Lactuca sativa L.) cultivars with high flavonoid and phenolic acid content and demonstrated their anti-diabetic effect. Here we report on developing three fertile and true-breeding lettuce lines enriched with flavonoids with reported beneficial health effects. These lines were identified in a segregating population of EMS-mutagenized red lettuce and characterized biochemically and genetically. Change in red coloration was used as a visual indicator of a mutation in a flavonoid pathway gene, leading to accumulation of flavonoid precursors of red anthocyanins. Pink-green kaempferol overproducing kfoA and kfoB mutants accumulated kaempferol to 0.6–1% of their dry weight, higher than in any vegetable reported. The yellow-green naringenin chalcone overproducing mutant (nco) accumulated naringenin chalcone, not previously reported in lettuce, to 1% dry weight, a level only observed in tomato peel. Kfo plants carried a mutation in the FLAVONOID-3′ HYDROXYLASE (F3′H) gene, nco in CHALCONE ISOMERASE (CHI). This work demonstrates how non-GMO approaches can transform a common crop plant into a functional food with possible health benefits.We previously developed red lettuce ( Epidemiological studies suggested that diets high in fruits and vegetables confer beneficial effects on chronic metabolic and cardiovascular diseases4. With some exceptions5, these benefits were confirmed by meta-analyses of cohort studies8. However, the average dietary flavonoid consumption in Europe and the US may be too low to confer health effects10.Fruits and vegetables are good dietary sources of phenolics, ubiquitous phytochemicals that include flavonoids and phenolic acidsLactuca sativa, family Asteraceae) is a crop domesticated more than ten thousand years ago11. It is the third most commonly consumed vegetable in the US after potato and tomato, with US per capita consumption estimated at 11.7 kg/year12, and is considered a good source of fiber, iron, folic acid and vitamin C13. Common lettuce phenolics are caffeic acid derivatives, predominantly chicoric, chlorogenic, caffeoyltartaric and caffeoylmalic acids; and flavonol glycosides, predominantly quercetin 3-O-malonylglucoside, quercetin 3-O-glucoside and quercetin 3-O-glucuronide15. In addition, red varieties contain the anthocyanin cyanidin 3-O-malonylglucoside15. Flavonoid and total phenolics levels vary widely between lettuce types: crisphead varieties, commonly consumed in the US, have low levels of phenolics, while red leaf and red oak lettuces have the highest levels15. Thus, there is potential to develop cultivars with enhanced nutritional or functional value (see e.g.16). Earlier we have developed three Rutgers Scarlet Lettuce (RSL) lines from existing red cultivars using tissue culture selection for deep purple color, an indicator of high anthocyanin content17. In addition to the anthocyanin cyanidin 3-O-malonylglucoside, RSL lines accumulated high levels of common phenolics reported in lettuce18, such as quercetin glycosides and chlorogenic acids, resulting in a total phenolic content of >9% dry leaf weight, the highest reported in the literature17. RSL leaf and extract showed in vivo anti-diabetic effect in a mouse model of type 2 diabetes18. Specifically, daily oral administration of RSL extract to obese C57BL/6 mice kept on High Fat Diet (HFD) for 28 days resulted in improved oral glucose tolerance and decreased liver lipid levels compared to control18. Thirteen-week diet supplementation with RSL powder resulted in improved glucose tolerance in obese C57BL/6 mice on HFD, even though other measured physiological parameters did not change19. In another study, 4-week supplementation with red leaf lettuce powder resulted in decreased levels of total blood cholesterol and triglycerides in HFD-fed mice20.Lettuce  and FLAVONOID-3′ HYDROXYLASE (F3′H) genes responsible for the novel phenotypes. These mutations increased the levels of targeted flavonoids to levels higher than in any other vegetable. Recent sequencing of the lettuce genome26 and identification of loci associated with the flavonoid biosynthesis pathway in lettuce11 allowed us to compare the CHI and F3′H sequences we determined to those putatively identified by Zhang et al.11.As flavonoid biosynthesis genes have been characterized in ana Fig.  for revicv. Firecracker red leaf lettuce segregating population derived from seeds of self-pollinated mutagenized plants was screened for anthocyanin  loss manifested by changes in color. 1522 mutagenized (M1) plants were grown from seed mutagenized by 0.10 or 0.15% EMS, selfed, and the mature dry inflorescences collected to obtain the M2 segregating population. 136 M1 lines were sterile. Seed from the remaining 1386 M1 lines were planted  in growth chambers equipped with cool fluorescent lights emitting high levels of both photosynthetically active radiation and ultraviolet (UV); lighting conditions known to induce strong anthocyanin accumulation, and, thus, red color. Forty-three lines harboring color variants were identified visually. Methanolic extracts of the twenty most prominent color mutants were biochemically profiled using an Ultra Performance Liquid Chromatography - Tandem Mass Spectrometer (UPLC-MS/MS) system. Three mutants were selected for further studies.An ethyl methanesulfonate (EMS)-mutagenized kfoA had high levels of kaempferol glycosides  but lacked quercetin or cyanidin derivatives. Another kaempferol overproducer, kfoB, accumulated the same kaempferol derivatives as kfoA, and had low but detectable cyanidin and quercetin glycoside content. The yellow-green naringenin chalcone overproducer nco line had high levels of glycosylated compounds  , a trait previously described in A. thaliana flavonoid biosynthesis mutants30.Pink-green kaempferol overproducer red Fig. , kfoA , the predominant anthocyanin in strawberries35, than cv. Firecracker (<0.2 mg pelargonidin/g dry weight). KfoB plants accumulated >6 mg kaempferol/g dry weight, lower than kfoA. However, they accumulated more pelargonidin than kfoA, and contained quantifiable cyanidin and quercetin. To our best knowledge, this is the first report on the accumulation of pelargonidin in lettuce leaves.The anthocyanin cyanidin and the flavonol quercetin were detected in Nco acid hydrolyzed extracts lacked cyanidin, kaempferol or pelargonidin, but contained >10 mg naringenin/g dry weight, and, on average, 0.6 mg quercetin/g dry weight. As naringenin chalcone glycosides, but not naringenin glycosides were observed in non-hydrolyzed nco extracts (see previous section), we tested the effect of acid hydrolysis on pure naringenin chalcone and observed full conversion to naringenin. Therefore, the levels of naringenin in hydrolyzed extracts of nco correspond to the levels of naringenin chalcone glycosides in the plant. Small amounts of quercetin observed in nco were also likely derived from naringenin formed spontaneously in planta from naringenin chalcone, as naringenin chalcone can spontaneously isomerize by C ring closure to naringenin36. To our best knowledge, naringenin chalcone has not been described in lettuce before.17. Wild type cv. Firecracker and kfoB both had 45 mg gallic acid equivalent/g dry weight. KfoA and nco plants had somewhat lower total polyphenol levels: 23 and 36 mg gallic acid equivalent/g dry weight, respectively , an enzyme that converts naringenin chalcone to naringenin37. Therefore, primers designed based on lettuce Expressed Sequence Tags (ESTs) homologous to the A. thaliana CHI gene (TAIR AT3G55120) were used to amplify the full coding sequence (CDS) of the putative lettuce CHI from cDNA in cv. Firecracker and nco. The wild type cv. Firecracker CHI  was predicted to code for a 235-amino acid protein, and the CDS was identical to XM_023891334, a predicted CHALCONE ISOMERASE from green crisphead lettuce cv. Salinas. Additionally, it was identical to LG9_805610, identified as the only CHI expressed (of two putative CHI genes) in the lettuce genome11. Nco plants were homozygous for an allele  that harbors a premature stop codon caused by a G to A mutation in codon 120, truncating the CHI enzyme. The CHI1 truncated protein lacks two conserved residues of the naringenin binding cleft, as well as a residue of the active site hydrogen bond network38; therefore, it is expected to be nonfunctional.The le Table  resemblenco mutant and 4 wild type siblings were genotyped. The mutant was homozygous for the chi1 allele, whereas wild type plants were heterozygous or homozygous for CHI+ allele. The M2 mutant and its wild type red siblings were selfed, and segregation ratios in M3 individuals were observed. In addition, selfed seed from two M3 mutants were planted.  and found that only yellow-green mutants were homozygous for chi1  gene (TAIR AT3G51240). F3H converts naringenin to dihydrokaempferol  phenotype, and that nco is a chi mutant.We then genotyped 5 mutants and 14 wild-type siblings from self-pollinated offspring of hi1 Fig. . AdditiokfoA flavonoid profile  enzyme41. Therefore, primers designed based on lettuce ESTs homologous to the A. thaliana F3′H gene (TAIR AT5G07990) were used to amplify the full CDS of the putative lettuce F3′H in cv. Firecracker, kfoA and kfoB. The wild type cv. Firecracker F3′H  was a gene containing three exons, predicted to code for a 512-amino acid protein, and was identical to XM_023887166, a predicted FLAVONOID 3′-MONOOXYGENASE-LIKE gene from green crisphead lettuce cv. Salinas. Additionally, it was identical to LG5_471950, one of five putative F3′H genes in the lettuce genome identified by Zhang et al.11.The le Table  resembleKfoA plants were homozygous for an allele  harboring a G to A mutation in the splice acceptor site of intron 2, while kfoB plants were homozygous for an allele  harboring a premature stop codon caused by a C to T mutation in codon 233. Translated kfo F3′H proteins harbor the CR1 active site (amino acids 171–186) responsible for the hydroxylating activity, but lack three substrate recognition sites as well as the EXXR motif necessary for core stabilization42, thus, it is expected that both kfoA and kfoB mutant F3′H proteins are nonfunctional. Of the M2 population, one kfoA mutant and seven wild type siblings were genotyped, as well as one kfoB and five wild type siblings. Mutants were homozygous for f3′h1 or f3′h2, while wild type plants were heterozygous or homozygous for the wild type F3′H+. Mutant plants and wild type red siblings were selfed, and segregation ratios in M3 and M4 individuals were observed , we found that only pink-green mutants were homozygous for f3′h1 , and that this gene is predominantly responsible for the synthesis of dihydroquercetin, a precursor in anthocyanin biosynthesis in wild type cv. Firecracker.