HMs) gene families. However, knowledge of HMs genes is limited in horticultural deciduous trees, including apple .Histone methylation and acetylation regulate biological processes in plants through various histone modifications  using transcriptome sequencing data, and most MdHMs were involved in flower induction processes. Subsequent quantitative real-time PCR was then performed to confirm the expression levels of candidate MdHMs under different flowering-related circumstances.Here, a comprehensive study of identifying and investigating MdHMs were involved in, and responsive to, flower induction in apple. This study established an MdHMs platform that provided valuable information and presented enriched biological theories on flower induction in apple. The data could also be used to study the evolutionary history and functional prospects of MdHMs genes, as well as other trees.The online version of this article (10.1186/s12870-018-1388-0) contains supplementary material, which is available to authorized users. HMs gene family members, including histone methyltransferases (HMTs), histone demethylases (HDMs), histone acetylases (HATs), and histone deacetylases (HDACs). Similarly, HMTs, HDMs, HATs and HDACs regulated various biological processes in plants [Histone modifications (HMs), which repressed or promoted gene expression, affected various processes and played important roles during plant growth and development. Methylation, demethylation, acetylation and deacetylation were common histone modifications processes. These modifications depended on four different n plants , 2.HMTs family included two subfamilies, and they were SDG (set domain group) and PRMT (protein arginine methyltransferases). HDMs family also included two subfamilies, HDMA  and JMJ (JmjC domain-containing proteins). As for HATs family, four kinds of subfamilies  were contained. I): HAG types included GCN5-, ELP3-, and HAT1-like histone acetylases domain structure; II): HAM types included a MOZ-YBF2 domain; III): HAC types included a p300/CREB-binding protein structure; IV): HAF types included a TATA binding protein-associated factors TAFII250. HDACs family shared three subfamilies, including HAD (RPD3/HDA1 superfamily), SRT (silent information regulator 2) and HDT (HD2 families) [These four gene family contained different subfamilies. HMs genes was also different in plants. A total of 136 HMs  have been identified in sweet orange, and they played important roles in fruit development [HMs  were also identified from tomato genome [SDGs members have been identified in the grape genome and some were up-regulated during grape softening [HMs gene functions were partially characterized, especially in the model plant Arabidopsis. They played important roles in plant growth and development, including in photomorphogenesis, seed germination and dormancy, embryo development, flowering-related processes, fruit development, stress and defense, and hormonal signaling [HMs can directly function in regulating flowering through their over expression or down expression. They can also affect the expression of flowering related genes. For example, an Arabidopsis thaliana HDA family member, AtHDA9 (AT3G44680), repressed flowering by affecting the acetylation of AGAMOUS-LIKE 19 (AGL19) [AtHDA19 (AT4G38130) influenced flower development together with A-class organ identity gene AP2 (APETALA2), similar as AtHDA6 (AT5G63110), which showed late flowering in the HDA6-RNAi plants [AtHAM1 (At5g64610), AtHAM2 (At5g09740), AtHAC1 (At1g79000) were also responsible for flowering time [AtHAM1 and AtHAM2 showed earlier flowering time, while overexpression AtHAM1 flowered later and had more rosette leaves [Solanum lycopersicum), SlHAG22, SlHAG8 and SlHAG18 were involved in vegetative or reproductive development, and SlSRT2 participated in flowering [HM genes can also regulate the expression level of flowering-related genes, such as FLOWERING LOCUS C (FLC), LEAFY (LFY), MADS AFFECTING FLOWERING4 (MAF4) and (MAF5) [HAM1 resulted in a higher H4 hyperacetylation and H4K5ac at FLC in Arabidopsis [Arabidopsis, an enriched level of histone H3 acetylation and H3K4 trimethylation at FLC and MAFs occurred in the histone deacetylase6 mutant (had6) [FLOWERING LOCUS T (FT) was also influenced by HMs. The Arabidopsis JmjC family protein T-DNA insertion mutant lines , showed earlier flowering, which might enrich FT mRNA and H3K4me3 levels within FT chromatin [FLC and FT were the main well researched genes that associated with HMs [HMs affect or interact with their downstream or upstream flowering genes to control flowering.Apart from their different structures, the number of ignaling –15. HMs i plants , 18. Othing time . For exae leaves . In tomad (MAF5) , 22, 23.bidopsis . In Arabt (had6) . Meanwhihromatin . Among vwith HMs –27. ThesMalus domestica) is an economically important fruit tree in temperate regions worldwide, and flower induction is an important issue, which restricted fruit yield and economic incomes [INDETERMINATE DOMAIN (IDD), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL), MADs-box, and GIBBERELLIC ACID STIMULATED ARABIDOPSIS (GASA), have also been well identified and reported to regulate flower induction in apple [HMs and their potential involvement in apple flower induction. In 2017, with the publication of a new apple genome [HMs gene family in apple and help us to make a comprehensive investigation about their characterizations and potential response to flower induction.Apple , 44 MdHDMs (16 MdHDMAs and 28 MdJMJs), 57 HATs  and 26 MdHDACs . Additionally, their chromosomes locations, gene and protein structures, gene phylogeny, synteny analysis and protein-protein interaction network were also performed. Meanwhile, transcriptomic sequencing of 6BA treated trees and different flowering varieties (Nagafu No.2 and Yanfu No.6) were performed to investigate their potential involvement during apple flower induction. Furthermore, quantitative real-time PCR (qRT-PCR) was employed to investigate the expression levels of candidate MdHMs in different tissues  and different flowering circumstances , and various hormones  stress treatment. The results revealed valuable information of HMs genes in apple, which might be applicable to other fruit trees.In this study, we identified 198 HMs gene family members in the apple genome, a HMM file of each domain was obtained from Pfam database (http://pfam.sanger.ac.uk/), as previous studies [Arabidopsis HDT genes, AtHDT1 (At3g44750), AtHDT2 (At5g22650), AtHDT3 (At5g03740), and AtHDT4 (At2g27840), were downloaded from the TAIR database  and used as a query to search against the Genome Database for Rosaceae [apple genome , HDMs (HDMAs and JMJs), HATs  and HDACs  were manually checked to confirm their highly conserved segments. The relative locations of HMs were obtained from the apple genome [To identify  studies , 7. Thes studies . The dete genome . They weT2 At5g2250, AtHDTHMs between apple and Arabidopsis. The Arabidopsis and apple HMs protein sequences were aligned by the ClustalW program with default parameters. The multiple sequence alignment generated files were analyzed and then used to build phylogenetic trees with Maximum Likelihood method, pairwise deletion for sequences analysis, and a bootstrap value of 1000 times. For the gene structural analysis, gene models were downloaded from apple genome database (https://iris.angers.inra.fr/gddh13/) [http://gsds.cbi.pku.edu.cn/) for structural analyses [http://meme-suite.org/), and 10 motifs were found within each gene family. Protein-protein interactions were analyzed with http://string-db.org/. For orthologous genes identification, each pair of gene was used to BLAST with sequences homology more than 60% and e-value less than 1e-20. Gene Ontology (GO) terms analysis were performed with online database (http://www.geneontology.org).For phylogenetic analysis, MEGA 7.0  was usedgddh13/) . They weanalyses . Additiohttp://circos.ca/) was employed to investigate their tandem duplication and synteny relationships as previous methods [MdHMs genes were identified according to their physical locations on individual chromosomes in the apple genome. Adjacent homologous HMs genes on the same apple chromosome with no more than one intervening gene were considered tandem duplicates. The Plant Genome Duplication Database (http://chibba.agtec.uga.edu/duplication/) was used to identify synteny blocks between Arabidopsis and apple. For orthologous gene pairs, synonymous (Ks) and non-synonymous (Ka) nucleotide substitutions were calculated according to the comparative synteny map between the apple and Arabidopsis thaliana genomes, with ClustalX and PAL2NAL programs for protein and coding sequences alignment. They were calculated with DNASP v5.10 software.Circos v. 0.63 , which had been planted at the Apple Demonstration Nursery of Yangling Modern Agriculture Technology Park , were used in this study. They were all grafted T337/ elopment . Trees welopment , 35. TheMalus robusta Rehd were used for sucrose treatment experiments in the same orchard. They were also divided into six blocks. Three of them were sprayed with 15,000 and 20,000 mg L− 1 sucrose at 29 and 36 DAFB [− 1 6BA was performed at 27 and 30 DAFB. They were all sprayed on the whole trees with a low-pressure hand wand sprayer in a clear morning. Samples were collected at 30, 50 and 70 DAFB and stored for further use. Meanwhile, 100 mM GA3, 300 μM ABA, 100 μM SA and 50 μM MeJA were treated on 2-year-old ‘Nagafu No.2’ trees, as water as control; leaves were collected at 0, 3, 6, and 12 h for each treatment as previous study [An additional 18 uniform ‘Fuji’/ T337/  36 DAFB , and theSix14-year-old alternate bearing ‘Fuji’ trees were used in Tiandu Town, Fufeng, Baoji, Shaanxi  were sampled. Samples were collected from trees in their ‘ON’ years (with a higher flowering rate) and ‘OFF’ years (with a low flowering rate) in 2014 at 30, 90, and 150 DAFB in the morning. Terminal buds of current spurs were collected from trees in their ‘ON’ or ‘OFF’ years and stored for further use.M. robusta Rehd. Flowers were collected on April 9, 2015 during the full-bloom period. Additionally, stems were collected from new shoots with diameters of 2–3 mm, while mature leaves were collected from the adjacent buds. Fruits with diameters of 2–3 cm were also collected. All samples were immediately frozen in liquid nitrogen and stored at − 80 °C until used in the gene expression analyses.For the tissue-specific expression analyses, various tissues or organs were collected from ‘Fuji’/T337 v/v) was added. Each tube was shaken and inverted vigorously and then centrifuged at 12,000×g for 10 min at 4 °C. For each sample, the supernatant was collected into a new tube and re-extracted with an equal volume of chloroform:isoamyl alcohol . The resulting supernatant was then transferred into a new 2-mL tube and LiCl  was added. The mixture was incubated at − 20 °C for 4 h and the RNA was selectively pelleted by LiCl after centrifugation at 18,000×g for 20 min at 4 °C. The pellet was resuspended in 