Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity. Here, we show that ribonucleotides are incorporated by the hyperthermophilic archaeon Thermococcus kodakarensis both in vitro and in vivo and a robust ribonucleotide excision repair pathway is critical to keeping incorporation levels low in wild-type cells. Using pre-steady-state and steady-state kinetics experiments, we also show that archaeal RNaseH2 rapidly cleaves at embedded ribonucleotides (200-450 s−1), but exhibits an ∼1,000-fold slower turnover rate (0.06–0.17 s−1), suggesting a potential role for RNaseH2 in protecting or marking nicked sites for further processing. We found that following RNaseH2 cleavage, the combined activities of polymerase B (PolB), flap endonuclease (Fen1), and DNA ligase are required to complete ribonucleotide processing. PolB formed a ribonucleotide-containing flap by strand displacement synthesis that was cleaved by Fen1, and DNA ligase sealed the nick for complete repair. Our study reveals conservation of the overall mechanism of ribonucleotide excision repair across domains of life. The lack of redundancies in ribonucleotide repair in archaea perhaps suggests a more ancestral form of ribonucleotide excision repair compared with the eukaryotic pathway.Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as  DNA polymerases limit rNTP binding in the active site by a steric clash between the rNTP 2′-hydroxyl and an amino acid side chain –3. Howev–000-fold –9. There00-fold –, 9–12. FOnce incorporated by DNA polymerases, embedded rNMPs have severe consequences for genome integrity. The reactive 2′-hydroxyl group on rNMPs renders the strand highly susceptible to cleavage, making RNA much less stable than DNA for the storage of genetic information , 14. Fur15–To protect genome stability, cells require repair mechanisms for removing these unstable, embedded rNMPs. A specific repair pathway termed ribonucleotide excision repair (RER) serves this function and was recently described in eukaryotic and bacterial systems , 20. In Thermococcus kodakarensis (Tko) for genetics and Thermococcus sp. 9°N (9°N) for biochemistry  but many also express an essential, archaea-specific Family D polymerase (PolD) and the division of labor between these two polymerases in DNA replication and repair is not yet clear. Our study makes use of two closely related, hyperthermophilic archaea: in vivo frequency of rNMP incorporation by DNA polymerases during archaeal chromosome replication using a Tko strain deleted for RNaseH2. In both eukarya and bacteria, RER is initiated by the enzyme RNaseH2, which nicks DNA 5′ to a single, embedded rNMP (24–A). In the absence of RNaseH2 (ΔRNaseH2), rNMPs incorporated by DNA polymerases remain in genomic DNA and reveal the underlying frequency of rNMP incorporation by DNA polymerases. Tko ΔRNaseH2 genomic DNA migrated as lower molecular weight fragments indicative of alkali-sensitive ribonucleotide sites that remain in the genomic DNA (A). The size distribution of the fragments indicates an approximate ribonucleotide incorporation frequency of 1 in 1,500 nucleotides in the absence of RNaseH2, which corresponds to at least 1,000 genomic rNMPs incorporated per genome copy. These results demonstrate that archaeal DNA polymerases incorporate ribonucleotides in genomic DNA and that RNaseH2 is an important enzyme for repair.We examined the ded rNMP , 24–26.  rNMP 24–, 28. We  rNMP 24–A. In theomic DNA A. The siin vitro synthesis with Thermococcus sp. 9°N DNA polymerases B and D. We monitored the extension of primed M13 DNA with [α-32P]dCTP in the presence of dNTPs or predicted concentrations of dNTPs and rNTPs. Similar to the previous experiment, sensitivity to NaOH hydrolysis was a measure of rNTP incorporation (B). Both polymerases fully extended M13 DNA under both conditions as assayed by neutral agarose electrophoresis . Alkaline hydrolysis showed that both polymerases incorporate rNTPs at a high frequency with 1 in 2,500 and 1 in 1,500 nucleotides for PolB and PolD, respectively   . Togethetk_rs03985) showed no detectable growth defects in Thermococcus.Thermococcus that nicks DNA at rNMPs other than RNaseH2. Although there were abundant rNMPs in the genomic DNA of cells deleted for RNaseH2, loss of a compensatory enzyme that nicks at rNMPs could result in an even greater load of embedded, toxic rNMPs. To test whether a different enzyme is capable of the same cleavage reaction as RNaseH2, we designed a substrate to mimic the product resulting from polymerase incorporation errors: a DNA strand containing a single embedded rNMP. Using a dual-labeled (5′-FAM and 3′-MAX) 50-bp double-stranded DNA substrate with a single, embedded rGMP nucleotide, we monitored Thermococcus extracts for cleavage activity at the rGMP over time using capillary electrophoresis (CE) (C) (D), and observed rapid conversion of the 50-bp, dual-labeled DNA substrate to individual DNA fragments with wild-type extracts (C and D). However, ΔRNaseH2 extracts incubated even for extended time points showed no cleavage activity at rG (C and D). Addition of purified Thermococcus sp. 9°N RNaseH2 in the presence of ΔRNaseH2 cell extracts yields the expected cleavage products, indicating no inhibitory activities present in the extract (data not shown). These results, combined with the lack of repair of genomic DNA in ΔRNaseH2 strains, indicate that RNaseH2 is the major enzyme to initiate repair of embedded rNMPs and that there is no detectable enzyme activity in Thermococcus that can compensate for it.Eukaryotes use RNaseH2-initiated RER as the primary pathway for excising embedded rNMPs, however, due to the critical need for this repair, they also possess a redundant pathway that instead begins with incision by topoisomerase I at the ribonucleotide . As in ysis (CE) C 29). W. Wtk_rs0(CE) (C) D, and obextracts , C and Dty at rG , C and DThermococcus RNaseH2 on embedded rNMPs must be efficient. Tko extract experiments showed rapid cleavage activity by RNaseH2 on embedded ribonucleotides (C). Using steady-state and pre-steady-state kinetic experiments, we aimed to quantify the rate of cleavage of RNaseH2 on an embedded rNMP, identify the substrate preferences of RNaseH2 among the different potential embedded rNMPs, and determine what might be the rate-limiting steps in RER. First, we performed steady-state kinetic experiments to determine the turnover rate of Thermococcus sp. 9°N RNaseH2. To monitor enzyme turnover, we used the fluorescently-labeled 50-bp, double-stranded DNA substrate containing a single embedded rGMP in the labeled strand  (kss) and active enzyme concentration (A) were determined. The active enzyme concentration, 5.5 nm , was used to calculate the concentration of enzyme used in subsequent pre-steady-state kinetic experiments. The steady-state turnover rates (kss) were 0.06, 0.08, 0.17, and 0.14 s−1 for rG, rA, rC, and rU substrates, respectively  . The con2 using RQF (D). The concentration of the 5′-FAM product detected using CE was graphed as a function of time and fit to the pre-steady-state equation to obtain the rate of cleavage (E). RNaseH2 showed a rapid rate of cleavage with 220, 200, 450, and 430 s−1 for rG, rA, rC, and rU substrates, respectively . Two fluorescently-labeled oligonucleotides are annealed to single-stranded M13 DNA: a 44-nt primer and a 90-nt primer with a 5′ phosphate-rGMP. Before addition of extract or purified components, the substrate oligonucleotides appear as two distinct fluorescent peaks of 44- and 90-nt on CE (A). Over time, repair intermediates such as extension of the 44-nt primer by DNA polymerase strand displacement (B) or flap cleavage of the 90-nt primer (C), can be observed. Finally, a completely repaired product is a dual-labeled, sealed DNA product (134 nt) (D). As a control to confirm repair, adding purified RNaseH2 at the end of the reaction did not result in detectable rNMP cleavage products, indicating that the rNMP was repaired and replaced with a dNMP and not simply sealed by Ligase .After RNaseH2 nicks 5′ to an rNMP, additional enzymes are required to complete repair. To identify and characterize the other enzymes required for RER in cleavage A. Two flnt on CE A. Over tlacement B or flapt primer C, can be(134 nt) D. As a cm with Thermococcus cell extracts from wild-type and mutant strains to identify putative pathway components. All extracts used were normalized by protein concentration and Ligase activity, ensuring that the extracts were otherwise functional. Wild-type extracts repaired the RER substrate within 60 min, although the overall level of repaired product was low (∼3 nm) due to competing nuclease activities in extracts (A and B). Extracts deleted for Fen1 (tk_rs03985) and PolB (tk_rs00010) showed greatly reduced (ΔFen1) or no (ΔPolB) repair (A and B). Addition of purified Thermococcus sp. 9°N Fen1 and PolB to extracts deleted for these genes complemented RER indicating that these were the key activities lacking and preventing the complete repair of this substrate . These results indicate that Fen1 and PolB play critical roles in RER.First, we incubated this RER substrate at 10 nextracts , A and B) repair , A and Bin vitro using the same DNA substrate as before , repaired product was detectable within 5 min (data not shown) and complete repair occurred within 30 min (B and C). Similar to previous studies on the effect of PCNA on Tko replication, PCNA and RFC were not required for repair under these conditions and no requirement was observed until salt concentrations were raised to greater than 100 mm NaCl (B). PolB extends the 5′-MAX-labeled primer (green) and displaces the downstream 3′-FAM-labeled strand (blue) creating a 5′ flap that is sequentially cleaved by Fen1. PolB continues to extend the 5′-MAX-primer using M13 as a template strand and synthesizes long products. When the synthesis length is longer than the CE detection range (up to 800 nt), the MAX signal disappears from view. In summary, following DNA nicking by RNaseH2, PolB, Fen1, and Ligase alone are sufficient to support RER.Experiments with Tko extracts suggested key roles for PolB and Fen1 in the RER pathway. Next we aimed to reconstitute the RER pathway s before  but withn 30 min . The samn 30 min , B and C mm NaCl B. PolB ein vitro (B) and in vivo (A). Similar rNMP densities have been reported in budding and fission yeast and in mouse cells deficient in RER  and PolD (the archaea-specific Family D polymerase), and the specific roles for each of these polymerases in DNA replication and repair are not fully understood. PolD subunits are essential genes (tk_rs09520 and tk_rs09525), and the PolD enzyme is known to interact with other components of the replication machinery, while PolB is nonessential in several archaeal species and is not known to form complexes with other replisome proteins . Additionally, the lack of known replisome-binding partners for PolB suggests that RER occurs outside of the assembled replisome, although the timing and coordination of repair remains to be determined.proteins –42. Therturation . Howeverturation C. AdditiOther DNA repair pathways such as base excision repair and nucleotide excision repair are also poorly understood in archaea. Given the extreme environments that these organisms inhabit, archaea likely accumulate substantial amounts of DNA damage that needs to be repaired to protect genome integrity. We speculate that the overall mechanism proposed here may be generalizable to other repair pathways as well, but making use of different enzymes to initiate each specific process. Future studies are needed to identify the initiating enzymes and determine whether PolB, Fen1, and Ligase are the primary repair enzymes required for these other types of repair