COI1) gene is the core member of jasmonate signaling pathway, which is closely related to plant biotic and abiotic resistance. However, there have been no reports on COI1 in sugarcane (Sacharum spp.). Hence, systematically investigating the characteristics of the COI1 multigene family in sugarcane can provide a means to study and manipulate the jasmonic acid signaling pathway.The coronatine insensitive 1  analysis demonstrated that these three ShCOI1 genes had divergent expression profiles in response to salicylic acid, abscisic acid, polyethylene glycol, cold, and S. scitamineum.A total of 156 COI1 proteins were obtained from the genomes of 19 land plants, while none were obtained from five algae species. A phylogenetic tree demonstrated that these COI1 proteins were classified into four groups, while 31 proteins of SsCOI1 from COI1 genes may act in sugarcane growth, development, and response to various stresses via different regulatory mechanisms, which laying a foundation for the functional identification of the sugarcane COI1 gene.These results suggest that The online version contains supplementary material available at 10.1186/s12864-021-08255-0. COI1  E3 ubiquitin ligase, jasmonate ZIM-domain (JAZ) repressor proteins, and myelocytomatosis2 (MYC2) transcription factor, have been defined as the COI1/JAZs/MYC2 module [As signal molecules, jasmonics, including jasmonic acid (JA) and its cyclopentanous derivatives, play an important role in plant growth , develop2 module .COI1, making JAZs ubiquitinate and degrade via the 26S proteasome pathway, releasing DNA-binding transcription factors (such as MYC2) and inducing the expression of JA response genes [COI1 locus or its functionally deficient mutations can lead to the elimination or weakness of plant responses to JA [In recent years, the mechanistic details of the JA signal transduction pathway and its regulatory network have gradually been revealed –9. Resease genes . In addies to JA , 17. TheCOI1 is not only involved in plant developmental processes, such as leaf senescence [COI1 belongs to a multigene family. There are one F-box domain and 16 leucine-rich repeats (LRR) in COI1 protein [COI1 E3 ubiquitin ligase complex involved in the ubiquitin-dependent proteolytic pathway [COI1-mediated stabilization and 26S proteasome pathway-mediated degradation [Arabidopsis thaliana [Arabidopsis coi1 mutants were observed to be male-sterile [coi1–37) [Arabidopsis coi1–1 mutant plants were infertile and showed a stay-green phenotype under dark-induced senescence conditions, but those phenotypes could be rescued in mutants overexpressing 35S:OsCOI1a or 35S:OsCOI1b due to the fact that the JA signaling insensitivity of coi1–1 mutants was complemented. This finding suggests that coi1 plays a key role in leaf senescence and fertility [Pst DC3000. However, different mutations in the COI1 gene have distinct effects on COI1 function in regulating male fertility. GhCOI1 silencing in Gladiolus hybridus impaired inducible defense and increased susceptibility to the necrotrophic pathogenic fungus Alternaria brassicicola [COI1 gene during the processes of plant growth and development, as well as defense responses. However, there have been no reports on the COI1 gene in sugarcane (Sacharum spp.). Hence, a systematic investigation of the characteristics of the COI1 multigene family in sugarcane should provide an efficient basis for the study and manipulation of the JA signaling pathway.It has been reported that nescence , 19, seenescence , flowerinescence , male fenescence , anthocynescence , and roonescence , 23, butnescence , 24, 25. protein , 27. The pathway , and reg pathway , 29. As radation , 31. To -sterile , apical coi1–37) , susceptcoi1–37) , insensicoi1–37) , and laccoi1–37) . Arabidoertility , 34. In ertility , amino aSsCOI1, three ShCOI1, and seven SbCOI1 genes were identified from the genomes of the sugarcane-related wild species Saccharum spontaneum [-related cultivated species Saccharum spp. hybrid cultivar R570 [Sorghum bicolor [cis-acting elements, tissue-specific expression, and expression profiles of the COI1 gene family under methyl jasmonate (MeJA), cold, drought, and Sporisorium scitamineum stresses were analyzed [ShCOI1 genes  were isolated