500 μL of SSTE buffer  that had been preheated to 65 °C and an equal volume of chloroform:isoamyl alcohol. The mixture was then centrifuged at 12,000×g for 10 min at 4 °C. The supernatant was transferred to a new microcentrifuge tube, and the RNA was precipitated with 2.5 volumes of cold ethanol at − 80 °C for at least 30 min and centrifuged at 1,2000×g for 20 min at 4 °C. Finally, the pellets were washed with 70% ethanol and resuspended in diethylpyrocarbonate-treated water. Total RNA integrity levels were verified by running the samples on 2% agarose gels. First-strand cDNA was synthesized from 1 μg of total RNA using a PrimeScript RT Reagent kit with gDNA Eraser  following the manufacturer’s instructions.Total RNA was extracted from plant tissue samples using the cetyltrimethyl ammonium bromide method with slight modifications . BrieflyHM genes were analyzed using quantitative real-time PCR (qRT-PCR). Primers were designed to span an intron-exon junction with Primer Premier 6.0 software. And they were designed with the preferred values to specific amplification with high yield as follows. (1) Length of PCR primers (18–24 bp); (2) Melting temperature (60 °C); (3) GC content (40–60%); (4) GC Clamp: more than 3 G’s or C’s should be avoided in the last 5 bases at the 3′ end of the primer. (5) Avoided hairpins, self and cross dimer, and repeats. (6) Avoid template secondary structure and cross homology. The qRT-PCR mix (20 μL) consisted of 2-μL cDNA samples (diluted 1:8), 10 μL 2× SYBR Premix ExTaq II (Takara Bio), 0.8 μL of each primer (10 μM)  was used as an internal control to normalize all mRNA expression levels under different treatments and different tissues [−ΔΔCt method was used to calculate the relative amount of template present in each PCR amplification mixture [The expression levels of the 12 candidate  tissues . Experim mixture .Gene expression data of RT-qPCR were subjected to an analysis of variance (ANOVA) at the 5% level with the SPSS 11.5 software package . Figures were constructed using Origin 8.0 .HMs were identified in the apple genome, including 71 HMTs, 44 HDMs, 57 HATs and 26 HDACs . Additionally, 16 MdHDMAs and 28 MdJMJs were widely distributed throughout the apple genome, except on chromosome 2, 11, 13 and 17. The 50 MdHAGs were distributed throughout 16 of the 18 chromosomes; however, chromosomes 0 and 17 lacked MdHAGs genes. The remaining groups of HATs, including MdHAMs, MdHACs, and MdHAFs, had partial distributions, similar to those of the MdHDACs.To clearly identify the ons Fig. , Table 1HMs Fig. , followeHMs genes, four rooted phylogenetic trees, including HMTs, HDMs, HATs and HDACs genes, were built with Arabidopsis and apple HMs proteins  were clustered and surrounded by HAGs gene members, and other HAMs and HACs were also tightly grouped with themselves  were also clustered  was used to evaluate the selection pressure during duplication. The Ka and Ks value was smaller in apple than betwen Arabidopsis and apple. However, the Ka/Ks values between gene pairs in apple less than 1, which was similar to apple and Arabidopsis .Additionally, a syntenic map of HMs gene families to confirm the present of each domain in apple, and a random gene was selected and to analyze their DOMAIN structure , which shared similar structures, were closely clustered. Additionally, 13 MdSDGs family members  shared the greatest number of motifs compared with the other MdSDGs proteins. In addition, MdPRMT05, 02, 07 and 01 also shared similar protein motifs   were built based on protein sequences  geneMdHMs regulated downstream genes or were regulated by their up-regulated genes to participate in various processes.To further predict their biological interactions, we visualized HM proteins with Cytoscape v3.5.1 , 46. As MdHMs expression profiles [p value were then adjusted with Benjamini and Hochberg’s approach for controlling the false discovery rate, and a corrected P value of 0.05 and log2 value (fold change) of 1 were set as the criteria for identifying DEGs. [MdHMs expression levels in response to exogenous 6-BA treatments and in two varieties with different flowering capabilities, Nagafu No.2 and Yanfu No.6. As shown in Figs. MdHMs, 197 genes, with the exception of MdHAG29, were detected in our transcriptome sequencing , 1 MdPRMT (MdPRMT4), 3 MdJMJs , 13 MdHAGs , 1 MdHAC (MdHAC03), and 3 MdHDAs  showed no or very low expression levels (less than 1). The non-existent or low expression levels of these MdHMs indicated that they did not function to great degrees in flower development. On the contrary, the expression levels of six genes, MdJMJ16, MdHAG08, MdHAG01, MdHAM01, MdHDT01, and MdHDT03, were extremely high (greater than 100), indicating that they may have important roles during the flower-induction period  were more highly expressed in variety Nagafu No.2  were more highly expressed in variety Nagafu No.2  were selected and their expression levels assessed using qRT-PCR under different flowering-related circumstances and in different tissues . These candidate MdHMs showed different expression patterns in the five tissues, and 11 of them showed higher levels in leaves and buds, with the exception of MdSDG55, which was higher in flowers  were also investigated . We investigated their expression levels in alternate-bearing ‘Fuji’ trees. The 12 MdHMs were expressed during the flowering periods of both the ‘ON’ and ‘OFF’ tree buds , 44 MdHDMs (16 MdHDMAs and 28 MdJMJs), 57 HATs , and 26 MdHDACs , were identified in the apple genome. They were further characterized, including gene phylogeny, protein-protein interactions, and expansion and synteny analyses. In addition, we investigated their potential expression levels and roles in response to flower induction. These results will add to the knowledge in this field.t traits , 47, 48.HMs genes, including HMTs (SDGs and PRMTs), HDMs (HDMAs and JMJs), HATs , and HDACs  have been systematically or partially reported in Citrus, S. lycopersicum, Arabidopsis, Z. mays, and O. sativa [HMs gene families in the important economic apple trees. With the republication of apple genome, it is useful for us to explore more information for genomic analysis. In present study, we totally identified 198 putative MdHMs , and they belonged to 11 different subfamilies, which were similar to those of other species [HMs, SDGs were the most conserved among the species. The number of MdSDGs was nearly 1.5-fold greater than the numbers of SlSDGs, CsSDGs, AtSDGs, OsSDGs, and ZmSDGs. Additionally, the number of MdHDMAs genes was nearly two to four times greater than those of other species. The number of HAGs in apple was greatly different from other species, especially Arabidopsis, O. sativa and Z. mays. This great difference was partially diminished when the AT1 domain was used as the query in a BLAST algorithm-based search, which led to 33 HAGs being identified in Arabidopsis [JMJs, HADs, and HDTs, were also present two times more in apple than in other species  were constructed using the all of the gene members from apple and the model plant Arabidopsis. Interestingly, one PRMT gene, AtPRMT16 was clustered with other SDG genes , HDMs (HDMAs and JMJs), HATs , and HDACs . The emergence of four different trees from HM subfamilies was useful to investigate their complex phylogenetic interactions.To better understand their phylogenetic interactions, four phylogenetic trees , neutral selection (Ka/Ks = 1), or purifying selection (Ka/Ks < 1) , 55. Intn 1 Fig. , which wstachyon , indicatArabidopsis and apple was also determined of the HMs. Because Arabidopsis is a model plant and functions of the AtHMs are better understood. A comparative genomic comparison investigation helped us understand information on AtHMs to MdHMs, and possible functions of MdHMs can be well inferred [Arabidopsis and apple genome . Similarly, MdHDA16, an orthologs of AtHDT15; MdHAM01, an orthologs of AtHAM1; and MdHAF01, an orthologs of AtHAF1, were expressed higher in ‘Yanfu No.6’ than in ‘Nagafu No.2’. However, MdHDT04, an orthologs of AtHDT1, was more highly expressed in ‘Nagafu No.2’ , which indicated their involvement in flowering. We analyzed their expression patterns in two varieties Nagafu No.2 and Yanfu No.6. ‘Yanfu No.6’ is a ‘Nagafu No.2’ mutant that has a higher proportion of spurs, shorter shoots, larger buds and a higher flowering rate [MdHMs were expressed and showed consistent patterns during the three developmental stages. A majority of the MdSDG genes were higher in ‘Yanfu No.6’ during the flower development stages , in different hormones stresses , and different flowering related circumstances . Totally, our identification and characterization of HMs genes in apple provided useful information and enriched biological theories, which could be foundation for further analysis.In this study, we systematically identified Additional file 1:Table S1. List of Pfam accession number of each HMs gene family (DOCX 13 kb)Additional file 2:Table S2. Primer information for gene expression analysis (DOCX 13 kb)Additional file 3:Table S3. Table S3. Detailed annotations of MdHMs according to the Gene Ontology (GO) terms (XLS 354 kb)Additional file 4:Table S4. Synteny analysis of MdHMs genes (DOCX 26 kb)Additional file 5:Table S5. Synteny information of MdHMs and AtHMs genes (DOCX 25 kb)Additional file 6:Figure S1. Average Ka, Ks values of duplication gene pairs. (A) gene pairs of apple; (B) gene pairs of apple and Arabidopsis (TIF 367 kb)Additional file 7:Figure S2. Diagram of HMs typical conserved domains (TIF 846 kb)Additional file 8:Figure S3. Gene structure and protein motifs analysis of MdSDGs gene family members (TIF 3270 kb)Additional file 9:Figure S4. Gene structure and protein motifs analysis of MdPRMTs gene family members (TIF 571 kb)Additional file 10:Figure S5. Gene structure and protein motifs analysis of MdHDMAs gene family members (TIF 639 kb)Additional file 11:Figure S6. Gene structure and protein motifs analysis of MdJMJs gene family members (TIF 1445 kb)Additional file 12:Figure S7. Gene structure and protein motifs analysis of MdHAGs gene family members (TIF 1174 kb)Additional file 13:Figure S8. Gene structure and protein motifs analysis of MdHDAs gene family members (TIF 1253 kb)Additional file 14:Figure S9. Gene structure and protein motifs analysis of MdHDTs gene family members (TIF 429 kb)Additional file 15:Table S6. Motif sequences of MdHMs proteins (DOCX 16 kb)Additional file 16:Figure S10. Interaction networks analysis of MdHMs genes (TIF 1052 kb)Additional file 17:Table S7. Predication protein-protein interaction information according to their orthologous in Arabidopsis (XLS 59 kb)Additional file 18:Figure S11. Transcript levels of 12 MdHMs genes following GA3, ABA, SA, and MeJA in by qRT-PCR. Leaves were collected after 0, 3,6 and 12 h after treatment. Each value represents the mean ± standard error of three replicates. Means followed by small letters are significantly different at the 0.05 level (the same below). (TIF 3532 kb)Additional file 19:Figure S12. Transcript levels of 12 MdHMs genes following sugar treatment and in Yanfu No.6. Terminal buds were collected from 30, 50, and 70 DAFB. (TIF 734 kb)Additional file 20:Table S8. Summary of HMs in different species (DOCX 13 kb)