as well.B). Furthermore, Thermococcus lack a redundant pathway to repair rNMPs. This contrasts with eukaryotes where redundant enzymes exist for several steps. Yeast RNaseH2 is the major enzyme to initiate RER yet the topoisomerase I-initiated RER pathway  . Overall, the three domains of life have utilized a similar mechanism for excision of erroneous nucleotides, such as rNMPs, while using slightly different machinery to achieve it. Although we depict the RER pathway as a stepwise process of individual enzymes  . BacteriThermococcus sp. 9°N PolD was purified as previously described , and cloned into pAII17 plasmid vector  of isopropyl β-d-thiogalactopyranoside. Cells were allowed to grow for 3 h in the presence of isopropyl β-d-thiogalactopyranoside, and were collected by centrifugation at 4000 rpm for 15 min. Cell pellets were suspended in buffer A  containing 0.25 m NaCl, and lysed by sonication and heating at 70 °C for 30 min. Centrifugation was used to remove cell debris. The supernatant was passed through a DEAE column and flow through was collected and diluted with buffer A to 50 mm NaCl. Diluted flow through was loaded onto a Heparin TSK column, pre-equilibrated with buffer A containing 50 mm NaCl, and eluted with a buffer A gradient from 50 mm to 1 m NaCl. Peak fractions were identified by SDS-PAGE analysis, pooled, and dialyzed to 10 mm Tris-HCl, pH 7.5, 100 mm KCl, 0.1 mm EDTA, 0.1% Triton X-100, and 50% glycerol.The d vector  cleaved T. kodakarensis (Tko) strains were grown as previously described (3 and strains deleted for PolB were generated previously (escribed . Tko streviously .m NaOH (or 5 m NaCl as negative control) was added to DNA at 0.3 m final concentration and heated at 55 °C for 2 h to hydrolyze at ribonucleotide positions in DNA. 6× Purple loading dye  was added to samples before loading onto a 1% alkaline-agarose gel . The gel was run at 5 V for 16 h, stained with SYBR Gold (Thermo Fisher Scientific), and visualized on a UV imager.Ribonucleotides in DNA are visualized using alkaline-agarose electrophoresis. Treatment with NaOH fragments DNA 3′ to an embedded rNMP, resulting in DNA that migrates faster by alkaline-agarose electrophoresis. 2 m) 2 SO4, 10 mm KCl, 2 mm MgSO4, 0.1% Triton X-100, pH 8.8, at 25 °C), additional MgSO4 (10 mm), [α-32P]dCTP (as a radioactive tracer), 9°N PCNA (40 nm as a trimer), and 9°N RFC (80 nm as a dimer). Reactions were performed with either dNTPs (100 μm) or a physiological concentration of rNTPs and dNTPs of 16 μm dATP, 14 μm dCTP, 12 μm dGTP, 30 μm dTTP, 3000 μm ATP, 500 μm CTP, 700 μm GTP, and 1700 μm UTP (m) and incubated at 65 °C for 30 min. Reactions were resolved by neutral or alkaline-agarose gel electrophoresis, exposed to a storage phosphor screen  and imaged on a Typhoon 9400 scanner. ImageQuant software was used to plot fragment distribution.The frequency of PolB and PolD rNMP incorporation was determined by primer extension on a single-stranded M13 substrate followed by alkali treatment and analysis by alkaline-agarose gel electrophoresis as described . Primer m) , ThermoP0 μm UTP . Reactiom) (m) 2 SO4, 10 mm KCl, 0.1% Triton X-100, pH 8.8, at 25 °C) by heating to 95 °C for 5 min and cooling to room temperature. A 600-μl dilution of the rG DNA substrate (50 nm) was made in ThermoPol buffer (1 × concentration) and a 600-μl dilution of the Thermococcus sp. 9°N RNaseH2 (10 nm) in ThermoPol buffer (1 ×). The two solutions were mixed in a RQF instrument  set to 62.5 °C with a circulating water bath to achieve a final 60 °C reaction temperature. Time points were mixed for 0.004–10 s and quenched with 50 mm EDTA. After mixing, the final reaction concentrations were 5 nm RNaseH2 and 25 nm DNA substrate in 1× ThermoPol buffer for a 5-fold excess of DNA substrate. The no enzyme (0 s) control was performed by rapidly mixing 1× ThermoPol buffer with the DNA substrate in the RQF for 10 s. To ensure that steady-state conditions had been achieved in these experiments, control experiments were performed with a final reaction condition of 5 nm RNaseH2 and 50 nm DNA substrate in 1× ThermoPol buffer (10-fold excess of substrate to enzyme) (data not shown). Reaction products were separated by capillary electrophoresis using a 3730xl Genetic Analyzer (Applied Biosystems) and the fluorescent peaks were analyzed using Peak Scanner software version 1.0 (Applied Biosystems). The concentration of 21-nt FAM product was graphed as a function of time and fit to kss) was obtained by dividing the linear rate (k2) by the amplitude (A). All kinetic assays were performed at least three times for reproducibility.The rGMP RER substrate used for steady-state kinetics was prepared by annealing the 50-nt 5′-FAM/3′-MAX rGMP oligonucleotide (1 μm)  to the 5m) (m)  in Thermm) to its 50-nt unlabeled complement (1.5 μm)  control was performed by rapidly mixing 1× ThermoPol II buffer with the DNA substrate-RNaseH2 mixture in the RQF for 5 s to show that the enzyme is fully chelated and has no activity in the absence of MgSO4. To ensure that pre-steady-state conditions had been achieved, control experiments were performed where the enzyme was in 6-fold excess to DNA substrate with a final reaction condition of 90 nm RNaseH2 and 15 nm DNA substrate (data not shown). Reaction products were separated by capillary electrophoresis and analyzed as described above. The concentration of 21-nt FAM product was graphed as a function of time and fit to The rGMP, rAMP, rCMP, and rUMP substrates were prepared by annealing the 50-nt 5′-FAM rNTP oligonucleotide (1 μ(1.5 μm)  in Thermm), 90-nt 3′-FAM rG oligonucleotide (410 nm), and ssM13mp18 (480 nm) in ThermoPol buffer II by heating to 95 °C for 5 min and cooling to room temperature. T. kodakarensis (Tko) KW128 (wild-type) and mutant strains  were grown as previously described . Suspended cells were sonicated for 2 min and centrifuged for 10 min at 14,000 × g to remove cell debris. The extract supernatant was mixed with glycerol to 50% final concentration, and stored at −20 °C. All extracts were normalized by DNA ligase activity as all strains contained the essential gene for DNA ligase. The rG RER substrate (15 nm) was incubated with Tko extracts as well as 1× ThermoPol buffer, 1 mm ATP, and 0.1 mm dNTPs at 60 °C over a time course from 0 to 60 min. Reactions were quenched with 50 mm EDTA and products were separated by capillary electrophoresis. The conversion of the 90-nt FAM oligonucleotide to the 134-nt MAX/FAM dual-labeled repair product was quantified for each Tko extract at the 60-min time point using Peak Scanner software as described above. Three biological replicates were performed for the extract experiments. For RNaseH2 assays (C), Tko extracts (wild-type and ΔRNaseH2) were prepared as described above and incubated with 10 nm rG 50-nt FAM/MAX DNA substrate, 1 mm ATP, 0.1 mm dNTPs, and 1× ThermoPol buffer at 60 °C over a time course from 0 to 20 min. Extracts were complemented with purified proteins (1 nm) . Reactions were quenched with 50 mm EDTA, products were separated by capillary electrophoresis, and results were quantified using Peak Scanner software.The rG RER substrate was prepared by annealing the 44-nt 5′-MAX oligonucleotide 400 n0 nm, 90-2 assays C, Tko exm) with purified Thermococcus sp. 9°N proteins at the following concentrations: 1 nm Fen1, 1 nm Ligase, 1 nm PolB, 1 nm PolD, 20 nm PCNA, 40 nm RFC. All reactions were performed in 1× ThermoPol buffer with 1 mm ATP and 0.1 mm dNTPs. The RER reactions were carried out at 60 °C over a time course from 0 to 30 min with different protein components omitted from the mix to assay their role on RER. The conversion of the 90-nt FAM oligonucleotide to the 134-nt MAX/FAM dual-labeled repair product was quantified for each reaction condition at the 30-min time point using Peak Scanner software as described above. Experiments were performed three times for reproducibility.Repair reactions were performed by mixing the rG RER substrate (10 nThermococcus strains. T. J. S. analyzed results and wrote the manuscript. A. F. G. designed experiments, analyzed results, and wrote the manuscript. All authors analyzed the results and approved the final version of the manuscript.M. R. H. designed and conducted experiments, analyzed results, and wrote the manuscript. B. W. B. constructed and grew


Chronic obstructive pulmonary disease (COPD) is a major public health problem and gives arise to severe chronic morbidity and mortality in the world. Inflammatory response and oxidative stress play dominant roles in the pathological mechanism of COPD, and have been regarded to be two important targets for the COPD therapy. Traditional Chinese medicines (TCMs) possess satisfying curative effects on COPD under guidance of the TCM theory in China, and merit in-depth investigations as a resource of lead compounds.One hundred ninety-six of TCMs were collected, and extracted to establish a TCM extract library, and then further evaluated for their potency on inhibitions of oxidative stress and inflammatory response using NADP(H):quinone oxidoreductase (QR) assay and nitric oxide (NO) production assay, respectively.Our investigation observed that 38 of the tested TCM extracts induced QR activity in hepa 1c1c7 murine hepatoma cells, and 55 of them inhibited NO production in RAW 264.7 murine macrophages at the tested concentrations. Noteworthily, 20 of TCM extracts simultaneously inhibited oxidative stress and inflammatory responses.The observed bioactive TCMs, particularly these 20 TCMs with dual inhibitory effects, might be useful for the treatment of COPD. More importantly, the results of the present research afford us an opportunity to discover new lead molecules as COPD therapeutic agents from these active TCMs.The online version of this article (doi:10.1186/s12906-016-1347-y) contains supplementary material, which is available to authorized users. Chronic obstructive pulmonary disease (COPD) is a disease characterized by progressive and not fully reversible airflow limitation, which is associated with abnormal inflammatory response of the lung to noxious particles and gases . TobaccoCumulative evidences indicate that inflammatory response, oxidative stress, and protease imbalance play dominant roles in the pathological mechanism of COPD , 4. BriePlenty of traditional Chinese medicines (TCMs) have been used clinically to treat COPD in the form of single or compound prescription under guidance of the TCM theory in China, and demonstrated satisfying curative effects , 9. TheiSulforaphane  was purchased from Sigma-Aldrich . Didox (purity >98 %) was purchased from MedChem Express . Solvents used for extraction were of analytical grade and obtained from Tianjin Fuyu Chemical Company .Traditional Chinese medicine (TCM) materials were purchased from the Jinan Jianlian TCM Co. Ltd in Shandong province, Anguo TCM market in Hebei province, and Bozhou TCM market in Anhui Province. These TCMs were identified by Prof. Lan Xiang, School of Pharmaceutical Sciences, Shandong University, through comparing their characteristics in plant morphology and taxonomy with that described in Chinese Pharmacopoeia. Voucher specimens  were extracted under reflux for 2 h with 75 % ethanol , and then EtOH was removed under reduced pressure. The yield of each extract was presented as a percentage of weight of dried plant material, and has been summarized in Table 2.Hepa 1c1c7 murine hepatoma cells  were maintained in Eagle’s minimal essential medium  supplemented with 10 % fetal bovine serum . RAW 264.7 murine macrophages (ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10 % FBS. All cells were incubated at 37 °C in a humidified incubator containing 5 % CO4 cells/well) were seeded in 96-well plates and treated