1.84 ± 0.17 to 6.94 ± 0.27 µM, respectively. With mean IC50 on human cancer cells of 6.85 ± 3.66 and 3.76 ± 1.87 µM, respectively, [G10a]2-SHa and [G10a]3-SHa are thus 2.6 and 4.8-times more active than [G10a]-SHa at inhibiting multiplying cancer cells. In addition, with IC50 values of 22.27 ± 0.40 and 13.94 ± 0.42 µM on normal human fibroblast (IMR-90 cells), dendrimers [G10a]2-SHa and [G10a]3-SHa were also found to be more selective than [G10a]-SHa at inhibiting the proliferation of human cancer cells over normal ones . Finally, dendrimer Jeff-[G10a]2-SHa was found to be less active than [G10a]-SHa or other dendrimers, with IC50 on cancer cells ranging from 33.92 ± 5.90 to > 100 µM and a mean activity of 52.13 ± 14.37 µM. In addition, with an IC50 of 66.24 ± 4.15 µM on normal human fibroblast cells, Jeff-[G10a]2-SHa was also found to be the least selective antiproliferative molecule, with a 1.2-fold difference between IC50 on human cancer and normal cells.Temporin SHa was found to be inactive in terms of preventing cell proliferation with low to no effect up to 100 µM (ICescribed . The 2-SHa and [G10a]3-SHa were, however, found to be highly hemolytic with complete hemolysis at 100 µM and an estimated HC50 of 11.55 ± 0.38 and 10.46 ± 0.36 µM, respectively. Interestingly, dendrimer Jeff-[G10a]2-SHa was shown to be the least hemolytic compound, with 24.01 ± 5.06% hemolysis at 100 µM and an estimated HC50 of 1926 ± 242 µM.In order to evaluate the safety of the dendrimers, hemolysis assay was performed using human red blood cells . WhereasH. pylori and S. aureus) peptides [2-SHa and [G10a]3-SHa, respectively. Initially, a dimer of [G10a]-SHa was synthesized to study the effect of branching on the chemical and biological properties of [G10a]-SHa. In addition, a trimer of [G10a]-SHa was synthesized, where branching was carried out through two Lysine residues at the C-terminal. In addition, temporin peptide [G10a]2-SHa was conjugated with the trifunctional polymer Jeffamine triamine linker to produce a novel hybrid material Jeff-[G10a]2-SHa, which represents the diverse functionality of peptides and flexibility of polymers. This polymer was earlier used in the identification of anticancer peptidomimetics from one-bead-one compound combinatorial libraries. 2-SHa was found to be the least active and the least selective analogs in terms of anticancer activity.Previously, [G10a]-SHa was found to be active against different human breast cancers, non-small cell lung cancers, and cervical cancers . However2-SHa and [G10a]3-SHa display high hemolytic activity at concentrations close to the ones active on bacteria or cancer cells, dendrimer Jeff-[G10a]2-SHa possesses limited hemolytic effect, making it suitable for potential medical uses.Finally, the safety of the dendrimers was measured and compared to the temporin SHa and its derivative G10a-SHa using hemolysis assay. Data obtained demonstrated that, although G10a, [G10a]Overall, although the antibacterial and anticancer activities of dendrimers of [G10a]-SHa were significantly amplified as compared to monomer [G10a]-SHa, the high hemolytic activity of these dendrimers is limiting their use to topical treatment such as in the case of skin infection. On the contrary, a Jeffamine-based dendrimeric conjugate displays antibacterial and anticancer activities associated with low hemolytic action, making it potentially suitable for systemic treatment. Further studies will be required to confirm the therapeutic values of these newly synthesized peptide-dendrimers.


The structure and chemical composition of the resultant hybrid material (LDH-KDA3) were characterized by powder X-ray diffraction (PXRD), FT-IR, FT-Raman and solid-state 13C{1H} NMR spectroscopies, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), thermogravimetric analysis (TGA), and elemental analysis (CHN). PXRD showed that the dicarboxylate guest molecules assembled into a monolayer to give a basal spacing of 18.0 Å. TGA revealed that the organic guest starts to decompose at a significantly higher temperature (ca. 330°C) than that determined for the free ketodiacid (ca. 230°C). Photochemical experiments were performed to probe the photoreactivity of the ketoacid in the crystalline state, in solution, and as a guest embedded within the photochemically-inert LDH host. Irradiation of the bulk crystalline ketoacid results in photodecarbonylation and the exclusive formation of the radical-radical combination product. Solution studies employing the standard myoglobin (Mb) assay for quantification of released CO showed that the ketoacid behaved as a photoactivatable CO-releasing molecule for transfer of CO to heme proteins, although the photoreactivity was low. No photoinduced release of CO was found for the LDH system, indicating that molecular confinement enhanced the photo-stability of the hexasubstituted ketone. To better understand the behavior of 3 under irradiation, a more comprehensive study, involving excitation of this compound in DMSO-d6 followed by 1H NMR, UV-Vis and fluorescence spectroscopy, was undertaken and further rationalized with the help of time-dependent density functional theory (TDDFT) electronic quantum calculations. The photophysical study showed the formation of a less emissive compound (or compounds). New signals in the 1H NMR spectra were attributed to photoproducts obtained via Norrish type I α-cleavage decarbonylation and Norrish type II (followed by CH3 migration) pathways. TDDFT calculations predicted that the formation of a keto-enol system (via a CH3 migration step in the type II pathway) was highly favorable and consistent with the observed spectral data.A ketodiacid, 4,4′-dicarboxylate-dicumyl ketone ( R-(+)-laurolenal to give R-(+)-1,2,3-trimethylcyclopentene -furanones  of the ketodiacid in the crystalline state, in solution, and as a guest embedded within the LDH host.Among the hexasubstituted ketones studied by Garibay and co-workers, several of the photoreactive substrates were functionalized with carboxylate groups . These mN,N′-dicyclohexylcarbodiimide, 4-(dimethylamino)pyridine, potassium hydride , methyl iodide, periodic acid, chromium(VI) oxide, Zn(NO3)2·6H2O , Al(NO3)3·9H2O , 1 M NaOH (Fluka), Na2S2O4 (Panreac), phosphate buffered saline (PBS) tablet, and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid  were obtained from commercial sources  and used as received. Lyophilized horse heart myoglobin and PBS solution were acquired from Sigma-Aldrich. THF (Aldrich) was dried over 4 Å molecular sieves. A nitrate-form Zn-Al LDH (denoted LDH-NO3) with the composition Zn4Al2(OH)12(NO3)2·2.5H2O was prepared by using the standard method of coprecipitation of the Zn2+ and Al3+ hydroxides  in the presence of nitrate ions at a constant pH of 7.5–8 under nitrogen, followed by aging of the gel at 80°C for 20 h  or  a Bruker-AMX spectrometer. Solution 13C{1H} NMR spectra were recorded on a Bruker-AMX spectrometer with an operating frequency of 101 MHz. Solid-state 13C{1H} cross-polarization (CP) magic-angle spinning (MAS) NMR spectra were recorded using a wide-bore Bruker Avance III 400 spectrometer (9.4 T) at 100.62 MHz with 3.7 µs 1H 90° pulses, 3.5 ms contact time, spinning rates of 12 kHz, and 5 s recycle delays.FT-IR spectra were collected using KBr pellets and a Mattson-7000 infrared spectrophotometer. Solution α radiation (λ = 1.54178 Å). Intensity data were collected by the step-counting method (step 0.02°), in continuous mode, in the 2θ range 3–70°. Scanning electron microscopy (SEM) images were obtained on a Hitachi SU-70 microscope at 15 kV. Samples were prepared by deposition on aluminium sample holders followed by carbon coating using an Emitech K 950 carbon evaporator. Thermogravimetric analysis (TGA) was performed using a Hitachi STA300 system at a heating rate of 5°C min−1 under air.Microanalyses for C, H, and N were carried out with a Truspec Micro CHNS 630-200-200 elemental analyzer. Powder X-ray diffraction (PXRD) data were collected at ambient temperature on a Philips Analytical Empyrean diffractometer equipped with a PIXcel 1D detector, with automatic data acquisition (X’Pert Data Collector software version 4.2) using monochromatized Cu-KBaseline-corrected absorption spectra for the Mb assays were measured from 200 to 800 nm at a scanning rate of 600 nm/min on a Cary 5000 UV-Vis-NIR spectrometer. Alternatively, a Shimadzu 2600 was used to obtain the UV-Vis absorption spectra, and a Horiba-Jobin-Yvon Spex Fluorolog 3-2.2. spectrophotometer, corrected for the instrumental response of the system, was used to record the fluorescence spectra.Structural models and representations were generated using CrystalMaker software .2Cl2 (45 ml) under inert atmosphere. A solution of p-tolylacetic acid  in CH2Cl2 (40 ml) was then added dropwise with stirring. The resultant yellow-orange mixture was stirred for 48 h at room temperature, and then the precipitated solid was removed by filtration, washed with CH2Cl2, and volatiles were removed from the filtrate by evaporation under reduced pressure. Purification of the residue by chromatography (hexane/dichloromethane 97:3) gave the substituted dibenzyl ketone 1 . 1H NMR : δ = 2.27 , 3.75 , 7.03 , 7.11 .The procedure described by 1  in dry THF (30 ml) was added dropwise to a magnetically stirred and cooled  suspension of potassium hydride  in dry THF (50 ml). After dropwise addition of methyl iodide , the yellow suspension turned pale yellow, and was heated to 85°C and refluxed for 4 h, during which time a further color change to orange occurred. The mixture was cooled to 0°C in an ice bath and an additional amount of methyl iodide  was added. After evaporation of the solvent under reduced pressure, the residue was washed with water and extracted with diethyl ether. Concentration of the organic phase yielded the derivative 2 as a pale yellow powder . 1H NMR : δ = 1.17 , 2.28 , 7.06 , 7.12 .The procedure described by 2  was added and the resultant yellow suspension was stirred for 30 min. At this point a second addition of a freshly prepared H5IO6/CrO3/MeCN mixture  was made and the mixture was stirred for a further 30 min. The solvent was then removed under reduced pressure and the resultant solid was washed several times with Milli-Q water. Purification of the product by recrystallization from acetone:water (20 ml: 40 ml) gave the ketodiacid 3 as a white powder . 