′h1 Fig. . Our datNco lettuce plants have a characteristic yellow-green leaf color due to the accumulation of yellow-colored naringenin chalcone glycosides and the lack of red anthocyanins. Naringenin chalcone has not been reported in lettuce before. Tomato (Solanum lycopersicum) skin is the best-known food source of this compound, where it accumulates up to 1% dry weight43, a level similar to that of nco lettuce. Naringenin chalcone is anti-inflammatory, anti-allergic (e.g.45) and anti-obesity46in vitro and in vivo, and was found to improve symptoms of perennial allergic rhinitis in a clinical trial47. Therefore, nco lettuce could be a useful dietary source for naringenin chalcone, with one US leaf lettuce serving of 85 g containing ~79 mg of the compound.nco lettuce is caused by a nonsense mutation in the CHI gene. To our knowledge, nco is the first chi mutant in lettuce. While CHI is ubiquitous in higher plants, chi mutants have been characterized from just a handful of species, with individual flavonoids from these mutants not quantified. Chi mutants have been described in ornamental flowers such as Petunia hybrida48, Callistephus chinensis49 and Dianthus caryophillus50, in crops such as barley (Hordeum vulgare)52, rice (Oryza sativa)53 and onion (Allium cepa)54 and inA. thaliana55. In all species, the chi mutant phenotype results in yellowish tissues: hull and internodes in rice53, bulb color in onion54, petals in C. chinensis49 and D. caryophillus50, seed coat in A. thaliana56 and pollen in P. hybrida48. P. hybrida chi mutants accumulate naringenin chalcone as aglycone48, while C. chinensis49, D. caryophillus50 and barley (Hordeum vulgare)51chi mutants accumulate naringenin chalcone 2′-glucoside . Detailed metabolome analysis of A. thaliana chi mutants revealed the presence of multiple naringenin chalcone glycosides37, while in chi onion54 and rice53 the compound responsible for the yellowish or golden color was not identified. We found that nco lettuces accumulate naringenin chalcone hexoside and malonylhexoside, but not the aglycone, similarly to most chi mutants.The color phenotype in nco lettuce, chi mutants of A. thaliana37, P. hybrida48, C. chinensis49 and D. caryophyllus50 had low but detectable levels of flavonols. In Arabidopsis, Peer et al.39 hypothesized that spontaneous isomerisation of naringenin chalcone in planta to naringenin, the substrate of the next enzyme in the anthocyanidin biosynthesis pathway, F3H, was responsible for the presence of flavonols.Like KfoA and kfoB lettuce plants are pink-green and accumulate high levels of kaempferol glycosides. Kaempferol has well-documented anti–diabetic, pancreatic β-cell protecting and anti-inflammatory effects59. It has been reported in lettuce; however, quercetin is the dominant flavonol in most cultivars34. Reported kaempferol levels in lettuce range from 0.0–2.36 mg/100 g fresh weight34, while kfoA and kfoB lettuce has 103 and 83 mg /100 g fresh weight, respectively, two orders of magnitude higher. Unlike naringenin chalcone, kaempferol is a ubiquitous flavonoid, described from over 400 species . However, kaempferol accumulation in kfo lettuces is higher than the amounts reported in vegetables considered high in kaempferol, e.g. endive (Cichorium endiva), 1.5–9.560; leek (Allium porrum), 1.1–5.660 and 11.834; shallot (Allium fistulosum), 11.734; potherb mustard (Brassica juncea), 48.234; kale , 5.134, 21.160 and 47.061; broccoli , 2.134 and 6.061; choi sum cabbage (Brassica rapa var. parachinensis), 2.0–3.732; turnip tops (Brassica campestris), 3.1–6.460; spinach (Spinacia oleracea), 4.9–9.032; radish root (Raphanus sativus), 0–4.134; toona leaf (Toona sinensis), 41.734 and 60.433; Chinese boxthorn shoot (Lycium barbarum), 44.633, rocket (Eruca sativa) 36.533 and water cress  35.133 mg kaempferol/100 g fresh weight. The only natural source higher in kaempferol than kfo lettuce is caper flower buds (Capparis ssp.), which have 85–295 mg kaempferol /100 g fresh weight, providing 8.5–29.5 mg kaempferol per 10 g serving62. Therefore, kfo lettuces could be valuable dietary sources of kaempferol with one US leaf lettuce serving (85 g) providing ~71–87 mg kaempferol.Kfo phenotypes were caused by mutations in the F3′H gene: a mutation of the intron 2 splice acceptor site in kfoA, and a nonsense mutation in kfoB. In lettuce, no f3′h mutant has been described, but in a study of 240 lettuce accessions five genes were identified as F3′H, three of which were expressed and two of which (including the F3′H gene mutant in kfoA and kfoB) carried expressed Qualitative Trait Loci (eQTL) for flavonoid composition11. The lettuce f3′h phenotype is very similar to A. thaliana f3′h mutants , which accumulate kaempferol, and the anthocyanin pelargonidin41 that differs from cyanidin by the lack of 3′-hydroxylation. Kfo f3′h mutant lettuces also accumulate pelargonidin, although pelargonidin levels in kfo are much lower than cyanidin levels in red parent line cv. Firecracker  accumulate pelargonidin derivatives producing magenta, pink or fuschia flowers63. In carnation (D. caryophillus), f3′h mutants have pink petals, accumulating a pelargonidin glycoside, while plants with functional F3'H have purple petals accumulating a cyanidin glycoside64.65. Flavonoids are hypothesized to act as UV absorbers and reduce the levels of damaging reactive oxygen species66. In lettuce, exposure to UV or blue light increases flavonoid levels, but reduces yield (e.g.73). This effect was observed during different months in the field growth season71, and in field70 and greenhouse68 experiments, where levels of UV exposure were controlled using UV-blocking cover foils, as well as in controlled growth chambers supplemented by UV or blue light emitting LED diodes73. UV-induced increase in flavonoid and total phenolic content was observed across different green and red cultivars74, indicating that it is a universal phenomenon in lettuce. Armas Gutierrez75 reported that continuous exposure to cool fluorescent lights resulted in high accumulation of total phenolics, total antioxidants and total anthocyanins. Therefore, in our experiment, we replicated the growth conditions optimal for high phenolic content determined by Armas Gutierrez75.In plants, many environmental stresses trigger the accumulation of antioxidants including flavonoids and other phenolicsA. thaliana flavonoid biosynthesis mutants are more sensitive to high UV76 and visible light stress77 than wild type plants. As in lettuce, wild type A. thaliana plants have a decreased rate of biomass accumulation under high UV stress compared to low UV conditions, but the effects are more severe in flavonoid biosynthesis mutants30. This sensitivity has been attributed to enhanced photoinhibition77 and increased lipid and protein peroxidation77 in mutants lacking flavonoids that absorb UV and scavenge reactive oxygen species. However, the UV sensitivity of the different A. thaliana mutants is not equal. The kaempferol-accumulating f3′h mutant is less UV sensitive than chs, chi and f3h mutants, which accumulate low levels of flavonols30. We found that nco and kfo lettuces grew somewhat slower than wild type cv. Firecracker plants under UV-emitting cool fluorescent lights, though we did not observe visible growth retardation under greenhouse conditions . Under cool fluorescent lights, kfo (f3′h) lettuce grew faster than nco (chi) but not as fast as wild type cv. Firecracker, similarly to A. thaliana f3′h and chi mutants30. Potentially, desirable high biomass and flavonoid levels could be obtained by growing nco and kfo under low UV conditions, and subjecting them to higher levels UV or blue light before harvest. 3-day supplemental UV treatment for 16 h/day has significantly increased total anthocyanin and antioxidant levels in red leaf lettuce, with no effect on the total leaf biomass73. Similarly, 6-day pre-harvest exposure to UV resulted in a 4.6x increase in total anthocyanin content and 2.3x increase in total phenolic content in red leaf lettuce field grown under UV-blocking foil70, while total biomass of these plants was not significantly different from those not exposed to UV.In conclusion, we created and characterized flavonoid biosynthetic mutants in lettuce with potential health benefits. The modified flavonoid profile characterized by record high accumulation of kaempferol and naringenin chalcone may transform lettuce into a food with health benefits. However, animal studies and human clinical trials will be needed to confirm the health benefits of the high flavonoid lettuce varieties described here. Innovative mutagenizing and selection strategies producing higher levels of beneficial phytochemicals could be an important strategy for adding value added output traits to common crops.cv. Firecracker (Johnny’s Selected Seeds) seeds were mutagenized with 0.10 or 0.15% EMS, using the protocol in75. In short, seeds (M0) were soaked in distilled water containing 0.1% or 0.15% (v/v) EMS and incubated for 12 h at room temperature in a rotary shaker. Thereafter, the EMS solution was decanted, the seeds were washed five times with 50 ml of distilled water and dried. Mutagenized M1 seeds were planted and grown under standard greenhouse conditions at the Rutgers New Jersey Agricultural Experiment Station (NJAES) glass research greenhouse under the following settings: 25 °C/19 °C day/night temperature, 16 h light/8 h dark photoperiod with natural light supplemented with 400 W high pressure sodium lamps. Inflorescences were individually collected from 1,522 mature M1 plants, and the M2 seed was threshed, dried out in the greenhouse and placed in paper coin envelopes. The envelopes were placed in re-sealable plastic storage bags with desiccant and stored at 4 °C.Lettuce 2d and UV light intensity of 0.7 mol/m2d75. Growth chambers were programmed for 16 h light/8 h dark photoperiod, 18 °C/15 °C day/night temperature and 60% relative humidity. Under these conditions, wild type cv. Firecracker develops a deep red color. At 4–6 weeks, segregating putative mutants exhibiting color variations, and their siblings with wild-type phenotype were identified and transplanted to 10 cm diameter pots filled with Sun Gro Professional Growing Mix . At 12–18 weeks, tissue samples were collected from putative mutants for genetic and phytochemical analysis, and the plants were transplanted to 23 cm diameter, 6 l pots  filled with Sun Gro Professional Growing Mix , and placed in the Rutgers New Jersey Agricultural Experiment Station (NJAES) research greenhouse under the following conditions: 25 °C/19 °C day/night temperature, 16 h light/8 h dark photoperiod, with natural light supplemented provided by 400 W high pressure sodium lamps. Individual inflorescences were collected from mature M2 plants; the seeds (M3 generation) were dried, threshed, and stored at 4 °C under the same conditions as M2 seeds.Selfed and segregated M2 seeds were planted in Sun Gro Propagation Mix  in plastic trays. At least 12 M2 seeds were planted if there were more than 12 seeds per M2 line. The trays were placed in growth chambers equipped with cool fluorescent lights , providing a PAR light intensity of 26.3 mol/mcv. Firecracker, and from homozygous mutant kfoA, kfoB and nco plants were planted in Sun Gro Propagation Mix  in plastic trays. The trays were placed in growth chambers under conditions described above. 26 days after planting, seedlings were transplanted to 10 cm-diameter pots filled with Sun Gro Professional Growing Mix , and kept in the growth chamber. 130 days (18 weeks) after seeding, a tissue sample was taken from 6 cv. Firecracker, 10 kfoA, kfoB and nco plants, weighed, frozen at −80 °C, then lyophilized.Seeds from selfed 18. In short, leaves were kept at −80 °C prior to lyophilization. Freeze-dried leaves were ground to a fine powder with a mortar and pestle. 