from Saccharum spp. hybrid cultivar ROC22 using a homologous cloning method. In addition, the real-time quantitative PCR (RT-qPCR) technique was used to analyze the gene expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6 under cold, drought, salicylic acid (SA), abscisic acid (ABA), and S. scitamineum stresses [COI1 gene family in sugarcane, understand their sequence characteristics and gene expression patterns, and thus provide candidate gene resources for sugarcane resistance molecular breeding.Sugarcane is an important sugar and biofuel crop in the world , 36. Howvar R570 , and the bicolor , respectanalyzed –42. Thirstresses . This stMedicago truncatula; seven in A. thaliana, Capsella rubella, and Vitis vinifera; and four in Fragaria vesca), 87 COI1s in 10 monocots , four COI1s in one basal angiosperm (Amborella trichopoda), 28 COI1s in two mosses , and four COI1s in one lycophyte (Selaginella moellendorffii). However, no COI1 was identified in five algae plants that belonged to Rhodophyta  and Chlorophyta (Coccomyxa subellipsoidea C169 and Micromonas pusilla CCMP1545)  and two SbCOI1s (SbCOI1–5 and SbCOI1–6). Group B included five SsCOI1s , two SbCOI1s (SbCOI1–2 and SbCOI1–4), and ShCOI1–2. There were seven SsCOI1s , three SbCOI1s , and two ShCOI1s (ShCOI1–1 and ShCOI1–3) in group D.On the basis of the topology of phylogenetic trees and the conserved amino acid sites of F-box and JAZ-binding sites on COI1 proteins  values of 31 COI1s varied from 5.25 to 8.40. The results of the instability index suggested that 30 of 31 COI1s were unstable proteins (instability index > 40), and the grand average of hydropathicity (GRAVY) showed that 22 of 31 COI1s were hydrophilic proteins (GRAVY < 0). There were no signal peptides or transmembrane structures in these COI1 proteins, suggesting that they were all non-secreted proteins  and random coils (28.80–37.89%) were the main components, the extended chain (10.90–14.60%) was secondary, and beta turns (3.20–5.84%) accounted for the smallest proportion /segmental duplications, were analyzed , three dispersed (14.29%), two proximal repeat genes (9.52%), one tandem (4.76%) duplication, and one singleton gene (4.76%) were found. Interestingly, all three ShCOI1 genes in R570 had dispersed duplications. Among seven SbCOI1 genes, five were detected as dispersed genes (71.43%), and two were WGD/segmental duplications (28.57%)  of collinearity relationships of 21 SsCOI1s were observed  of homoeologous genes that were distributed in different chromosomes. In R570, there was no collinear relationship among three ShCOI1 genes in R570 /synonymous (Ks) ratios of all duplicated COI1 genes in S. bicolor, R570, and S. spontaneum were < 1, indicating that the COI1 gene family might have experienced strong purifying selective pressure during evolution , except for SbCOI1–5, 26 (83.87%) contained abscisic acid response elements (ABRE), and 24 (77.42%) contained anaerobic induction elements (ARE), while 22 (70.97%) contained drought-inducibility (MBS) and low-temperature response elements (LTR). In addition, the numbers of COI1 promoter regions that contained auxin (IAA), gibberellin (GA), and SA response elements, were 16, 18, and 12, respectively. The existence of these functional elements indicates that COI1 genes may participate in the induction of multiple stress responses and thus play a role in sugarcane defense against various environmental stresses.The ent Fig. . The ligSsCOI1 genes were constitutively expressed in the root, bud, leaf, stem pith, and epidermal tissues of sugarcane cultivar ROC22 (Saccharum spp. hybrid), but with various expression levels  were the highest in the bud and the lowest in the leaf. SsCOI1–2a and SsCOI1–2b had the lowest expression levels in the bud and the highest in the stem pith. The expression levels of SsCOI1–6a, −6b, −6c, −7a, −7b, −8a, −8b, and −8c were the highest in the stem pith and the lowest in the root. The highest expression levels of SsCOI1–4a, −4b, −4c, and −5 were observed in bud tissues, followed by those in the epidermis, stem pith, root, and leaf. There were different expression patterns between SsCOI1–4e and its duplicated gene, SsCOI1–4c. It was noteworthy that the SsCOI1 genes that clustered to group D