1H NMR : δ = 1.25 , 7.28 , 7.85 . 13C NMR : δ = 27.5, 53.0, 126.0, 129.0, 129.5, 148.9, 167.1, 211.7.The procedure described by 3 was prepared by dissolving 3  in decarbonated deionized (DD) water (30 ml) along with 2 equiv. of 0.25 M NaOH. A solution of Zn(NO3)2·6H2O  and Al(NO3)3·9H2O  in DD water (30 ml) was added dropwise to the above solution, under nitrogen atmosphere, and 0.25 M NaOH was added simultaneously to maintain the pH of the reaction mixture at 8. Once addition of the Zn2+/Al3+ solution was complete, the resultant white suspension was stirred for 18 h at 65°C . The solid product was recovered by filtration, washed several times with DD water (0.5 L), and dried at room temperature under reduced pressure in a vacuum desiccator. Anal. Calcd for Zn4.3Al2(OH)12.6(C21H20O5)0.92(NO3)0.16(H2O)5 (973.62): C, 23.83; H, 4.24; N, 0.23. Found: C, 23.84; H, 4.24; N, 0.23%. EDS gave an average Zn/Al atomic ratio of 2.15 ± 0.05. TGA revealed a mass loss of 8.9% from ambient temperature up to 150°C .A solution of the sodium salt of the diacid 6 solutions of 3 in an NMR tube.Photolysis experiments were carried out using a 158.5 W medium pressure Peschl Ultraviolet mercury arc lamp  equipped with a circulating water cooled pyrex jacket. 2CO3  in deionized water (7 ml), and the suspension was stirred overnight at rt. The resultant solid  was recovered by filtration, washed with deionized water (2 × 5 ml), and vacuum-dried at rt. A second solid designated as iKDADI was recovered from the filtrate by evaporation of the solution to dryness. This procedure was also performed for non-irradiated LDH-KDA3, giving the solids LDHDI and KDADI. 2) In two parallel experiments, LDH-KDA3 (20 mg) was incubated in 0.01 M PBS or 0.01 M HEPES buffer solutions at rt for 5 h. The resultant solids, designated as LDHPBS and LDHHEPES, were recovered by filtration, washed with deionized water (2 × 10 ml), and vacuum-dried at rt.The stability of LDH-KDA3 in different aqueous media was explored through the following experiments: 1) A sample of LDH-KDA3 (27 mg) was irradiated in the solid-state for 12 h. The solid (iLDH) was then added to a solution of NaThe photoinduced release of CO from the synthesized compounds was assessed by using the Mb assay, in which the conversion of deoxymyoglobin (deoxy-Mb) to carbonmonoxy-myoglobin (MbCO) can be spectrophotometrically measured .The heme group functions as the active site of Mb, originating two π → π* electronic transitions: one, very intense, at about 400 nm (Soret or B band), and a second at 500–600 nm (the Q bands). The wavelengths corresponding to these wavelength maxima are governed by the oxidation, spin, and coordination states of the heme iron. As a result, it is possible to distinguish between different forms of Mb by the respective peak positions and relative optical density values of the absorption spectra.2 free) 0.1 M PBS (pH = 7.4). An aliquot of this solution was taken and bubbled with gaseous nitrogen (99.99% pure), to which freshly prepared 1 M Na2S2O4 (300 μl) was added to promote conversion of met-myoglobin into deoxy-Mb. While the solution was being bubbled, a solution (2 ml) of the ketone was added and the solution made up to 3 ml with 0.1 M PBS. After 15 min of bubbling, this solution was stored in quartz cuvettes with a magnetic stirring bar, with an optical path of 1 cm, and sealed with a Teflon stopper and parafilm to prevent any escape or entry of gas. An absorbance spectrum was immediately acquired after preparation of the samples. These were subsequently irradiated at room temperature and absorption spectra of the irradiated solutions were recorded periodically.All experiments were performed by using a stock solution of Mb, which was freshly prepared by dissolving the protein in degassed  functional, as implemented in GAMESS-US  were performed to predict the vertical electronic excitation energies.All theoretical calculations were of the DFT type, carried out using version R3 of GAMESS-US . A rangeAMESS-US , was use3 was prepared by a direct coprecipitation method. The PXRD pattern of the resultant solid, designated as LDH-KDA3, is typical of LDHs containing organic guests (l) reflections are observed at 2θ angles below 30°. The sharpness of the basal reflections indicates that the layer structure is well-ordered, i.e., the interlayer spacing is highly regular. On the other hand, the presence of several weak, broad and asymmetric non-basal reflections at 2θ angles above 30° indicates disorder in the layer stacking, such as a turbostratic distortion or an intergrowth of the rhombohedral and hexagonal polytypes (c0) can be calculated from averaging the positions of the five 00l harmonics: c0 = (d001 + 2d002 + 3d003 + 4d004 + 5d005)/5 = 18.0 Å. This is almost exactly double the value of c0 (8.89 Å) for the reference material LDH-NO3  (CSD Refcode WIVHOZ). A realistic model for the molecular structure of the deprotonated form of 3 was created by extracting a molecule of 2 from the crystal structure and replacing the p-tolyl methyl groups by carboxylate groups with a fixed C‒O bond length of 1.25 Å and a <OCO angle of 125°. ca. 52° to the hydroxide surface of the layers. Apart from the fact that this arrangement gives a gallery height of 13.2 Å, there are other features that support this model: 1) hydrogen-bonding interactions between the carboxylate oxygen atoms and the layer hydroxyl groups are fortified, while positioning the hydrophobic dicumyl ketone section of the molecule towards the center of the interlayer region; 2) the inclined orientation may allow an efficient guest packing mode in which molecules can interlock as represented in 2, when viewed down the crystallographic b axis, as shown in Subtracting the hydroxide layer thickness of 4.8 Å from the interlayer spacing of 18.0 Å for LDH-KDA3 gives a gallery height of 13.2 Å. To the best of our knowledge, no crystal structure has been reported for the ketodiacid Database  for the 3 and the intercalated material LDH-KDA3 are shown in 3 is dominated by the very strong absorption at 1,686 cm−1 assigned to overlapping ν(C=O) bands of the ketone and carboxylic acid groups. LDH-KDA3 displays this band at the same frequency, albeit with reduced relative intensity, and it is therefore assigned to ν(C=O) of the ketone group. The deprotonation of the carboxylic acid groups of the guest molecules in LDH-KDA3 is confirmed by the appearance of two strong bands attributable to νsym(CO2)  and νasym(CO2)  vibrations, together with the absence of a ν(C‒O) band . A weak, sharp band at 1,385 cm−1 that overlaps with the νsym(CO2) band may be due to an asymmetric ν3 stretching mode of nitrate ions . Bands at 1,586 and 1,609 cm−1 for the intercalated LDH are assigned to aromatic ring stretching vibrations. Below 700 cm−1, the three main bands observed at 426, 559 and 621 cm−1 are attributed to the characteristic Zn/Al-OH lattice translation modes of Zn-Al LDHs (3 (−1 (νring) and a strong band at 1,399 cm−1 [(νsym(CO2)] (The FT-IR spectra of the free ketodiacid -Al LDHs . Similar LDHs (3 . The Ramym(CO2)] .13C{1H} CP MAS NMR spectrum of LDH-KDA3 is shown in 3 in DMSO-d6. The LDH displays eight distinct resonances that have a clear counterpart in the solution spectrum. Thus, the signals for the aliphatic (20–60 ppm), aromatic (120–150 ppm) and carbonyl (170–220 ppm) carbon atoms of LDH-KDA3  are attributed as follows:  CH3 at 27.4 (27.5), C(CH3)2 at 53.4 (53.0), phenylene-CH at 126.5, 129.4, 133.6, and 147.2 , ‒CO2− at 174.1 (167.1), and C=O at 215.1 (211.7). Hence, as expected, the most significant change is the downfield shift of the carboxylate resonance owing to deprotonation.The solid-state 3  curve from 335°C for 3 to 470°C for LDH-KDA3. Comparable increases in thermal stability of organic carboxylates  have been reported for other systems, including Zn-Al LDHs containing intercalated benzenecarboxylate derivative anions, e.g., positive shifts of ca. 150°C for 5,5′-methylenedisalicylic acid , in agreement with the PXRD data.The phase purity of the material LDH-KDA3 was verified by SEM and EDS . The mor4.3Al2(OH)12.6(C21H20O5)0.92(NO3)0.16(H2O)5 is proposed for the material LDH-KDA3. As discussed above, the presence of a small amount of nitrate ions was indicated by FT-IR spectroscopy, and the nitrate content in the formula is consistent with microanalyses for N. Since neither the IR spectrum nor the solid-state 13C{1H} CP MAS NMR spectrum of LDH-KDA3 show peaks characteristic of carbonate ions, any interference from such species is deemed to be insignificant, and hence the presence of CO32− (in addition to NO3−) is not contemplated in the proposed formula.From CHN, EDS, and TGA analyses, together with the other characterization data, the formula ZnTo assess the stability of LDH-KDA3 in aqueous media, the material was incubated in two different biological buffers, 0.01 M PBS and 0.01 M HEPES, for 5 h at room temperature. No significant alterations in the IR spectrum or PXRD pattern of LDH-KDA3 were observed after these treatments , indicat3 and LDH-KDA3 were irradiated with UV light for 12 h at room temperature, giving materials designated as i3 and iLDH. The 1H NMR spectrum of i3 in DMSO-d6 was recorded and compared with that for 3 . Differences in sample crystallinity and structure may account for the contrasting photoreactivities observed. 3 (Solid samples of erved. 3  matches ν(C=O) of the ketone group, suggesting that very little or no photodecarbonylation had occurred. This was investigated further by performing deintercalation (DI) reactions in which LDH-KDA3 and iLDH were incubated overnight in a solution of Na2CO3 at room temperature. The PXRD patterns and FT-IR spectra of the resultant solids (designated as LDHDI and iLDHDI) confirmed that complete exchange of organic guest ions for carbonate ions had taken place to give Zn-Al-CO3 LDHs with the characteristic interlayer spacing of 7.6 Å (exemplified in DI). Second solids designated as KDADI and iKDADI were recovered from these experiments by evaporation of the exchange solutions to dryness. 