50 or 100 mg lyophilized leaf powder was placed in a 15 ml plastic tube, protected from light, then, respectively, 1.5 ml or 3 ml solvent , was added. The leaf powder was vortexed with the solvent for 30 sec, sonicated for 5 min, vortexed for another 30 sec, kept for 10 min at room temperature, and centrifuged at 1700 rcf for 5 min. The supernatant was decanted, then the extraction was repeated twice, and the decanted extracts pooled. The decanted solution was centrifuged at 1700 rcf for 8 min and filtered through 0.45 µm polytetrafluoroethylene (PTFE) filters (Fisher Scientific) for UPLC-MS/MS analysis.To determine the flavonoid composition of putative color mutants, extracts were prepared from lyophilized and ground leaf tissue, using the method described in78. Since this protocol results in the co-elution of chlorogenic acid and cyanidin 3-O-malonylglucoside, we used a modified gradient elution to separate these two compounds. For this protocol, the mobile phase consisted of two components: Solvent A , and Solvent B (100% acetonitrile). The initial conditions of the gradient were 95% A and 5% B; for 20 minutes the proportion reached linearly 80% A and 20% B. Within the next 3 minutes the proportion was 5% A and 95% B, which was maintained for 4 minutes. Within the following 3 minutes the gradient was adjusted to initial conditions, and 5 additional minutes were included for equilibration before subsequent injections.Extracts were separated and analyzed by a UPLC-MS/MS system using the protocol described incv. Firecracker, kfoA, kfoB and nco plants were ground using a mortar and pestle. Fifty mg leaf powder was placed in a plastic tube and subjected to acid hydrolysis, based on the method of Hertog et al.31. In short, 4 ml solvent  was added, then the mix was acidified with 1.0 ml 6 M HCl, vortexed for a few seconds, and placed in a 90 °C water bath for 2 h. Afterwards, 100% methanol was used to make up the volume of the extract to 10 ml. The extract was then sonicated for 5 min, centrifuged at 2500 rpm for 8 min, and filtered through 0.45 μm PTFE filters (Fisher Scientific) for UPLC-MS/MS analysis. Total polyphenol content of the extracts was measured by a modified Folin-Ciocalteu assay17 based on79 and80.Lyophilized lettuce leaves from 18-week old ®’s SII LC control software, solvent rack/degasser SRD-3400, pulseless chromatography pump HPG-3400RS, autosampler WPS-3000RS, column compartment TCC-3000RS, and photodiode array detector DAD-3000RS. After passing through the photodiode array detector, the eluent flow was guided to a Q Exactive Plus Orbitrap high-resolution high-mass-accuracy mass spectrometer (MS). Mass detection was full MS scan with low energy collision induced dissociation (CID) from 100 to 1000 m/z in either positive, or negative ionization mode with electrospray (ESI) interface. Sheath gas flow rate was 30 arbitrary units, auxiliary gas flow rate was 7, and sweep gas flow rate was 1. The spray voltage was 3500 volts (−3500 for negative ESI) with a capillary temperature of 275 °C. The mass resolution was 140,000. Column and run conditions were identical to78 apart from that the average pump pressure was 3900 psi for the initial conditions.Extracts were separated and analyzed by a UPLC-MS/MS consisting of the Dionex® UltiMate 3000 RSLC ultra-high-pressure liquid chromatography system, consisting of a workstation with ThermoFisher Scientific’s Xcalibur v. 4.0 software package combined with DionexPutative formulas of flavonoids and other compounds were determined by isotope abundance analysis on the high-resolution mass spectral data with Xcalibur v.4.0 software and reporting the best fitting empirical formula. Database searches were performed using reaxys.com  and SciFinder .Quantification was based on external standards of commercially available compounds. Naringenin and naringenin chalcone were purchased from Cerilliant, quercetin from Tocris, kaempferol and cyanidin chloride from Sigma Aldrich. Standards were dissolved in anhydrous methanol  or ethanol . Additionally, pelargonidin was quantified in cyanidin equivalents.81. Total RNA was isolated using the QIAGEN RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s instructions. Nucleic acids were quantified using a NanoDrop UV-Vis spectrophotometer (Thermo Fisher Scientific). cDNA synthesis was performed from total RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. Primers , F3′H (TAIR AT5G07990) and F3H (TAIR AT3G51240). PCR-amplification of the full CDS of F3H and CHI was performed on nco cDNA, and of F3′H was performed on kfoA and kfoB genomic DNA, with the following PCR program: 5 min at 94 °C; 34 cycles of 30 sec at 94 °C, 30 sec at 60 °C, 90 sec at 72 °C; 10 min at 72 °C. PCR products were treated with ExoSAP-IT (Affymetrix), and Sanger sequenced. Raw sequence reads were assembled using SeqMan Pro (DNASTAR). Of the M2 generation, one kfoA mutant and seven wild type siblings, as well as one kfoB and five wild type siblings were genotyped for their F3′H alleles. Fifteen kfoA mutants and five wild-type siblings were genotyped for their F3′H alleles in the M3 generation. One M2 generation nco mutant and four wild type siblings were genotyped for their CHI alleles. Five nco mutants and fourteen wild-type siblings were genotyped for their CHI alleles in the M3 generation.Total cellular DNA was isolated from leaves of lettuces grown in growth chambers, using a modified cetyltrimethylammonium bromide (CTAB) methodSupplementary Information


A 30‐year‐old oligoasthenozoospermia man was found to have unbalance mosaic translocation between chromosome 22 and four other chromosomes  during the investigations for a couple with infertility for 3 years, which is a rare event in human pathology.DNA.Classical cytogenetics analysis, fluorescence in situ hybridization (FISH), and chromosome microarray analyses (CMA) were performed on peripheral blood lymphocytes; copy number variation sequencing (CNV‐Seq) analysis was performed on sperm XY, der (13) t,‐22[10]/46, XY, t[6]/45, XY, der(15)t,‐22[4]/46, XY, t[1]/45, XY, der(5)t,‐22[1]/45, XY, der(6)t[1]. FISH and CMA performed on peripheral blood cells showed the presence of a 6.9 Mb mosaic 22q11 deletion (approximately 50% of cells); it is unexpected that the phenotypes of this man were merely oligoasthenozoospermia, mild bradycardia, and mild tricuspid regurgitation. CNV‐Seq analysis performed on sperm DNA revealed the rate of 22q11 deletion cells was obviously lower compared with peripheral blood cells. And the frequency of gametes exhibiting a normal or balance chromosomal equipment was above 80% in sperm samples.Classical cytogenetics analysis showed the presence of six cell lines on peripheral blood lymphocytes: 45, To the best of our knowledge, this report is the first case of a de novo gonosomal mosaic of chromosome 22q11 deletion just associated with male infertility. Chromosomal defects are the most common genetic abnormalities in infertile men, with an incidence of cytogenetic abnormalities ranging from 2.1% to 15.5% , cat eye syndrome (CES), and 22q11.2 distal deletion syndrome  disclosed that no Y chromosome microdeletions existed; the levels of FSH, LH, free T, and PRL were within normal limits. Two different semen analyses of patient were carried out according to the World Health Organization criteria (1999), and the results showed severe oligoasthenozoospermia: Sperm volume was only 1.5 ml, sperm concentration was only 0.8 million/ml, and sperm progressive motility  was 15.7%. Electrocardiogram showed that his heart rate was 58 beats per minute, and ultrasonic cardiogram showed that he had mild tricuspid regurgitation.This study was approved by the ethics board at West China Second University Hospital, Sichuan University, China. Informed consent of the legal representatives of this patient was obtained.2.2GTG‐banded chromosomes from cultured peripheral blood lymphocytes were tested. The two lymphocytes samples were obtained at different time with more than 6 months interval. Briefly, whole blood was cultivated for 72 hr at 37°C in lymphocyte culture medium. Twenty‐four metaphases were analyzed, and the karyotype results were described according to the International System for Human Cytogenetic Nomenclature . FISH was performed on peripheral blood lymphocytes using the 22q11.2 probe  and 13q14 probe as control.2.3We performed CMA analysis of DNA extracted from peripheral blood of this patient. Microarray testing was undertaken using the Affymetrix CytoScan 750K array. Data were analyzed using Chromosome Analysis Suite Software with copy number changes reported at a resolution of 200 kb.Y‐axis) versus chromosome pos (X‐axis); a blue line was used to indicate the mean copy number across each chromosome to identify the nature and map position of any deleted regions.Sperm DNA of this patient was analyzed by CNV‐seq, gDNA (50 ng) was fragmented, and DNA libraries were subjected to massively parallel sequencing using the NextSeq CN500 Platform (Illumina) to generate 5 million raw sequencing reads with 36‐bp genomic DNA sequences. Chromosome profiles were finally plotted as copy number (https://decipher.sanger.ac.uk/).Identified and mapped CNVs were interrogated using publicly availed DECIPHER databases t,‐22[10]/46, XY, t[6]/45, XY, der(15)t,‐22[4]/46, XY, t[1]/45, XY, der(5)t,‐22[1]/45, XY, der(6)t[1]/45, XY, der(13)t,‐22[1]  showed only one signal for 22q11.2 probe  mosaic 22q11 deletion (approximately 50% of cells)  analysis performed on sperm DNA revealed the rate of 22q11 deletion cells was obviously lower compared with peripheral blood cells Figure d. And th4In the present study, we found an oligoasthenozoospermia male with complex chromosomal abnormality in peripheral blood cells. The two lymphocytes samples were obtained at different time with more than 6‐month interval, and classical cytogenetics analysis showed the presence of six cell lines in peripheral blood: 45, XY, der (13) t,‐22[10]/46, XY, t[6]/45, XY, der(15)t,‐22[4]/46, XY, t[1]/45, XY, der(5)t,‐22[1]/45, XY, der(6)t, ‐22[1]/45, XY, der(13)t,‐22[1]. FISH and CMA showed the presence of a 6.9 Mb mosaic 22q11 deletion (approximately 50% of cells) in peripheral blood. These rearrangements not only changed the order, but also the amount of genetic material. Analysis through DECIPHER databases the 6.9M deletion could lead to two syndromes: 22q11 deletion syndrome  and 22q11.2 distal deletion syndrome . Additionally, symptoms such as Tetralogy of Fallot (Chen et al., The patient complained that he had not been exposed to radioactive source and other substances that may cause chromosomes aberrations. We assumed that this patient might be a mosaic individual. The generation of genetically distinct cells from a single zygote necessitates postzygotic de novo mutational events (Veltman & Brunner, It was reported that some mosaic disorders are caused by mutations that were seen only in mosaic form and that were incapable of germ line transmission Happle, , 1987. TThe main purpose of this patient was to consult the cause of infertility, so we tested sperm DNA by CNV‐seq analysis to evaluate the genetic material of gametes. The result showed that the rate of 22q11 deletion sperms was obviously lower than peripheral blood cells; the frequency of gametes exhibiting a normal or balance chromosomal equipment was above 80% in sperm samples. Fortunately, this patient decided to try an IVF with microinjection and had a chance to get a normal offspring.To the best of our knowledge, our report is the first case of a de novo gonosomal mosaic of chromosome 22q11 deletion associated with male infertility, meanwhile, without other severe clinical manifestations.None.H. Z. performed the FISH assay; T. H. and Z. Z. performed the CMA assay; J. W. performed the CNV‐seq assay; X. Z., Y. L., J. Z., D. X., B. X., L. Q., and L.X. performed the karyotype assay; Y. L. and H.S. analyzed the data; S. L. and H.W. coordinated the project. Y.L. and H.S. wrote the paper.