showed a higher expression level compared with the other group in general. These results suggest that SsCOI1 genes may play a role in sugarcane growth and development, but with different function modes.The results of transcriptome sequencing (RNA-seq) showed that 21 els Fig. . Among tSsCOI1s, their expression profiles under drought and cold stresses were analyzed to further investigate the function of SsCOI1 genes. As shown in Fig. SsCOI1 responded to both drought and cold stresses, but with different expression patterns.Due to the fact that the drought-inducibility (MBS) and low-temperature response elements (LTR) were observed in most of the promoter sequences of SsCOI1–1a, −1b, −3a, −3b, −3c, −8a, −8b, and −8c had the highest transcription levels at 10 d and the lowest expression levels after water recovery. The expression levels of SsCOI1–2a and SsCOI1–2b were down-regulated at 6 d, but up-regulated at 10 d and after water recovery. Compared with the control, the expression levels of SsCOI1–4a, −4b, −4c, −4e, −5, −7a, and −7b were increased and reached a single peak at 2 d, but decreased to the lowest after 10 d water recovery. The transcription levels of SsCOI1–6a, −6b, −6c, and −6d were increased and had the highest levels at 10 d after drought treatment, but all of them were decreased after water recovery. Under cold stress, the expression levels of all SsCOI1s were elevated. For instance, SsCOI1–4b and SsCOI1–4e showed the highest expression levels at 0.5 h and the lowest expression levels at 1 h. The expression levels of SsCOI1–1a, −1b, −4c, −5, −8a, −8b, and −8c were all enhanced to a peak at 0.5 h and then decreased. The transcription levels of SsCOI1–2b, −3a, −3c, −4a, −6b, −6c, and −6d were continuously up-regulated from 0 to 6 h after cold treatment, while SsCOI1–2a, −3b, and −6a decreased first and then increased and showed the highest levels at 6 h. In addition, SsCOI1–7a and SsCOI1–7b were up-regulated at 0.5 h and remained stable at 1 h after cold treatment.Under drought stress, SsCOI1s in response to smut pathogen infection, the gene expression patterns during the interaction between two different sugarcane genotypes and S. scitamineum were analyzed. As shown in Fig. Saccharum spp. hybrid cultivar YC05–179, SsCOI1–3a had no significant expression change while SsCOI1–1a and SsCOI1–1b were down-regulated. SsCOI1–4a and SsCOI1–6b were up-regulated and reached the highest expression levels at 1 day post-inoculation (dpi), while SsCOI1–2a, −2b, −3b, −3c, −4b, −4c, and −4e were up-regulated and reached a single peak at 2 dpi. The transcription levels of SsCOI1–5, −6b, −6c, and −6d were increased to a peak at 5 dpi. In addition, the expression levels of SsCOI1–7a, −7b, −8a, −8b, and −8c were decreased at 1–2 dpi and then increased at 5 dpi. In sugarcane smut-susceptible Saccharum spp. hybrid cultivar ROC22, the transcription levels of SsCOI1–1a, −1b, −2a, −2b, −3a, −3c, −4a, −4b, and −4e were inhibited after smut pathogen inoculation. The expression levels of SsCOI1–3b, −4c, and −5 were up-regulated at 1 dpi, and then down-regulated from 2 dpi to 5 dpi. The expression levels of SsCOI1–6a, −6b, −6c, −6d, −8a, −8b, and −8c were decreased and reached the lowest point at 2 dpi, and then increased at 5 dpi. The expression levels of SsCOI1–7a and SsCOI1–7b remained stable from 1 dpi to 2 dpi, and then were up-regulated at 5 dpi.To study the function of SsCOI1 gene family could be induced during the interaction between sugarcane and smut pathogen, and these allele genes showed similar expression patterns.These results indicated that all 21 members of the COI1 gene is the core member of the JA signaling pathway. To understand the expression pattern of the COI1 gene in response to JA, the expression levels of seven SsCOI1 haplotype genes under MeJA stress were evaluated via RT-qPCR  remained unchanged from 0 h to 24 h. The expression levels of the SsCOI1–1 gene (a member of group B) decreased significantly at 3 h, but increased significantly at 12 h and remained at a high level at 24 h, at levels 1.62- and 1.92-fold that of the control, respectively. The expression levels of SsCOI1–3 (a member of group B) were increased remarkably at 3 h and 12 h, at levels 1.54- and 1.40- fold higher than