with the indicated doses of tested extracts for 24 h. The medium was decanted, and the cells were incubated with 40 μL of lysing solution [0.8 % digitonin and 2 mM EDTA solution (pH 7.8)] for 15 min at 37 °C. Then, 170 μL of a complete reaction mixture containing bovine serum albumin, 3--2,5-diphenyltetrazoliumbromide (MTT), 1.5 % Tween 20, 0.5 M Tris–HCl, 7.5 mM flavin adenine dinucleotide (FAD), 150 mM glucose-6-phosphate, 10 units/μL glucose-6-phosphate dehydrogenase, 50 mM NADP, and 50 mM menadione was added into each well. After incubation for 4 min, a blue color was developed and the reaction was arrested by adding 50 μL per well of a 0.3 mM dicoumarol solution (pH 7.4). Absorbance was measured at 630 nm on the Model 680 plate reader (Bio-rad). SF (2.0 μM) was adopted as a positive control.NADP(H):quinone oxidoreductase (QR) assay was modified from previously described method . Hepa 1c4 cells/well) were seeded in 96-well plates and treated with 1 μg/mL LPS, in the absence or presence of tested TCM extractions for 24 h. Then, 100 μL of supernatant was removed to a new 96-well plate and added with 100 μL of Griess reagent (0.1 % naphthylethylenediamine and 1 % sulfanilamide in 5 % H3PO4 solution) at room temperature for 15 min. Absorbance was measured at 570 nm on the Model 680 plate reader (Bio-rad). Nitrite concentration was calculated from a NaNO2 standard curve. Didox (100 μM) was used as a positive control.Inhibition of NO production by LPS-stimulated RAW 264.7 murine macrophages was applied to evaluate anti-inflammatory functions of TCM extracts. RAW 264.7 cells -2,5-diphenyltetrazolium bromide  assay. Briefly, 100 μL of DMEM media containing 0.4 % MTT were added to each wells, after removing 100 μL of supernatant as described in NO production assay. Then, the cells were incubated at 37 °C for 3 h, and absorbance was measured at 570 nm on the Model 680 plate reader (Bio-rad).P < 0.05 was considered to be significant.One way analysis of variance (ANOVA) and post hoc multiple comparison Bonferroni test were applied to compare the significant difference between two groups. Results are presented as the mean ± SD. To establish a TCM extract library for biological screening, we firstly selected 196 TCMs based on Chinese Pharmacopoeia (Edition 2015) and TCM literatures, and collected TCMs from Jinan local TCM drugstore, as well as the two biggest Chinese TCM markets, Anguo and Bozhou TCM markets. After plant material authentication, TCMs were extracted with 75 % EtOH to prepare their EtOH extracts, and then the concentrations of 200, 100, 50, 25, 12.5, 6.25 μg/mL were selected as tested doses for bioassays. Names, origin, extract yields and biological activities of investigated TCMs were summarized in Table We adopted a bioassay measuring QR activity in hepa 1c1c7 murine hepatoma cells to evaluate the ability of TCM extracts on inhibiting of oxidative stress . AlthougAndrographis paniculata , Aucklandia lappa , Cimicifuga heracleifolia , Glycyrrhiz uralensis , Pyrrosia sheareri , Saposhnikovia divaricate , Siegesbeckia orientalis , and Tussilago farfara . Twenty-two extracts are moderate QR inducers (1.8 > MQI ≥1.5), containing Albizia julibrissin , Cassia angustifolia , Cirsium setosum , Citrus limon , Curculigo orchioides , Cyperus rotundus , Eucommia ulmoides , Illicium difengpi , Inula helenium , Isatis indigotica , Ligusticum chuanxiong , Lindera aggregate , Lithospermum erythrorhizon , Misla chinensis , Perilla frutescens , Physalis alkekengi L. var. franchetii , Pinellia ternata , Pogostemon cablin , Rhaponlicum uniflorum , Rosa laevigata , Sparganium stoloniferum , and Zanthoxylum schinifolium . Moreover, eight extracts possessed weak QR inducing effect (1.5 > MQI ≥1.3), including Angelica sinensis , Areca catechu , Arisaema erubescens , Artemisia scoparia , Morus alba , Rabdosia rubescens , Salvia miltiorrhiza , and Sophora japonica . QR inducing effects of 38 bioactive TCM extracts in hepa 1c1c7 cells have been detailedly summarized in Additional file Ultimately, 38 TCM extracts demonstrated the QR inducing activities with MQI ranging from 1.33- to 2.38- folds under the tested concentrations , Aucklandia lappa , Callicarpa macrophylla , Chrysanthemum morifolium , Cimicifuga heracleifolia , Fraxinus rhynchophylla , Glycyrrhiza uralensis , Inula helenium , Oraxylum inddicum , Physalis alkekengi L. var. franchetii , and Scutellaria baicalensis . Moreever, 25 extracts displayed moderate inhibitory effect of NO production (80 % > MIR ≥ 50 %), and 19 extracts weakly inhibited NO production (50 % > MIR ≥30 %). Inhibitory effects on NO production of 55 bioactive TCM extracts in RAW 264.7 cells have been detailedly summarized in Additional file Our investigation indicated that 55 TCM extracts inhibited the LPS-induced NO production with MIRs between 30.7 % and 100 % under the tested nontoxic concentrations , Aucklandia lappa , Cassia angustifolia , Cimicifuga heracleifolia , Cirsium setosum , Curculigo orchioides , Eucommia ulmoides , Glycyrrhiza uralensis , Inula helenium , Ligusticum chuanxiong , Lithospermum erythrorhizon , Morus alba , Perilla frutescens , Physalis alkekengi var. franchetii , Pogostemon cablin , Rabdosia rubescens , Rosa laevigata , Salvia miltiorrhiza , Siegesbeckia orientalis , and Zanthoxylum schinifolium  simultaneously inhibited oxidative stress and inflammation (Table Aucklandia lappa (31), Cimicifuga heracleifolia (40), and Glycyrrhiza uralensis (85) are labelled as the level of strong. In addition, the extracts of Inula helenium (92) and Physalis alkekengi L. var. franchetii (132) also demonstrated the potencies that are closed to the strong level.Since oxidative stress and inflammatory response have the synergistic reactions in the pathophysiology of COPD, TCMs having dual inhibitions on the two targets are apt to be the resource for discovering lead molecules , 7. Our a root, 3, Cassia a root, 3, Cassia Andrographis paniculata (16) , (Zsis 20) , dehydrosis (85) , isoalanium (92) , 2’,3’-d , dehydrns (154) , danshenza (162) . These dza (162) .Agrimonia pilosa (8)  have not been investigated in the field of inflammation. Significantly, little research on the therapeutic effect of COPD has been performed, and thus these active TCMs are still being researched.TCMs have been adopted for the therapy of inflammation-related diseases with a long history in China. Compared with QR inducing assay, NO inhibitory effect assay and other in vitro and in vivo anti-inflammatory models are classical and commonly adopted biological research methods, and accordingly more literatures concerning inflammation of TCMs have been published. Based on our findings, we carried out a systemic search of reported inflammation-related literatures of our observed 55 active TCM extracts, and concluded that: some TCMs have been comprehensively investigated for their anti-inflammatory property and resulted in the discovery of diverse types of natural products, covering agrimonolide from losa (8) , (-)-nyades (17) , alantolppa (31) , berberisis (53) , forsythnsa (79) , resveraum (141) , etc. BeIn the present screening assay, we only adopted two typical markers, QR and NO, to evaluate the potential of TCMs as oxidative stress and inflammation inhibitory agents. Based on our preliminary results, active TCM extracts could be subjected to further research in the field of phytochemistry and pharmacology, however, solid evidences on their biological functions are required before a systemic investigation . With reAdditionally, the pivotal regulators for oxidative stress and inflammation should be sufficiently investigated to verify action of mechanism of the active TCMs. The transcription factor Nrf2 plays a dominant role for regulating oxidative stress. It is ubiquitously expressed in human organs, particularly rich in lung, and counteracts oxidative injury through activating intracellular redox-balancing proteins  and up-regulating endogenous antioxidants (e.g. GSH) , 34. NF-Although the present research indicates that some TCMs possessed inhibitory effects on inflammation and oxidative stress, further pharmacological investigations in vitro and in vivo models are warranted. Furthermore, bioassay-guided fractionations and identifications of active ingredients should be launched to help us illustrate the mechanism of these active species, and discover new lead molecules with unknown mechanisms and potent functions on oxidative stress- and inflammation-related diseases, especially COPD. Accordingly, these results may give new insight in research and development of COPD therapeutic agents.


AbstractJiaolong. These represent a new species of Ophryotrocha that is here described as Ophryotrochajiaolongisp. n. This is the first dorvilleid described from vents on the Southwest Indian Ridge. It most closely resembles another vent species, Ophryotrochaakessoni Blake, 1985 from the Galapagos Rift, but can be distinguished by its antennae, palps, jaw structure. The new species has particularly distinctive mandibles, which allow it to be easily identified.Dorvilleids were collected from hydrothermal vents on the Southwest Indian Ridge by manned submersible  Ophryotrocha Claparède & Mecznikow, 1869 is a diverse dorvilleid genus with more than 70 species described to date. These are distributed world-wide in diversified habitats from shallow water to deep-sea. A number of species in this genus are opportunistic or stress tolerant, can reach high abundance in reducing environments, such as hydrothermal vents and cold seeps, as well as whale and wood fall ecosystems  cruise DY35 was carried out by the research vessel Xiangyanghong 9, visiting the Southwest Indian Ocean. Sampling from the vents field was undertaken by the manned submersible Jiaolong. Specimens collected from two sites of the Longqi vent field, were sieved through a 250 μm mesh sieve, sorted, and preserved in 95% ethanol on board. The holotype and most paratypes are deposited in the repository of the Second Institute of Oceanography (RSIO), Hangzhou, China; additional paratypes are deposited in the Scripps Institution of Oceanography Benthic Invertebrate Collection (SIO-BIC), La Jolla California, USA.In January 2015, the China Ocean Mineral Resource R&D Association . Jaws from both holotype and paratype were obtained after digesting anterior decapitated ends with a proteinase K solution at room temperature. Once the tissue was digested, the jaw elements were cleaned with distilled water and transferred to a glass cover slip. All elements for SEM were mounted on stubs and sputter coated with platinum-palladium and imaged using a Hitachi TM1000 scanning electron microscope.2O, 1µl each primer (10 µM), 2 µl template DNA, 0.5 U of Taq polymerase , 2.5 µl of buffer solution (supplied by the polymerase manufacturer) and 0.5 µl of 2.5 mM dNTPs solution in a mixture of total 25µl. The temperature profile was as follows: 96°C/240s - (94°C/30s - 50°C/30s - 72°C/60s) * 35cycles - 72°C/420s. PCR products were purified with QIAquick PCR purification kit  following the protocol supplied by the manufacturer. Sequencing was performed by Sangon Biotech  on an ABI 3730XL DNA analyser (Applied Biosystems). Alignments of the three genes  were performed using the program MAFFT (ML) analysis was conducted by RAxML  following the protocol supplied by the manufacturer. About 680 bp of CO1, 500 bp of 16S and 350 bp of H3 were amplified using primers LCO1490 and CO-E  for 16S am MAFFT  with allby RAxML  using coTaxon classificationAnimaliaAnnelidaDorvilleidaehttp://zoobank.org/60CF9A6D-DBB9-4048-9C1C-501E94C580E0Jiaolong Dive 94, 49.6495°E; 37.7835°S, 2760m depth, 11 January 2015: ~ 10 mm long, 58 chaetigers; Paratypes: 21 specimens (RSIO35302) from same location as holotype; 7 specimens (RSIO35303) from Southwest Indian Ridge, Longqi vent field, 49.6501°E; 37.7836°S, 2737m depth; 8 specimens (SIO-BIC A6729) same locality as holotype.(RSIO35301) Southwest Indian Ridge, Longqi vent field, HOV PageBreakinserted laterally, similar in length with the parapodia on the last chaetigers, a small median papilla ventrally placed  is currently available, which is located in a different clade from the new species. Further DNA data is being acquired from other vent Ophryotrocha species, which will help us to get a better understanding of the relationship among vents Ophryotrocha species in the near future .Sequences of