Overexpression of Ku80, however, promoted melanoma growth and increased the insensitivity of melanoma cells to melatonin. Mechanistically, we found that Ku80 bound to the PDK1 promoter and activated the transcription of PDK1. Moreover, we showed that the binding of Ku80 at the PDK-1 promoter was HIF1-α dependent, and melatonin degraded HIF1-α in melanoma cells. Furthermore, clinical data revealed that the expression of Ku80 and PDK-1 proteins were positively correlated and elevated in the tumor tissues of melanoma patients, and high expression of Ku80 predicted a poor prognosis in melanoma. Collectively, our study demonstrated that Ku80 promoted melanoma growth and regulated antitumor activity of melatonin by targeting HIF1-α dependent PDK-1 signaling pathway, suggesting that Ku80 may be a potential molecular target for melanoma treatment.Melanoma is one of the most malignant and aggressive cancers with high cancer-related deaths. However, it is unclear whether Ku80 regulates tumor growth in human melanoma. In this study, we screened a siRNA library targeting 6024 human genes and identified Ku80 as a potential therapeutic target in melanoma cells. Knockdown of Ku80 significantly suppressed melanoma cell proliferation and induced apoptosis, as well as enhanced the antitumor effect of melatonin in melanoma  Due to c-myc origin were more active as a result of this increased association [Ku80 is a subunit of the conserved DNA binding heterodimer Ku70/Ku80, which plays a critical role in guarding chromosome stability by repairing DNA double strand breaks via the non-homologous end joining (NHEJ) pathway . Besidesociation . Moreoveociation . And Komociation . Similarociation . In non-ociation . In melaociation ,33. NoneN-acetyl-5-methoxytryptamine), an indoleamine compound produced in the pineal gland and secreted into circulating blood. Melatonin has a wide range of reported biologic effects including circadian rhythm, sleep, mood, sexual maturation, stress, insulin secretion, and reproduction [Melatonin , cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum (FBS), 100 μg/ml penicillin and 100 μg/ml streptomycin, and maintained in a humidified atmosphere with 5% CO2.2p-AKT (#4060), ERK (#9102), p-ERK (#9102), mTOR (#2972), p-mTOR (#2971), MMP2 (#4022), MMP9 (#3852), PARP (#9542), cleaved-caspase 3 (#9661) and cleaved-caspase 9 (#9505) were purchased from Cell Signaling Technology . Melatonin (S1204) was purchased from Selleck Chemicals , dissolved in DMSO, and added into cell culture medium for a final concentration of 1 mM.IgG (#2729) and antibodies for Ku80 (#2753), PDK1 (#3820), HIF-1α (#3716), Histone H3 (#4499), GAPDH (#5174), PI3K (#4255), p-PI3K (#4228), AKT (#4691), 2.33 cells per well in 96-well plates, and the viability of the cells was assessed by the MTS assay  48 h later. The absorbance value at 490 nm in each well was measured with a microplate reader. All experiments were performed in 6 replicates per trial, with 3 independent trials in total and the average percentages of cell viability were shown.Cells were seeded at a density of 3 × 102.4Cells were seeded at a density of 200–400 cells per well in 6-well plates and cultured for 2 weeks. The colonies were then stained with 1% crystal violet and counted. All experiments were performed in 3 independent trials.2.5Whole cell lysates or nuclear extracts were prepared using Complete Lysis-M reagent  and RIPA lysis buffer . Protein concentration was determined by BCA assay . The proteins were separated in 8%–10% SDS-PAGE gels and transferred onto PVDF membranes for detection.2.65 cells/ml) were stained with 5 ml AnnexinV-FITC and 5 ml PI (propidium iodide), incubated in room temperature for 15 min in the dark, and then analyzed by flow cytometry . Apoptosis was determined as FITC-positive in cells. Cell cycle analysis was performed using PI staining.Cells , and cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix . The SYBR Green PCR master mix (Toyobo) was then used for qPCR, which was followed by detection with a Bio-Rad CFX96 and analyzed with the Bio-Rad Manager software . Each sample was tested in triplicate.2.8g to pulldown the DNA-protein complex.The PDK1 promoter binding proteins were analyzed by streptavidin-agarose pulldown assay. Briefly, 1 mg of nuclear protein extracts from human melanoma cells were incubated with 10 μg of biotin-labeled double-stranded DNA probes corresponding to nucleotide −875 ∼ +40 of the PDK1 promoter region  and 100 μl of streptavidin-agarose beads (Sigma-Aldrich) at 4 °C over night. The mixture was then centrifuged at 500×2.9Cells were fixed with 1% formaldehyde, and the cross-linking was quenched by adding in 1.375 M glycine (100 μl/ml of culture). The samples were sonicated on ice to shear the DNA into 300 to 1000 bp fragments. For each total cell lysate, one third was used as the DNA input control, another third was immunoprecipitated with Ku80 antibody, and the last third was subjected to non-immune rabbit IgG. DNA fragments were purified by spin columns , and PCR was performed to amplify the promoter region of PDK1 with the following primer pair - Forward: 5′-ACG CAG ATT GGT GGT TC-3′, Reverse: 5′-AGA GAA GCC ACA GCC AGT-3’. The PCR products were resolved by electrophoresis in a 2% agarose gel and visualized by Gel-Red staining.2.10SacI and HindІІІ sites of the firefly luciferase vector pGL4.10 , and Renilla luciferase reporter vector pRL-TK was used as a control. Cells (2 × 104 cells/well) were seeded in a 96 well plate. After the cell confluence reached 50%, pGL4.10-hTERT-387 or pRL-TK was transfected into melanoma cells with Ku80 stable knockdown or overexpression with Lipofectamine 3000  (30:1 ratio). 72 h after transfection, dual-luciferase assay was performed using the Dual-Luciferase® Reporter Assay System .A fragment containing the promoter region of PDK1 (−875 ∼ +40) was inserted between the 2.11For confocal microscopy analysis, cells grown on chamber slides were washed with PBS, fixed with paraformaldehyde and permeabilized with pre-cooled methyl alcohol for 15 min at −20 °C. The samples were then pretreated with 10% bovine serum albumin (BSA) in PBS for 30 min, and specific antibodies were added and incubated overnight at 4 °C. Following five 5 min washes with PBS, secondary antibodies were added and incubated for another hour. After five additional 5 min washes, samples were examined using the confocal microscope.2.12Equal amounts of nuclei protein extracts prepared from different cell lines were incubated with the indicated antibodies. Then, the agarose-conjugated protein-A/G beads  were added and the mixture was incubated at 4 °C overnight. After extensive washing with cold phosphate-buffered saline (PBS), the beads were mixed with loading buffer and boiled. The proteins in the supernatant were detected by Western blotting analysis.2.136 cells in 100 μl PBS) subcutaneously into the left flank of each mouse. When the formed tumor reached 100 mm3, the animals were randomly divided into groups of 5. For melatonin treatment, mice were intratumorally injected with melatonin (25 mg/kg) every other day for 3 weeks, while the control group received PBS. The tumor size was measured using vernier calipers once every three days and volumes were calculated as V = (length × width × height)/2. At the end of the experiment, the mice were sacrificed, and the tumors were excised, weighed and processed for immunohistochemical analyses.All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals  and the Institutional Ethical Guidelines for Animal Experiments developed by Sun Yat-sen University. Female athymic nude mice aged with 3- to 4-weeks were purchased and housed in the animal center of Sun Yat-sen University. A375 cells with control shRNA, Ku80 shRNA, vector, Ku80 or PDK1 overexpression plasmid were injected . Detailed clinical information, including age, gender and location of the melanoma, was available for all cases. For prognosis analysis, the melanoma tissue microarray with overall survival information of each case was purchased from Novus Biologicals .2.15Briefly, tissue sections were deparaffinized, rehydrated and then immersed in target retrieval solution  and boiled at medium baking temperature for 20 min once in a microwave. After blocking the slides with 3% BSA, the sections were then incubated with primary antibody against Ku80 (1:100) and PDK1 (1:50). Then, the sections were incubated with rabbit and mouse conjugated-second antibody for 45 min at 37 °C and the specimens were counterstained with hematoxylin and the target-positive cells were counted in 3–4 different fields and photographed using CellSens Dimension . The staining intensity was measured with ImagePro Plus .2.16P < 0.05 was considered statistically significant.Data were presented as mean ± standard deviation from at least three independent experiments. Statistical analysis was carried out using SPSS 11.0 software . 33.1In order to discover proteins involved in the growth of melanoma, we screened a siRNA library targeting >6000 genes in human melanoma A375 cells, and found that 4780 genes whose knockdown inhibited cell viability . Of the We then investigated whether Ku80 inhibited apoptosis of melanoma cells. FACS analysis revealed that A375 and MEWO cells with Ku80 knockdown had a higher apoptosis rate than the control cells A and B. 3.2To further clarify the downstream proteins that mediated the effects of Ku80 in melanoma cells, we established Ku80 knockdown and control stable cell lines, and performed transcriptome analysis. The result showed that PDK1 (pyruvate dehydrogenase kinase 1) was one of the candidates regulated by Ku80, as its expression was dramatically decreased in Ku80 knockdown cells A. RT-qPCRenilla luciferase reporter vector as internal control for dual luciferase reporter assay. The results showed that knockdown of Ku80 significantly decreased the PDK1 promoter activity in A375 and MEWO cells  staining in a tissue microarray that contained 99 melanoma samples and 8 normal samples A. Our an4Melanoma is a highly malignant tumor with very poor prognosis. Although the application of targeted therapies and immunotherapy has significantly improved the overall survival of patients, the prognosis is still not so favorable. Also, the development of drug resistance after a period of treatment has been an unneglectable problem. Therefore, it is still urgent to investigate the mechanism of melanoma development, and seek for potential therapeutic targets.In this study, we screened a siRNA library in A375 melanoma cells to identify the genes that affected cell viability, and found that knockdown of Ku80 significantly suppressed the growth of melanoma cells. Ku80 is best known for its function in DNA damage repair in its heterodimer with Ku70 , while aMechanistically, we found that Ku80 regulated the expression of PDK1 by binding to its promoter as a transcription factor. Knockdown of Ku80 down-regulated the transcription of PDK1, whereas overexpression of Ku80 up-regulated the transcription of PDK1. PDK1 is a member of the pyruvate dehydrogenase kinase (PDK) family that also includes PDK2, PDK3 and PDK4 . PDK1 isPrevious studies have reported that the Ku heterodimer could generally displace transcription factors when bound to DNA and thus inhibit transcription , or specMelatonin is a hormone mainly produced by the pineal gland. It has been found to be a pleiotropic molecule that functions in gene expression regulation, anti-proliferation, immunity modulation, anti-inflammation, anti-oxidation and anti-angiogenesis, and its effects for cancer management have been under clinical investigation . PreviouOur analysis of clinical samples showed that high expression of Ku80 and PDK1 was associated with poor prognosis in melanoma patients. This is in consistence with the findings by Song et al. that the expression of Ku80 increased as melanoma developed from benign naevi to different stages of primary and metastatic tumors, and was inversely associated with favorable survival of patients . It is cIn summary, our study found that Ku80 bound to the promoter of PDK1 to enhance its transcription via a HIF1-α dependent manner. Melatonin promoted the degradation of HIF1-α, thus weakening the combination of Ku80 with PDK1 promoter . FurtherThe authors declare no potential conflicts of interest.This work was carried out in collaboration between all authors. TL, TK and WD defined the research theme and designed the experimental approach. TL, LJ, MC, ZZ, WL, WF, LL, FZ, QZ, HQ, JL, MC, CT, ZH, CZ, YL carried out the experiments. TL, ML JL, LY, YL and WD analyzed the data and interpreted the results. TL, MC and WD wrote the manuscript.