Essential tremor (ET), a prevalent neurological disorder, is featured by postural and kinetic tremors in upper limbs. Studies of twins and families indicate an important role for genetic factors in ET development. There are substantial overlaps between ET and Parkinson's disease (PD). The aim of this study was to examine the possible roles of genetic variants in ET development.A total of 200 Han Chinese ET patients and 432 ethnically matched normal controls were enrolled, and genetic analysis of 23 variants in 15 genes was performed.MC1R) variant rs34090186 showed statistically significant differences in ET patients and controls . No statistically significant difference was revealed in either genotypic or allelic distributions of other variants or haplotypes .Genotypic and allelic frequencies of the melanocortin 1 receptor gene (MC1R gene, some variants of which were reported to be related to increased risk of PD and melanoma, may play a risk role in ET, confirming a potential association between ET and PD. Evidence supporting ET‐PD link will continue to accumulate and improve our understanding of any underlying mechanisms for both disorders.The discrepancies found in this study indicate the variant rs34090186 in the  Prevalence increases with age , 2p24.1 (ETM2), 6p23 (ETM3), 16p11.2 (ETM4), 11q14.1 (ETM5), 2p13.1, 1p13.3, 17q23.3, 5q35, and 3p22.2, have been reported to be related to ET development. By linkage analysis, whole‐exome sequencing and genomewide association studies, several genes have been identified to be associated with monogenic inherited ET or ET susceptibility, such as the FUS RNA‐binding protein gene (FUS), the HtrA serine peptidase 2 gene (HTRA2), the sortilin 1 gene (SORT1), the teneurin transmembrane protein 4 gene (TENM4), the sodium voltage‐gated channel alpha subunit 4 gene (SCN4A), SCN11A, the dopamine receptor D3 gene (DRD3), the leucine‐rich repeat and Ig domain containing 1 gene (LINGO1), the solute carrier family 1 member 2 gene (SLC1A2), the heme oxygenase 1 gene (HMOX1), and HMOX2. However, further confirmation is needed , LINGO1, LINGO2, HTRA2, the leucine‐rich repeat kinase 2 gene (LRRK2), and the DnaJ heat‐shock protein family (Hsp40) member C13 gene (DNAJC13), have been shown to exert either a risk or protective effect on ET and PD, or to be responsible for both disorders  of the cases were screened for the FUS gene coding variants, and all patients were tested for seven variants, including rs1721100, rs1989754, rs10089600, rs12720208, rs17550360, rs3758549, and rs4919621  from mainland China participated in the genetic study. There were 432 unrelated normal controls of similar gender, age, race, and residence . Of the 200 patients, the age, in years, at sample collection was 50.8 ± 15.3, and at ET onset was 41.5 ± 18.6. ET patients were consecutively enrolled from the Department of Neurology, the Third Xiangya Hospital of Central South University, Changsha, China. Two senior independent neurologists using the internationally accepted clinical criteria made the clinical diagnoses  and predicted deleterious effects by bioinformatics  and Online SHEsis program . p‐Values, odds ratios, and 95% confidence intervals were generated for statistical results. The discrepancies are statistically significant with the obtained two‐sided p‐values less than 0.05 . Eighteen variants showed monomorphism , including rs2227843, rs2227849, rs28924121, rs72470545, rs10115304, rs73672607, rs74942016, rs80127039, rs3207505, rs34184838, rs375681722, rs538881762, TENM4 c.4100C>A (p.T1367N), rs75932628, rs74654177, rs13071187, rs2230149, and rs35693565. Genotypic and allelic distributions for the other five variants in ET patients and controls appear in Table MC1R) variant rs34090186 . The patient group had a higher A allele frequency  compared to the control group. No statistically significant differences were found in genotypic or allelic frequencies of the other variants, including rs33932559, rs2254562, rs2076485, and rs7757931  or rs2076485–rs7757931 , as all p‐values were greater than 0.05 , located in chromosome 16q24.3, was initially known as an intronless gene spanning over 3 kb. It encodes a G‐protein‐coupled receptor with seven transmembrane domains, consisting of 317 amino acids , which is a transcription factor primarily expressed in the central nervous system, including dopaminergic neurons related to PD pathogenesis and is essential for dopaminergic phenotype induction and late dopaminergic precursor neuron survival , with ET. This indicates a common underlying mechanism or potential link between PD and ET, at least in our Han Chinese population. Several MC1R gene variants, not including rs34090186, were found to contribute to the PD development, suggesting a converging and distinguishable genetic basis for these two disorders (He et al., Using a case–control methodology in a well‐defined Han Chinese cohort, we demonstrated a potential association of a single nucleotide variant in a known risk gene of PD, None declared. Click here for additional data file.


It could be determined that blue light significantly diminished the induced α-SMA protein expression in both normal palmar fibroblasts and Duypuytren’s fibroblasts. The beneficial effect mediated by this irradiance, radiant exposure and wavelength was associated with an elevated reactive oxygen species generation. Furthermore, the data underlines the potential usefulness of blue light irradiation as a promising therapy option for Dupuytren’s disease, especially for relapse prevention, and may represent a useful strategy to treat further fibrotic diseases, such as keloids, hypertrophic scarring, and scleroderma.Dupuytren’s contracture is a fibroproliferative disorder affecting the palmar fascia of the hand. Most affected are the ring fingers, and little fingers of middle-aged men. Symptomatic for this disease is the increased proliferation and differentiation of fibroblasts to myofibroblasts, which is accompanied by an elevated α-SMA expression. The present study evaluated the therapeutic benefit of blue light (λ = 453 nm, 38 mW/cm The cause of Dupuytren’s disease (DD) is believed to be multifactorial with a genetic disposition and concerns predominantly middle-aged Caucasian men . It’s a Furthermore, TGF-β1 seems to play a superordinate role in the formation and maintenance of DD, especially because TGF-β1 induces the expression of α-SMA, collagen III and fibronectin, and has been shown to increase the contraction of myofibroblasts , 10. So,One therapy option for advanced DD is the partial fasciectomy, which is unfortunately accompanied by significant recurrence rates . This suThe use of photobiomodulation-based therapies as a treatment option for DD is a promising alternative, which has not been applied to this day. Visible light could mediate its therapeutic effect via the interaction with endogenous photoreceptors, chromophores such as porphyrins, cytochrome c oxidase, nitrosated and flavo- proteins, opsins, and ion-gate channels , 17, dep2) an interesting tool for the treatment of Dupuytren’s fibroblasts. Therefore, the therapeutic benefit of blue light (with these parameters) on Dupuytren’s disease was evaluated in the present work.It has already been shown, that blue light, dependent on the wavelength, intensity (irradiance) and radiant exposure, has anti-inflammatory effects in keratinocytes  and suppStudy approval was obtained from the Ethics Review Board of the Medical Faculty, Heinrich-Heine-University Düsseldorf (Study No. 3634). Of the 23 patients with Dupuytren’s disease (DD), 3 were female and 20 were male (average age 62 years), all of whom underwent fasciectomy at the Department of Trauma and Hand Surgery, University Hospital, Düsseldorf, Germany. Patients who suffered from carpal tunnel syndrome served as normal palmar fascia control (NPF). Their tissue was dissected from the palmar fascia and served as control group, if it had to be removed anyway . The usage of human material was in compliance with the Declaration of Helsinki Priniciples. Informed written consent was gathered from all patients. During surgery, tissue specimen were deposited in PBS (1% penicillin/streptomycin) and cooled to 4°C. Samples were processed within 24 h. All data were anonymized prior to analysis.All chemicals were obtained from Sigma-Aldrich, and cell culture materials from Cellstar (Greiner bio-one), if not mentioned otherwise.2.Minced tissue was incubated at 37°C in 10 ml collagenase-solution  for 45 min, filtrated through a 100 nm nylon strainer, centrifuged  and the pellet resuspended in 30 ml 0.9% NaCl-solution. After centrifugation (5 min at 1200 rpm at 4°C) the pellet was resuspended in standard medium  and incubated at 37°C, 5% CO4/6-Well and irradiation was started after 24 h, so that procedure was started under subconfluent conditions. Fibroblasts and NPF were either treated with 2 ng/ml TGF-β1 (ImmunoTools rh TGF-β1), irradiated (0–80 Joule/cm2), treated and irradiated, or neither of those. The irradiation was performed in PBS. The appropriate control cells were put beside the irradiation unit and incubated with PBS equally for the same time. After that, PBS was replaced by culture medium. Temperature was checked with a thermometer, and the achieved maximum temperature was 37°C. Cells were harvested by cell-scraping and the α-SMA protein expression was measured with Western Blot Analysis as described down below. No technical repeats could be performed due to limited biological tissue samples.Fibroblasts and NPF were used in passage 3 – 8. Cells were seeded in 3 × 102. The cells were continuously irradiated from above in PBS, in 6-Well-Plates. 60 (6 × 10) LEDs were equally distributed over a total of 10–12 cm2. The blue light-emitting device used in our experiments was provided by Philips GmbH, Innovative Technologies, Aachen, Germany.All irradiation experiments were accomplished with a prototype of a narrow-band light-emitting diode (LED), which emits light of the wavelength λ = 453 nm (16.7 min). The power density was 38 mW/cm2). Then, effects were analyzed with CellTiter-Blue Assay . CellTiter-Blue uses an indicator dye to measure the metabolic capacity of cells, as an indirect evidence for cell viability. CellTiter-Blue was added at a ratio of 1:20 into the medium. After an incubation time of 1 h, the fluorescence (540 EX/590EM) was measured in a 1420 Multilabel Counter .Fibroblasts were irradiated with different doses  and analyzed with the Quantity One 1-D Analysis Software Version 4.6.5 (BioRad).In order to verify the differentiation from fibroblasts to myofibroblasts, the α-SMA protein expression was measured with Western Blot analysis. Protein concentration was determined with the Pierce BCA Protein Assay Kit in line with the manufacturer’s instructions. 