1H NMR analysis of the phenyl group hydrogens of iKDADI confirmed, through comparison with the spectra for KDADI and the disodium salt of 3, the presence of the unchanged ketodiacid , in agreement with the FT-IR spectrum of iLDH . There was no detectable alteration in the relative intensity of the band assigned to  of iLDH .3 in DMSO-d6 was irradiated at room temperature with UV light and 1H NMR spectra were recorded for different irradiation times  shown in 3, the final products could include the dimethylbenzocyclobutenol derivative D (via Norrish-Yang cyclization) and the keto-enol system E (via a methyl migration reaction)  , which c3 under irradiation, a more comprehensive photophysical study, involving excitation of this compound in DMSO-d6 followed by UV-Vis and fluorescence spectroscopy, was undertaken. To better understand the behavior of λexc) at 290 nm to avoid the superimposition of the Raman peak of the solvent. The dependence of the absorption spectra of ketoacid 3 on the irradiation time shows an increase of the 304 nm band and a decrease of the ∼314 nm and ∼327 nm bands. Although an isosbestic point could not be selected for excitation to obtain the emission spectra (to avoid the overlap of the Raman peak of the solvent in the emission spectra), with the λexc = 290 nm used for excitation (close to the peak maximum of 304 nm) the absorbance value increases with the irradiation time, whereas the emission band  decreases with the irradiation time. This shows that a new and less emissive compound is being formed at the expense of photodegradation of ketoacid 3 (see The emission spectra were collected with excitation (id 3 see .3 and of the photoproducts (A–E). Indeed, for ketoacid 3 and photoproduct E, an absorption band  with maxima at ca. 316–317 nm  matches with the longest wavelength vibronically resolved absorption band  do not show evidence of theoretically predicted low energy n,π* forbidden transitions. Moreover, the oscillator strength associated with the emission at 380 nm (3 (f = 0.01) is higher than that of photoproduct E (f = 0.002), thus attesting, once more, that formation of this photoproduct is highly favorable and takes place through the proposed Norrish type mechanism , which displayed the methine resonance in the range 4.7–4.8 ppm with CDCl3 as solvent  in the two experiments with different Mb concentrations.With ketodiacid  for 7 h  and one  for 7 h . Data in3 is inhibited.Regarding the material LDH-KDA3, suspensions of the solid in 0.1 M PBS buffer solution (0.8 g/L) were irradiated in the presence of two different concentrations of Mb (30 and 60 μM). As shown in 2, the ditolyl precursor of 3, was performed. Due to the low solubility of 2 in aqueous solution, the Mb assay was performed with a solution of 1% DMSO to solubilize 2  host-guest interactions, the compound, thus inhibiting triplet formation and α-cleavage to give an acyl-alkyl radical pair, followed by loss of CO from the acyl radical fragment. As mentioned above, other studies with crystalline dicumyl ketone derivatives are consistent with this observation since they showed that intermolecular hydrogen bonds can quench photodecarbonylation  via a methyl migration reaction is proposed, although the co-formation of photoproduct D (through Norrish-Yang cyclization) cannot be discarded. In future work it would be desirable to study more photoreactive ketones to probe the influence of the supramolecular ordering on the photochemistry of the guest molecules.In the present work we have demonstrated the enhanced thermal and photo-stability of a para-substituted dicumyl ketone intercalated in a layered double hydroxide. Due to the diminished photoreactivity, the study of the photochemistry of the ketodiacid (with respect to photoproduct selectivities) was limited to a comparison of results obtained for the compound in solution and in the crystalline state. The work has, nevertheless, worked as a proof of concept concerning the use of LDHs as hosts for the supramolecular organization of hexasubstituted ketones. Concerning the photoreactivity of


It was demonstrated the addition of NO3− (50 to 800 μmol L−1) significantly promoted denitrification rates, as expected by Michaelis-Menten kinetics. Ponds with higher NO3− concentrations in the overlying water responded more greatly to NO3− additions. Moreover, N2O production was also promoted by such an addition of NO3−. Denitrification was significantly inhibited by the elevation of DO concentration from 0 to 2 mg L−1, after which no significant increase in inhibition was observed. Denitrification rates increased when organic C was introduced. Due to the abundant NH4+ in pond sediments, the addition demonstrated little influence on nitrogen removal. Moreover, anammox rates showed no significant changes to any amendment.Denitrification and anammox occur widely in aquatic ecosystems serving vital roles in nitrogen pollution removal. However, small waterbodies are sensitive to external influences; stormwater runoff carrying nutrients and oxygen, flows into waterbodies resulting in a disruption of geochemical and microbial processes. Nonetheless, little is known about how these short-term external inputs affect the microbial processes of nitrogen removal in small waterbodies. To investigate the effects of NO Durinerbodies . They caerbodies , leadingerbodies . Furthererbodies , but theerbodies . The nuterbodies ,13,14.3− and NH4+ provide substrates for denitrification and anammox processes, which can directly affect the microbial nitrogen removal [The nitrogen cycle is recognized as one of the most important biogeochemical processes on earth . Environ removal ,21. The  removal ; while a removal . The con removal ,25. Alth removal ,27, the 15N tracer techniques were used in this study to investigate the influence of NO3−, NH4+, DO and TOC on nitrogen removal rates in pond sediments. The objective of this study was to (i) identify factors influence the denitrification process and to (ii) quantify their effects on the sediments of small waterbodies.What are the short-term effects of runoff into waterbodies on the microbial nitrogen removal processes? In this context, slurry incubation experiments combined with Samples were collected from three ponds  located in Chongqing, southwest China  in July 2021. Precipitation in July was 189 mm which was relatively high throughout the year with annual mean of 109 mm in 2021 and resulted in runoff confluence into ponds regularly in this season. At each pond, three parallel surface sediments (0–10 cm) and overlying water were sampled. Field samples were stored in sterile plastic bags and transported to the laboratory in a cooler box (4 °C) for subsequent analysis. One subsample was stored in 4 °C refrigerator for microbial nitrogen removal regulatory experiments, and the second subsample was used for physicochemical analyses. All samples were analyzed in triplicate, and the values were averaged to represent site conditions.4+, NO2−, NO3− in overlying water were determined via a spectrophotometric detection assay [4+, NO2−, NO3− were extracted from 5 g of fresh sediment/soil with 25 mL of 2 M KCl (1:5 w/v). The supernatant was filtered through a 0.22 μm membrane filter and the compounds were determined via a spectrophotometric detection assay. Moisture content was measured by oven-drying at 105 °C until a constant weight was achieved. The pH was determined in 1:2.5 sediment/water (w/v) suspensions after shaking and centrifugation, with a Mettler Toledo pH analyzer . Organic matter (OM) was measured as loss on ignition at 550 °C (LOI 550) using a Muffle furnace. The TN and TP were determined with the potassium persulfate oxidation-ultraviolet spectrometry method [NHon assay . pH in oy method , using aw/v) agarose gel electrophoresis.DNA was extracted from sediment samples (approximately 0.5 g) using the FastDNA SPIN Kit for Soil  following the manufacturer’s protocol. The concentration of extracted DNA was measured with a NanoDrop Lite , and the DNA quality determined by means of 1%  were defined by Usearch . Rare OTUs with less than 0.01% of the total sequences were excluded. To avoid biases arising from sequencing depth and to make samples comparable, sequences were rarefied to a uniform sequencing depth based on the sample with the lowest sequences. The raw sequences of nirS and Anammox-specific 16S rRNA genes used in this study were deposited in the Sequence Read Archive  of NCBI under the accession numbers PRJNA844121.The ommunity . Primersommunity . More deommunity  and Mothommunity . OTUs winir gene (nirS and nirK), nosZ gene (nosZ I and nosZ II) and anammox-specific 16S rRNA genes in sediments were quantified by a LightCycler® R480 II Real-Time PCR . Each sample was analyzed in triplicates. The standard curves used for calculation were achieved with plasmid DNA with known concentrations and copy numbers. Results of qPCR with high amplification efficiency (90–110%) and correlation coefficient values of the standard curve (r2 > 0.97) were included in the analysis. The specificity of PCR amplifications was defined by melting curve analysis and gel electrophoresis. Primers, reaction systems and procedures are shown in The abundance 3−, NH4+, dissolved oxygen (DO) and organic C (as glucose) concentrations on the rates of denitrification, anammox and N2O production in pond sediments. In the nitrogen addition experiments, five concentration gradients of 15NO3−  and five concentration gradients of NH4+  were set up in the incubations. The regulation of DO was achieved through replacement, by injecting oxygen-rich water (12 mg L−1) to replace the supernatant to reach different DO gradients . The organic C experiments were set up at 0, 100, 200, 400 and 600 μmol glucose L−1, respectively. Four sets of parallel regulation experiments were conducted on three different ponds  and each gradient was incubated in triplicate.Four parallel incubations were performed to investigate the effects of NOx− (NO3− and NO2−) and DO, the slurries were pre-incubated in the dark at an in situ temperature (28 ℃) for 36–48 h. After pre-incubation, the slurries were transferred to 12.5 mL tubes . The tubes were injected with the designed substrates and incubated in the incubator at in situ temperature. The final concentration of NO3− was fixed at 100 μmol L− for treatments concerning addition of DO, organic C and ammonium. The slurries incubation was thereafter terminated by adding 200 μL of 50% ZnCl2 at 0 and 2 h from the beginning of incubation. 