Originating from the complicated redox of lithium polysulfide intermediates, Li–S batteries suffer from several problems, restricting their application and commercialization. Such problems include the shuttle effect of polysulfides (Li2Sx (2 < x ≤ 8)), low electronic conductivity of S/Li2S/Li2S2, and large volumetric expansion of S upon lithiation. In this study, a lotus root-like nitrogen-doped carbon nanofiber (NCNF) structure, assembled with vanadium nitride (VN) catalysts, was fabricated as a 3D freestanding current collector for high performance LSBs. The lotus root-like NCNF structure, which had a multichannel porous nanostructure, was able to provide excellent  conductive networks, which promoted ion transport and physical confinement of lithium polysulfides. Further, the structure provided good electrolyte penetration, thereby enhancing the interface contact with active S. VN, with its narrow resolved band gap, showed high electrical conductivity, high catalytic effect and polar chemical adsorption of lithium polysulfides, which is ideal for accelerating the reversible redox kinetics of intermediate polysulfides to improve the utilization of S. Tests showed that the VN-decorated multichannel porous carbon nanofiber structure retained a high specific capacity of 1325 mAh g−1 after 100 cycles at 0.1 C, with a low capacity decay of 0.05% per cycle, and demonstrated excellent rate capability.Lithium–sulfur batteries (LSBs) are regarded as one of the most promising energy-recycling storage systems due to their high energy density (up to 2600 Wh kg LSBs are considered a promising potential energy storage system for the future because of their evident advantages, including high energy and power density, environmental friendliness and low cost as a result of the sulfur-rich nature of Earth ,2,3. Howi anodes ,5,6,7. Ii anodes ,12,13,14i anodes ,16,17,18i anodes ,20,21, ai anodes ,26,27,286 S m−1 at room temperature), can be a good choice to effectively anchor lithium polysulfide intermediates and to restrict the shuttle effect as a polar host, thereby improving sulfur utilization and enhancing cycling stability through high electron mobility and the catalytic effect [Nanostructured porous carbon materials were first investigated as lithium polysulfide traps by way of physical confinement. Such materials as graphene, carbon nanosheets, carbon spheres, carbon nanotubes, and microporous carbon materials, provide excellent electron transport networks for sulfur intermediates of the redox reaction and high specific surface areas to accommodate volumetric expansion ,37,38,39c effect ,65,66,67−1 at 0.1 C with a low capacity decay of 0.05% per cycle was obtained, as well as a favorable rate capability. In this study, we prepared a lotus root-like nitrogen-doped carbon nanofiber (NCNF) structure, assembled with VN catalysts, to act as a self-supported current collector in LSBs. With more reaction sites, the freestanding 3D nanostructure enhanced the contact area with the electrolyte, providing good electron and ion transport networks. The VN/NCNF structure was found to be capable of trapping lithium polysulfides by both physical confinement and chemical affinity, and accelerate conversion of lithium polysulfides to enhance the energy storage of LSBs. Finally, a highly reversible capacity of 1325 mAh g2) were mixed with 15 mL DMF overnight, forming the electrospun solution. At an environmental temperature of 50 °C, electrospinning was carried out with a 19G needle, distance of 20 cm to the Al-foil collector, 2.4 mL h−1 feed rate and 13.5 kV applied voltage. The collected nanofiber disks (diameter of 19 mm) were annealed at 280 °C  and subsequently 800 °C  under Ar. After annealing, VN-decorated multichannel porous CNF (denoted as MPVN) disks with diameters of approximately 9 mm were obtained. MVN (VN-decorated multichannel CNF), PVN (VN-decorated porous CNF) and NCNF (nitrogen doped CNF) were also obtained with same procedure without PVP, PS or VO(acac)2, respectively. VN-decorated CNFs were fabricated by electrospinning. Under magnetic stirring (70 °C), 1.2 g of polyacrylonitrile (PAN), 0.3 g of polystyrene (PS), 0.3 g of polyvinyl pyrrolidone (PVP) and 0.9 g of vanadium (IV) oxy(acetylacetonate) (VO(acac)2S6 catholyte was prepared by dissolving sublimed sulfur and lithium sulfide with a molar ratio of 5:1 in an electrolyte solution (1.0 M LiTFSI in a 1:1 (v/v) 1,3-dioxolane (DOL)/dimethyl ether (DME) mixture with 1.0 wt % LiNO3) by stirring . To study the adsorption capability of VN, the same weight (20 mg) of MPVN, MVN and PVN were mixed with 10 uL of 1 M Li2S6 catholyte and 1mL electrolyte (as described above). After 8 h of adsorption, the solution was removed, and MPVN, MVN and PVN with adsorbed Li2S6 were dried for XPS analysis. All of the above operations were carried in a glovebox filled with argon .Li2S6 (1 M) catholyte. The ratio of electrolyte to S was 15 uL mg−1 and the corresponding areal density was 3 mg cm−2 (based on sulfur). Symmetric batteries were constructed with VN as the work and counter electrodes and 20 μL of 1 M Li2S6 catholyte as the active material, wherein the dosage of electrolyte was 10 uLelectrolyte mgs−1. All of the above procedures were performed in a glovebox filled with argon .Coin cells (2032-type) were assembled using lithium metal (counter electrode and reference electrode), a celgard 2500 (separator) and 1.92 mg S , derived from the 10 μL Liα, λ = 0.154056 nm; Bruker, Karlsruhe, Germany) and X-ray photoelectron spectroscopy (XPS) spectra were measured using a Thermos SCIENTIFIC ESCALAB 250Xi  with a monochromatic Al Kα X-ray source . Specific surface area (SSA) was derived from the nitrogen adsorption–desorption isotherms (based on Brunauer–Emmett–Teller method) using ASAPA 2020 . The morphology of the current collector was characterized by a field-emission scanning electron microscope . Images from a transmission electron microscope (TEM), a high-resolution TEM (HR-TEM) and energy-dispersive spectrometer (EDS) mapping were recorded with JEM-2100HR . X-ray diffraction (XRD) patterns were obtained by BRUKER D8 ADVANCE . Cyclic voltammetry (CV) measurements were performed with a CHI660E electrochemical workstation using a different scan rate for the Li–S batteries (0.05 mV s−1) and symmetric batteries (10 mV s−1). Electrochemical impedance spectroscopy (EIS) tests were also performed using CHI660E  at a potential of about 2.3 V before and after CV, in a frequency range from 100 kHz to 0.01 Hz and an amplitude of 5 mV.Galvanostatic charge–discharge (GCD) electrochemical performance was tested in a NEWARE battery testing system at a confirmed voltage interval, 1.8 to 2.8 V vs Li/ Li3COO)2 was the in situ nitride, en route to be VN [In the electrospun solution, PAN served as the nitrogen source and a continuous template for the droplet phase of PS and PVP because of surface tension differences. During electrospinning, PAN formed a surrounding phase to eliminate Rayleigh instability, entraining discontinuous PS to be a continuous fluid . After sto be VN ,66,67. M−1 may be ascribed to carbon (D band and G band) under 532 nm laser excitation, reflecting disordered and graphitic carbon, respectively. However, the peaks of VN were very weak as a result of the homogenous distribution of VN and the VN peaks being covered by CNF peaks. As depicted in 2 g−1 and pore volume at 0.137 cm3 g−1 for MPVN, which was higher than those of MVN  and PVN . As depicted in The crystal structure of materials was investigated by XRD, as presented in 2S6 dissolution into the electrolyte using MPVN, MVN and PVN was investigated by a visual adsorption test of Li2S6 solution , V–N–O(V4+) and V–O(V5+) in the MPVN [3+ of VN, further confirming the VN phase in MPVN. After interaction with Li2S6, the V–N of MPVN underwent more negative shifts (0.34 eV to 516.76 eV) than both MVN (0.21 eV from 517.47 to 517.26 eV), outlined in x2− (4 ≤ x ≤ 8) to positive V3+ of VN [2Sx (4 ≤ x ≤ 8) are anchored on the surface of VN by enhanced polar binding, strengthened during the charging and discharging process. VN depicts excellent catalytic effect to accelerate the conversion of lithium polysulfides and comparable electron conductivity to facilitate electron transport, which induces fast redox kinetics and increases sulfur utilization [2S6, further demonstrating the poor adsorption of nonpolar carbon to polar lithium polysulfides. In other words, MPVN can be a favorable sulfur host material with efficient restriction of the shuttle effect by strong chemical binding to Li2Sx (4 ≤ x ≤ 8), thereby ensuring excellent durable cycle stability and high sulfur utilization for superior and stable performance of LSBs. The restriction of Lisolution a. After eraction . As a reeraction ,69. As dthe MPVN . In addi3+ of VN . Benefitlization . Moreove−2 at a potential of 1.8 to 2.8 V (vs Li/Li+). The CV curves in 2S2/Li2S) to soluble long-chain lithium polysulfides  and to S8, respectively. In the cathodic scan, the distinct reduction peaks at 2.31 and 2.06 V belong to a multistep transformation from S8 to Li2S2/Li2S. The LSB with MPVN exhibited higher cathodic peak potential (Epc) at 2.06 V and lower anodic peak potential (Epa) at 2.36 V, with a low polarization of 300 mV, compared to the 340 mV of MVN , 340mV of PVN  and 350 mV of NCNF . Furthermore, the LSB with MPVN exhibited noticeably higher current density. The low polarization and high current density of MPVN indicates excellent VN catalysts, which accelerate the redox kinetics of lithium polysulfides. Impressively, the onset potentials depicted in 2Sx (6 < x < 8) to short-chain Li2Sx (4 < x < 6). Besides, Tafel plots, which are an effective tactic for identifying the catalytic effect of VN on the conversion of lithium polysulfides, showed that MPVN had a decreased Tafel slope of 52 mV dec−1, compared to 55, 58 and 65 mV dec−1 of MVN, PVN and NCNF, respectively, further confirming the accelerated redox kinetics of lithium polysulfides (ct) within cathodes [ct of MPVN (30 Ω cm−2) was evidently lower than those of MVN (39 Ω cm−2), PVN (56 Ω cm−2) and NCNF (77 Ω cm−2), indicating the enhanced redox kinetics of MPVN. The slope of the short line (low frequency region) is related to the solid-state diffusion process of Li+ into the electrode materials, where the smaller slope of MPVN indicates increased ion diffusion efficiency. After 3 cycles, the Nyquist plots had two semicircles in the high and medium frequency regions and a short line in the low frequency region  of MPVN, MVN, PVN and NCNF was measured with sulfur areal loading of 3 mg cmsulfides . EIS wassulfides  of Li–S cathodes . The Rcty region . The sem−1, which was much higher than those of MVN (1294 mAh g−1), PVN (1021 mAh g−1) and NCNF (516 mAh g−1), indicating a highly reversible solid–liquid–solid reaction of S↔Li2Sx (4 ≤ x ≤ 8)↔Li2S2/Li2S. Notably, LSBs with MPVN had the lowest