10 μg protein were mixed with the Laemmli buffer , denaturated for 5 min at 95°C, and separated on a 12% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE)). Separated proteins were transferred to a nitrocellulose membrane (25 V– 1.0 A– 30 min) in a trans-blot system (BioRad Trans-Blot Turbo). Successful transfer was verified by Ponceau red S staining. The membrane was saturated with 5% BSA in TBST for 1 h (RT) and immunolabeled with mouse anti-human-α–SMA (Abcam/ab7817) (1:1000 in 3% BSA in TBST) over night at 4°C and mouse anti-human Glyerinaldehyd-3-phosphat-Dehydrogenase (GAPDH) antibody (BioRad/#12004167) (1:6000 in 3% BSA in TBST) for 1 h (RT). After washing with TBST, the membrane was incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse secondary antibody (Dako/P0447) for 1 h (RT). Bound antibodies were detected using Clarity 4 cells/6-Well, ROS detection was performed using Dihydrorhodamine 123 . DHR123 is oxidized to its fluorescent derivative Rhodamine 123 by unspecific ROS. In order to trigger a strong reaction, the irradiation set up was first conducted with 60 Joule/cm2, and then fibroblasts were incubated for a minimum of 30 min in DHR123 (10 μM) in standard medium. Cells were washed with PBS, and fluorescence was measured (485EX/535EM) in the 1420 Multilabel Counter .24 h after seeding 3 × 102), treated and irradiated, or neither of those over the course of 5 days. After that, fibroblasts were treated with staurosporine (STS) (Sigma-Aldrich/S5921) (0.075 μM) for 18 h. Cells were then harvested and centrifuged (1200 rpm for 5 min). The supernatant was removed, and the pellet resuspended in 500 μl FACS buffer (cell wash + 3% FBS). Cells were set on ice, fixed with 70% EtOH for 20 min and centrifuged (1200 rpm for 5 min). Pellets were washed two times with FACS buffer and stained with 200 μl propidium iodide solution . Cells were incubated protected from light for 30 min (RT) and measured with FACS Calibur (BD Biosciences).Fibroblasts and NPF were either treated with 2 ng/ml TGF-β1, irradiated . The level of significance was considered to be p ≤ 0.05.Statistical analysis was performed with two-tailed 2 on, metabolic activity was significantly reduced in NPF fibroblasts and in DD fibroblasts metabolic activity was notably reduced. From 80 Joule/cm2 on DD fibroblasts activity was significantly down regulated and in NPF fibroblasts highly significantly degraded, compared to their appropriated untreated controls (0 Joule/cm2) . From the beginning metabolic activity of DD fibroblasts was higher compared to all NPF  fibroblasts . From 60ule/cm2) . Because-SMA protein expression  has already been proven to inhibit TGF-β-induced α-SMA expression in differentiated human dermal fibroblasts [Partial fasciectomy, enzyme injection, and needle fasciotomy are todays treatment options for Dupuytren’s disease (DD) . Due to roblasts . Especia2), and as the most promising radiant exposure 40 Joule/cm2 was used for the subsequent experimental procedures  fibroblasts were irradiated  [It was assumed that the effect of the blue light irradiation is mediated by the generation of reactive oxygen species (ROS), such as singlet oxygen  or hydro2O2) . And it 2O2) , cytochr2O2) , or flav2O2) . In fact2O2) . These f2O2) .2, and a radiant exposure of 40 Joule/cm2, does not reinforce apoptosis in neither Dupuytren’s fibroblasts nor in NPF fibroblasts.The excessive proliferation of myofibroblasts , combineThe present study highlights, that the application of blue light could inhibit the differentiation of Dupuytren's fibroblasts into myofibroblasts and the accompanied α-SMA expression. Our data suggests that, especially in the proliferative and involutional stages, or after surgery when TGF-β concentration is increased, an irradiation with blue light could be beneficial. Consequently, blue light therapy could be a promising therapy option for DD, and especially for relapse prevention. Our results suggest that the usage of blue light is a promising tool for therapeutic treatment for further fibrotic diseases such as keloids, hypertrophic scarring, and scleroderma.


However, there are still no reports in the literature about inflammation and marinobufagenin, a cardiotonic steroid from the bufadienolide family endogenously found in mammals. Therefore, the aim of this work was to analyze, in vivo and in vitro, the role of marinobufagenin in acute inflammation. Swiss mice were treated with 0.56 mg/kg of marinobufagenin intraperitoneally (i.p.) and zymosan  was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for counting cells by optical microscopy and proinflammatory cytokine quantification  by immunoenzymatic assay (ELISA). Zymosan stimulation, as expected, induced increased cell migration and proinflammatory cytokine levels in the peritoneum. Marinobufagenin treatment reduced polymorphonuclear cell migration and IL-1β and IL-6 levels in the peritoneal cavity, without interfering in TNF-α levels. In addition, the effect of marinobufagenin was evaluated using peritoneal macrophages stimulated by zymosan (0.2 mg/mL) in vitro. Marinobufagenin treatment at different concentrations  showed no cytotoxic effect on peritoneal macrophages. Interestingly, the lowest concentration, which did not inhibit Na+/K+-ATPase activity, attenuated proinflammatory cytokines IL-1β, IL-6, and TNF-α levels. To investigate the putative mechanism of action of marinobufagenin, the expression of surface molecules (TLR2 and CD69) and P-p38 MAPK were also evaluated, but no significant effect was observed. Thus, our results suggest that marinobufagenin has an anti-inflammatory role in vivo and in vitro and reveals a novel possible endogenous function of this steroid in mammals.Cardiotonic steroids, such as ouabain and digoxin, are known to bind to Na Marinoba marina . More rea marina , 4. The a marina , 6. Consa marina , 8, but Some studies suggest that cardiotonic steroids, such as ouabain, digoxin, and bufalin, have immunomodulatory activity because they interfere in several inflammatory parameters such as cell migration, vascular permeability, and proinflammatory cytokines –14. The Leishmania amazonensis [β (IL-1β) and tumor necrosis factor α (TNF-α), which could be associated with p38 mitogen-activated protein kinase (MAPK p38) and nuclear factor kappa B- (NF-κB-) reduced activity [Our group has demonstrated the ability of ouabain to act as an anti-inflammatory substance. Ouabain can reduce cell migration to the inflamed site by several stimuli, such as zymosan , concanazonensis . Besidesactivity , 23. Recactivity . Furtheractivity , 12.in vivo and in vitro.Although many works have investigated marinobufagenin's role in cardiovascular homeostasis, there are still no reports about marinobufagenin's effect on inflammation. Therefore, this work is aimed at analyzing marinobufagenin's effect in the acute inflammation process Swiss albino mice (6-8 weeks) were obtained from the Thomas George animal house of Federal University of Paraíba (UFPB). Animals were kept under standard laboratory conditions on a constant 12 h light/dark cycle with temperature (21 ± 1°C), and food and water were given ad libitum. All procedures adopted in this study were approved by the Ethics Committee on Animal Use of UFPB (Protocol: 125/2016).Female Rhinella schneideri toad venom at the Institute of Biomedical Sciences (ICB) of the Federal University of Rio de Janeiro (UFRJ) as reported elsewhere [in vivo and in vitro experiments, DMSO maximum concentrations used were 0.6% and 0.016%, respectively. Ouabain was purchased from Sigma and diluted in water.Marinobufagenin was purified from the lsewhere . A marinn = 5): saline, DMSO, marinobufagenin (MBG), zymosan (ZYM), and MBG + ZYM. MBG and MBG + ZYM groups were treated with marinobufagenin (0.56 mg/kg) intraperitoneally (i.p.) for three consecutive days. All groups received 0.5 mL of each solution. MBG dose and administration route were chosen based on the previous work from our group, wherein a dose-response curve was performed using 0.10, 0.31, and 0.56 mg/kg of ouabain, another physiological cardiotonic steroid, for three consecutive days in a model of ZYM-induced paw edema [To compare ouabain's effect described by our group and marinobufagenin's effect, the same dose (0.56 mg/kg) and experimental protocol (pretreatment for three consecutive days) were used –24. Miceaw edema .Peritoneal inflammation was performed as described in the literature , 27. Zymg for 10 minutes (4°C). Cytospin slides from each sample were stained by Fast Panoptic Method. Differential cell count was performed by optical microscopy, and each slide was analyzed until the count of 100 cells can be reached using an oil-immersion objective. Finally, the supernatant was collected and stored at −20°C for cytokine analysis.Exudates collected were pooled, and the total number of leukocytes was determined by optical microscopy using Turk's solution . For differential cell count, samples were centrifuged at 350β, IL-6, and TNF-α cytokines present in peritoneal fluid were quantified by sandwich ELISA according to the manufacturer's instructions (eBioscience). Optical density was read at 450 nm using a microplate spectrophotometer .IL-1Swiss albino mice were used for in vitro experiments. Thus, peritoneal exudate was elicited in mice with an i.p. injection of 4% thioglycolate (Sigma-Aldrich). Four days after thioglycolate injection, animals were euthanized by cervical displacement and peritoneal cavity was washed with 5 mL of complete RPMI-1640 medium (Gibco) , supplemented with 10% fetal bovine serum (FBS) (Gibco). Cell suspension obtained from peritoneal lavage was centrifuged at 350g for 5 minutes (4°C). The supernatant was discarded, and the pellet was resuspended in 2 mL of complete RPMI medium. Viable cells were counted with a Neubauer chamber (Hemocytometer L. Optik ATC-111020) using a Trypan Blue solution (Merck). Macrophages were then enriched by adherence to plastic. For that, viable peritoneal cells were seeded in 96-well plates at a concentration of 4 × 105 cells/well in a final volume of 200 μL and incubated overnight (18 h) in an atmosphere of 5% CO2 at 37°C. Then, nonadherent cells were removed by aspiration. Remaining cells were stimulated with zymosan (0.2 mg/mL) and treated with different marinobufagenin concentrations , the same used in ouabain experiments [β, IL-6, and TNF-α) by ELISA, as previously described.Peritoneal macrophages of eriments . After 2μL of RPMI medium containing 10% of MTT solution (5 mg/mL) was added to each well and cells were maintained in an atmosphere of 5% CO2 at 37°C. After 4 h of incubation, the MTT-containing medium was removed, and the precipitate was solubilized in the DMSO solution (100 μL). Optical density was read at 570 nm using a microplate spectrophotometer .Cell viability was estimated by MTT  assay. For that, 100 6/well) and treated with marinobufagenin (10 nM) in the absence or presence of zymosan (0.2 mg/mL) for 24 h. After this period, cells were collected and placed in a 96-well plate (U bottom). Then, cells were blocked to prevent nonspecific binding using anti-CD16/32 and labeled separately with antibodies anti-TLR2 (PE) and anti-CD69 (PE), according to the manufacturer's instructions (BD Biosciences). Finally, cells were suspended in PBS and evaluated in a flow cytometer—FACSCanto II (Becton & Dickinson).Peritoneal macrophages isolated as described in The same culture conditions were performed for P-p38 MAPK evaluation, as described in Briefly, peritoneal macrophages were visualized using size (FSC), and granularity (SSC) parameters and a gate were performed in the cell population. Macrophages were analyzed by fluorescence intensity quantification corresponding to antibody labeling. These data were evaluated using FlowJo software version 10.