29N2 and 30N2 signals in the tubes were detected with a membrane inlet mass spectrometry . Detailed methods are described in Cai, et al. [The potential nitrogen removal rates of sediments were measured using slurry incubation and isotope pairing techniques . Fresh s, et al. .2O production rates were measured with headspace equilibrium gas chromatography using the samples prepared as described above [2O in the headspace gas was detected by means of gas chromatography . Detailed methods are described in Cai, et al. [Ned above . The tub, et al. .One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis was used to test significant differences in potential rates among different concentration gradients . Linear and Michaelis-Menten kinetics were fitted to the data using the regression function of the Prism 8 software (version 8.0.2).3− was highly varied with values of 58.3 ± 5.8, 103.8 ± 7.3 and 10.5 ± 1.7 μmol L−1 in pond 1#, 2# and 3# respectively. NH4+ concentrations were determined at 7.8 ± 0.7, 24.4 ± 3.5 and 7.6 ± 1.8 μmol L−1 while the NO2− concentrations in the overlying water were 3.5 ± 1.2, 7.3 ± 1.1 and 12.6 ± 4.1 μmol L−1, respectively.The overlying water of the three ponds studied had pH values ranging from 7.4 ± 1.2 to 7.6 ± 0.9 . The con4+ concentration was measured to be 13.0 ± 2.4, 21.2 ± 1.9 and 8.6 ± 1.5 mg kg−1 in the three ponds, and was the main form of dissolved inorganic nitrogen (DIN) accounting for 93.5, 96.4 and 93.5% of DIN, respectively. The contents of TN and TP were 2.2 ± 1.1, 3.0 ± 0.8, 6.7 ± 1.2 g kg−1 and 0.2 ± 0.0, 1.1 ± 0.3, 1.5 ± 0.2 g kg−1, in the three respective ponds. All ponds had a high OM, although the lowest value was found in pond 2# (65.2 ± 11.2 g kg−1), while the other two ponds were higher, i.e., 113.0 ± 15.7 g kg−1 in pond 1# and 105.0 ± 12.8 g kg−1 in pond 3#.In sediments, pH in pond 1# (6.9 ± 1.0) was lower than the two other ponds (7.5 ± 0.8 and 7.6 ±0.5). The NHnir genes, genes related to denitrification, was the group of genes most highly abundant in all three ponds. nirS gene abundance was observed at (1.8 ± 0.0) × 108, (6.4 ± 0.2) × 108 and (5.2 ± 0.0) × 108 copies g−1, for pond 1#, 2# and 3#, respectively. The abundance of nirK gene was determined to be (1.5 ± 0.1) × 108, (5.2 ± 0.2) × 107 and (5.5 ± 0.3) × 107 copies g−1 in the three ponds, respectively. The genes related to N2O-reduction were of lower abundance than nir gene, of which nosZ I was found to be present at (1.1 ± 0.0) × 106, (1.2 ± 0.7) × 106 and (4.2 ± 0.0) × 106 copies g−1. The abundance of the nosZ II gene in pond surface sediments was determined to be (2.8 ± 0.5) × 107, (1.4 ± 0.3) × 107 and (12.1 ± 1.7) × 107 copies g−1. Additionally, the abundance of the anammox bacterial 16S rRNA gene was 2–4 magnitudes lower than the denitrification functional genes, which varied from 1.5 × 104 to 2.8 × 104 copies g−1.The abundance of denitrification- and anammox-related genes in the three ponds are reported in For denitrifiers, the 40 most abundant OTUs, containing 92.4% of nirS sequences, all affiliated to Proteobacteria. At the genus level, three genera including Steroidobacter, Azoarcus and Dechloromonas were identified across the pond sediments a. Azoarc−1 h−1 with the addition of NO3− from 50 to 200 μmol L−1 . The response of potential denitrification rates to increasing NO3− concentrations was in accordance with the Michaelis-Menten kinetics, demonstrating a maximum denitrification rate (Vmax) of 39.0 nmol N g−1 h−1 and an affinity constant (Km) of 109.5 μmol L−1. Ponds with higher NO3− concentrations in the overlying water had a greater response to NO3− addition in sediment , and remained low at 0.3 ± 0.4 nmol N g−1 h−1  of 41.8 μmol L−1 and a maximum rate (Vmax) of 8.8 nmol N g−1 h−1. N2O production rates in pond 2#, the pond with the highest NO3− concentration in the overlying water, were enhanced 1.6-fold, while the pond with the lowest NO3− concentration, pond 3#, showed only a 0.3-fold increase . The relative contribution of anammox to nitrogen removal (ra%) was low, varying from 4.8 to 11.6%. Similarly, the addition of NH4+ exerted no effect on the N2O production rates and the relative proportions of N2O to N2 and N2O .The addition of NHp = 0.011, R2 = 0.40, −1, the introduction of DO significantly decreased denitrification rate from 20.0 to 15.1 nmol N g−1 h−1 , but hereafter no significant change was observed when the DO concentration was further increased . There was no significant correlation between DO and anammox rates , whose rates ranged from 1.1 to 1.2 nmol N g−1 h−1. Furthermore, the relative contribution of anammox to nitrogen removal was always lower than 10%. As the DO concentration increased, N2O production rates showed no significant change, varying from 3.4 to 5.4 nmol N g−1 h−1 . An increase in DO also had no effect on the relative proportions of N2O .Generally, denitrification in the sediment was observed to be inhibited by the elevation of oxygen . No significant changes were observed with further glucose addition beyond 200 μmol L−1 . However, the response of the potential denitrification rate to the addition of glucose in the various pond sediments was related to the OM contents in sediments  which had lower OM content, than in pond 1# and 3#, which both had higher OM contents . Anammox rates ranged from 1.6 to 1.8 nmol N g−1 h−1 , but showed no response to the addition of glucose. In contrast to the group without glucose addition, the N2O production rates were significantly higher when glucose concentration was 200 μmol L−1 . With the addition of glucose, no significant change to the relative proportions of N2O was observed .Potential denitrification rates can be enhanced by the addition of glucose b. Until ediments . With thSmall waterbodies frequently receive high loads of pollution from human activities and stormwater runoff due to their small area and close proximity to human settlements ,38. Comp3− was the most important parameter altering the microbial nitrogen cycle in sediment. The artificial addition of NO3− significantly increased the potential denitrification rates 1.8-fold when the NO3− concentration increased from 50 to 800 μM. These results suggest that the denitrification process in such small waterbodies was limited by NO3−, which are consistent with studies conducted on rivers, estuary sediments and paddy soils as the addition of NO3− alleviates the limitation of substrate deficiency process [nirS, nirK, nosZ I and nosZ II) were detected with high copy numbers (106–109 copies g−1) in pond sediments, being higher than those reported for marine and freshwater sediments [3− concentrations the better the denitrification genes expressed [3− may induce more expression of denitrification genes, leading to more NO3− being reduced to gas and released. Since the anammox rates remained low in the present study, we can conclude that the nitrogen removed mainly occurred through added NO3− via denitrification. In the meantime, denitrification functional genes were 2–4 magnitudes more abundant than the anammox functional genes, which could explain the domination of denitrification process. Moreover, the sediment with a lower NO3− concentration in the overlying water (pond 3#) showed a lower Vmax for denitrification with NO3− addition. This can be explained by microbial denitrifiers in poor NO3− habitats being well-adapted to such environments, and so their denitrification rates respond weakly to NO3− additions [3− addition of the three ponds sediment may be ascribed to their community compositions which showed significant difference [4+ exerted no effect on denitrification and anammox rates in pond sediments. This is probably due to NH4+ usually being in surplus and is therefore not a limiting factor for denitrification in sediments [Our study showed that the NO process ,42,43. H process . Furtherediments ,47,48,49dditions . There wfference . The addediments .−1. Organic C acts as an electron donor in heterotrophic denitrification [15NO3− was also added into the simulation system  to measure the denitrification and anammox rates in the test of organic C addition. As the substrate of denitrification, NO3− often acts as a limiting factor in such habitats [3− is deficient in situ. On the contrary, DO elevation exerted negative effects on denitrification. Lower DO levels create ideal reduction conditions for the denitrification process [2− reductase (encoded by nirS and nirK gene) could be inhibited by high DO concentrations, leading to the accumulation of NO2−, which consequently limits denitrification [2−) for denitrification and anammox. Although the nitrogen removal from the sediment was inhibited immediately after the elevation of DO, the nitrogen could still be removed in the long term.The addition of glucose was found to promote the denitrification rates 0.8-fold when the concentration was increased to 200 μmol Lfication . Therefofication ,54. Howehabitats . Therefo process ,57. Furtfication ,59,60. H2O emission during denitrification is also of concern. The denitrification genes nir (N2O producer) and nosZ (N2O reducer) are often used as genetic markers for N2O production [2O-producing and N2O-reducing microorganisms (nir/nosZ ratio) can partly explain N2O emissions, which were positively correlated in some studies [nir/nosZ and N2O/(N2O  +  N2) ratio were also observed in previous studies [2O emissions are not only controlled by genetic potential, but also by transcriptional regulation and enzymatic activity [3− also significantly influenced the N2O production not only denitrification. Although N2O production increased, the proportion of N2O production in nitrogen loss (N2O/(N2 + N2O)) decreased. The addition of NO3− may promote the expression of N2O production and reduction genes [2O increased less than N2. In other words, the addition of NO3− not only promoted nitrogen removal, but also reduced greenhouse gas emissions, which is more environmentally beneficial. It was found that the addition of C sources such as glucose increased the abundance and expression of nosZ gene, but had no effect on nirS gene [2− reductase, which consequently reduced production of N2O and N2 [Noduction . The pro studies ,63. Neve studies ,65. Theractivity . Howeveron genes , so N2O irS gene ,68. FurtO and N2 ,59,60.4+, DO, NO3− and organic C from domestic pollution, rainfall runoff, and agricultural pollution to waterbodies. The present results showed that NO3− and organic C promoted denitrification and nitrogen removal from water sediments until a certain extend. Although DO inhibited denitrification in the short term, nitrification could be activated over time, which supplies substrates for the denitrification process and can thereby promote nitrogen removal. Since NH4+ in the sediment was in surplus, the addition of NH4+ showed no significant effect on nitrogen removal from the pond. Small waterbodies play an essential role in agricultural watersheds to store nitrogen and mitigate the pollution output from the watersheds [3− is the dominant form of nitrogen in runoff [3− [External inputs usually carry NHtersheds . NO3− isn runoff , primarin runoff , and thenoff [3− . Our stu