Most importantly, the Au nanoparticle-functionalized graphene-modified electrode could be successfully applied for the detection of sulfanilamide in animal meat, and exhibited good stability, acceptable recovery, and offered a promising platform for point-of-care detecting in real samples.In this paper, we present a simple and feasible electrochemical sensor based on Au nanoparticle-functionalized graphene for the determination of sulfanilamide. Au nanoparticles were deposited on graphene, which acted as a platform to prepare excellent nanocomposites. Attributed to the graphene’s large surface area and the Au nanoparticles’ strong conductivity, many sulfanilamide molecules were enriched on the sensor surface and the signal response became more sensitive. Under the optimal conditions, the electrochemical sensors could be used for the efficient detection of sulfanilamide. Good linearity was observed in the range of 0.1–1000 μmol·L Sulfonamides (SAs) are one of the oldest classes of antibiotic drugs with widespread applications in prophylactic, therapeutic, and growth promoting ,2 treatm3O4/Gr/GCE electrochemical sensors ), potassium ferrocyanide (K4[Fe(CN)6]), and reagents for preparing Britton-Robinson (BR) buffer solution  were purchased from Luoyang Chemical Reagent Factory . AuNPs/Gr nanocomposites were dissolved in DMF. SAM solutions were prepared in 0.04 mol·L−1 Britton-Robinson (BR) buffer solution. Any other solvent used was double distilled water, if not clearly indicated otherwise. All chemicals were of at least analytical grade and were used without further purification. Gr was purchased from Ningbo Institute of Industrial Technology. The morphology of materials was investigated via transmission electron microscopy (TEM) on an HT7700  and energy dispersive X-ray (EDX) spectroscopy. Ultraviolet spectrophotometer (UV-vis) absorption spectra were recorded using a UV-6100s Double beam spectrophotometer . Differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) measurements were carried out using a CHI660E electrochemical workstation .−1 chloroauric acid solution was added to a glass beaker containing 400 μL of 1.0 g·L−1 Gr. The mixture was mixed evenly after the addition of 7.8 mL double distilled water and the homogeneous suspension was formed by sonication for 5 min. Meanwhile, 1 mL ascorbic acid solution (0.1 mol·L−1) was slowly added to the abovementioned glass beaker and kept for 2 days after 40 min of treatment. Centrifugation and washing were executed to remove most of the matrices, including ascorbic acid. Lastly, products were collected for further use after drying.AuNPs/Gr nanocomposites were prepared according to a previously reported method . At room3, ethanol, and water to obtain a cleaned GCE surface. The cleaned GCE was activated by CV scanning in a 0.5 mol·L−1 H2SO4 solution before use. AuNPs/Gr (1.0 g·L−1) suspensions were prepared by ultrasonically dispersing 1 mg AuNPs/Gr nanocomposites in 1 mL DMF. Then, 2 μL as-prepared AuNPs/Gr (1.0 g·L−1) suspensions were dropped onto cleaned GCE and dried for measuring CV and EIS, which were conducted in 5 mM [Fe(CN)6]3− solution containing 0.10 mol·L−1 KCl and 10 mM [Fe(CN)6]3−/4− solution containing 0.10 mol·L−1 KCl as a redox probe, respectively 3−/4− solution [The characterization of the electron transfer properties of the different modified electrodes is always carried out using EIS, which is measured in [Fe(CN)solution . FerrocyAs shown in −3 mol·L−1 SAM at the surface of AuNPs/Gr/GCE. As shown in I(A) = − 3.914 + 0.772 Lgv(V·s−1) (R2 = 0.970). The slope was 0.772, illustrating an adsorption-controlled process of SAM on AuNPs/Gr/GCE. Therefore, in order to prevent excessive charging current and obtain a better peak shape, we chose a scanning rate of 100 mV·s−1.The effects of pH on the performance of AuNPs/Gr/GCE were studied systematically. As can be seen from −1. The linear regression equation was: I (μA) = − 9.837 × 10−2 + 16.39c (mmol·L−1) (R2 = 0.998). From the plot, the calculated limit of detection (LOD) was 0.011 μmol·L−1 on the basis of the 3SD/n method, where ‘SD’ is the standard deviation of the blank solution and ‘n’ is the slope of the standard curve. The experimental result indicated that our proposed method possessed a wider dynamic range and a lower limit of detection relative to previous methods  method, as shown in  methods .−3 mol·L−1 SAM solution containing interference species. Specifically, 200-fold of glucose and sucrose, and 300-fold of NaCl, KCl, CaCl2, and MgSO4 were added into the SAM solutions, respectively. It was found that 300-fold of NaCl, KCl, CaCl2, MgSO4 had little interference effect on the detection of SAM, but 200-fold of glucose and sucrose had a great impact. As shown in To evaluate the effects of interfering substances on the detection of SAM, CV was performed in 1.0 × 10−3 mol·L−1 was continuously scanned 12 times. In this work, the change of oxidation peak current that occurred the first three times was small, but afterwards it changed obviously. The reason for this may be the surface passivation of AuNPs/Gr/GCE under acidic conditions, which hindered the electron transfer to the surface of AuNPs/Gr/GCE. The AuNPs/Gr/GCEs were observed for 0–15 h, respectively. The peak current of newly prepared AuNPs/Gr/GCE was 32.23 μA. After 15 h, the peak current was 29.01 μA with a reduction of 9.71% and the oxidation peak potential was almost unchanged. Therefore, AuNPs/Gr/GCE had high electrocatalytic activity and good stability.To evaluate the repeatability and stability, the corresponding experiments were studied. Under optimized conditions, SAM of 1.0 × 10To prove the feasibility of the AuNPs/Gr/GCE in real sample, the calibration experiment was studied. Pork extract was selected as the sample, and the content and recovery of SAM in the diluted sample solutions were determined via the standard adding method. The established standard curve equation in the above experiment was used to calculate the content of SAM. The results are shown in A simple and feasible electrochemical sensor based on AuNPs/Gr nanocomposites was provided for detecting SAM. The AuNPs/Gr nanocomposites have a better performance compared with pure Gr for the detection of SAM owing to their high conductivity and large surface area. Furthermore, the prepared AuNPs/Gr/GCE possessed a certain stability, good selectivity, and acceptable recovery abilities. Based on the abovementioned characteristics, it could be promising in the practical application of detecting in a real sample.


A) accounting for 46.3%, common or shared environmental parameter (C) accounting for 26.3% and unique/nonshared environmental parameter (E) accounting for 27.5% for females and 29.9, 33.1, and 37.0% for males, respectively. Although no SUA-related genetic variants reached genome-wide significance level, 25 SNPs were suggestive of association (P < 1 × 10−5). Most of the SNPs were located in an intronic region and detected to have regulatory effects on gene transcription. The cell-type specific enhancer of skeletal muscle was detected which has been reported to implicate SUA. Two promising genetic regions on chromosome 17 around rs2253277 and chromosome 14 around rs11621523 were found. Gene-based analysis found 167 genes nominally associated with SUA level (P < 0.05), including PTGR2, ENTPD5, well-known SLC2A9, etc. Enrichment analysis identified one pathway of transmembrane transport of small molecules and 20 GO gene sets involving in ion transport, transmembrane transporter activity, hydrolase activity acting on acid anhydrides, etc. In conclusion, SUA shows moderate heritability in women and low heritability in men in the Chinese population and genetic variations are significantly involved in functional genes and regulatory domains that mediate SUA level. Our findings provide clues to further elucidate molecular physiology of SUA homeostasis and identify new diagnostic biomarkers and therapeutic targets for hyperuricemia and gout.Serum uric acid (SUA), as the end product of purine metabolism, has proven emerging roles in human disorders. Here based on a sample of 379 middle and old-aged Chinese twin pairs, we aimed to explore the magnitude of genetic impact on SUA variation by performing sex-limitation twin modeling analyses and further detect specific genetic variants related to SUA by conducting a genome-wide association study. Monozygotic (MZ) twin correlation for SUA level (rMZ = 0.56) was larger than for dizygotic (DZ) twin correlation (rDZ = 0.39). The common effects sex-limitation model provided the best fit with additive genetic parameter ( Serum uric acid (SUA), as the end product of purine metabolism, has proven emerging roles in human disorders, such as kidney disease , 2, diabSLC22A7, SLC2A9, SLC22A11, SLC22A12, ABCG2, etc., and a number of additional intriguing genetic networks , locus missing (>0.05), and minor allele frequency (<0.05). Finally, a total of 1,365,181 SNPs was included for subsequent GWAS analysis.DNA samples of 139 DZ pairs were genotyped on the Illumina’s Infinium Omni2.5Exome-8v1.2 BeadChip platform . Strong quality control was performed using the genome-wide efficient mixed-model association (GEMMA)  by removData preparation and descriptive analyses as well as genetic analyses were performed with SPSS version 22.0 and Mx program,A), common or shared environmental (C), and unique/nonshared environmental (E) parameters. After the general sex-limitation ACE model was firstly fitted, we then fitted its sub-models: the common effects sex-limitation models by setting the male-specific additive genetic effects (mC and fC) to 0 and the scalar sex-limitation models by constraining the variance components for females to be equal to a scalar multiple of the variance components for males, respectively.Then, standard structural equation modeling methods were used for sex-limitation twin modeling based on the classical twin methods. The variation was decomposed into sources of additive genetic  was calculated and a lower AIC indicated a better fit when no statistical difference was observed between two models . The covP < 5 × 10−8 and suggestive evidence level of P < 1 × 10−5 for this association were adopted (P < 1 × 10−5) was submitted. The enrichments of cell-type enhancers with uncorrected P < 0.05 were reported.The association of SUA level with SNP genotypes was tested using the GEMMA . The cov adopted . We furt adopted , 29. A sP < 2.63 × 10−6  was considered to be genome-wide significant for the association as 19,001 genes being evaluated.We performed gene-based tests on GWAS summary results by using Versatile Gene-based Association Study-2 (VEGAS2) which uses 1,000 genomes data to model SNP correlations across the autosomes and chromosome X , 31. In P < 0.05) were included to compute the over-represented gene sets in the gene sets-based analysis using the online version of gene set enrichment analysis (GSEA) programP-value, i.e., false discovery rate (FDR) <0.05.A list of significant genes .The final sample contained a total of 379 twin pairs (240 MZ and 139 