polarization (0.25 V), indicative of fast sulfur-based active material electrochemical dynamics. The GCD measurement can be also applied to analyze the self-discharge rate resulting from the soluble Li2Sx shuttle effect, which can damage the lifespan of Li–S batteries. Accordingly, soluble Li2Sx content reached a maximum when the discharging potential was set to 2.1V. After 19 stable cycles, the discharge process was interrupted at about 2.1 V for 48 h, where Li2Sx diffused and then reduced to Li2S2/Li2S, leading to capacity decay (The GCD (0.1 C rate) of LSBs with potential from 1.8 to 2.8 V was also tested, in which the typical multi-plateau behavior was consistent with the CV tests. The initial discharge capacity with MPVN was 1392 mAh gty decay . LSBs wity decay  had almoty decay  and PVN ty decay . The low2S6 without lithium foil to further investigate the catalytic kinetics of VN. CV curves of MPVN in 2S6 is firstly reduced to Li2S2/Li2S on the working electrode for the first cycle; and an anodic peak at 0.37 V, related to the multistep oxidation of Li2S2/Li2S to higher order Li2Sx and then conversion to S8 on the counter electrode. The high overlap of the subsequent cycle indicates a highly reversible electrochemical conversion of the lithium polysulfides (ct). The symmetric cells with MPVN exhibited a small Rct of 16 Ω cm−2, compared to the 20 Ω cm−2 of MVN, 48 Ω cm−2 of PVN and 83 Ω cm−2 of NCNF, suggesting an enhanced redox dynamic by rapid charge transportation in the electrochemical process. MPVN not only provides strong affinity capability with lithium polysulfides to promote S utilization, it also catalyzes the conversion of lithium polysulfides, enabling enhanced redox kinetics.Symmetric cells were also assembled by sandwiching the current collector containing Lisulfides . Moreove−1 (458 Wh kg−1 based on MPVN and Li2S6), and 1325 mAh g−1 (436 Wh kg−1) was maintained after 100 cycles (high capacity retention of 95.2% and low capacity decay of 0.05% per cycle), superior to those of MVN (0.09%), PVN (0.06%) and NCNF (0.31%). This is because there are more exposed catalytic sites on MPVN to enhance the contact area between VN and lithium polysulfides. Moreover, the coulombic efficiency of MPVN was more stable (about 99%) compared to the other host materials, further confirming its excellent catalytic ability to restrict the shuttle effect of lithium polysulfides. It should be noted that the bare MPVN without Li2S6 active materials displayed a low specific capacity of merely 39 mAh g−1 for lithium storage . In contrast, the batteries with MVN and PVN displayed a poorer rate performance than MPVN, related to their fewer catalytic sites for VN decorating. Without VN, NCNF showed the lowest electrochemical performance, further confirming the vital vole of VN in the lithium–sulfur system. Demonstrating the synergistic effect between the appropriate porous channel design and the VN catalytic effect, LSBs with MPVN exhibited an obvious S electrochemical reaction plateau, even at a high current density of 1.5 A g−1 (−1 (6.1 mAh cm−2)—even at a high sulfur loading of 6 mg cm−2, which is higher than the 4 mAh cm−2 of commercial Li-ion batteries. After 200 cycles, the LSBs with MPVN still delivered a high capacity, retaining 811 mAh g−1, and high capacity retention (80%) (As shown in .5 A g−1 . As depion (80%) . The att2Sx↔Li2S2/Li2S, thereby realizing fast redox kinetics and promoting the utilization of S for high cycling capability and rate performance. Furthermore, the multichannel porous nanostructure was beneficial for Li ion transfer and as an electrolyte–electrode contact interface. LSBs with an MPVN current collector displayed a high reversible capacity of 1325 mAh g−1 at 0.1 C, with a high capacity retention of 95% and excellent rate performance (985 mAh g−1 at 1.5 A g−1).Herein, we designed a lotus root-like NCNF structure assembled with VN catalysts as a self-supported current collector for superior LSBs. In the experimental host material, MPVN not only trapped lithium polysulfides by strong chemical affinity but also electro-catalyzed the conversion of S↔Li


The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST.Hydrogen sulfide (H It was recently demonstrated that these species are not only much more abundant in the cell than previously thought but also, due to their elevated nucleophilicity [2S or their parental thiol compounds [2S are currently believed to be mediated by sulfane sulfur species [Hydrogen sulfide . Moreov species ,16,17. 2S and related species in blood and tissues are kept in balance through a tight regulation of H2S biosynthesis and catabolism [2S is enzymatically produced by the two predominantly cytosolic enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) [2S. MST is therefore a recognized source of both H2S and sulfane sulfur species in the cell. Unlike H2S biosynthesis, which takes place both at the cytosolic and mitochondrial level, H2S catabolism occurs essentially in the mitochondrion, where sulfide:quinone oxidoreductase (SQR)—a flavoenzyme associated to the inner membrane—catalyzes the oxidation of H2S. The reaction leads to formation of a protein-bound persulfide [2S oxidation, SQR-mediated breakdown of H2S stimulates oxidative phosphorylation and, thus, ATP synthesis, which makes H2S the first known inorganic substrate of the mitochondrial electron transport chain [2S levels produced by the gut microbiota [2S oxidation pathway enzymes, whereas this localization pattern is lost in the epithelium of colorectal cancer [Steady-state levels of Htabolism . In mammse (CSE)  from these (CSE) . Two isose (CSE) . MST conse (CSE) ,21—leadise (CSE) , that carsulfide ,23 that rsulfide  or glutarsulfide . SQR is rt chain ,27. Thisrt chain , the latcrobiota . Indeed,l cancer . 2S metabolism , and th targets ,32,33. H biology ,35. In cycolysis . Accordiycolysis . Furtherycolysis ,38. MST ycolysis , renal cycolysis , astrocyycolysis . Howeverycolysis . Recentlycolysis . Moreoveycolysis . N-acetylcysteine (NAC), which has been extensively studied not only for its ROS scavenging action [2S metabolism in colorectal cancer cells. By combining work on the SW480 cell line and on recombinant human MST, we observed that NAC not only promotes increased expression and activity of MST and SQR, both involved in the production of sulfane sulfur species, but is also an effective substrate for MST. The association between altered mitochondrial metabolism, dysregulated cell redox homeostasis and carcinogenesis is well documented (reviewed in ). The mig action , but alsg action ,56 and cg action ,58,59. Dg action . Accordig action . Recentlg action . This st1 , d-lactic dehydrogenase , 5,5′-dithiobis-(2-nitrobenzoic acid) , l-cysteine , N-acetylcysteine , metaphosphoric acid (239275), 2-mercaptoethanol (805740), sodium 3-mercaptopyruvate dihydrate , N-ethylmaleimide , oxaloacetate (O7753), perchloric acid , protease inhibitor cocktail (P8340), rabbit polyclonal antibody against human SQR (HPA017079) and CBS (SAB1411562), anti-rabbit peroxidase secondary antibody (A6154), rotenone (R8875), sodium sulfide nonahydrate , sodium sulfite (S0505) were purchased from Sigma . The bicinchoninic acid assay (BCA) kit and rabbit polyclonal antibody against human MST (PA528779) were purchased from Thermo Fisher Scientific . Mouse monoclonal antibody against human CSE (SC-365381) was purchased from Santa Cruz Biotechnology . Fetal bovine serum (FBS), l-glutamine, streptomycin and penicillin were purchased from Biowest . Mini-PROTEAN TGX Stain-Free Precast Gels, the Clarity Western ECL substrate and the Laemmli protein sample buffer were purchased from Bio-Rad . Bovine serum albumin was purchased from AppliChem .The human colon cancer cell line SW480 was purchased from the American Type Culture Collection (ATCC No. CCL228™). Acetyl coenzyme A (A2056), 7-azido-4-methylcoumarin , reduced β-nicotinamide adenine dinucleotide , Dulbecco’s Modified Eagle Medium (DMEM) cell culture media (D6546), the cell lysis reagent CelLytic™MT (C2978), coenzyme Q2S crystals in degassed ultra-pure  water under a N2 atmosphere, as reported in [412 = 14,150 M−1·cm−1 as recommended by the manufacturer. The concentration of sulfide was then adjusted to 50 mM by dilution with degassed ultra-pure (Milli-Q®) water in a gas-tight glass syringe. For simplicity, sulfide solutions, though containing H2S, HS− and S2− in different proportions depending on the pH, here will be referred to as “H2S solutions”.Sulfide stock solutions were prepared by dissolving Naorted in . Sulfideorted in , using t−1 glucose, supplemented with 2 mM l-glutamine, 10% (v/v) heat-inactivated FBS, 100 U·mL−1 penicillin and 100 μg·mL−1 streptomycin. For each studied condition, cells were seeded in five 25 cm2 flasks (1 × 106 cells/flask) and grown at 37 °C and 5% CO2. After 24 h, the culture medium was replaced with fresh medium (control) or medium supplemented with 10 mM NAC, and further incubated for 24 h. After trypsinization, cells were washed with fresh culture medium, counted using the trypan blue dye exclusion test, and centrifuged at 1000× g for 5 min, yielding usually 2–3 × 107 cells per condition. Under the tested conditions, 10 mM NAC proved not to be cytotoxic.SW480 colorectal cancer cells were cultured in DMEM containing 4.5 g·L2, 1% v/v protease inhibitors cocktail) and kept on ice for 1 h. The sample was then transferred to a pre-chilled glass-teflon tissue grinder and homogenized with 100 strokes. Immediately afterwards, 100 µL of hypertonic buffer  were added to the homogenate, which was carefully resuspended, followed by centrifugation at 1000× g for 5 min at 4 °C to pellet unbroken cells, debris and nuclei. The supernatant was kept on ice, while the pellet was resuspended in isotonic buffer  and centrifuged a second time at 1000× g for 5 min at 4 °C, to minimize heavy contaminants. The supernatants from both centrifugations were pooled and centrifuged at 20,000× g for 30 min at 4 °C. The mitochondria-enriched pellet was solubilized in 150 µL of 10 mM Tris-HCl pH 7.4, 250 mM sucrose, 1 mM EDTA, 0.5% (w/v) N-lauryl maltoside, as reported in [Mitochondrial preparations were obtained as described in  with modorted in .Recombinant human MST was expressed and purified as described in Yadav et al. .2 atmosphere for a few minutes. The reaction mixture contained 30 µL of mitochondrial extract , 58 µM CoQ1, 1 mM sodium sulfite, 2 mM sodium cyanide (to inhibit complex IV) and 4 µM rotenone (to prevent H2S-derived reverse electron flow through complex I). The reaction was triggered by adding 96 µM H2S and the reaction rates were determined by following the reduction of CoQ1 at 278 nm (Δεox-red = 12 mM−1cm−1). Data were normalized to total protein (quantified with the BCA method) and were expressed as nmol of CoQ1 reduced per minute per mg of protein.The SQR activity assay was adapted from . Measure2S and