De novo initiation by viral RNA-dependent RNA polymerases often requires a polymerase priming residue, located within a priming loop, to stabilize the initiating NTPs. Polymerase structures from three different non-segmented negative strand RNA virus (nsNSV) families revealed putative priming loops in different conformations, and an aromatic priming residue has been identified in the rhabdovirus polymerase. In a previous study of the respiratory syncytial virus (RSV) polymerase, we found that Tyr1276, the L protein aromatic amino acid residue that most closely aligns with the rhabdovirus priming residue, is not required for RNA synthesis but two nearby residues, Pro1261 and Trp1262, were required. In this study, we examined the roles of Pro1261 and Trp1262 in RNA synthesis initiation. Biochemical studies showed that substitution of Pro1261 inhibited RNA synthesis initiation without inhibiting back-priming, indicating a defect in initiation. Biochemical and minigenome experiments showed that the initiation defect incurred by a P1261A substitution could be rescued by factors that would be expected to increase the stability of the initiation complex, specifically increased NTP concentration, manganese, and a more efficient promoter sequence. These findings indicate that Pro1261 of the RSV L protein plays a role in initiation, most likely in stabilizing the initiation complex. However, we found that substitution of the corresponding proline residue in a filovirus polymerase had no effect on RNA synthesis initiation or elongation. These results indicate that despite similarities between the nsNSV polymerases, there are differences in the features required for RNA synthesis initiation. Mononegavirales, the non-segmented, negative strand RNA viruses (nsNSVs). Like other viruses in this order, RSV encodes an RNA dependent RNA polymerase, which is responsible for transcribing and replicating the viral genome. Due to its essential role during the viral replication cycle, the polymerase is a promising candidate target for antiviral inhibitors and so a greater understanding of the mechanistic basis of its activities could aid antiviral drug development. In this study, we identified an amino acid residue within the RSV polymerase that appears to stabilize the RNA synthesis initiation complex and showed that it plays a role in both transcription and RNA replication. However, the corresponding residue in a different nsNSV polymerase does not appear to play a similar role. This work reveals a key feature of the RSV polymerase but identifies differences with the polymerases of other related viruses.RSV has a significant impact on human health. It is the major cause of respiratory disease in infants and exerts a significant toll on the elderly and immunocompromised. RSV is a member of the  The non-segmented negative strand RNA viruses (nsNSVs) are a large and diverse order of viruses, currently comprising 11 virus families. These families include, amongst others, the filoviruses, such as Ebola and Marburg virus, the paramyxoviruses, such as parainfluenza viruses types 3 and 5 (PIV-3 and PIV-5), the rhabdoviruses, rabies and vesicular stomatitis virus (VSV) and the pneumoviruses, respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) , 3. The leader (le) promoter region. The key promoter residues are contained within the first 12 nt of the 44-nt le region  ATP included as the radioactive tracer. CTP was omitted to prevent initiation from position 1U  and the α-phosphate of the incoming nucleotide (NTP2 during initiation) enabling the nucleophilic attack required for phosphodiester bond formation -GTP tracer were performed in 25 μL reactions with the following conditions: 2 μM RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MgCl2, 5 mM DTT, 10% glycerol, 500 μM (each) ATP, CTP, GTP, and UTP with 5 μCi [α-32P]-GTP (3000 Ci/mmol). Reaction mixtures were preincubated at 30°C for 5 minutes prior to addition of L-P complexes to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h and then heat inactivated at 90°C for 3 minutes then allowed to cool on ice for 2 minutes. RNA was extracted with phenol-chloroform and ethanol precipitated. Pellets were resuspended in RNase free water, and an equal volume of stop buffer  was added. Products were migrated alongside molecular weight ladders representing products initiated from 1U or 3C sites, prepared as described previously -CTP (3000 Ci/mmol). Reactions to detect pppGpA and elongation products generated from position 3 of the promoter were performed in a volume of 25 μL with the following conditions: 2 μM RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MgCl2, 5 mM DTT, 10% glycerol, 10 or 500 μM ATP, 10 or 500 μM GTP, and 500 μM UTP with 5 μCi [α-32P]-ATP (3000 Ci/mmol). In all cases, reaction mixtures were preincubated at 30°C for 5 minutes prior to addition of L-P complexes to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h and then heat inactivated at 90°C for 3 minutes then allowed to cool on ice for 2 minutes. Following inactivation of the polymerase, all reactions were adjusted to contain 500 μM GTP  and RNA was extracted with phenol-chloroform and ethanol precipitated. Pellets were resuspended in RNase free water, and an equal volume of stop buffer  was added. The samples were subjected to denaturing electrophoresis alongside T7 triphosphorylated RNA markers by electrophoresis on 25% polyacrylamide gels containing 7 M urea in Tris-taurine-EDTA buffer. Gels were analyzed directly (without drying) by autoradiography and phosphorimage analysis.Reactions to detect pppApC formation from position 1 of the promoter were performed in a volume of 25 μL with the following conditions: 2 μM 2 or MgCl2, 5 mM DTT, 10% glycerol. The pppGpC reactions contained le 1–14 2G/A RNA template, 20 μM (each) GTP and CTP, and 5 μCi [α32P]-CTP (3000 Ci/mmol) as the radioactive tracer. The pppApC reactions contained tr 1–16 RNA template, 20 μM (each) ATP and CTP, and 5 μCi [α32P]-CTP (3000 Ci/mmol) as the radioactive tracer. The pppGpA reactions contained tr 1–16 RNA template, 20 μM (each) GTP and ATP, and 5 μCi [α32P]-GTP (3000 Ci/mmol) as the radioactive tracer. Reaction mixes were preincubated prior to addition of L-P protein to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h, heat inactivated at 95°C for 5 minutes, and allowed to cool on ice for 2 minutes. Reactions were adjusted to contain 500 μM GTP after inactivation of the polymerase and RNA was purified and analyzed as described above.Reactions were performed in a volume of 25 μL with the following conditions: 2 μM RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MnClin vitro transcription. Annealed oligonucleotides were prepared by mixing 4 μM short oligonucleotide and 4 μM long overhang (template) oligonucleotide in Tris-EDTA pH 8.0, incubated at 90°C for 3 minutes, then allowed to cool to room temperature. 40 μL in vitro transcription reactions were prepared with the following conditions: 40 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 2 mM spermidine, 10 mM DTT, 0.1 mg/mL bovine serum albumin, 2 mM ATP, 1 mM CTP, 1 mM GTP with 1 μCi [α-32P] ATP, [α-32P] CTP, or [α-32P] GTP depending on the marker being synthesized, 0.1 μM DNA annealed oligos and 1 unit/μL T7 RNA polymerase (New England Biolabs). Reactions were incubated at 37°C for 5 hours. Samples were treated with 1 μL DNase I (New England Biolabs) for 10 minutes at 37°C. 1 μL 0.5 M EDTA was added to the reactions. Products were subjected to phenol chloroform extraction and ethanol precipitation prior to use as markers.Markers consisting of 5´ triphosphorylated RNA used to characterize small RNA products were generated using a method adapted from . DNA olile 1–30 RNA oligonucleotide (Dharmacon), 50 mM Tris, pH 7.4, 8 mM MgCl2, 5 mM DTT, 10% glycerol, 500 μM (each) ATP and GTP, 10 μM CTP, and 100 μM UTP, with 10 μCi [a-32P]-CTP (3000 Ci/mmol) as the radioactive tracer. Reaction mixes were preincubated prior to addition of L-VP35 complexes to a concentration of 8 nM L protein. Reaction mixes were incubated at 30°C for 1 h, heat inactivated at 95°C for 5 minutes, and allowed to cool on ice for 2 minutes. RNA was purified and analyzed on 25% Tris-taurine gels, as described above.Reactions were performed in a volume of 25 μL with the following conditions: 2 μM MARV 2PO4, 20 mM imidazole) for 15 minutes on ice, treated with Turbo DNase (Invitrogen) for 5 minutes at 37°C, and then migrated on a 10% SDS polyacrylamide gel and analyzed by Western blot analysis using a monoclonal antibody specific for the FLAG tag (Sigma). Proteins were detected using a goat anti-mouse IgG coupled to IRDye 800CW and LI-COR analysis.To confirm that each of the mutant L proteins could be expressed in mammalian cells, BSR-T7/5 cells were transfected with pTM1 plasmids containing FLAG-tagged versions of wt or mutant L plasmids. Cells in a 6-well plate were transfected with 2 μg of pTM1 P and 1 μg wt or mutant L using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The cells were incubated at 30°C or 37°C (as indicated) and at 48 h post-transfection, they were collected and incubated with lysis buffer Click here for additional data file.S2 Figle promoter (shown in (A) L mutant proteins containing an N-terminal FLAG tag were expressed in mammalian cells at 30°C. At 48 h post transfection, cell lysates were analyzed by Western blot analysis using a FLAG-specific antibody. The image is representative of three independent experiments. (B) Analysis of RNA products generated from the 1U and 3C sites in a minigenome containing a shown in  by mutanshown in .(TIF)Click here for additional data file.