DZ pairs) with a median age of 50 years (95% range: 41–69 years). The median (95% range) of SUA level for all participating individuals was 256 µmol/L (143–468 µmol/L), with males having higher SUA level than females [298 (179–509) vs. 226 (130–374), rMZ = 0.56, 95% CI: 0.47–0.64) was larger than for DZ twin , indicating the presence of genetic influence  As described, we firstly fitted the general sex-limitation ACE model and its sub-models and then compared their performances. For the variance in SUA, the common effects sex-limitation model (Model II) provided the best fit  with A parameter accounting for 46.3% (95% CI: 15.0–73.4), C parameter accounting for 26.3% (95% CI: 0–55.8), and E parameter accounting for 27.5% (95% CI: 19.8–37.9) for females and 29.9% (95% CI: 0–60.0), 33.1% (95% CI: 5.4–63.4), and 37.0% (95% CI: 26.9–50.3) for males, respectively  (Table P = 2.50 × 10−7) in an intronic region of EXOC3L2 on chromosome 19q13.32.A total of 1,365,181 SNPs genotyped from a sample of 139 DZ twin pairs were included for the GWAS of SUA level. The relationship between the observed and expected GWAS 5) Table . The strP = 3.25 × 10−6–7.29 × 10−6) and two SNPs (P = 1.30 × 10−6–8.93 × 10−6) were located at or near TNRC6C and TMC6/TNRC6C-AS1 genes, respectively. At chromosome 14q24.3, four SNPs rs2270073, rs2270074, rs11621523, and rs2159179 (P = 2.77 × 10−6–7.59 × 10−6) were positioned within or closest to PTGR2 gene that was involved in terminal inactivation of prostaglandins. The rs2336742 (P = 7.02 × 10−6) was located at the intronic region of ENTPD5 gene, which was involved in the pathway of purine metabolism. The number of SNPs mapping to ZNF410 and FAM161B was five (P = 3.39 × 10−6–7.64 × 10−6) and one (P = 7.64 × 10−6), respectively. All the abovementioned genes showed nominal association with SUA level (P < 0.05) from the following VEGAS2 analysis.Among these top signals, two chromosomal loci (17q25.3 and 14q24.3) showed nominal association with SUA level as the locus zoom plots illustrated  of brain angular gyrus (P = 0.003) and skeletal muscle  (P = 0.008) were identified for the set of 25 query SNPs . Most of the SNPs were located in intronic regions. Several SNPs were detected within regions with promoter histone marks or enhancer histone marks and could change DNA motifs for DNA-binding proteins, and thus would have regulatory effects on gene transcription . We compared previously reported 2,368 significant SUA-associated SNPs in a series of studies with our results. Although no genome-wide significant SNPs were identified in our study, we defined our SNPs with P < 0.05 as supportive to the reported SNPs. And 57 SNPs located in genes SLC2A9, ABCG2, LRRC16A, LOC107986260, GLUT9, SCGN, LOC107986971, TFCP2L1, TET2, KCNQ1, FRAS1, SLC16A9, RAF1P1, LOC100129344, LOC107986581, LRRC16A, LOC100287951, WDR1, PDZK1, and LOC107986260 could be replicated .As predicted by HaploReg v4.1, two cell-type specific enhancers (uncorrected P < 0.05) from VEGAS2 analysis. Genes of TNRC6C-AS1, ZNF410, TNRC6C, FAM161B, PTGR2, SESTD1, RGS20, ENTPD5, EXOC3L2, DNAH3, and TMC6 had already been indicated in the SNPs-based analysis  were presented in Table q-value = 0.020). And the GO gene sets were involved in ion transport, regulation of catabolic process, transmembrane transporter activity, hydrolase activity acting on acid anhydrides, cellular process, regulation of immune system process, etc.In the gene sets-based analysis using GSEA program, one REACTOME gene set and 20 GO gene sets (FDR P > 0.05), indicating that there was no evidence for sex-specific additive genetic effects. We then considered whether the common or shared environmental effects for males (mC) (Model III) and further for females (fC) (Model IV) could also be fixed to 0. However, the goodness-of-fit statistics indicated that these two models provided a significantly worse fit (P < 0.05). Thus, these two models were rejected and model II remained the favored one. Finally, we considered the scalar sex-limitation models (Model V and Model VI) by constraining the variance components of females to be equal to scalar multiples of the variance components of males. Both of the models provided a significantly worse fit than Model II (P < 0.05). Hence, we concluded that the Model II was the best fitting model, in which additive genetic parameter (A) explained larger proportion of SUA variation for females (46.3%) than for males (29.9%), whereas the environmental parameters together (C and E) explained smaller proportion (53.7 vs. 70.1%) . In fitting the sex-limitation ACE models, the common effects sex-limitation model of dropping The sex-difference in the genetic and environmental effects on SUA variation obtained here was in line with the previous Boyle et al.’s genetic study. Based on a sample of 112 twin pairs, they also found a more significant genetic component in control of SUA variation in females than males, whereas a stronger role of environmental component in males . We specPTGR2 and ENTPD5 genes around the rs11621523 have been emphasized for their roles in SUA level. Prostaglandin reductase 2 (PTGR2) is the enzyme involved in terminal inactivation of prostaglandins . Additionally, rs11621523 and rs2159179, which were located in an intergenic region at 14q24.3 and closest to PTGR2 gene, should also be candidates to be further studied. The protein encoding by ENTPD5 gene was involved in the pathway of purine metabolism in which SUA was a by-product of oxidation. As SNP rs2336742 was located at the intronic region of ENTPD5 gene and could change its DNA motifs, it might be associated with purine metabolism as well as SUA level . However, the association of novel TNRC6C, TMC6, and TNRC6C-AS1 genes around the other SNP rs225327 with SUA level still needs to be validated. Finally, the enhancer of skeletal muscle was predicted by submitting the set of 25 query SNPs to HaploReg v4.1 . And the relationship between SUA level and skeletal muscle strength/volume has been fully researched currently  from VEGAS2 analysis. The results of GO gene sets from using GSEA program indicated that these genes might be associated with process of generation, catabolism, transport, intracellular signal transduction, and hydrolase activity during SUA metabolism. Besides, several genes were enriched in pathway of transmembrane transport of small molecules, including solute carrier family  and ATPase family (ATP6V1H and ATP11B), which strengthened their significance in regulating SUA transport process and thus further influencing SUA level  and thus influences the GPCRs activity. And the GPCRs could regulate the assembly of a multienzyme complex for purine biosynthesis (SLC2A9 (P = 0.001) with SUA level has previously been reported  (Accession No. PRJEB23749).All participants provided written informed consent for participating in the study which was approved by the local ethics committee at Qingdao CDC, Qingdao, China.DZ and WW contributed to the conception and design. HD and CX organized the collection of samples and phenotypes. WW and YW contributed to sample data and sequencing data management. QT and SL analyzed the sequencing data and WW and CX interpreted the analysis results. WW and DZ drafted the manuscript, YW and HD were involved in the discussion, and QT, SL, and DZ revised it. All the authors read the manuscript and gave the final approval of the version to be published. All the authors agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The emergence of pathogens with decreased susceptibility to available therapies has become an emerging problem often associated with treatment failure. Hence, there is an urgent need for novel broad-spectrum antimicrobial agents. The purpose of this study was to assess the feasibility of chlorquinaldol as an alternative approach to currently used topical antibiotics for the treatment of skin and soft tissue infections. The activity of chlorquinaldol was investigated against a collection of bacterial isolates responsible for skin infections, including strains resistant to fusidic acid and gentamicin. After determination of MIC and MBC, time-kill experiments were carried out by counting colonies grown after 0, 3, 6, 9, 24, and 48 h of incubation with concentrations equal to ¼×, ½×, 1×, 2×, and 4× MIC of chlorquinaldol, gentamicin, or fusidic acid. Staphylococci resulted the Gram-positives most sensitive to chlorquinaldol, with MIC-values ranging from 0.016 to 0.5 mg/L. A lower activity was observed against Gram-negative bacteria, with 77% of the isolates being inhibited at concentrations ranging from 128 to 512 mg/L. Generally, in time-kill studies, chlorquinaldol showed a bactericidal activity at the higher concentrations  after 24–48 h of incubation. In conclusion, chlorquinaldol may represent a valuable alternative to conventional topical antibiotics for the treatment of skin and soft tissue infections.Skin and soft tissue infections  Skin and soft tissue infections (SSTIs) are commonly occurring diseases with a wide-range of clinical manifestations varying from minor superficial to life-threatening infections , is a derivative of 8-hydroxyquinoline which is usually administered topically for the treatment of skin infections, alone or in combination with diflucortolone valerate , methicillin-susceptible S. aureus , methicillin-resistant Staphylococcus epidermidis , Streptococcus pyogenes (n = 6), Enterococcus faecalis (n = 13), Propionibacterium acnes (n = 10), Proteus mirabilis (n = 11), P. aeruginosa (n = 12), Escherichia coli (n = 12), and Enterobacter spp. (n = 12) were used. Identification of the isolates was carried out on a Vitek2 Compact  and further confirmed by pyrosequencing .Clinically relevant strains isolated from SSTIs at the Laboratory of IRCCS Galeazzi Orthopedic Institute were used. In particular, methicillin-resistant Stock solutions of chlorquinaldol , fusidic acid , and gentamicin  were prepared in 95% ethanol  or sterile water (fusidic acid and gentamicin) at concentrations of 5,000 mg/L and stored in aliquots at −20°C until use.The bacteriostatic and bactericidal properties of chlorquinaldol and its spectrum of activity were evaluated in comparison with gentamicin and fusidic acid.8 CFU/ml). For P. acnes broth was supplemented with 3% of defibrinated sheep blood . After obtaining a concentration of 105 CFU/ml using appropriate dilutions, each suspension was inoculated in a 96-wells microtiter plate containing a serial 2-fold dilution of chlorquinaldol, fusidic acid, or gentamicin. MIC-values, corresponding to the lowest concentration exhibiting no visible bacterial growth, were read after 24 h of incubation at proper conditions (except for P. acnes that required 48 h). The MBC was determined by plating 10 μl from each well showing no turbidity onto agar plates. After incubation at proper conditions, MBC was read as the lowest concentration able to kill 99.9% of the initial inoculum.The antimicrobial activity was assessed by determining the MIC and the MBC-values against the microbial strains described above. The MIC was determined by broth microdilution method, in accordance with EUCAST standard8 CFU/mL). An aliquot was inoculated into test tubes containing 2 ml of growth medium alone (growth control) or containing each testing substance (see above) at different concentrations . Final concentration of bacteria was 5 × 105–5 × 106 CFU/mL. Tubes were incubated at 37°C in proper conditions. Microbial counts were performed after 0, 3, 6, 9, 24, and 48 h of incubation by plating 0.1 mL of a proper dilution of bacterial suspension onto agar plates. Results were expressed as Log10 (CFU/mL). The limit of count detection was 200 CFU/mL. Bactericidal activity was defined as a 3 Log10 decrease