+/K+-ATPase α2 and α3 isoforms, in rodents) and kidneys  were performed as described by Müller et al. [g for 60 min in an Optima XE-90 ultracentrifuge (Beckman Coulter). The pellet was resuspended in the same buffer without PMSF and stored at -80°C. Protein concentration was determined by the Lowry protein assay method. According to Noël et al. [2, 3 mM ATPNa2, 1 mM EGTA, and 20 mM maleic acid/Tris (pH 7.4), brain and kidney preparations were incubated at 37°C with increasing concentrations of marinobufagenin or ouabain. After 2 h, the reaction was stopped by the Fiske & Subbarow solution to measure the Pi released from ATP hydrolysis by a colorimetric method.Crude homogenates of naive mouse brain  was estimated. Data were expressed as mean ± SEM and analyzed by GraphPad Prism 7.0 software. Results were submitted to one-way analysis of variance (ANOVA) followed by Tukey's test. Results were considered statistically significant when p < 0.05.Inhibition curves were fitted by nonlinear regression analysis  assuming a sigmoidal concentration-response curve model of one site (kidney/ouabain and brain/marinobufagenin) or two sites (brain/ouabain), and the mean inhibitory concentration  compared to the saline group. Marinobufagenin pretreatment in zymosan-challenged animals induced a reduction in IL-1β (62%) and IL-6 (48%) but not TNF-α levels when compared to the zymosan group. In DMSO and marinobufagenin groups , cytokine levels remained similar to the saline group  and mostly α2 and α3 (brain preparation) isoforms are depicted in α2/α3-sensitive isoforms (IC50 = 55 ± 9 nM) and a minor one which reflects the α1-resistant isoform (IC50 = 25 ± 12 μM). The latter was similar to the potency estimated for the mouse kidney, known to harbor only the α1-resistant isoform (IC50 = 53 ± 5 μM). In contrast, just a single sigmoidal curve was observed for the mouse brain when marinobufagenin was employed, reaching a maximal inhibitory effect at 70% (IC50 = 3.8 ± 0.9 μM), which reflects a 70-fold lower potency of marinobufagenin to inhibit α2/α3 isoforms and failure to inhibit α1 (α1 isoform could not be inhibited at the highest concentration of marinobufagenin (1 mM).Inhibition curves representing the effect of increasing concentrations of marinobufagenin  on mouse Nahibit α1 . Consistin vivo analysis, the effect of marinobufagenin on proinflammatory cytokines was also evaluated in vitro. Zymosan-stimulated peritoneal macrophages showed increased proinflammatory cytokine levels  compared to the control group. Marinobufagenin treatment at the lowest concentration (10 nM) decreased IL-1β (45%), IL-6 (17%), and TNF-α (20%) levels when compared to the zymosan group. However, other concentrations did not alter the level of these cytokines. In the DMSO (data not shown) and marinobufagenin groups (unstimulated cells), cytokine levels remained similar to the control group  when compared to the zymosan group Figures . Also, zin vivo) and cultured peritoneal macrophages (in vitro).Several works have demonstrated that cardiotonic steroids have a role in the immune system acting as an anti-inflammatory substance –13. MariLeishmania amazonensis [Zymosan-induced peritonitis represents an acute inflammation model characterized by increased vascular permeability and production of inflammatory mediators, leading to migration of leukocytes, mainly polymorphonuclear cells (neutrophils) in the peritoneum for the first four hours . In accozonensis  and alsozonensis . This abzonensis . Besideszonensis .Peritoneal macrophages (mononuclear cells) are resident in the peritoneum, and their activation leads to the release of several mediators and results in increased vascular permeability and leukocyte extravasation . Zymosanβ, IL-6, and TNF-α in zymosan-induced peritonitis [β and IL-6 levels present in peritoneal exudate without changing TNF-α levels. In this same model, Leite et al. [β and TNF-α but not IL-6 levels. Differences of the affected cytokines may be associated with the pharmacological profile of these steroids. We have recently shown that structurally related bufadienolides generate distinct cellular effects probably by a particular selection of intracellular signaling pathways, which is called functional selectivity [in vivo.It has been well described that leukocyte recruitment and inflammation maintenance involve proinflammatory cytokines such as IL-1itonitis . Our date et al.  describeectivity . At lastμM marinobufagenin for 24 h displayed no cytotoxicity. This is compatible with our data using ouabain, which did not alter macrophage viability at least up to 100 nM [+/K+-ATPase inhibition [+/K+-ATPase α1 isoform [+/K+-ATPase α1 isoform, and the inhibition of the other isoforms, α2 and α3, only begins at around 1 μM (IC50 = 3.8 μM). Marinobufagenin has been considered a unique cardiotonic steroid regarding its selectivity to the ouabain-resistant rodent α1 isoform; namely, in contrast to any other bufadienolide or cardenolide, it was shown to be more potent to inhibit the rat Na+/K+-ATPase α1 isoform than α2/α3 isoforms [+/K+-ATPase α1 isoform ligand [+/K+-ATPase α isoforms similar to all known cardiotonic steroids (α2/α3 > α1) and is a poor α1 ligand. The reason of these discrepancies is unknown and is now being better investigated. Interestingly, the lowest marinobufagenin concentration (10 nM) was the single one that partly hampered zymosan-induced peritoneal macrophage IL-1β, IL-6, and TNF-α increased levels in vitro. Besides Na+/K+-ATPase inhibition, cardiotonic steroids may modulate intracellular signaling cascades by protein-protein interaction mediated by Na+/K+-ATPase but independent on changes in ionic gradients [α, IL-1β, and IL-6. In addition to its role as a cell activation marker, CD69 is also involved in inflammatory signaling pathways that result in the release of inflammatory mediators, such as cytokines [+/K+-ATPase leads to different conformational changes in this enzyme [+/K+-ATPase may trigger different cellular signaling pathways by functional selectivity [in vitro.Incubation of cultured peritoneal macrophages with 10 nM-10 o 100 nM . Micromohibition , 37. Rod isoform . This is isoform . Additio isoform , 40 thatisoforms , and it m ligand . Neverthm ligand , 43 and m ligand  have fouradients . Namely,radients , 47. MAPradients , and proytokines , 50. Ourytokines , 51. Theytokines . Furthers enzyme . Thus, iectivity . Therefoin vivo and in vitro. This study contributes to clarifying the physiological functions of cardiotonic steroids. Finally, more studies are needed to better understand the mechanism of action of marinobufagenin.In conclusion, our findings demonstrate the role of marinobufagenin in the immune system, evidencing its anti-inflammatory effect


Controls were exposed to curcumin or light alone or remained untreated. Curcumin plus light, but not curcumin or light exposure alone altered growth, proliferation, and apoptosis of all three RCC tumor cell lines. Cells were arrested in the G0/G1 phase of the cell cycle. Phosphorylated (p) CDK1 and pCDK2, along with their counter-receptors Cyclin B and A decreased, whereas p27 increased. Akt-mTOR-signaling was suppressed, the pro-apoptotic protein Bcl-2 became elevated, and the anti-apoptotic protein Bax diminished. H3 acetylation was elevated when cells were treated with curcumin plus light, pointing to an epigenetic mechanism. The present findings substantiate the potential of combining low curcumin concentrations and light as a new therapeutic concept to increase the efficacy of curcumin in RCC.The anti-cancer properties of curcumin in vitro have been documented. However, its clinical use is limited due to rapid metabolization. Since irradiation of curcumin has been found to increase its anti-cancer effect on several tumor types, this investigation was designed to determine whether irradiation with visible light may enhance the anti-tumor effects of low-dosed curcumin on renal cell carcinoma (RCC) cell growth and proliferation. A498, Caki1, and KTCTL-26 cells were incubated with curcumin (0.1–0.4 µg/mL) and irradiated with 1.65 J/cm Cancer is the second most common cause of death in Europe. The incidence of renal cell carcinoma (RCC) is, compared to other cancer types, relatively rare, but both incidence and mortality are steadily increasing at a rate of approximately 2% to 3% per decade. Already, approximately 3% of all adult cancer patients suffer from malignant kidney tumors ,2. RCC iCurcuma longa, commonly known as tumeric. Apart from its use as a food additive and spice, anti-inflammatory and anti-oxidative qualities have been attributed to curcumin. It promotes the function of the immune system and acts as an antioxidant to capture free radicals, protecting living cells [These unsatisfactory therapeutic options have opened patients’ minds to complementary and alternative medicine (CAM) approaches to actively contribute to treatment, coupled with the hope of prolonging survival or even curing their disease. Up to 50% of cancer patients worldwide are applying CAM . Howeverng cells ,8,9. Recng cells . Curcuming cells . Thus, cng cells .Low bioavailability, however, remains a serious problem with curcumin treatment. Due to poor water solubility, low absorption, and fast metabolization and clearance, a transfer to clinical use is hampered ,14. HumaBernd et al. recently discovered that irradiation of tumor cells with visible light dramatically enhances the antitumor properties of curcumin . ApplicaCurcumin (0.1–0.4 µg/mL) plus light (5500 lx) or curcumin alone did not impair the cell membrane integrity of all cell lines evaluated  and 32.06 ± 5.68% (0.2 µg/mL curcumin plus light).Low dosed curcumin combined with visible light induced a cell cycle arrest at the G0/G1-phase . G0/G1-pThe expression of cell cycle regulating proteins is depicted in Akt and pAkt were reduced in the presence of curcumin plus light. The mTOR complexes Raptor and Rictor were also diminished by the treatment regimen in all cell lines, compared to controls.The amount of deacetylated histones was not significantly influenced by light or curcumin alone compared to the untreated control in all three cell lines. However, 0.2 µg/mL curcumin in combination with visible light induced a significant overexpression of aH3 in Caki1 cells, pointing to an epigenetic mechanism. This finding was confirmed by the histone deacetylase (HDAC) expression assay pointing to a reduction of HDAC of about 