Smyd5, but not the C-terminal domain-truncated Smyd5, restores H3K36me3 at promoters in Smyd5 knockout cells. Furthermore, elevated Smyd5 expression contributes to tumorigenesis in liver hepatocellular carcinoma. Together, our findings identify SMYD5 as the H3K36me3 methyltransferase at promoters that regulates gene expression, providing insights into the localization and function of H3K36me3.Histone marks, carriers of epigenetic information, regulate gene expression. In mammalian cells, H3K36me3 is mainly catalyzed by SETD2 at gene body regions. Here, we find that in addition to gene body regions, H3K36me3 is enriched at promoters in primary cells. Through screening, we identify SMYD5, which is recruited to chromatin by RNA polymerase II, as a methyltransferase catalyzing H3K36me3 at promoters. The enzymatic activity of SMYD5 is dependent on its C-terminal glutamic acid-rich domain. Overexpression of full-length  SETD2 methylates histone H3K36me3 in gene bodies in mammalian cells. Here the authors show H3K36me3 is also enriched at the promoter regions, and that this methylation is carried out by SMYD5, which is recruited by RNA polymerase II. They furthermore show SMYD5 is elevated in liver cancer and is correlated with changes in gene expression. Four core histones are extensively modified posttranslationally at their N- and C-terminal tails, including methylation, acetylation, phosphorylation, and ubiquitylation. Histone tails are heavily enriched with positively charged amino acids to interact with negatively charged DNA, and their modification can alter chromatin structure to modulate gene expression5.Nucleosomes, the basic repeating units of chromatin, consist of one core histone H3–H4 tetramer and two core histone H2A–H2B dimers, wrapping around with 146–147 base pairs of DNA9. Beyond the findings that H3K36me3 is associated with actively transcribed genes9, emerging evidence indicates that H3K36me3 participates in the regulation of several cellular activities. Before DNA damage occurs, H3K36me3 could pre-recruit the DNA repair machinery to set the actively transcribed chromatin into a ‘ready’ state to quickly initiate the repair response upon DNA lesion10. In yeast, H3K36me3, once deposited at the active transcriptional gene bodies, recruits histone deacetylation complex to reset the activation of chromatin, leading to the prevention of cryptic transcription12. In mammalian cells, this prevention of cryptic transcription is not only mediated by the interplay between H3K36me3-mediated histone deacetylation and associated H3K4/H3K9 methylations14, but also facilitated by H3K36me3-guided DNA methylations17. Besides the regulation of mRNA levels, H3K36me3 can be recognized by METTL14 to enable the co-transcriptionally deposition of RNA m6A modification18. ZMYND11 binds H3K36me3 as an H3.3 specific reader to regulate RNA splicing and intron retention20. In addition, other regulatory functions are proposed for H3K36me3, including three-dimensional chromosome organization21, X-chromosome dosage compensation22, and pre-mRNA splicing23. Because of the diverse roles of H3K36me3 in cellular processes, H3K36me3 misregulations are identified in many human diseases, particularly renal cell carcinoma24. The H3K36me3 methyltransferase was originally identified in yeast as Set226, and then recognized in mammalian cells as the homology SETD228. Furthermore, meiosis-specific histone methyltransferase PRDM9 can catalyze both H3K4me3 and H3K36me3 in testis29.H3K36 trimethylation (H3K36me3), which is deposited onto chromatin following RNA polymerase II (Pol II) elongation, usually marks active gene body regions with a gradual increase from 5’ ends toward 3’ ends of genes30. The target protein of interest is recognized by its specific antibody which is subsequently bound with a Protein A linked Tn5 transposome. The target protein-bound DNA is cut and tagged by Tn5 transposomes to incorporate specific DNA sequences on both sides, which are then used to amplify target DNA for high-throughput sequencing. With this approach, cells are not crosslinked or sonicated so that the chromatin is maintained at a native state.CUT&Tag was developed to efficiently profile chromatin binding elements in cellsSmyd5 is overexpressed in Smyd5 knockout (KO) cells. Moreover, elevated Smyd5 expression contributes to the liver hepatocellular carcinoma tumorigenesis. Together, our observations uncover SMYD5 as a histone methyltransferase for H3K36me3 at promoters to control gene expression. These data expand our understanding of how H3K36me3 is catalyzed and regulated in cells.Here, we profile the distributions of H3K36me3 in primary cells and find that H3K36me3 is also enriched at promoters beyond gene body regions. Through a targeted gene screening, we find that SMYD5 catalyzes the methylation of H3K36me3 in vivo and in vitro. SMYD5 is recruited to chromatin by Pol II, resulting in the enrichment of H3K36me3 at promoters. The C-terminal domain of SMYD5 is important for its binding with histone H3 and methylation of H3K36me3. Depletion of the C-terminal domain reduces the reestablishment of H3K36me3 at promoters when Setd2 with two independent sgRNAs. Two clones were established from independent sgRNAs  to the transcription end site (TES) in genes that were identified by NCBI RefSeq in WT cells  and performed CUT&Tag to exclude the possibility of nonspecific binding of the antibodies used33. Two replicates with high correlations were conducted and merged for analysis , we used the CRISPR/Cas9 system to knock out lls Fig. . This H3lls Fig. . It’s unThe Tn5 transposome, which is also used in ATAC-seq (assay for transposase-accessible chromatin using sequencing) to profile the accessibility of chromatin, can attack open chromatin regions, leading to nonspecific signals at promoters in an open state. To investigate this possibility, we analyzed the H3K27ac and H3K27me3 CUT&Tag results in WT mESCs from the GEO dataset GSE169049. Because H3K27me3 was largely enriched on chromatin, and the antibody against H3K27me3 was unlikely to cause open chromatin bias, we performed H3K9me3 CUT&Tag as a complementary analysis of repressive histone mark. Moreover, we included the RNA-seq results to compare gene expression levels. H3K27me3 and H3K9me3 were detected at the promoters of weakly expressed genes, and H3K27ac was found at the promoters of genes with high expression levels Fig. . More imSetd2 KO cells. Chromatin was digested by MNase without crosslinking or sonication. Chromatin from Drosophila S2 cells was used as the exogenous spike-in genome to normalize the sequencing signals. The two biological replicates for each sequencing run were highly correlated and were merged for further analysis  and found weak enrichment at promoters, further supporting the idea that H3K36me3 is enriched at promoters  revealed that H3K36me3 was enriched at promoters in the CUT&Tag and N-ChIP-Rx data but not in the X-ChIP-Rx data  in WT and lls Fig. . Interesata Fig. . These data Fig. .36. To elucidate whether Pol II can affect the enrichment of H3K36me3 at promoters, we used CDK inhibitors or a high concentration of salt (300 mM NaCl) to remove Pol II from chromatin after antibody and Tn5 transposome binding and then proceeded to perform tagmentation  reduced the nonspecific tagmentation by the Tn5 transposome at active histone marksion Fig. 30. CellsSetd2 with two repeats, showed a relatively increased in H3K36me3 at promoters and that cells with individual knockout of 14 genes, including two repeats of Smyd5 and Zmynd1, exhibited decreased H3K36me3 at promoters  assay. Since the specific H3K36me3 was detected at the promoters and found to undergo highly dynamic changes, it is possible that H3 was not assembled into nucleosomes when it was methylated by SMYD5. We used both reconstituted core nucleosomes and core octamers as the substrate. The SET domains of SETD2 and SUV420H1 were used as positive controls for H3K36me3 and H4K20me3, respectively. SMYD5 catalyzed H3K36me3 using core octamers but not core nucleosomes as the substrate Fig. . As prevtes Fig. . We and ted Fig. . Methyltted Fig. . To furtted Fig. . TogetheSmyd5 KO #1 mESCs . No specific signals were detected in gene bodies or at promoter regions after the high salt treatment  to quantify nascent RNA in WT and Smyd5 KO mESCs. Two replicates with yeast RNA as the spike-in control were conducted and showed high correlations in the WT cell line and two Smyd5 KO cell lines  > 0.5 and P < 0.05 were set as the cutoff thresholds  using anti-FLAG beads. Pol II and H3 were copurified with FLAG-SMYD5, as detected by Western blotting Fig. . In addi42, both complexes induced S5P of the Pol II CTD, and the CDK7-Cyclin H complex showed lower enzymatic activity for S2P in the Pol II CTD in vitro  in the Pol II C-terminal domain (CTD). After treatment with 1 μM THZ1 for 30 min, S5P was inhibited without significant suppression of serine 2 phosphorylation (S2P) in the Pol II CTD Fig. . The intSince the Pol II CTD can bind to SMYD5, we next sought to determine whether this interaction affects the enzymatic activity of SMYD5. The H3K36me3 methyltransferase activity of SMYD5 increased when the Pol II CTD was added to the in vitro HMT system Fig. . The act37  and SMYD5-ΔC (R = 0.61) signals   Fig. . MoreoveSmyd5 expression was elevated in liver hepatocellular carcinoma (LIHC) tissues compared to normal liver tissues  . The expression level of Smyd5 increased from patients with stage I disease to patients with stage III disease, with a decrease in patients with stage IV disease  database. Interestingly, GEPIA analysis showed that ues Fig. 43. FurthIHC Fig. . We also45. Our previous GO enrichment analysis results showed that Smyd5 KO affected the expression of genes associated with Ras signaling, Wnt signaling, and liver development  to induce high expression of Nras and Ctnnb1 in the mouse liver. To further demonstrate the function of Smyd5 in tumor formation, we knocked down Smyd5 and rescued its expression with Smyd5-FL or Smyd5-ΔC. Eight shRNAs were designed for knockdown of Smyd5 and tested in the mouse liver cell line H2.35. Two shRNAs (#3 and #6) showing the highest knockdown efficiency were selected for further experiments  immediately before tagmentation, enrichment of H3K36me3 at promoters was quickly depleted. It’s unlikely that a demethylase specifically removes the H3K36me3, since these H3K36me3 marks are very dynamic. We speculate that this specific enrichment of H3K36me3 may set histones into a “ready