in CFU/mL (99.9% kill) of the initial inoculum. Bacteriostatic activity was defined as <99.9% kill.Killing curves were performed against one representative strains of each species described above. Briefly, a suspension in growth medium was prepared for each bacterial strain with an optical density equal to 0.5 McFarland . Modest inhibitory activity was observed against S. pyogenes and P. acnes.Among Gram positives, staphylococci resulted the most sensitive cocci to chlorquinaldol, which was able to inhibit bacterial growth at concentrations ranging between 0.016 and 0.5 mg/L. MBC-values were highly variable: in 47% of cases they were equal or 2-fold the MIC, in 37% 4–8-folds the MIC, and in the remaining 16% they were 16–32-folds the MIC. Chlorquinaldol was effective at low concentrations against P. acnes presented MIC and MBC-values higher than 512 mg/L. Staphylococci displayed the highest variability among the tested strains with MIC and MBC-values ranging from 0.25 to 1,024 mg/L.All staphylococci with the exception of five MRSE strains resulted resistant to gentamicin. All S. aureus strains were susceptible to fusidic acid, presenting MIC-values varying from 0.016 to 0.032 mg/L, with the exception of one MSSA isolate (MIC 2 mg/L). Eight strains of S. epidermidis were susceptible to fusidic acid, while five strains were found to be resistant. Propionibacteria were inhibited by low concentrations of fusidic acid, while higher MIC-values were observed for E. faecalis and S. pyogenes. Generally, MBC of fusidic acid were 4–128 times higher than the corresponding MIC.All E. coli, 75% of P. aeruginosa, 33% of Enterobacter spp., and 81% of P. mirabilis. MBC-values were close to MIC-values , except for one P. aeruginosa strain .Gram-negative strains revealed a high heterogeneity in distribution of gentamicin resistance, so that susceptible and resistant strains are reported separately in Table E. coli isolates showed a MIC-value of 8 mg/L, while 1 P. mirabilis and 2 P. aeruginosa isolates showed a MIC-value of 32 mg/L. MBC-values were ≥512 mg/L for 77% of tested strains , MSSA (4 × MIC), MRSA (4 × MIC), and E. faecalis (4 × and 2 × MIC), and after 24 h and 48 h for S. pyogenes (4 × and 2 × MIC). 2 log10 reductions were observed for MRSA (2 × MIC), P. acnes (4 × MIC), and E. faecalis (1 × MIC) after 48 h of exposure. Gentamicin (4 × and 2 × MIC concentrations) was bactericidal against S. epidermidis after 24 and 48 h. Fusidic acid was essentially bacteriostatic against staphylococci and P. acnes.Time-kill curves for Gram-positive strains are reported in Figures P. aeruginosa after 48 h exposure (4 × MIC) and against P. mirabilis after 24 h (4 × MIC) and 48 h (2 × and 4 × MIC). 2 log10 reductions were observed for E. coli (4 × MIC) and E. cloacae (4 × MIC) after 48 h of exposure, and for P. mirabilis after 24 h (2 × MIC). Gentamicin was bactericidal against E. coli after 6 h (4 × MIC) and 9 h (2 × MIC), against E. cloacae after 6 h (2 × and 4 × MIC) and 9 h (1 × MIC), and against P. aeruginosa after 6 h (4 × MIC) and 9 h (2 × MIC).Time-kill curves for Gram-negative strains are reported in Figures S. pyogenes, are the dominant organisms isolated in the acute infectious process, whereas Gram-negative organisms are more often seen in chronic or postoperative wounds  for the treatment of SSTIs. Its percutaneous absorption is very small, narrowing the risk of systemic effects  than those observed in the present manuscript  in SSTI (Moet et al., Considering that, usually, topical formulations contain high concentrations of antibiotics, it may be hypothesized that chlorquinaldol levels at the infection site might be much higher than the MIC observed in this study.In conclusion, chlorquinaldol, due to its wide spectrum of activity, would provide a valuable alternative for the treatment of SSTIs. Moreover, its use in combination with other antibiotics could be hypothesized in order to enhance antimicrobial activity and to limit the increasing resistance to topical antibiotics. Further, studies aiming to define a clinical breakpoint, evaluate synergy with other compounds and the ability to select for resistance after antibiotic exposure are advisable in the next future. Old studies pointed out chlorquinaldol efficacy against mycotic infections, so additional investigations to update its spectrum of activity against fungi would be also of great interest.LD conceived and designed the experiments. MB, AB, and MA performed the experiments. MB, AB, and ED analyzed the data. MB and AB prepared figures and graphs and wrote the manuscript. LD and ED revised the manuscript. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brassicaceae family. Exogenous application of MeJA has shown to increase tissue GS concentrations and the formation of myrosinase-mediated GS hydrolysis products (GSHPs). In vitro and in vivo assays have demonstrated the potential health-promoting effects of certain GSHPs. MeJA is also known to elicit and induce genes associated with defense mechanisms to insect herbivory in Brassica species. To investigate the relationship between MeJA-induced GS biosynthesis and insect defense, three treatments were applied to “Red Russian” kale (Brassicae napus var. pabularia) seedlings: (1) a 250 µM MeJA leaf spray treatment; (2) leaf infestation with larvae of the cabbage looper (Trichoplusia ni (Hübner)); (3) control treatment . Samples of leaf tissue from the three treatments were then assayed for changes in GS and GSHP concentrations, GS gene biosynthesis expression, and myrosinase activity. Major differences were observed between the three treatments in the levels of GS accumulation and GS gene expression. The insect-damaged samples showed significantly lower aliphatic GS accumulation, while both MeJA and T. ni infestation treatments induced greater accumulation of indolyl GS. The gene expression levels of CYP81F4, MYB34, and MYB122 were significantly upregulated in samples treated with MeJA and insects compared to the control group, which explained the increased indolyl GS concentration. The results suggest that the metabolic changes promoted by MeJA application and the insect herbivory response share common mechanisms of induction. This work provides potentially useful information for kale pest control and nutritional quality.Methyl jasmonate (MeJA), synthesized in the jasmonic acid (JA) pathway, has been found to upregulate glucosinolate (GS) biosynthesis in plant species of the  Brassica oleracea, while others are classified as Brassica napus. The popularity of kale has recently increased, as more information has been published regarding its high nutritional value  from kale leaf samples  across all 10 hydrolysis products from insect-damaged samples ranged from 34% to 136%, while the CV of MeJA-treated samples was relatively small (10% to 27%). These results suggest that the stimulus from the insect herbivory process might be more dynamic than the one-time MeJA application, because insects can be exposed to significantly different levels of GS depending on kale leaf location and respond accordingly .T. ni larval feeding on the expression of genes related to GS biosynthesis or their hydrolysis products in “Red Russian” kale, we investigated gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) to measure the abundance of transcripts associated with the above-mentioned pathways under the two treatments. We measured six genes involved in the aliphatic GS biosynthesis pathway, including MAM3, SOT17, SOT18, GSL-OH, MYB28, and MYB29, and nine genes involved in indolyl GS biosynthesis, including SUR1, CYP79B2, SOT16, CYP81F1, CYP81F2, CYP81F3, CYP81F4, MYB34, and MYB32 , and epithiospecifier modifier 1 (ESM1), were measured to investigate the potential changes in the levels of hydrolysis products (To understand the different effects of exogenous 250 μM MeJA application and nd MYB32 . In addiproducts .MAM3 (chain elongation), SOT17 (core biosynthesis), SOT18 (core biosynthesis), GSL-OH (secondary modification), MYB29 (transcription factor), TGG1 and TGG2, ESP, and ESM1, between 1.7- and 9.4-fold. T. ni-induced damage only significantly upregulated SOT17, while there was a significant downregulation of MAM3, SOT17, SOT18, MYB28, and MYB29. These results indicated that a spray application of 250 μM MeJA is sufficient to induce the majority of aliphatic GS biosynthesis genes in B. napus kale. It has been reported that a 400 μM MeJA application to the heads of the broccoli cultivar “Green Magic” can significantly increase SOT17, SOT18, TGG1, TGG2, and ESM1, but this is not observed in the doubled haploid inbred “VI-158” [MYB28 was the only gene that was significantly downregulated by the 250 μM MeJA treatment. Different gene homologues encoding MYB28 may have differential sensitivity to exogenous MeJA, since it was reported that these genes can be differentially expressed in allopolyploid Brassica juncea [B. oleracea kale, found that the relative gene expression of MYB28 was inconsistent among all measured homologues encoding MYB28. The authors reported that a 250 μM MeJA application significantly increased, decreased, or did not change the levels of MYB28 transcripts. The transcript we measured was Bol036286, and our result agrees with their results regardless of the fact that different species were used in the two studies. Another possible reason is that the primers used in B. oleracea did not bind or effectively amplify the transcripts of MYB28. B. napus kale is an allopolyploid which contains the genome from B. rapa (AA) and B. oleracea (CC) and many gene duplications, and subsequent functional divergence has occurred during polyploidization [MYB28 homologs might not have been accurately measured when we analyzed the data. Interestingly, even with increased gene expression levels, the total aliphatic GS concentration in MeJA-treated kale was not significantly greater compared to control kale samples  expression in “Red Russian” kale of multiple genes related to indolyl GS biosynthesis, such as CYP79B2 (core biosynthesis), SOT16I (core biosynthesis), and CYP81F1, CYP81F2, CYP81F3, and CYP81F4 (side chain modification), and the transcription factors MYB34 and MYB122  [Brassica oleracea ssp. italica) [MYB34 in MeJA-treated kale resulted in a significantly higher accumulation of indolyl GS  , and twoitalica) . Our findolyl GS . CYP79B2aldoxime . This coaldoxime . The intbidopsis . The hig cabbage . In Yi e cabbage , the sigspecific . The higrassicin . The genre of GS . ESP procultures . Isothiocultures , and werT. ni feeding on “Red Russian” kale plants was not strong enough to induce the expression of CYP79B2. However, considering the indolyl GS changes in T. ni damage may be more location-specific than MeJA treatment and, therefore, it might not be as powerful as MeJA application in triggering gene regulation. As a generalist insect, T. ni is generally exposed to a broad array of plants. The chemical composition of T. ni’s saliva can be altered when T. ni confronts different plant species, as suggested by a recent study [T. ni was not as effective as the MeJA treatment in activating genes involved in the indolyl GS biosynthesis pathway. Noticeably, the expression levels of MYB34, CYP79B2, and CYP81F1 in insect-damaged were slightly higher than those in control kale, but not significantly different. Compared to the previous exogenous MeJA application study, significantly increased levels of gene expression were measured 2 days after MeJA application on broccoli [In the present study, insect-damaged kale did not show the same pattern of gene expression, suggesting that larval feeding was not perceived or did not provide as intense a response as observed following exogenous application of 250 μM MeJA