the control, and returned to the control level at 24 h. The transcription levels of SsCOI1–2 and SsCOI1–7 (members of group D) were up-regulated at 3 h and then stayed at a relatively stable level. For the SsCOI1–8 gene (a member of group D), its expression levels increased with a single peak at 12 h, which was 1.67-fold higher than the control. The above results show that various SsCOI1 genes might play different roles in response to MeJA.The PCR Fig. . After MCOI1 genes in a sugarcane cultivar, three candidate COI1 genes, SsCOI1–4b (clustered into subgroup A), SsCOI1–1b (clustered into group B), and SsCOI1–3b (clustered into group B), were cloned from ROC22 and termed as ShCOI1–4, ShCOI1–5, and ShCOI1–6, respectively. The full lengths of the cDNA of the ShCOI1–4, ShCOI1–5, and ShCOI1–6 genes were 1953 bp, 2152 bp, and 2322 bp, respectively, with 592, 600, and 661 encoding amino acids, respectively. The amino acid sequence similarities of ShCOI1–4, ShCOI1–5, and ShCOI1–6 to SsCOI1–4b, SsCOI1–1b, and SsCOI1–3b, respectively, were 99.50, 99.70, and 99.2%, respectively , and drought  treatments  were evaluated using RT-qPCR , but were significantly decreased in FN40 at 7 dpi. Except for the increased expression levels of ShCOI1–4 in LC05–136 at 7 dpi and in GT02–467 at 3 dpi, the expression levels of the ShCOI1–4 and ShCOI1–6 genes were both significantly increased in YZ03–258 and YT96–86 and decreased or remained stable in the other four sugarcane cultivars. In summary, ShCOI1–4, ShCOI1–5, and ShCOI1–6 can be induced by smut pathogen attack, but their expression patterns vary during the interaction between different sugarcane cultivars and the smut pathogen.In the sugarcane-smut pathogen biosystem, the expression patterns of PCR Fig. . CompareCOI1 has received increasing attention in recent years. It has been reported that the expression levels of AsCOI1 in Aquilaria sinensis can be significantly induced by MeJA, mechanical wounding, and heat [HbCOI1 in latex can be induced by JA and tapping [O. sativa, the mutation of OsCOI1b can delay leaf senescence, down-regulate several senescence-associated genes , and result in significant decreases in spikelet fertility and grain filling [OsCOI1 was inhibited by RNA interference (RNAi), O. sativa plants showed a decrease in resistance to Cnaphalocrocis medinalis and activity declines of trypsin protease inhibitor (TrypPI), polyphenol oxidase (PPO), and peroxidase (POD) [According to previous reports, jasmonic signal molecules can induce plants to activate resistance-related genes and systematically accumulate defense-related proteins to resist biotic and abiotic stresses , 8, 49. and heat . The tra tapping . In O. s filling . When Osse (POD) .COI1 gene has been identified and reported in many plants, such as A. thaliana [O. sativa [Artemisia annua [T. aestivum [Hevea brasiliensis [S. spontaneum, three ShCOI1s from R570, and seven SbCOI1s from S. bicolor, but no COI1 was obtained from five algae plants. It is speculated that COI1 may only exist in terrestrial plants because the COI1 gene family originated after the divergence of the algae and the ancestor of terrestrial plants. Of four groups, only group D in the phylogenetic tree was present in all terrestrial plants, indicating that a common ancestor was shared among the COI1 gene family from terrestrial plants after the divergence from algae  [A. thaliana  [C. rubella  [V. vinifera  [F. vesca  [COI1 gene, while the polyploid or paleopolyploid dicots may possess a number of COI1 orthologues in their genomes [ShCOI1s, SbCOI1s, and SsCOI1s are common with those in other plants, such as the TaCOI gene family [AsCOI1 [HbCOI1 [Currently, as a polygenic family, the thaliana , O. sati. sativa , Artemisia annua , T. aestaestivum , and Heviliensis . In thisgae Fig. . In the raploid) ; seven idiploid) , C. rubediploid) , and V. diploid) ; and foudiploid)   and three ShCOI1s from R570 were clustered into two groups (B and D), revealing that the COI1 gene family exhibited differences in evolution among species.As it shown in phylogenetic tree Fig. , the COICOI1 clustered into group B and group D had two introns, while there were more intron numbers in group A. Furthermore, the number, length, and positions of