to incorporate” states for incorporation into chromatin. Histones are methylated by SMYD5 before they are deposited onto chromatin. Thus, this process, once inhibited, can lead to the release of histones from chromatin, in turn leading to a quick reduction in H3K36me3 at promoters. Consistent with this idea, SMYD5 favors histone core octamers over DNA-containing nucleosomes as its substrate. The enriched DNA signals in the CUT&Tag and N-ChIP-Rx data likely arose from histones that were ready to be incorporated into chromatin and located very close to chromatin. It would be interesting to investigate whether H3K36me3 at promoters affects the interactions between histones and histone chaperones and, subsequently, the incorporation of histones into chromatin.55. Determination of whether SMYD5 preferentially uses H3.1 or H3.3 as the substrate and how H3.3S31ph may affect SMYD5-mediated H3K36me3 would shed light on the regulatory mechanism of SMYD5 enzymatic activity.The canonical histone H3.1/H3.2 is incorporated into chromatin during S phase. The histone H3 variant H3.3 is deposited into chromatin during gene transcription throughout the cell cycle. When catalyzing H3K36me3, SETD2 preferentially uses H3.3 as the substrate if H3.3S31 is phosphorylated. Via an in vitro HMT assay and structural analysis, Armache et al. found that the enzymatic activities of SETD2 are promoted by H3.3S31ph44. The reduction in Setd2 expression modulated the DNA damage response and lipid metabolism in the liver. Smyd5 regulates the Wnt and/or Ras signaling. These two genes may regulate two different mechanisms during liver tumorigenesis. H3K36me3 at promoters is not totally absent when Smyd5 is knocked out in mESCs, suggesting that there are other enzymes responsible for enrichment of H3K36me3 at promoters. When we performed screening for H3K36me3 methyltransferases, the target genes were based on the known histone methyltransferases. Other proteins that have not been previously identified as histone methyltransferases could be responsible for this specific enrichment of H3K36me3. An expanded screening library could help to identify undiscovered enzymes. Through years of study, many histone methyltransferases have been found to decorate core histone proteins. An increasing number of the enzymes identified initially have been proven to be responsible for methylating other amino acid residues of histones and even other proteins50. A reanalysis of known histone methyltransferases seems warranted.Li et al. recently reported that SETD2 deficiency in the liver results in HCC2. K562 cells were cultured in RPMI1640 supplemented with 1% glutamax, 1% antibiotic solution (penicillin/streptomycin) and 10% FBS.E14 mouse embryonic stem cells (mESCs) were cultured on 0.1% gelatin-coated dishes in DMEM supplemented with 15% fetal bovine serum (FBS), 1% antibiotic solution (penicillin/streptomycin), 1% glutamax, 1% MEM nonessential amino acids, 1% sodium pyruvate, 0.1 mM-mercaptoethanol and 1000 U/ml recombinant leukemia inhibitory factor (LIF). HEK 293 T cells and NIH3T3 cells were cultured in DMEM supplemented with 1% glutamax, 10% FBS and 1% antibiotic solution (penicillin/streptomycin). H2.35 cells were cultured in DMEM supplemented with 1% glutamax, 200 nM Dexamethasone, and 4% FBS. All cells were grown at 37 °C and 5% COSetd2, Smyd5 KO, were constructed by sgRNAs, which were cloned into pSpCas9(BB)-2A-Puro from Dr. Feng Zhang (Addgene plasmid #48139).mESCs KO cell lines for screening, including mESCs were seeded into 96 well plates in growth medium for 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. CellTiter-Blue kit  was used to measure cell viability at different time points. Fluorometric signals were recorded by a microplate reader .mESCs were fixed with 1% paraformaldehyde before alkaline phosphatase staining. The activity of alkaline phosphatases in mESCs was detected by INT/BCIP (Brown) kit  following the manufacturer’s instructions. NIH3T3 cells were used as negative controls.2) with 1000 units of MNase  by incubating at 37 °C for 20 min. The reaction was stopped by adding 2X STOP buffer . Sonication was performed in X-ChIP-Rx, but not N-ChIP-Rx, for 5 cycles of 30 s on and 30 s off in Bioruptor. After centrifuged at 15,000 × g at 4 °C for 10 min, the supernatant was used for chromatin immunoprecipitation. Same amounts of Drosophila S2 cell chromatin were spiked in for normalization. 10 µg of H3K36me3 antibodies  or IgG was added into each sample and rocked at 4 °C overnight. Protein G beads were added and incubated for 3 h. After the beads were extensively washed, the bound DNA was eluted by elution buffer . Reverse-crosslinked at 65 °C overnight. The eluted DNA was treated with proteinase K and RNase A, and then purified with the Min-Elute PCR purification kit . Chromatin contents were measured by Qubit assay , and subjected to library preparation with TruePrep DNA library prep kit  following the manufacturer’s instructions. The library was sequenced on an Illumina NovaSeq platform with pair-end reads of 150 bp.Cells for X-ChIP-Rx were crosslinked with 1% paraformaldehyde for 10 min and then quenched in 125 mM glycine for 5 min at room temperature with rotation, while cells for N-ChIP-Rx were processed without paraformaldehyde crosslink. After washed with cold PBS, cells were lysed in lysis buffer  on ice for 10 min. The nuclei were spun down and then digested in 500 µl MNase digestion buffer  and on ice for 10 min. ConA beads were pre-washed and re-suspended by binding buffer . 10 µl beads were added to each sample and incubated at room temperature for 10 min. The beads were washed with washing buffer  and resuspended in blocking buffer  at room temperature for 5 min. Primary antibodies were added by 1:100 dilution and incubated at room temperature for 2 h. After washed with washing buffer, secondary antibodies were added by 1:100 dilution and incubated at room temperature for 30 min. 1.2 µl PA-Tn5 transposomes were added to each sample and incubated at room temperature for 30 min. After washed with washing buffer, if indicated, beads were treated with Pol II inhibitors or high salt (300 mM NaCl) in washing buffer for 10 min. Beads were resuspended in 30 µl washing buffer with 10 mM MgCl2 and incubated at 37 °C for 1 h. Reactions were stopped by adding 5.5 µl stop buffer  and incubated at 55 °C for 30 min, and then 70 °C for 20 min to inactivate Proteinase K. 0.9X of VAHTS DNA clean beads  were added to each sample to extract the tagmentated DNA.CUT&Tag was performed as previously describedPrimary antibodies were Rabbit polyclonal anti-Histone H3K36me3  if indicated; H3K36me3 antibodies ; Mouse monoclonal anti-RNA polymerase II ; Mouse monoclonal anti-FLAG M2 ; Rabbit polyclonal anti-SMYD5 (this study).sgRNAs were designed for each picked gene and single clones were generated by the sgRNA with CRISPR/Cas9 knockout. The KO clones were tested for knockout by Sanger sequencing and then subjected to H3K36me3 CUT&Tag to test whether H3K36me3 at promoter regions were decreased. After the first screening, an independent sgRNA was designed for each pick gene and single clones were generated by this new sgRNA. The KO of genes were identified by Sanger sequencing and cells were subjected to H3K36me3 CUT&Tag in the first screening.56. Plasmids carried histone proteins, pRSFDuet1-hH3.3/H4 and pRSFDuet1-hH2A/H2B which were kind gifts from Dr. Ruiming Xu were used. For the nucleosome construction, the ‘601’ 167 bp DNA and histone octamers were titrated at a molar ratio of 0.9:1, and mixed together in initiation buffer . The mixture was step-wisely dialyzed to the final buffer  at 4 °C for 36 h. The final mixture included nucleosomes and a small proportion of free DNA. The fractions containing mononucleosomes were collected by gel filtration and further loaded to centrifugal filters for purification and concentration.Histone extraction was performed as previously describedSmyd5 cDNAs were cloned into the GST-tagged pGEX-6p-1 vector and HIS-tagged pET28a-smt3 vector, respectively. The CTD domain of RNA Pol II in the GST-tagged pGEX-6p-1 vector was a kind gift from Dr. Huasong Lu. Briefly, the plasmids were transformed into E.coli BL21 cells (Weidi Biotechnology) and grew to OD600 at around 0.6. 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce the expression of recombinant proteins. The lysates were bound to GST  or NI-NTA Seflnose Resin beads  at 4 °C for 2–4 h. The beads were extensively washed and beads-bound proteins were eluted by 100 mM L-glutathione or 250 mM imidazole respectively. The eluted proteins were dialyzed to storage buffer  before usage.The wild-type and mutant Plasmid pRK5M-Flag-CDK7/cyclin H and pRK5M-Flag-CDK9/cyclin T1 which were kind gifts from Dr. Huasong Lu were co-transfected into HEK 293 T cells respectively. After 48–72 h, cells were harvested and lysed in whole-cell extraction buffer  at 4 °C for 20 min. The supernatant was collected after high-speed spinning. FLAG M2 agarose beads  were added into supernatant to bind CDK7/cyclin H or CDK9/cyclin T1 complex at 4 °C for 3 h. Beads were extensively washed in washing buffer  5 times and stored in PBS at 4 °C.2, 50 mM NaCl, 50 mM HEPES, pH 7.4) by adding 2 µg CTD protein, 2 µl of 1 mM ATP, and 10 µl kinase-bound beads for 25 µl mixture in total. The reaction was incubated at 30 °C for 1 h. After high-speed spinning, the supernatant containing phosphorylated CTD protein was saved for the Pull-down assay immediately.The kinase reaction was performed in BF buffer  were added for HIS-Pull down. The pre-washed GST Seflnose Resin beads  were added for GST-pull down. Beads were added into the binding reaction and incubated at 4 °C for 4 h in a rotisserie-style tube rotator. The beads were extensively washed by TBS, and boiled with SDS loading buffer before being analyzed by Western blotting.2, 2.5 mM DTT). 