in kale plants. It is possible that the intensity of the response caused by six-second instar of nt study . Therefobroccoli  and pak broccoli , or threbroccoli .Athalia rosae on Sinapis alba (Family Brassicaceae) and found a strong increase in myrosinase activity in day-one tissue samples, decreased activity in day-two samples, and no difference in control and insect-treated samples on day four after feeding initiation [The greatest myrosinase activity was detected in control samples (4.86 ± 0.16 Unit/g DW), while reductions in the activity were observed in both MeJA-treated samples (3.42 ± 1.00 Unit/g DW) and insect-damaged samples (2.63 ± 1.31 Unit/g DW), with 30% and 45% lower activity than in the control, respectively A. In genitiation . The effitiation . This suT. ni were found to show feeding preference for nitrile-producing Arabidopsis lines [Nitrile formation (%) from sinigrin was found to be increased only by MeJA treatment. Both MeJA spray application and larval feeding significantly decreased nitrile formation following hydrolysis of gluconasturtiin B. The cois lines . HydrolyIn addition to the change of secondary metabolites under the various treatments, changes in several primary metabolite concentrations  were measured via gas chromatography–mass spectrometry (GC–MS) to further illustrate the effects of insect damage and MeJA treatment on kale plants.Principal component analysis (PCA) was utilized to compare the changes in metabolites and identify significantly differences in metabolites between control and treatment groups. According to PCA scores plot, the distribution of metabolites in MeJA-treated and insect-damaged kale was clearly different compared to the control . PrincipNicotiana attenuata plants at various developmental stages [Brassica crops [The most significantly different concentrations in metabolites among the three treatments were selected by partial least-squares discriminant analysis (PLS-DA) using variable importance in projection (VIP) to estimate the importance of variables in the model. VIP score 1.2 was set as a threshold. Glucose, galactose, sucrose, fructose, and alanine  were abol stages . Mechadol stages  presenteca crops ,42. The ca crops . The monca crops ,74. In t® LC1  professional soil mix. The plants were grown in a greenhouse at the University of Illinois at Champaign-Urbana at 25°C/18°C and 14 h–10 h day–night temperature regime, with supplemental high-intensity discharge (HID) lighting and 50–70% relative humidity. Four weeks later, the plants in the vegetative growth stage were transferred to 1 L pots in the greenhouse under the same conditions. Kale plants with eight fully developed leaves were selected for the experiments.The kale variety used in this experiment was “Red Russian” . Seeds of “Red Russian” kale were germinated in 32-cell plant plug trays filled with SunshineT. ni (Hübner). T. ni was cultured in the Department of Entomology at the University of Illinois Urbana-Champaign. Six larvae of T. ni per plant were employed, with three plants per treatment, and the leaf tissue was scored for damage and then harvested for the analyses after four days of feeding.The control plants were sprayed with a 0.1% Triton X-100  solution. MeJA-treated plants were sprayed with a solution of 250 µM JA  with 0.1% Triton X-100. MeJA-treated kale leaf samples were harvested four days after spray treatment. Using a paintbrush, insect-treated plants were infested with second-instar larvae of https://imagej.nih.gov/ij/). . . Area seg for 5 min at 4°C. The supernatant was saved, and the pellet was re-extracted with 2 mL 70% methanol at 95°C for 10 min, after which the two extracts were combined. A subsample (1 mL) from each pooled extract was transferred into a 2 mL microcentrifuge tube . The proteins were precipitated with 0.15 mL of a 1:1 mixture of 1 M lead acetate and 1 M barium acetate. After centrifuging at 12,000× g for 1 min, each sample was loaded onto a column containing 1M NaOH and 1M pyridine acetate-charged DEAE Sephadex A-25 resin  for desulfation with arylsulfatase  for 18 h, and the desulfo-GSs were eluted with 3 mL Millipore-filtered ddH2O. The samples (100 µL) were injected onto an Agilent 1100 HPLC system , equipped with a G1311A binary pump, a G1322A vacuum degasser, a G1316A thermostatic column compartment, a G1315B diode array detector, and an HP 1100 series G1313A autosampler. The UV detector was set at 229 nm wavelength. An all-guard cartridge pre-column  and a Kromasil RP-C18 column  were used for quantification. The flow rate was 1 mL/min with mobile phase A (water w/1% acetonitrile v/v and 1 mM ammonium acetate) and B (100% acetonitrile) with the following elution profile: 0 min 0% B, 7 min 4% B, 20 min 20% B, 35 min 25% B, 36 min 80% B, 40 min 80% B, 41 min 0% B, and 50 min 0% B. Benzylglucosinolate was used as an internal standard for quantification whose relative response factor (RRF) was set as 1 [All above-ground leaves of control, insect-infested, and MeJA-sprayed kale plants were harvested after four days of treatment for GS analysis. Above-ground aerial leaf and stem samples were frozen in liquid nitrogen and stored at −20°C prior to freeze-drying. Freeze-dried tissues were ground into a fine powder using a coffee grinder and stored at −20°C prior to GS analysis using high-performance liquid chromatography (HPLC). Extraction and quantification of GSs using HPLC were performed using a previously published method . An amouset as 1 . The UV set as 1 ,78,79 ap–1. Ten microliter of desulfo-glucosinolate extract was injected. The column was heated to 40°C with a column heater. Mass identification was acquired in both positive and negative ion mode. Full scan mode range was m/z 100.0–900.0. Nitrogen gas was used for ESI, collision Resolution was 70,000 FWHM (at m/z 200). Automatic gas control (AGC) value was 3×106 in full scan; maximum ion inject time was 200 ms. The ion spray voltage was 3.9 kV with capillary temperature at 320°C. Aux gas flow rate was S-Len, RF level was 55. MS/MS isolation window width was 4.0 m/z, with resolution at 17,500 in both positive and negative modes. AGC value was 2×105, and maximum ion inject time was 100 ms. The identification of desulfo-GS was based on Capriotti et al. with a slight modification , and usig for 2 min, and the lower organic layer was collected. A gas chromatograph  coupled to an MS detector system  and an autosampler . A capillary column  was used to determine GS hydrolysis products. A 1 μL sample of the dichloromethane extract was injected into the GC–MS with a split ratio of 1:1. After an initial temperature held at 40°C for 2 min, the oven temperature was increased to 260°C at 10°C/min and held for 10 min [Freeze–dried kale powder (50 mg) was suspended in 1 mL distilled water in a 2 mL microcentrifuge tube . Hydrolysis products were generated naturally by endogenous myrosinase in the absence of light at room temperature for 24 h. After adding 1 mL of dichloromethane, the samples were centrifuged at 12,000× r 10 min ,82. The r 10 min  or by coMyrosinase activity was estimated as the total amount of hydrolysis products produced within 60 min . One unig for 5 min. The supernatant of the horseradish root extracts was transferred to a beaker and boiled until the solvent was evaporated and then it was reconstituted with 50 mL of deionized water). After centrifugation at 12,000× g for 2 min, 0.6 mL of supernatant was transferred to a 1.5 mL Teflon centrifuge tube , and then 0.6 mL of dichloromethane was added. The tubes were placed upside down to minimize the loss of volatile compounds at room temperature for 10 min. Then, the tubes were vortexed and centrifuged at 12,000× g for 4 min. The dichloromethane organic layer was injected into a GC–MS  coupled to an MS detector system  and an autosampler . A capillary column  was used to determine GS hydrolysis products. After an initial temperature held at 40°C for 2 min, the oven temperature was increased to 320°C at 15°C/min and held for 4 min. Injector and detector temperatures were set to 270°C and 275°C, respectively. The flow rate of the helium carrier gas was set to 1.2 mL/min. Standard curves of allyl isothiocyanate, 2-phenthyl isothiocyanate, and 3-phenylpropionitrile  were used for quantification. A standard curve generated from allyl isothiocyanate was applied to quantify of 1-cyano-2,3-epithiopropane.Freeze–dried kale powder (75 mg) was mixed with 1.5 mL of concentrated “1091” horseradish root extract [v/v) for qRT-PCR. The primer sets of GS biosynthesis genes, hydrolysis genes, and transcription factor genes were designed on the basis of database-published sequences (http://www.ocri-genomics.org/bolbase/index.html) [BoACT1) [® Green RT-PCR Master Mix (QIAGEN) using an ABI 7900HT Fast Real-Time PCR System  according to the manufacturer’s instructions. The relative expression ratio was determined with the equation 2Ct−∆∆, using the BoACT1 normalized ∆Ct values generated by the ABI 7900HT Sequence Detection System Software 2.4 (Applied Biosystems) [Total RNA was isolated from control and insect- MeJA-treated freeze–dried kale powder samples using the RNeasy Mini Kit (QIAGEN), according to the manufacturer’s instructions. The quantity of RNA was measured using a NanoDrop 3300 spectrophotometer . One μg of total RNA was reverse-transcribed with Superscript™ III First-Strand Synthesis SuperMix for qRT-PCR , according to the manufacturer’s instructions. The resulting cDNA samples were diluted to 1/10 of their concentrations (ex.html) . A final[BoACT1) . The prisystems) .g for 3 min, and 0.7 mL of supernatants was transferred to new 2 mL microcentrifuge tubes. To fractionate polar compounds, 0.375 mL of cold chloroform (−20 °C) and 0.7 mL cold water (4°C) were added. After vigorous mixing, the extracts were centrifuged at 15,000× g for 3 min, and then 50 μL supernatant was transferred to 1.5 mL microcentrifuge tubes. The extracts were dried using VacufugeTM concentrator  with 10 μL of methanol to facilitate water evaporation. The dried extracts were derivatized with 50 μL of methoxyamine hydrochloride (40 mg/ml in pyridine) for 90 min at 37°C, then with 70 μL MSTFA + 1%TMCS at 37°C for 30 min. The metabolites were analyzed using a GC–MS  coupled to an MS detector system  and an autosampler . A capillary column  was used to detect polar metabolites. After an initial temperature held at 80°C for 2 min, the oven temperature was increased to 330°C at 15°C/min and held for 5 min. The injector and detector temperatures were set to 250°C and 250°C, respectively. An aliquot of 1 μL was injected with a split ratio of 70:1. The helium carrier gas was kept at a constant flow rate of 1.2 mL/min. The mass spectrometer was operated in positive electron impact mode (EI) at 70.0 eV ionization energy at m/z 40–500 scan range.Primary metabolites were extracted by published protocol  with modThe acquired chromatograms were converted to mzXML using the RawConverter . Peak deT. ni larval feeding affected “Red Russian” kale GS profiles, hydrolysis products formation, related gene expression profiles, and primary metabolites production. In general, both treatments significantly triggered the accumulation of GS. MeJA treatment induced higher accumulation of aliphatic GSs, while the insect feeding treatment induced higher accumulation of indolyl GSs  and one amino acid  between control and treatment groups. This variation is likely due the production of defense-related compounds associated with insect herbivory and the induction of the jasmonic acid pathway. The ideal sample size was suggested to be 1:10 [This paper demonstrated how 250 uM MeJA spray applications and olyl GSs . Increasolyl GSs . Both trolyl GSs , but the be 1:10 ,88 for P be 1:10 .