25% (KTCTL-26), 35% (Caki1), and nearly 50% (A498) .Since curcumin strongly modified CDK1 and 2 as well as Cyclin A and B in all tumor cell lines, the physiologic relevance of these proteins was evaluated by siRNA knock-down. Down-regulation of CDK1 and Cyclin B or CDK2 and Cyclin A A correla50 of 8 µM curcumin to block the growth of gastric cancer cell lines [50: 30–80 µM) [50: 30 µM) [50: 20 µM) [Light exposure greatly increased curcumin’s anti-tumor properties. As low as 0.1 µg/mL (Caki1 and KTCTL-26) or 0.2 µg/mL curcumin (A498) significantly reduced tumor cell growth following irradiation, while 0.1 or 0.2 µg/mL curcumin alone or with light alone did not lead to growth-blocking effects in any of these cell lines. To achieve similar effects on RCC cell growth without irradiation, Caki1 cells required curcumin at a concentration of 2.9 µg/mL (8 µM) (data not shown). This coincides with data from others reporting an ICll lines . Even hi0–80 µM) , neurobl: 30 µM) , or blad: 20 µM) .The photodynamic effect is not restricted to RCC cells. A study, using skin keratinocytes, has shown that 0.2 µg/mL curcumin causes a significant proliferation inhibition when combined with visible light. This concentration was more than one magnitude lower than the lowest curcumin (without light) concentration providing pharmacological effects . CurcumiReduction of cell number was not caused by toxic effects as demonstrated by the LDH assay. However, Bcl-2 decreased in the RCC cell lines treated with 0.2 µg/mL curcumin–light, which may indicate apoptotic events. Indeed, the number of apoptotic cells evaluated after 72 h significantly increased when tumor cells were subjected to curcumin plus light. Dujic et al. have reported a strong increase of apoptotic nuclei in the keratinocyte cell line, HaCaT, 24 h after treatment with light and 1 µg/mL curcumin . On the The analysis of cell cycle progression revealed distinct modulations caused by curcumin. The number of cells in the S-phase  and the G2/M-phase (A498 and KTCTL-26) decreased, whereas the number of G0/G1-phase cells increased. A similar effect has also been observed on breast , prostatBased on an investigation by Zhang et al., 10 µM curcumin blocked bladder cancer cells at the S-phase, whereas 15 µM led to a G2/M-phase arrest . The mesAlong with the G0/G1 block, the expression of CDK1 and CDK2 with their respective counterparts, Cyclin B and Cyclin A, as well as the Akt-mTOR signaling pathway, was significantly diminished in all cell lines in the presence of 0.2 µg/mL curcumin–light . Information about the influence of curcumin on the CDK-Cyclin-axis in RCC is sparse. Diminished expression of Cyclin B has been seen in the RCC cell line RCC-949 following curcumin exposure . TreatmeFrom a clinical viewpoint, loss of Akt and the mTOR-members, Rictor and Raptor, is of interest. The Akt/mTOR-pathway plays a crucial role in the pathogenesis of RCC, and various drugs targeting this signaling cascade have already been established and approved . UnfortuThe influence of curcumin on HDAC is difficult to interpret. There is little doubt that curcumin targets HDAC and that HDAC-suppression along with histone acetylation may contribute to the anti-cancer effects of curcumin . Based oThe exact mechanism underlying the advantageous effect of light is not totally understood. Speculatively, light-dependent energy transfer during curcumin–protein interactions may enhance the influence of curcumin on protein function and cell regulation . It has Ongoing studies must now deal with the technical aspect of curcumin–light application in renal cancer. Introducing an optical fiber into RCC tumors of a mouse model with subsequent laser illumination of the vascular-acting photosensitizer WST11, either at a single wavelength (750 nm) or multispectrally (700 to 800 nm), induced necrosis in the RCC tissue, as evidenced by histological analysis. . Baran eBased on pediatric epithelial liver tumor cell lines, evidence has been provided that irradiation with blue light (480 nm) amplifies the cytotoxic effects of low dosed curcumin. The authors concluded that combining low curcumin concentrations with light irradiation may compensate for low bioavailability and rapid degradation of curcumin in vivo . From a Curcumin is not the only compound shown to have enhanced effects when combined with an energy source. Hypericin’s anti-tumor effects have been shown to be enhanced when human RCC cells are exposed to radiation or light in vitro, pointing to a clinical relevance of radiotherapy and intraoperative photodynamic therapy . Ongoing2 incubator. Subcultures from passages 5–30 were selected for experimental use.Renal carcinoma Caki1 and KTCTL-26 cell lines were purchased from LGC Promochem . The A498 cells were derived from Cell Lines Service . Caki1 and KTCTL-26 cells were chosen since both lines are derived from a clear cell renal cell carcinoma, which is the most common renal carcinoma tumor type. A498 served as the “classical” RCC cell line used as a model of ccRCC as well . Both ce2; Waldmann UV 801AL, Villingen-Schwenningen, Germany). Curcumin was stored at −20 °C and was diluted in cell culture medium to a final concentration of 0.1–1 µg/mL (0.27–2.7 µM). Cells were treated for 1 h with curcumin and subsequently irradiated with visible light for 5 min . After irradiation PBS was replaced by cell culture medium containing no curcumin. Control cell cultures were exposed to visible light without curcumin, received curcumin without light exposure, or were treated with PBS alone. Tumor cells were then subjected to the assays listed below. 4 cells/0.33 cm2) and treated with curcumin and light as aforementioned. The next day, cell-free supernatants were incubated with NAD+, which is reduced by lactate dehydrogenase to NADH/H+. Consecutively, NADH/H+ reduces the yellow tetrazolium salt to a red-colored formazan salt. The amount of red color is proportional to the number of lysed cells. For quantification, the absorbance of the reaction product was measured at 490 nm using an ELISA reader.Membrane integrity was quantified using a cytotoxicity detection kit  based on the release of lactate dehydrogenase (LDH) from damaged cells. Briefly, the cells were cultivated in 96-well plates (2 × 104 cells/0.33 cm2) and treated with curcumin and light as mentioned above. After 24 h, the cytosolic fraction was subjected to a sandwich enzyme-linked immunosorbent assay with the primary anti-histone antibody coated to the microtiter plate and the secondary anti-DNA antibody coupled to peroxidase. Optical density was measured at 530 nm by an ELISA reader.DNA fragmentation was chosen as an indicator of apoptosis. Quantification was performed with the Cell Death Detection ELISA  according to the manufacturer’s instructions. In brief, cells were cultured in 96-well plates (2 × 10Expression of Annexin V/propidium iodide (PI) was evaluated using the Annexin V-FITC Apoptosis Detection kit . Tumor cells were washed twice with PBS-buffer, and then incubated with 5 µL of Annexin V-FITC and 5 µL of PI in the dark for 15 min at room temperature. Cells were analyzed on a FACScalibur . The percentage of vital, necrotic, and apoptotic cells (early and late) in each quadrant was calculated using Cell-Quest software (BD Biosciences).4 cells/mL) were plated into 96-well tissue culture plates. After 24, 48, and 72 h, MTT (0.5 mg/mL) was added for an additional 4 h. The reaction was stopped by lysing the cells in a buffer containing 10% SDS in 0.01 M HCl. After incubating the plates overnight at 37 °C and 5% CO2, the absorbance at 570 nm was measured for each well using a microplate proliferation enzyme-linked immunosorbent assay (ELISA) reader. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as mean cell number. Cell growth was measured using the 3--2,5-diphenyltetrazolium bromide (MTT) dye reduction assay . Tumor cells . Tumor cells, were seeded into 96-well tissue culture plates, incubated with 20 µL BrdU-labelling solution per well for 8 h, and fixed and detected using anti-BrdU mAb according to the manufacturer’s instructions. Absorbance was measured at 450 nm after 24 h.Cell cycle analysis was carried out with sub confluent tumor cells after 24 h cultivation with or without 0.2 µg/mL curcumin. Tumor cell populations were stained with propidium iodide, using a Cycle TEST PLUS DNA Reagent Kit  and then subjected to flow cytometry with a FACScan flow cytometer (BD Biosciences). In total, 10,000 events were collected for each sample. Data acquisition was carried out using Cell-Quest software and cell cycle distribution was calculated using the ModFit software (BD Biosciences). The number of gated cells in the G1, G2/M, or S-phase is presented as %. Histone deacetylation (HDAC) activity of renal cancer cells was quantified using the Color De Lys assay  according to the manufacturer’s instructions. Cells were cultivated with curcumin and/or light as aforementioned. All substances were plated on a 96-well plate and the reaction was initiated by adding substrate and stopped by Color De Lys developer. Optical density was measured at a wavelength of 405 nm using an ELISA reader.To investigate the level of the cell cycle regulating proteins in the three cell lines, tumor cell lysates were applied to a 7–12% polyacrylamide gel (depending on the proteins) and electrophoresed for 90 min at 100 V. The protein was then transferred to nitrocellulose membranes . After blocking with nonfat dry milk for 1 h, the membranes were incubated overnight with monoclonal antibodies directed against the cell cycle proteins: CDK1/Cdc2 , pCDK1/Cdc2 ), CDK2 , Cyclin A , Cyclin B , Cyclin D1 , p27 , CDK4 , p19 , pCDK2 . The mechanistic target of rapamycin (mTOR) pathway was investigated using the following monoclonal antibodies: Raptor , Rictor , PKBα/Akt (IgG1 clone 55), anti phospho Akt . aH3 (Lys9), aH4  and Bax (B-9:sc-7480), Bcl-2 . HRP-conjugated goat anti-mouse IgG and HRP-conjugated goat anti-rabbit IgG  served as the secondary antibody. The membranes were briefly incubated with ECL detection reagent  to visualize the proteins and then analyzed by the Fusion FX7 system . β-Actin  served as the internal control.5 cells/2.3 mL of medium were transfected with small interfering RNA (siRNA) directed against CDK1 , CDK2 , Cyclin A  or Cyclin B   or with an siRNA/transfection reagent  at a ratio of 1:6. Non-treated cells and cells treated with 5 nM control siRNA  served as controls. Subsequently, tumor cell growth was evaluated and Western blotting was done as indicated above.To determine whether CDK1, CDK2, Cyclin A, and Cyclin B impacted tumor cell growth in A498, Caki1, and KTCTL-26 cell lines, cells were transfected with the respective small interfering RNA (siRNA). Per batch, 3 × 10U test. Values are expressed as means ± S.D. Differences were considered statistically significant at a p value less than 0.05. All experiments were performed three to six times. Statistical significance was calculated with the Wilcoxon–Mann-Whitney