1 µg reconstituted histone octamers or nucleosomes were used as the substrates, 0.5 µl S-adenosylmethionine  was used as the methyl-donor, and 1 µg purified proteins were added as methyltransferases. The reaction was carried out at 30 °C for 1 h and stopped by adding SDS loading buffer. Samples were analyzed by Western blotting or Coomassie Brilliant Blue staining.Histone methyltransferase reaction was performed in HMT buffer  was added to convert SAH to ADP at room temperature for 30 min. And then the detection solution was added to convert ADP to ATP which was detected via a luciferase reaction. The Luminescence was detected by a microplate reader . The enzymatic activity of each substrate was normalized to its own negative control (reaction mix without incubation for HMT).The purified proteins after histone methyltransferase reaction were analyzed on an Xevo G2-XS QTOF MS System (Waters Corporation) equipped with an electrospray ionization (ESI) source in conjunction with Waters ACQUITY UPLC I-Class plus. Separation and desalting were carried out on a Waters ACQUITY UPLC Protein BEH C4 Column . Mobile phase A was 0.1% formic acid in water and mobile phase B was acetonitrile with 0.1% formic acid. A constant flow rate of 0.2 ml/min was used. Data was analyzed using Waters UNIFI software. Mass spectral deconvolution was performed using UNIFI software .Smyd5 were cloned into the pcDNA3.1-FLAG plasmid to be overexpressed in HEK293T cells for immunoprecipitation. The transfected HEK293T cells were lysed in IP buffer . After spun at 15,000 x g 4 °C for 10 min, the supernatant was used to bind with FLAG M2 agarose beads  at 4 °C overnight. The beads were extensively washed by washing buffer . Beads-bound proteins were eluted by boiling with SDS loading buffer and then analyzed by Western blotting.Human wild-type and mutant For endogenous protein immunoprecipitation, mouse monoclonal anti-RNA polymerase II  or IgG was added to the lysed samples and incubated at 4 °C overnight. Sheep-anti-mouse IgG Dynabeads  were added into the samples and incubated at 4 °C for 2 h. After being extensively washed with washing buffer , beads-bound proteins were eluted by boiling with SDS loading buffer. Eluted proteins were subjected to Western blotting analysis.57. In brief, 107 cells were used for nuclei isolation per sample. Nuclear Run-On was performed at 37 °C for 5 min. Brominated UTP was incorporated in the Nascent RNA during this step. RNA was extracted by Trizol reagent  and immunoprecipitated with mouse monoclonal anti-BrdU antibody  and sheep-anti-mouse IgG Dynabeads . The Purified nascent RNA was used for library preparation by RNA-seq library prep kit .The Global run-on sequencing (GRO-seq) was performed as previously describedCells were seeded on coverslips into 24 well plate and washed with 1 ml PBS three times. Cells were fixed with 4% PFA for 10 min, then washed with 1 ml PBS twice. Permeabilization was performed in 0.5% Triton X-100 solution with 5% normal goat serum for 1 h at room temperature. Cells were washed with PBS twice. Primary antibodies were diluted 1: 100 in PBS, 4 °C overnight. After washed with 1 mL PBS-T (1X PBS with 0.1% Tween 20) for 3 times, 1:1000 diluted with PBS, Alexa fluor 488 antibodies were added for 1 h in dark at room temperature. After washed with 1 mL PBS-T for 3 times in dark, 5 min each, 1:1000 diluted DAPI  in PBS was added for 5 min in dark. Cells were washed with PBST twice. Slides were sealed with mounting medium, and then analyzed under fluorescence microscope .58. Liver tumors were induced by transposon-mediated integration and expression of HA-tagged oncogenes Nras and Ctnnb1. For Smyd5 knockdown, two shRNAs were designed and efficiency was validated in mouse liver cell line H2.35. shRNAs were then expressed by the U6 promoter in tandem with Ras. For Smyd5 reexpression, OLLAS-tagged mouse Smyd5 was expressed by ACT promoter in an individual transposon plasmid. Plasmids used for hydrodynamic tail-vein injection were prepared by Qiagen EndoFreeMaxi Kit, and were dissolved in sterile Ringer’s solution  equal to 10% mice body weight. 62.5 μg of total transposon plasmids and 17 μg piggyBac transposase plasmids were delivered through the tail vein by hydrodynamic injection. Mice were sacrificed 90–100 days after injection. Livers were pictured, weighted, and fixed or snap-frozen in liquid nitrogen for further usage. In this study, four-week-old male specific-pathogen-free ICR mice were purchased by Shanghai SLAC Laboratory Animal Company, and kept in SPF facilities at Zhejiang University Laboratory Animal Center. All the mice study methods were approved by Zhejiang University Animal Care and Use Committee.The hydrodynamic tail-vein injection was performed as described previouslyThe paraffin slides were deparaffined and rehydrated by xylene and alcohol gradients. Hematoxylin-Eosin staining was carried out by HE staining Kit  following the manufacturer’s instructions. The slides were stained with Hematoxylin staining solution for 5 min, differentiated with 0.1% hydrochloric acid-ethanol for 10 s, and counterstained in Eosin staining solution for 30 s. Slides were dehydrated in alcohol and xylene gradients and then sealed with a mounting medium. The slides were analyzed and scanned under microscopy .59. In brief, the paraffin slides were deparaffined and rehydrate by xylene and alcohol gradients. Antigen retrieval was done by heating in citrate buffer, pH 6. Endogenous peroxidase activity was quenched by incubating slides in 2% H2O2/distilled H2O for 30 min. the slides were blocked in 10% goat normal serum with Avidin. Primary Rabbit monoclonal anti-HA-tag antibody  was added by 1:200 dilution with biotin and incubated at 4 °C overnight. A biotinylated secondary antibody was added by a 1:200 dilution and incubated at room temperature for 30 min. After washed with PBS 3 times, the slides were incubated with VECTASTAIN elite ABC reagent for 30 min. Slides were stained with peroxidase substrate solution, DAB , for 2–10 min until desired staining intensity was developed, and then stopped with PBS. In addition, slides were stained with hematoxylin, de-stained with hydrochloric acid-ethanol, and reversed blue in ammonium solution. Slides were dehydrated in alcohol and xylene gradients, sealed with mounting medium, and then analyzed under microscopy .Immunohistochemistry (IHC) staining of paraffin sections was performed by the avidin-biotin complex (ABC) method as previously describedThe normal livers and tumors were ground with steel balls, and RNA was extracted by Trizol reagent . cDNA was then synthesized by One-step gDNA Removal and cDNA synthesis SuperMix kit  following the manufacturer’s instructions.Sdha were used as the control to normalize the expressions of target genes. Primers used were listed in Supplementary Table Quantitative RT-PCR was performed using Hieff qPCR SYBR Green Master Mix  with Quanta gene q225 qPCR system . The expression levels of ® 488 AffiniPure Alpaca Anti-Rabbit IgG (H + L) , 1:1000 diluted for IF.Rabbit polyclonal anti-Histone H3 , 1:5000 diluted for Western blot; Rabbit monoclonal anti-Histone H3K36me3 , 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K4me3 , 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K9me3 , 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-Histone H3K27me3 , 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K27ac , 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit monoclonal anti-SETD2 , 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H4K20me3 , 1:1000 diluted for Western blot; Mouse monoclonal anti-Histone H4 , 1:1000 diluted for Western blot; Mouse monoclonal anti-BrdU , 1:100 diluted for GRO-seq; Mouse monoclonal anti-FLAG M2 , 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-RNA polymerase II CTD phospho S2 , 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II CTD phospho S5 , 1:1000 diluted for Western blot; Mouse monoclonal anti-RNA polymerase II , 1:1000 diluted for Western blot, 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 , 1:100 diluted for CUT&Tag; Rabbit polyclonal anti-Histone H3K36me3 , 1:100 diluted for CUT&Tag, 10 µg for ChIP-seq; Mouse monoclonal anti-GST Tag , 1:1000 diluted for Western blot; Mouse monoclonal anti-6x HIS Tag , 1:1000 diluted for Western blot; Rabbit monoclonal anti-HA-tag antibody , 1:200 diluted for IHC; Rabbit polyclonal anti-SMYD5 (this study); Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) , 1:5000 diluted for Western blot. Alexa Fluorhttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore [https://www.bioinformatics.babraham.ac.uk/projects/trim_galore]) was used with the parameter ‘–paired’. Trimmed reads were then mapped to the mouse reference genome mm9 using Bowtie260 (version 2.3.5.1). PCR duplicates were removed by GATK4 (version 4.1.4.0) (https://github.com/broadinstitute/gatk [https://github.com/broadinstitute/gatk]) with the parameter ‘–REMOVE_DUPLICATES = true’ in ChIP-seq data while not removed in the CUT&Tag. MACS261 (version 2.2.6) was used to call broad peaks for H3K36me3. Specially, FLAG-SMYD5 peaks were called by SEACR62 (version 1.3) for sparse enrichment analysis.To remove adaptors and low-quality reads, Trim Galore (version 0.6.4) (63 (version 2.92.2) and bedGraphToBigWig approach (version 4) (https://www.encodeproject.org/software/bedgraphtobigwig/ [https://www.encodeproject.org/software/bedgraphtobigwig/]) were used with the following parameters ‘genomecov–scaleFactor 10,000,00/’. deepTools64 (version 3.4.3) was used to draw heatmaps by function computeMatrix and plotHeatmap. Normalized signals were visualized in Integrative Genomics Viewer (IGV)65 (version 2.6.3). Peak distribution and annotation were generated by ChIPseeker66 (version 1.22.1). For downstream differential peaks analysis, multiBigwigSummary in deepTools was used to count average signals over each region. Differential regions analysis was performed by limma67 (version 3.42.2) with normalized signals. clusterProfiler68 (version 3.14.3) was used to perform GO analysis. The correlations between two repeats were summarized in Supplementary Table 69 (version 3.26.2).For visualizing epigenomic signals, normalizing mapped reads, and calculating the coverage of features across the genome, BEDtoolsPromoter regions were defined as 500 bp around TSS. Gene body regions were defined as 500 bp downstream of TSS to TES. To calculate the read density at promoters and gene body regions, computeMatrix was used with scale-regions mode. H3K36me3 index, defined as Index = 70 (version 2.5.4b) was used for mapping the filtered reads to mouse reference genome mm9. Expression results were counted using featureCounts71 (version 2.0.0) and normalized as reads per kilobase per million mapped reads (RPKM) by edgeR72 (version 3.28.1). edgeR was used to find genes with different expression levels, which were selected by the threshold abs(log2(foldchange)) > 0.5 and P < 0.05. The volcano plot showing differential genes was generated by ggplot273 (version 3.3.2).Trim Galore was used to remove sequencing reads with adaptors or low-quality bases with the parameter ‘–paired’. STARSupplementary Information