pyruvate in a 1:1 stoichiometry. MST activity was therefore evaluated by quantifying the amount of pyruvate produced over a given time (1 h) by MST in the presence of an excess of the substrates. Pyruvate was quantified spectrophotometrically from the amount of NADH enzymatically oxidized in the presence of LDH. Briefly, cells were harvested by centrifugation and lysed using the CelLytic™MT reagent and the protease inhibitors cocktail from Sigma, according to the manufacturer’s instructions. Cell lysates  were dispensed into eppendorf tubes containing 100 mM Tris-HCl pH 8.0, 50 mM sodium sulfite, 10 mM 2-mercaptoethanol . Data were normalized to total protein content (quantified with BCA method) and expressed as nmol of pyruvate generated per minute per mg of protein. The MST activity was assayed in cell lysates as described in  with modacceptor ) and 10 e of LDH . NADH ox2S-selective fluorescent probe 7AzC and a Thermo Scientific Appliskan plate reader. The reaction mixture, in 200 mM Tris-HCl pH 8.0, contained 10 μg of recombinant human MST per well, 50 μM 7AzC, and NAC (or Cys) at varied concentrations. The reaction was triggered by adding 3MP in a total assay volume of 250 μL. The increase in fluorescence  was monitored over 90 min at 37 °C. Kinetic parameters for Cys and NAC were determined at concentrations ranging from 0 to 40 mM in the presence of 0.5 mM 3MP. Conversely, kinetic parameters for 3MP were determined at concentrations ranging from 0 to 2 mM in the presence of 20 mM NAC or Cys. Prior to use, the solutions of NAC, Cys and 3MP were adjusted to pH 8.0 and their concentration determined by spectrophotometric titrations with DTNB, using the molar extinction coefficient ε412nm = 14,150 M−1·cm−1. Data were analyzed using Excel and the MST activity was calculated from the initial slope of the fluorescence increase.Activity assays on isolated recombinant human MST were adapted from ,33,69. TCells were harvested and lysed as described in v/v)) containing 3% bovine serum albumin  and then incubated overnight at 4 °C with antibodies against human SQR or MST. After three washing steps with PBS-T, the membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody, followed by three washing steps with PBS-T and detection by enhanced chemiluminescence . Specific bands were analyzed using Image Lab software , followed by normalization of the target protein band intensity to the total protein load determined as described above.Cells were lysed as described in ®) water, kept on ice for 30 min and then lysed by three cycles of freeze-thawing. Lysates were mixed with 250 µL of 3.34% meta phosphoric acid and, after vortexing, were incubated for 30 min on ice, followed by centrifugation at 12,000× g for 10 min at 4 °C. The supernatant was filtered and 120 µL of the deproteinized solution were mixed with 60 µL of 0.3 M Na2HPO4 and 90 µL of 1 mM DTNB. NAC quantification was performed at room temperature by injecting 100 µL of DTNB-derivatized samples into a PrevailTM C8 column (150 × 4.6 mm–5 µm)  connected to a double pump HPLC apparatus  equipped with an UV-Vis detector . Injections were performed automatically using an HT300L autosampler (HTA). The elution was monitored by recording the signal at 331 nm using the following mobile phase: 20 mM NaH2PO4 pH 4.9 (Buffer A) and 100% methanol (Buffer B). The flow rate was 1 mL·min−1 and the concentration of buffer B was varied during the elution as follows: 2% B for 10 mL, then the concentration of B was raised to 40% in a steep gradient of 2 mL, kept at 40% for 1 mL to allow the elution of the unreacted excess of DTNB, then brought back to 2% in a 2 mL reverse gradient and kept at 2% for 7 mL before injection of the next sample. Calibrations were performed by injecting standards of NAC, GSH and Cys at known concentrations after derivatization with DTNB. For each condition, two experimental duplicates were tested, and each duplicate was injected twice. The derivatization buffer and a derivatized lysate of untreated cells were injected as controls. This method was adapted from . Cells wSW480 colorectal cancer cells were grown in DMEM alone or supplemented with 10 mM NAC. After 24 h, the expression levels of CBS, CSE, MST and SQR were evaluated in the cell lysates by immunoblotting. Whereas cell exposure to NAC had no effect on the expression of CBS and CSE , as comp−1·mg−1 vs. 92.1 ± 7.9 nmol·min−1·mg−1, Prompted by the increased MST expression levels observed upon cell exposure to NAC, we evaluated the MST activity in lysates of NAC-treated cells. As described in 2S oxidizing activity of SQR was evaluated in mitochondrial preparations of NAC-treated and control cells by monitoring spectrophotometrically the H2S-induced reduction of exogenous CoQ1 .The HCoQ1 see . The effCoQ1 see . A repretransfer  or by Consfer [2 . The SQRn−1·mg−1 B. A blan−1·mg −1 and 10.4 ± 1.9 µM·min−1·mg−1, Citrate synthase (CS) activity measurements were carried out as a validated surrogate marker of mitochondrial mass . NAC-tre2S-synthesizing activity of recombinant human MST was evaluated in the presence of 3MP using the H2S selective fluorescent probe 7AzC [KM and Vmax values for NAC or Cys were independently determined at fixed concentration of 3MP. A hyperbolic dependence of MST activity on the substrate concentration was observed for both NAC and Cys . To evaluate the effect on MST activity of the simultaneous presence of the two substrates, H2S production by MST was determined in the presence of Cys and NAC together. As shown in Vmax and KM values for 3MP were in the same order of magnitude . Altere2S synthesis, such as CBS, CSE and MST [2S and related reactive sulfide species, including sulfane sulfur species, play a role in cancer biology [2S appears to favor cancer cell survival and proliferation by stimulating cell bioenergetics and neo-angiogenesis, among other effects, sulfane sulfur species could potentiate the cell antioxidant defense system, by both scavenging free radicals and enhancing the activity of antioxidant enzymes such as glutathione peroxidase, glutathione reductase and superoxide dismutase [Reprogramming of sulfur metabolism has been reported as a common mechanism to different cancer types . In  and MST  were fou and MST ,41,77,78 biology ,34. Whilismutase ,80.  and SQR, the enzyme catalyzing the limiting step of mitochondrial sulfide oxidation. Cell exposure to NAC resulted in increased expression levels of both MST (≈60%)  exposure to NAC on HT (≈60%) B and SQRT (≈60%) D, whereaT (≈60%) . Such chT (≈60%) , which acysteine . In this2S metabolism. With the aim of exploring the correlation between increased expression levels and enzymatic activity, a functional approach was undertaken by measuring MST and SQR activity in cell and mitochondrial extracts, respectively. As compared to controls, NAC-treated cells displayed, respectively, ≈20% and ≈50% increase in MST and SQR activity  (2S generation by MST and probably compete for the same binding site on the protein. Given the higher reactivity of (per/poly)sulfides with electrophiles compared to their thiolic counterpart [N-AceCysSSH)—the expected product of the reaction of MST with NAC—can directly contribute to ROS scavenging, as already reported for cysteine persulfide  . This hiVmax/KM) . CombineVmax/KM) . Therefonterpart ,88,89, i. It is he urine ,17. On tThe physiological relevance of the reaction between NAC and MST might be questioned because cell-internalized NAC was reported to be deacetylated into cysteine ,84, thou2S metabolism has been evaluated in colon cancer cells. The evidence collected with SW480 colorectal cancer cells shows that: (i) treatment with NAC promotes increased expression and activity of mitochondrial enzymes (MST and SQR) involved in the production and consumption of H2S and sulfane sulfur species and (ii) NAC can act as a direct sulfane sulfur-accepting co-substrate for MST, being likely persulfidated (2S and related reactive species.In summary, this is to our knowledge the first study in which the effect of prolonged cell exposure to NAC on Hlfidated . The rel


CDKAL1 rs10946398 is a type 2 diabetes (T2D)-associated variant. It is a new body mass index (BMI)-associated variant in Asian populations. We investigated the association between rs10946398 and T2D among 9908 participants aged 30–70 years based on BMI: normal weight; 18.5 ≤ BMI < 24 kg/m2, overweight; 24 ≤ BMI < 27 kg/m2, and obesity; BMI ≥27 kg/m2. The CC genotype conferred a higher risk of T2D than the CA genotype. The odds ratios (ORs) were 1.83; 95% confidence interval (CI) 1.49–2.26 and 1.20; 95% CI 1.02–1.40, respectively. The C allele was the significant risk allele compared with A allele . For normal, overweight and obese participants with CC genotype, the ORs were respectively 1.69; 95% CI 1.02–2.81, 2.34; 95% CI 1.50–3.66, and 1.58; 95% CI 1.02–2.45 among men and 1.22; 95% CI 0.67–2.22, 2.42; 95% CI 1.30–4.52, and 2.3; 95% CI 1.19–4.50 among women. The C allele ORs were higher in obese and overweight women. In conclusion, the rs10946398 CC/CA genotypes, as well as the C allele increased the risk of T2D. The ORs were higher in women who were overweight and obese than in those with normal weight. Nonetheless, significant results were prominent only among those with CC genotype and C allele. In Taiwan, the prevalence is reported to have increased from 5.8% in 2000 to 12.4% in 20143. High blood glucose is one of the significant risk factors for deaths among Taiwanese adults4.Type 2 diabetes is a complex metabolic disorder characterized by interaction between peripheral insulin resistance, hepatic glucose production, and beta-cell dysfunction. It remains an increasingly challenging public health issue. Globally, the prevalence of the disease is approximately 8.5% and nearly 592 million people are estimated to be infected by 20355. Modifiable factors such as physical activity, maintenance of normal BMI, and dietary intake are relevant to studies investigating type 2 diabetes. About 36% of individuals with type 2 diabetes were found to be obese6. Besides the traditional risk factors, genetic determinants play a vital role in the development of type 2 diabetes. Some of the loci that have been consistently associated with the disease risk include CDKAL1, HHEX, CDKN2A/2B, IGF2BP2, CDC123-CAMKID, TSPAN8-LGR5, THADA, SLC30A8, ADAMTS9, FTO, PCSK9, and NOTCH2, with odds ratios ranging from 1.09–1.7910. New independent risk variants have also been reported11. Most of the studies investigating the association between genes and T2D have been carried out in Europe12. However, only a few genetic markers for type 2 diabetes have been reported in Asia11.Traditional risk factors have not fully accounted for the higher prevalence of diabetesCDKAL1) is among the genes consistently investigated for diabetes susceptibility among Europeans. One of its variants (rs10946398) has been significantly associated