Tuberculous peritonitis (TBP) mimics peritoneal carcinomatosis (PC). We aimed to investigate the discriminative use of PET/CT findings in the parietal peritoneum.18F-FDG) uptake in a long beaded line (string-of-beads 18F-FDG uptake) or in a cluster (clustered 18F-FDG uptake) or focal 18F-FDG uptake. CT patterns included smooth uniform thickening, irregular thickening, or nodules.Parietal peritoneal PET/CT findings from 76 patients with TBP (n = 25) and PC (n = 51) were retrospectively reviewed. The lesion locations were noted as right subdiaphragmatic, left subdiaphragmatic, right paracolic gutters, left paracolic gutters, and pelvic regions. The distribution characteristic consisted of a dominant distribution in the pelvic and/or right subdiaphragmatic region  (SAPI distribution), a dominant distribution in the remaining regions  (LSAPI distribution), or a uniform distribution. PET morphological patterns were classified as F18-fluorodeoxyglucose (18F-FDG uptake (76.0% vs 7.8%), and (d) smooth uniform thickening (60.0% vs 7.8%) , whereas more frequent findings in PC compared with TBP were (a) SAPI distribution (78.4% vs 28.0%), (b) clustered 18F-FDG uptake (56.9% vs 20.0%), (c) focal 18F-FDG uptake (21.6% vs 4.0%), (d) irregular thickening (51.0% vs 12.0%), and (e) nodules (21.6% vs 4.0%) .More common findings in the parietal peritoneum corresponding to TBP as opposed to PC were (a) ≥4 involved regions (80.0% vs 19.6%), (b) uniform distribution (72.0% vs 5.9%), (c) string-of-beads Our data show that PET/CT findings in the parietal peritoneum are useful for differentiating between TBP and PC. Tuberculous peritonitis (TBP) is a rare form of extrapulmonary TB; its reported incidence varies from 0.1% to 0.7% among all forms of TB worldwide, and delay in treatment initiation can lead to high mortality.,3 Ascites cytology has low positive detection rates.,4 Ascites adenosine deaminase (ADA) measurement is helpful in the differential diagnosis of TBP and PC; however, ADA is a nonspecific inflammatory and immune response marker for TB, and false negative results or false positive results are frequent in various clinical situations, such as TBP in patients with liver cirrhosis or HIV infection, PC, and bacterial peritonitis.,5 A mycobacterial culture of ascitic fluid is problematic because tuberculous peritonitis-associated mortality is high among patients waiting for the results. A T-cell-based enzyme-linked immunospot assay (T-SPOT.TB) is useful for the diagnosis of latent tuberculosis infection but has a limited role in individuals with a previous history of TB.,8 Although peritoneal biopsy has a higher diagnostic accuracy, it is limited due to its invasiveness, sampling error, and complications .,9,10Unfortunately, it is challenging to differentiate TBP from peritoneal carcinomatosis (PC) because the clinical manifestations and conventional laboratory tests of TBP and PC frequently overlap.–14 In contrast, F18-fluorodeoxyglucose(18F-FDG) PET has a high sensitivity for detecting peritoneal lesions because 18F-FDG PET can clearly detect harboring lesions as high-uptake foci.–17 Furthermore, malignant and benign peritoneal involvement may manifest as various imaging patterns on 18F-FDG PET, and awareness of the disease-specific characteristics is important to optimize diagnostic accuracy.18F-PET/CT is performed for TBP patients to further diagnose after conventional clinical examinations present undetermined diagnoses or suspected PC, particularly in countries having medium-to-high TB burdens,19–22 and occasionally in other countries.,24Thus, medical imaging is crucial for differentiating TBP from PC. Specifically, peritoneal CT findings are valuable for this differential diagnosis but still yield an undetermined diagnosis or even a misdiagnosis.18F-FDG PET/CT is used in the evaluation of peritoneal diseases of undetermined causes, but special attention should be paid to TBP mimicking PC.,19 However, until recently, there has been a lack of comparison studies that discuss how to differentiate between TBP and PC using 18F-FDG PET/CT.,16,22 The present study aimed to investigate discriminative PET/CT findings in the parietal peritoneum between the 2 entities.22.1This study was approved by the Institutional Review Board of Nanfang Hospital, Southern Medical University. Because of the retrospective nature of the study, the requirement of informed consent was waived.18F-FDG PET/CT before therapy were retrospectively enrolled. TBP was confirmed by peritoneal pathology using surgery (n = 10) and laparoscopy (n = 2) or by diagnostic anti-TB therapy and at least 12 months of clinical follow-up (n = 13). PC was confirmed by peritoneal pathology using surgery (n = 13) and laparoscopy (n = 18) or by ascites cytology (n = 20). No patients had both TB and malignancy concomitantly. The clinical characteristics are shown in Table From July 2004 to January 2015, 76 consecutive patients with TBP (n = 25) and PC (n = 51) who underwent Among the 25 TBP patients, concomitant TB at other sites was present in the ovary (n = 9), mediastinal lymph node (n = 8), lung (n = 6), ileocecum (n = 3), endometrium (n = 1), and fallopian tube (n = 1).Among the 51 PC patients, the primary cancer was present in the ovary (n = 19), stomach (n = 9), colon and rectum (n = 7), bile duct (n = 3), liver (n = 1), fallopian tube (n = 1), and endometrium (n = 1); the primary cancer was undetermined in 10 cases.2.218F was produced in a PET trace cyclotron . The 18F-FDG was automatically synthesized in a chemical synthesis module  with a radiochemical purity >95%. After fasting for more than 6 h in a calm state, the patient was intravenously injected with 0.15 mCi (5.5 MBq)/kg of 18F-FDG followed by lying in a dark room for approximately 1 h. PET and nonenhanced CT imaging was performed after emptying the urinary bladder. Scanning was performed from the middle femur to the cranial vault. The PET images were reconstructed according to an iterative ordered subset expectation maximization method. The reconstruction thicknesses of the CT images were 4.25 mm and 3.0 mm, and the PET and CT images were individually transferred to Xeleris  or Syngo MMWP (Siemens) workstations, respectively, to display frame-on-frame fusion images.Eleven TBP patients and 30 PC patients underwent a Discovery LS PET/CT Scanner . Fourteen TBP patients and 21 PC patients underwent a Biograph mCTx PET/CT Scanner . The 2.3All of the following PET/CT findings were separately reviewed by 2 nuclear medicine specialists who were blinded to all laboratory data and other imaging examinations. If interpretive disagreements occurred, the final reports were decided by a third nuclear medicine specialist.On each PET image, the region of interest (ROI) was drawn along the margin of the lesion to measure the standardized uptake value (SUV). The SUV is commonly used as a relative measure of FDG uptake. The basic expression for SUV is r is the radioactivity activity concentration (kBq/mL) measured by the PET scanner within an ROI, a′ is the decay-corrected amount of injected radiolabeled FDG (kBq), and w is the weight of the patient (g), which is used a surrogate for a distribution volume of trace. Because the maximum of SUV (SUVmax) has a significantly improved reproducibility compared to the mean SUV (SUVmean), the SUVmax was used in this study. The confirmation of parietal peritoneal lesions was based on the requisite exclusion of physiologic bowel activity, retained urinary activity, misregistration artifacts, attenuation correction artifacts, blooming from highly hypermetabolic lesions, respiration artifacts at the hemidiaphragm, and surrounding abnormalities from the viscera, visceral peritoneum, omentum, mesentery, and other peritoneal structures. The location of the parietal peritoneal lesions was noted as the right subdiaphragmatic, left subdiaphragmatic, right paracolic gutter, left paracolic gutter, and pelvic regions.,26where –28 the distribution of the parietal peritoneum lesions was classified as the SAPI distribution, in which the lesions were completely or primarily localized in the pelvic and/or right subdiaphragmatic region; the LSAPI distribution, in which the lesions were completely or primarily localized in the remaining regions; or the uniform distribution, in which the lesions were uniformly distributed in the susceptible and less-susceptible areas.Because the pelvic and right subdiaphragmatic regions of the parietal peritoneum are susceptible areas for peritoneal implantation (SAPI),whereas the remaining regions are less-susceptible areas for peritoneal implantation (LSAPI),18F-FDG-avid lesions in the sectional PET images, the morphological patterns included the following: (1) lesions continuously disseminated beyond a single region showing18F-FDG uptake in a long beaded line (string-of-beads 18F-FDG uptake)   Figs. A and C. ,29On the CT imaging, the CT findings were classified as smooth uniform thickening Fig. B, irregu2.4max and the number of regions with parietal peritoneum involvement were compared between the 2 entities using a receiver operating characteristic (ROC) curve analysis. The lesion distributions and the morphological presentations were compared between these 2 entities using the χ2 test, the continuity correction χ2 test, or Fisher's exact test. The data were analyzed using MedCalc, version 13.0.0.0 . If P < 0.05, the difference was considered statistically significant.The SUV33.1max calculations for the peritoneal lesions with the most significant 18F-FDG uptake between TBP and PC were 7.8 ± 3.3 (2.8–14.8) and 7.0 ± 3.7 (2.0–15.5), respectively. The area under ROC curve (AUC) was 0.591 (P = 0.174).The SUV3.2The involvement of the parietal peritoneum was noted in 25 (100%) TBP patients and 44 (86.3%) PC using PET, 19 (76.0%) TBP patients and 41 (80.4%) PC patients using CT, and 25 (100%) TBP patients and 44 (86.3%) PC patients using both PET and CT.P < 0.001)  of regions with parietal peritoneal involvement in TBP and PC patients was 5 (1) and 2 (2), respectively, with an AUC = 0.863 and a cut-off≥4. In addition, ≥4 involved regions in the parietal peritoneum occurred in 20 (80.0%) TBP patients and 10 (19.6%) PC patients  occurred more frequently than in PC patients (5.9%), P < 0.001  occurred more frequently in TBP patients (76.0%) than in PC patients (7.8%), P < 0.001. Clustered 18F-FDG uptake (clustered sign) was observed in 20.0% of TBP patients and 56.9% of PC patients, P < 0.05. Focal 18F-FDG uptake was noted in 4.0% of TBP patients and 21.6% of PC patients, P>0.05.Table P < 0.001. Irregular thickening in TBP patients (12.0%) was less common than in PC patients (51.0%), P < 0.05. Nodules were noted in TBP patients (4.0%) and PC patients (21.6%), P > 0.05.With regard to CT patterns, smooth uniform thickening in the parietal peritoneum occurred more frequently in TBP patients (60.0%) than in PC patients (7.8%), 3.5P < 0.001).When TBP was diagnosed based on one of the differential findings between the 2 entities, the sensitivities and specificities were 80.0% and 80.4% , 72.0% and 94.1% (uniform distribution), 76.0% and 92.2% (string-of-beads sign), and 60.0% and 92.2% (smooth uniform thickening) , 56.9% and 80.0% (clustered sign) (P < 0.05), and 51.0% and 88.0% (irregular thickening) (P < 0.05).For the diagnosis of PC, the sensitivities and specificities were 78.4% and 72.0% (SAPI distribution) . TB lesions contain a large number of epithelioid cells, lymphocytes, and Langerhans cells that have a high expression of glucose transporter 1 (Glut-1) and Glut-3, which induced high 18F-FDG uptake. This might be one of the main reasons that published studies have reported that TBP mimics PC on 18F-FDG PET/CT when attempting to differentiate between the 2 entities.–23,30–32As a semiquantitative indicator for reflecting glucose metabolism in lesions, SUV proposed that diffuse peritoneal uptake of 18F-FDG may be indicative of TBP rather than PC. Other case reports also described diffuse 18F-FDG uptake in the peritonea of TBP patients.,23,31,32 Conversely, the most frequent pattern was abnormally intense focal 18F-FDG uptake, although diffuse and focal 18F-FDG uptake in the peritoneum was observed in PC patients.,16Shimamoto et alP < 0.001) and that a cut-off of ≥4 involved regions (extensive involvement) was a significant indicator for diagnosis of TBP with 80.0% sensitivity and 80.4% specificity.This study indicated that the number of regions with parietal peritoneal involvement was significantly higher in TBP patients than in PC patients  is the lowest point of the abdominal cavity in the standing position. This area had a high incidence of lesion implantation through ascites. Although the paracolic sulcus is the lowest point in the reclining position of the human body, the right subdiaphragmatic space is the area where ascites most easily accumulates when in a reclining position due to the influences of intrathoracic negative pressure. The main reason that ascites does not easily accumulate in the left subdiaphragmatic space is restriction by the left phrenicocolic ligament and hepatic falciform ligament. Another reason is that the left paracolic sulcus is shallower than the right paracolic sulcus; thus, ascites can easily reach the right subdiaphragmatic region through the right paracolic sulcus. Therefore, the right subdiaphragmatic region is another area that facilitates the colonization of implanted lesions through ascites.,28With the exception of the greater omentum, the sites most commonly involved in peritoneal implants were the right subdiaphragm and the pelvis.P < 0.001). These results also support the notions that the peritoneal lesion spread of PC is commonly restricted by the anatomical features of the abdominal cavity, that intraperitoneal implantation through ascites might be the most common route of PC, with the peritoneal lesion distribution perhaps differing according to the site of primary cancer, and that hematogenous spread is the most common route for TBP.This study showed that parietal peritoneal lesions, completely or primarily localized in the pelvic and/or right subdiaphragmatic regions (SAPI distribution), were significant indicators of PC, with 78.4% sensitivity and 72.0% specificity, and that a uniform distribution in the susceptible and less-susceptible areas for peritoneal implantation (uniform distribution) was a significant indicator of TBP, with 72.0% sensitivity and 94.1% specificity .Different examination methods demand different diagnostic criteria. Visual laparoscopic diagnosis of TBP was based on the presence of multiple yellowish-white miliary tubercles of uniform size  on the visceral and parietal peritonea. PC was diagnosed by the presence of large nodules (1 to 5 cm in diameter) on the parietal peritoneum, omentum, falciform ligament, or liver surface.,29 For PET imaging, blooming from these miliary lesions with high hypermetabolism causes an observed lesion size that is larger than its actual size. Consequently, these nodular characteristics identified by PET do not exhibit a smooth uniform presentation  to identify their nodular features by CT, which yields a CT pattern of smooth uniform thickening.ion Fig. .18F-FDG uptake. These patterns were differentially observed in TBP and PC patients. The string-of-beads sign was a significant indicator of TBP, with 76.0% sensitivity and 92.2% specificity (P < 0.001), and the clustered sign was a significant indicator of PC, with 56.9% sensitivity and 80.0% specificity (P < 0.05).In this study, parietal peritoneal involvement was classified into 3 distinct PET patterns: string-of-beads, clustered, and focal 18F-FDG PET/CT features in the parietal peritoneum for differentiating between TBP and PC. Extensive involvement, a uniform distribution, string-of-beads sign, and smooth uniform thickening might be significant differential features of TBP. A SAPI distribution, clustered sign, and irregular thickening might be significant differential features of PC. However, further investigation with a large patient enrollment is warranted to confirm our findings.In conclusion, this study has established some basic