Likewise, the major fatty acids were palmitic (23.52%–38.42%), linoleic (13.09%–18.97%), and linolenic (26.32%–51.80%) acids, which comprised the highest compositional ratio (more than 50%) of polyunsaturated fatty acids (PUFAs) in most cultivars. Among the antioxidants, total phenol exhibited the most significant positive correlation (r = 0.698 **) with antioxidant activity, followed by vitamin C (r = 0.522 **) and total flavonoid (r = 0.494 **), indicating their significant contributions to total antioxidant activity.This research was conducted to evaluate glucosinolate profiles, vitamin C, total phenol, total flavonoid, and free sugar  content, fatty acid composition, and antioxidant activity in floret and leaf of six cauliflower and broccoli cultivars. The level of chemical constituents as well as antioxidants significantly varied among crop types, cultivars, and their different parts, in that phytochemicals such as glucosinolate were statistically higher in florets compared with leaves in both broccoli and cauliflower cultivars. In contrast, total flavonoid and free sugar were found at higher levels in the leaf parts. The Asia purple cultivar exhibited statistically higher vitamin C (649.7 mg·100 g Brassica vegetables such as cauliflower and broccoli are popular and are among the most consumed vegetables in the world. Many epidemiological studies have indicated that a diet rich in these vegetables is associated with reduced risk of a several type of cancers, type 2 diabetes, and cardiovascular diseases [Brassicas are known to possess antioxidant activity [Brassica crops, a large group of sulfur-containing compounds possessing anticancer activity that are known to be responsible for the pungent flavor of the plants [Brassica crops [Brassica crops are responsible for masking the bitter taste of glucosinolates, promoting human consumption [diseases ,2,3. Addactivity ,5. Such activity ,7,8,9,10e plants ,12. Vitaca crops  that proca crops . Likewisca crops , often cca crops . Likewissumption . Additiosumption ,19. Brassica vegetables varies significantly depending on the genotypes of cultivars, the specific plant tissue, fertilization, growing season, and several other environmental factors [Brassica crops such as cauliflower and broccoli [The content of these compounds in  factors ,21,22,23 factors ,24. Therbroccoli ,9,15,25.broccoli . It is t−1 and from 298.6 to 454.3 mg·100 g−1, respectively. The cultivar AMaGi exhibited higher vitamin C content in floret and leaf tissues  and leaf (533.6 mg·GAE 100 g−1) parts  between florets and leaves in all the cultivars. Similar to vitamin C, the highest flavonoid content was present in AMaGi in both floret (317.1 mg·CE 100 g−1) and leaf (816.8 mg·CE 100 g−1). The presence of higher total flavonoid content in leaf suggests higher nutritional value of leaves, as flavonoids possess strong antioxidant activity and inhibit oxidative stress [Our results showed variation in antioxidant  contents with crop type, cultivar, and plant part A–D. In b tissues A than di parts B. Regard Hamauzu  and with[ et al. : 481–157−1), followed by Bridal (410.4 mg·100 g−1) and Asia white (396.7 mg·100 g−1)  and Bridal (471.9 mg·100 g−1). Likewise, total phenol content was highest in Asia purple (1345.2 mg·GAE 100 g−1) and lowest in Bridal in the floret part (−1) exhibited statistically higher total phenol content in leaves compared with florets. Similar to the broccoli cultivars, total flavonoid content in cauliflower cultivars was also statistically higher in leaf parts compared with floret parts, and Asia white and Bridal exhibited an approximately fivefold higher flavonoid content in leaf tissue compared with their respective floret parts. The presence of higher total flavonoid content in leaf parts in both broccoli and cauliflower crops suggest that its leaf can be used as an alternative source of total flavonoids, as flavonoids are responsible for various beneficial health effects [The vitamin C content in floret tissues of the three cauliflower cultivars was highest in Asia purple (649.7 mg·100 g100 g−1) A. This r  showed the highest total glucosinolate concentration compared with AMaGi (8.09 µmol·g−1) and Cheonjae (5.59 µmol·g−1)  . Furthermol·g−1) . The glu, followed by Bridal (7.41 µmol·g−1) and Asia white (1.97 µmol·g−1) , which revealed relatively higher proportions of glucoraphanin and gluconapin. However, the levels of gluconapin and glucoraphanin observed in this study appear to be higher than those in previous reports [Among cauliflower cultivars, Asia purple exhibited the highest total glucosinolate concentration (8.80 µmol·gmol·g−1) . With th . Compared with previous reports by Rosa et al. [−1 of total free sugars in the florets of various broccoli cultivars in different seasons, we observed comparatively higher total free sugar in all cultivars. The highest free sugar content was observed in Cheonjae (237.2 mg·g−1), followed by Baeridom (205.8 mg·g−1) and AMaGi (153.3 mg·g−1). In the case of individual free sugars, Cheonjae exhibited statistically higher glucose and fructose content compared with AMaGi and Baeridom  than in leaves (120.5 mg·g−1) in the Asia white cultivar, whereas Asia purple and Bridal exhibited statistically similar glucose contents in their plant parts. In contrast, all cultivars displayed statistically higher fructose content in florets compared with leaves. Similarly, sucrose content exhibited cultivar- and plant-part-dependent variation. Among the three cultivars and their parts, florets of Asia white showed higher glucose (161.8 mg·g−1) and fructose (127.0 mg·g−1) content but lower sucrose content (10.5 mg·g−1) compared with the other cultivars. Total free sugar content in the florets was statistically higher in Bridal (318.6 mg·g−1) than in Asia white (299.3 mg·g−1) or Asia purple (269.6 mg·g−1), and in all these cultivars, florets exhibited statistically higher total free sugar content compared with leaves  between plant parts. Of the two crops, cauliflower exhibited comparatively higher free sugar content than broccoli. All of these results suggest that the free sugar content in Brassica crops is dependent not only on the genotype of the crop, but also on the specific plant tissue.In cauliflower cultivars, glucose was present in a statistically higher quantity in florets  and polyunsaturated fatty acids (PUFA) ranged from 3.34% to 4.67% and from 47.90% to 65.89%, respectively. Compared with other cultivars, florets of AMaGi exhibited higher SFA (46.75%) and lower PUFA (47.92%). Similarly, in the case of cauliflower cultivars, 12 fatty acids were found, the major ones being palmitic (27.11%–38.42%), linoleic (13.09%–18.97%) and linolenic acid (26.32%–41.03%), accounting for more than 80% of total fatty acid content  value was calculated following a linear regression analysis of the observed inhibition percentage versus concentration, where a lower IC50 value shows higher antioxidant activity. Antioxidant activity in broccoli cultivars varied significantly in florets, whereas no such variation was observed in leaves . In the case of cauliflower, only the Asia purple showed statistically higher antioxidant activity in its floret  compared with leaves , while other cultivars exhibited higher antioxidant activity in their leaf parts than in floret parts. This might be due to the higher content of total phenols, as phenolics are major contributors to total antioxidant activity [In our experiment, we measured antioxidant activity by evaluating the 2,2,-diphenyl-1-picrylhydracyl (DPPH) radical-scavenging activity of different concentrations of methanolic extracts from n leaves D. Both Aactivity .50, thus indicating the highest antioxidant activity , followed by the correlation with total phenol (r = 0.522 **) and total glucosinolates (r = 0.494 **). Likewise, we found a significant positive correlation between antioxidant activity and total phenols (r = 0.698 **), vitamin C (r = 0.632 **), and total flavonoid (r = 0.456 **). These results are consistent with previous reports that described the antioxidant capacity of various Brassica vegetables [ et al. [ et al. [To determine the contribution of antioxidants to antioxidant activity and phytonutrients, we performed a correlation analysis on the relationships among vitamin C, phenolics, flavonoids, total glucosinolates, and antioxidant activity . In our ls r = 0.98 **, vigetables ,30,37. Ils r = 0.98 **, vi1 hybrid Brassica cultivars, including three cauliflower cultivars  and three broccoli cultivars , were used in this study. All were grown in an experimental field of the National Institute of Horticultural and Herbal Science (NIHHS), Rural Development Administration (RDA), Suwon, South Korea. For both the crop types, seedlings were transplanted to the cultivation field 33 days after sowing. Seedlings were planted in rows with 50 cm between plants and 60 cm in rows. The sowing date was 5 March 2011, and the plant materials were harvested at commercial maturity stage, which occurred between 65 and 75 days after sowing. During the field experiments, water, fertilizers and pesticides were applied according to standard cultural practices of NIHHS and RDA. Ten broccoli/cauliflower heads were used for each sample. After harvesting, florets and leaf parts were separated, cut into small pieces, and then freeze dried. The samples were ground into fine powder and then stored at −20 °C until used for the analysis of glucosinolates, vitamin C, total phenol, total flavonoid and free sugar content, fatty acid composition, and antioxidant activity.Six commercial FHelix pomatia, vitamin C, glucose, sucrose, fructose, gallic acid, DPPH , and catechin hydrate were purchased from Sigma-Aldrich . A standard for fatty acid methyl esters (FAME) was obtained from Supelco . Chemicals such as sodium hydroxide, sodium carbonate, sodium nitrite, aluminum chloride, Folin−Ciocalteu reagent, and 2,2-dimethoxypropane were purchased from Sigma-Aldrich . Benzene, n-heptanes, and sulfuric acid were acquired from Daejung Chemicals . Other chemicals, including acetonitrile (HPLC grade), methanol (HPLC grade), and formic acid (ACS reagent), were purchased from J. T. Baker .The glucosinolate standards, glucoiberin, progoitrin, glucoraphanin, sinigrin, gluconapin, glucobrassicanapin, glucoerucin, glucobrassicin, and gluconasturtiin, were purchased from Cfm Oskar Co. . Authentic standards for DEAE (Diethyl aminoethyl) Sephadex-A25, aryl sulfatase  from  et al. [Brassica samples (0.5 g) were extracted with 5% metaphosphoric acid solution. Then, after centrifugation and filtration (with a 0.20 μm syringe filter), the aliquot was analyzed using an H-Class UPLC (ultra performance liquid chromatography)  equipped with an Acquity UPLC® HSS T3  column and PDA (photo diode array) detector (Waters) at 254 nm in wave length. An isocratic mobile phase composed of aqueous 0.1% (v/v) formic acid at a flow rate of 0.3 mL·min−1 was used for separation of the vitamin C peak. An authentic ascorbic acid standard at various concentrations  was used for the identification and quantification of the peak and vitamin C content was expressed as