with T2D15, gestational diabetes16 and diabetic retinopathy17 in different subgroups. The C allele of rs10946398 is reported to be a T2D risk allele18. A meta-analysis of cohort studies has reported significant associations between CDKAL1 variant and T2D in general populations19. However, further analyses showed significant associations only in Asian but not African subgroups. This is an indication that results from individual studies are not consistent with one another.Cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1  is strongly associated with the risk of type 2 diabetesTable Table Table CDKAL1 rs10946398 and type 2 diabetes among Taiwanese individuals. The association was substantially stronger among CC compared to CA carriers, as well as in C allele compared to A allele carriers. Based on stratified analyses, the CC genotype was significantly associated with T2D in overweight and obese women, as well as in men regardless of their BMI. Several studies have provided evidence for the significant contribution of CDKAL1 rs10946398 to T2D risk23. According to findings from a global meta-analysis, 8 studies have reported a trend of elevated ORs for the C risk allele, whereas only two studies have found no associations14.In this study, we found a significant association between In our initial analysis, the CA genotype was a risk factor for T2D. However, after stratification by BMI, a significant odds ratio was found only among obese individuals . When stratified by sex, borderline associations were found among overweight  and obese  women. However, significant odds ratios were not observed in men.25. Post-menopausal women are believed to be more susceptible to T2D due to their greater percentage of body fat and intra-abdominal visceral fat26. In the present study, an increased risk of T2D was found among normal weight menopausal women .Menopause has been associated with certain unfavorable changes in the body that serve as predisposing factors for type 2 diabetes2 indicates normal weight, 24–26.9 kg/m2 indicates overweight while ≥27 kg/m2 indicates obesity27. The cut-point for T2D risk in different Asian populations varies from 22–25 kg/m2. Values above 26 kg/m2 are associated with a higher risk of T2D28. In a study conducted in Taiwan, Chang and colleagues reported that the rs10946398 C allele was associated with an increased risk of T2D. This association was attenuated in persons with a larger BMI12. Nonetheless, we found contrasting results. In their study, the sample size was comparatively small and stratifications did not include sex. In addition, there was no information about the total number of participants with a higher BMI (>26.9 kg/m2). In another study, ENPP1 rs1044498 was significantly associated with T2D but not with obesity29. It is worth mentioning that T2D had no significant association with smoking, physical activity, and alcohol drinking.Based on the criteria defined by the Department of Health, a BMI of 18.5–23.9 kg/mCDKAL1 gene influences T2D is not fully understood. However, it has been reported that it codes for the cyclin-dependent kinase 5 (CDK5) regulatory subunit-associated protein 1-like 1 which may affect the activity of the CDK5 protein15. This may subsequently lead to degeneration of β cells and development of type 2 diabetes mellitus14.The mechanism through which the CDKAL1 rs10946398 and type 2 diabetes. Such findings are necessary considering that T2D is one of the significant risk factors associated with mortality in Taiwan. Furthermore, noting that SNP rs10946398 is a new BMI-associated locus specifically for individuals of Asian ancestry, large dedicated studies are necessary to confirm such associations22. Genetic variants associated with BMI may also have associations with metabolic traits. Stratification of samples helps to clarify the role of BMI in modulating pancreatic beta-cell function possibly influenced by the rs10946398 variant. So far, such stratifications have not been widely considered in Asian populations13. Our study is limited in that we used a nonrandom or purposive sampling technique; hence, results may not be fully reflective of the general population. Second, there is a possibility of a response bias considering that information were collected using questionnaires. Moreover, there was no information regarding diabetes regimens. Finally, multiple stratifications might have affected our results. From this aspect, there is a need for additional research.In this Taiwan-based study, we have attempted to validate the association between CDKAL1 rs10946398 and type 2 diabetes among Taiwanese men and women. (2) The CC genotype is a risk factor for type 2 diabetes in women with BMI ≥ 24 kg/m2, as well as in men regardless of their BMI. (3) The CA genotype appears to be a risk factor for T2D mainly in obese individuals. In conclusion, we found that rs10946398 CC/CA genotypes and C allele increased the risk of T2D among Taiwanese adults. Overweight and obese women had higher odds ratios than normal women. However, the effect was significant only among those with CC genotype and C allele. These findings should serve as an incentive for larger dedicated studies.In summary, our study provides the following conclusions: (1) there is a significant association between 32. The project aims to recruit over 200,000 volunteers by 202732. All methods were carried out in accordance with the relevant guidelines and regulations. Participants obtained written informed consents prior to data collection. The study protocol was approved by the Institutional Review Board of Chung Shan Medical University.Data were obtained from Taiwan Biobank, a database with genetic information of over 89169 individuals aged 30–70 years2), overweight (24 ≤ BMI < 27 kg/m2), and obesity (BMI ≥ 27 kg/m2). The selected phenotypic characteristics included age, sex, body mass index, total cholesterol (T-CHO), triglycerides (TG), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), uric acid, systolic blood pressure (SBP), and diastolic blood pressure (DBP). Lifestyle factors included physical activity, smoking (never/former and current smoker), and alcohol consumption (150 c.c per week or on a regular basis for 6 months). Information on menopause was based on self-report. Women who reported a complete absence of menstrual period for 12 consecutive months without hysterectomy were categorized as naturally menopausal. These variables were selected based on their previous assessments in previous studies34.Overall, 10853 Taiwan Biobank participants aged 30–70 years were recruited. Excluded were individuals with missing information (n = 652) and those with BMI under 18 (n = 93). The final enrollment included 9908 participants with complete phenotypic and genotypic data. Taiwan Biobank has 29 recruitment centers. Participants were assessed in specific recruitment centers during the period 2012–2016. Among those selected, 355 women and 619 men had type 2 diabetes. Individuals were defined as having type 2 diabetes mellitus if they (1) had a fasting glucose level ≥126 mg/dl; (2) had a glycosylated hemoglobin A1c value of at least 6.5%); or (3) self-reported a history of diabetes based on the question, “have you ever been diagnosed with type 1 or type 2 diabetes by a doctor or health professional?” In general, 49.49% (n = 482) of patients with type 2 diabetes were diagnosed by physicians. Participants were grouped into BMI categories: normal weight  were chosen for analysis. However, only rs10946398 was included in the final analysis because of its highly significant association. Furthermore, it is the only CDKAL1 SNP associated with BMI among Asian populations. In addition, it has been shown to have a more striking replication signal. SNP genotyping was performed at the National Center for Genome Medicine in Academia Sinica using the Axiom-Taiwan Biobank Array Plate 31. The Axiom™ Genome-Wide ASI Array Plate maximizes genomic coverage of common and rare alleles of East Asian genome while the Axiom™ Genome-Wide CHB Array plate maximizes genomic coverage of common alleles of the Han Chinese genome. Only participants with call rates greater than 90% were included in the study. SNPs were excluded if the minor allele frequency (MAF) was <0.05. Also excluded were SNPs whose genotypes deviated from the Hardy-Weinberg equilibrium (HWE).We searched peer-reviewed literature databases  to identify common We used the PLINK 1.09 beta and SAS 9.3 software  for data management and statistical analyses. The distribution of variables were tested using Kolmogorov-Smirnov test and Shapiro-Wilk test. Data with normal distributions were analyzed by t-test and ANOVA while those not normally distributed were compared by Wilcoxon rank-sum test and Kruskal-Wallis test. Normally distributed variables were presented as mean and standard deviation (SD) while non-normally distributed variables  were presented as median and interquartile range (IQR). Genotypic associations of the SNP with T2D were determined using the Chi-square test. The risk allele/genotype-specific odds ratios and 95% confidence intervals were calculated using multivariate logistic regression models. Associations of genotypes with T2D were represented as co-dominant models. The interaction between BMI and rs10946398 on T2D risk was tested using a dominant model.


Enterococcus gilvus CR1, isolated from raw cow’s milk, can produce carotenoids. The complete genome sequence of this strain was determined using the PacBio RS II platform. Enterococcus gilvus CR1, isolated from raw cow’s milk, can produce carotenoids. The complete genome sequence of this strain was determined using the PacBio RS II platform. The assembly was found to contain a circular chromosome, including carotenoid biosynthesis genes, and comprises 2,863,043 bp, with a G+C content of 41.86% and three plasmids. Enterococcus gilvus was first isolated from clinical specimens from humans in 2002  technology . SMRT teDe novo assembly was constructed using the hierarchical genome assembly process (HGAP) workflow  supplemented with 0.5% glucose at 30°C under static condition. The genomic DNA was extracted as previously reported  and puriworkflow  implemenworkflow  with itsspx genes encoding transcriptional regulators involved in carotenoid biosynthesis  and three plasmids with sizes of 919,333 bp (G+C content of 42.94% and 935× coverage), 80,244 bp , and 82,704 bp . The PacBio RS II platform produced 1,665,885 reads with a mean length of 2,876 bp. The chromosome contains the isoprenoid biosynthesis pathway. The ynthesis  were fouFurther investigation into the CR1 genome will provide more insight into the regulation of carotenoid biosynthesis and milk fermentation.Enterococcus gilvus CR1 has been deposited at DDBJ/ENA/GenBank under accession numbers CP030932 (chromosome), CP030933 (pCR1A), CP030934 (pCR1B), and CP030935 (pCR1C).The complete genome sequence of