milligrams per 100 gram (mg·100 g−1) of dry weight.Vitamin C content was analyzed according to the methods described by Spinola et al.  with modTM microplate spectrophotometer  at 640 nm. Gallic acid standards at different concentrations  were used for the calibration. Total phenol content was expressed as milligrams of gallic acid equivalent per 100 g (mg·GAE 100 g−1) dry weight. Total phenolic content was estimated by the Folin–Ciocalteu colorimetric method based on the procedure previously described by Singleton and Rossi  using ga et al. [3 was added to the solution. At 6 min, 2 mL of 1 M NaOH was added, and the sample was brought to final volume 10 mL using distilled water. The solution was mixed carefully, and the absorbance was measured at 510 nm using an EONTM microplate spectrophotometer . Catechin hydrates at different concentrations  were used as the standard compound. Total flavonoid was expressed as milligrams of catechin hydrate equivalent per 100 g (mg·CE 100 g−1) of dry weight.The vegetable extracts obtained for total phenol analysis were also used for total flavonoid analysis using a colorimetric method described by Zhishen et al. . Only 1  et al. [. The column was capped and left for 24 h at room temperature, and the desulfoglucosinolates were eluted with 1.5 mL distilled water, filtered through a 0.2-µm syringe filter, injected into H-Class UPLC (Waters) using an Acquity UPLC® BEH-C18 column , and measured at 229 nm with a PDA detector. Solvent A (100% distilled water) and solvent B (20% acetonitrile) were used for the elution of compounds at the flow rate of 0.2 mL·min−1. The gradient programs were as follows: a linear step from 1% to 99% of solvent B within 6 min, followed by constant conditions for up to 10 min and then a quick dropdown to 1% of solvent B at 12 min and isocratic conditions with 1% of solvent B up to 15 min. Authentic standards of glucosinolates were used for the identification and quantification of the peaks. Total and individual glucosinolates were expressed as micromole per gram (µmol·g−1) of dry weight.Sample preparation and glucosinolate analysis were performed according to methods described by Lee et al. . Briefly et al. [Brassica samples (1.0 g) were extracted with distilled water by shaking for 20 min in a water bath at 80 °C, centrifuged at 3500 rpm for 5 min, filtered through a 0.45 µm syringe filter, and analysed using an HPLC system (Waters) with a Kromasil® 100-NH2 column  and RI (refractive index) detector (Waters) with acetonitrile/distilled water  for the mobile phase at a flow rate of 1.5 mL·min−1. Authentic standards of free sugars at different concentrations were used for the identification and quantification of the peaks. Total as well individual free sugars were expressed as milligram per gram (mg·g−1) of dry weight.Free sugars  were analyzed according to Bhandari et al.  with som et al. [2SO4 = 39:20:5:2) and 400 µL of heptane. After vigorous mixing and heating for 2 h at 80 °C in a water bath, samples were cooled at room temperature, and then a heptane layer was collected and injected into a gas chromatograph  equipped with a flame ionization detector and a capillary column: CP SIL 88 CB FAME . The temperature was set 210 °C for both the injector and FID (flame ionization detection) detector . The injection volume was 1 µL with a split ratio of 1:50 on a constant column flow (1 mL·min−1) of helium gas. The oven temperature was initially maintained at 100 °C for 5 min, and FID was increased up to 160 °C at a rate of 5 °C min−1, maintained for 5 min, and then increased again at a rate of 4 °C min−1 up to 180 °C. All the fatty acids were expressed as relative percentage (%) of dry weight.Samples for fatty acid composition analyses were prepared and analyzed according to Bhandari et al. . Briefly et al. [−1) and 100 μL of methanol in 96-well plates. After 30 min, the absorbance of the resultant solution was measured using an EONTM microplate spectrophotometer  at 517 nm wavelength against a blank, which was 80% methanol without DPPH. Similarly, the absorbance of samples was measured after mixing 100 μL samples with 100 μL of 80% methanol. Free radical-scavenging activity (%) was calculated using the following equation:The antioxidant activity of vegetable extracts taken from different plant parts was determined using the 2,2,-diphenyl-1-picrylhydracyl (DPPH) radical scavenging method described in Koleva et al. , with mo50 value, which is the concentration required to obtain 50% antioxidant capacity, was calculated and used to compare the antioxidant activity of sample extracts.The IC® 9.2 software .For each sample, three independent replicate measurements were used in all statistical analyses. To determine differences among crop types, cultivars, and plant parts, one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test (DMRT) was performed at a significance level of 0.05 using SASBrassica are affected in different ways depending on the nature of the compounds. Furthermore, the major fatty acids were palmitic, linoleic, and linolenic acids with a high compositional ratio of unsaturated fatty acids, thereby signifying the nutritional value of these fatty acids that are responsible for the promotion of human health in a number of different ways. In conclusion, the results of our study showed that the levels of chemical constituents and antioxidant activity are significantly dependent on crop type, cultivar, and plant part. Most of the compounds as well as the antioxidant activity were higher in plant florets; however, phytonutrients, such as flavonoids in both broccoli and cauliflower and free sugar in broccoli cultivars, revealed statistically higher values in leaves, indicating that leaves are a good source of those phytochemicals. Similarly, the highest levels of vitamin C, total phenol, total flavonoids, free sugar, and antioxidant activity were observed in the cauliflower cultivars, whereas the highest total glucosinolate was present in the broccoli cultivars; however, no specific cultivar had significantly higher quantities of all the phytochemicals. All of these results suggest that phytonutrients in


Furthermore, this aptasensor showed high selectivity toward OTA against other structural analogs and other mycotoxins, and was successfully applied in Astragalus membranaceus samples. The presented aptasensor for OTA detection could be a promising tool for the field monitoring of food and TCM.With the widespread contamination of ochratoxin A (OTA), it is of significant importance for detecting OTA in foods and traditional Chinese medicine (TCM). In this study, a novel label-free fluorescent aptasensor utilizing the interaction between OTA-triggered antiparallel G-quadruplex and (N-methyl-4-pyridy) porphyrin (TMPyP) for the rapid and sensitive determination of OTA was established. The fluorescence of CdTe quantum dots (QDs) could be quenched by TMPyP. In the presence of analyte (OTA), the aptamer could recognize OTA and transform from a random coil to the antiparallel G-quadruplex. The interaction between G-quadruplex and TMPyP could release CdTe QDs from TMPyP, and thus recover the fluorescence of CdTe QDs. Under optimized conditions, the detection limit of the designed aptasensor was 0.16 ng mL Aspergillus ochraceus and Penicillium verrucosum  was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. . OTA, OTB, OTC, ZEA, and HT-2 were purchased from Romer Labs Inc.  and dissolved in acetonitrile to prepare 10 μg mL−1 of stock solutions. The stock solutions were stored in the dark at −20 °C. The OTA aptamers (5′-GAT CGG GTG TGG GTG GCG TAA AGG GAG CAT CGG ACA-3′) [2, and 20 mM of MgCl2) was prepared for the binding reaction between OTA and the aptamer. Astragalus membranaceus samples were purchased from Jiangxi Yaodutang Chinese Herbal Pieces Co., Ltd. .Sodium tellurite (Na ACA-3′)  were synCD spectra were measured on a JASCO J-1500 spectropolarimeter  in 10 mM of binding buffer. The fluorescence spectra were acquired by a Hitachi F-2700 fluorescence spectrophotometer . The experimental parameters were set as follows: emission wavelength (λem), 490 to 630 nm; excitation wavelength (λex), 360 nm; emission slit, 10 nm; excitation slit, 10 nm. The deionized water used throughout the study was prepared by a Milli-Q Reference water purification system .−1, 0.125 μmol L−1, 0.15 μmol L−1, 0.175 μmol L−1, and 0.2 μmol L−1. The fluorescence of CdTe QDs/TMPyP was performed as follows. Different concentrations of TMPyP were mixed with 80 μL of CdTe QDs (4.2 × 10−9 mol L−1) at room temperature. The fluorescence of the CdTe QDs/TMPyP/OTA aptamer was conducted as follows: 50 μL of OTA-aptamer (20 ng mL−1), different concentrations of TMPyP, and 80 μL of CdTe QDs (4.2 × 10−9 mol L−1). The incubation times were 1 min, 3 min, 5 min, 8 min, and 10 min. . The emission spectrum of the sample was measured, and each sample was measured three times.In order to obtain the best experimental results, the concentration of TMPyP and incubation time were both optimized. The concentration range of TMPyP was 0.1 μmol L−1) were placed in an optical chamber , which was deoxygenated with dry purified nitrogen (99.99%) prior to analysis and also insulated by a nitrogen atmosphere. The CD spectrum was run in triplicate with a 200-nm/min scan speed, 1-nm bandwidth, and 1-s time constant. The data were measured in a range of 230 to 340 nm at 0.1-nm intervals. The buffer solution was used as a blank to correct the background.The aptamer conformation was measured by a CD spectropolarimeter. The DNA aptamer (5 μM) and DNA aptamer (5 μM) with OTA (5 μg mL−7 mol L−1), 80 μL of CdTe QDs (4.2 × 10−9 mol L−1), and 50 μL of OTA aptamer were made up to 1 mL. After equilibrating at room temperature for 5 min, the fluorescence emission spectra were measured at a wavelength range from 490 to 630 nm.For the quantitative determination of OTA, different concentrations of OTA were mixed with 250-nM aptamers in binding buffer and heated at 95 °C for 5 min, and were then cooled down to room temperature for upcoming experiments. After that, samples containing 175 μL of TMPyP (1.75 × 10−1), HT-2 (25 ng mL−1), ZEA (25 ng mL−1), OTB (25 ng mL−1), and OTC (25 ng mL−1) were separately added into 250 nM of aptamer in binding buffer, and then heated at 95 °C for 5 min before being gradually cooled down to room temperature. Following this, samples containing 175 μL of TMPyP (1.75 × 10−7 mol L−1), 80 μL of CdTe QDs (4.2 × 10−9 mol L−1), and 50 μL of mycotoxins aptamer were made up to 1 mL. Multiple mycotoxins, including 175 μL of TMPyP, 80 μL of CdTe QDs, and 50 μL of OTA-aptamer, as well as other mycotoxins, were made up to 1 mL. After 5 min, the fluorescence intensity was measured.To verify the specificity of the aptasensor, HT-2, ZEA, OTB, and OTC were used as controls. Firstly, OTA . The samples were ultrasonicated for 10 min and then centrifuged at 4000 rpm for 10 min. The supernatant underwent a fivefold dilution with deionized water to minimize the matrix effect [To verify the feasibility of this aptasensor, x effect . Additio