introns exhibited diversity. Thus, we speculated that the intron loss or gain events occurred during the process of structural evolution of the COI1 gene family [COI1 gene family, such as the different numbers of motifs 4, 5, 7, 8, and 9 among various COI1 proteins that clustered into different clades. Therefore, the classification and evolution of COI1 genes might be related to their structural divergence and diversification.According to the phylogenetic tree, protein motifs, and gene structure analysis, those COI1 proteins in the same group showed a similar motif composition and exon/intron structure, but varied among different groups Fig. . For exae family , 60. MotSsCOI1 genes were unevenly distributed among 18 of 32 chromosomes of S. spontaneum, and not every COI1 had homoeologous genes on the homologous chromosomes A, B, C, and D, suggesting that some homologous COI1 genes may have been lost during the polyploidization of the genome [S. spontaneum, R570, and S. bicolor, and the Ka/Ks ratios of all duplicated COI1 genes were < 1, indicating that the COI1 gene family might have experienced strong purifying selective pressure during evolution , hormone responsiveness , light responsiveness, and growth and development  were the highest in the bud and the lowest in the leaf. Compared with the other group genes, SsCOI1 genes, which clustered into group D, showed abundant transcripts and had the highest expression levels in stem pith. These results indicate that COI1 genes exhibit a tissue-specific pattern, and the same expression pattern suggests a similar function in the growth and development process.Gene expression patterns are usually related to their function . As repotemporal , 57, 58. tissues . In Solaormation . In addiues Fig. . For exaCOI1 gene family played a positive role in sugarcane response to drought and cold stresses with different expression patterns  and drought (PEG) stresses, while it was up-regulated significantly after SA and ABA treatments. The expression levels of ShCOI1–5 were up-regulated remarkably under SA and drought stresses, and down-regulated under ABA stress, and remained unchanged under cold stress. The transcriptions of the ShCOI1–6 gene were increased under SA, ABA, and drought stresses, but remained unchanged under cold stress  and TaCOI6  were clustered into group A, while TaCOI5  was clustered into group D [TaCOI2 were up-regulated under the ABA, GA, and low-temperature treatments, but down-regulated under the IAA and MeJA treatments [TaCOI6 could be induced by ABA and MeJA, but were suppressed by IAA and PEG [TaCOI5 were increased under the GA, low temperature, and PEG treatments, while they were decreased after the ABA, IAA, MeJA, and salinity treatments [Z. mays, the expression levels of four ZmCOIs (clustered into group A) responded to plant hormones were detected, and the results showed that ZmCOI1a and ZmCOI1b were strongly induced by JA and ABA, while ZmCOI1c and ZmCOI2 were less-expressed in maize tissues and slightly induced by JA and ABA, but there was no significant induction for ZmCOI1a, ZmCOI1b, ZmCOI1c and ZmCOI2 by 1-Aminocyclopropane-1-carboxylic acid, GA, 1-Naphthylacetic acid, and SA [Arabidopsis mutant coi1–1 could result in plants that overexpressed ZmCOI1a, ZmCOI1b, or ZmCOI1c, but not ZmCOI2, indicating the successful complementation of coi1–1 sterility by ZmCOI1a, ZmCOI1b, and ZmCOI1c and the functional divergence of ZmCOIs [COI1 genes may exhibit inconsistent expression patterns under certain environmental stresses even if they are clustered into the same group.RNA-seq data revealed that the rns Fig. . InteresPCR Fig. . The resess Fig. . These rvergence , 57. Lik, and SA . In addif ZmCOIs . It shouCOI1 gene family may be involved in sugarcane response to pathogen infection. Transcriptome analysis showed that SsCOI1 genes could be induced during the interaction between sugarcane and the smut pathogen, and the alleles showed similar expression patterns. Among them, the expression levels of SsCOI1–4b and SsCOI1–3b were up-regulated in smut-resistant cultivar YC05–179, but down-regulated in smut-susceptible cultivar ROC22, while SsCOI1–1b was down-regulated in both YC05–179 and ROC22. Furthermore, the expression patterns of ShCOI1–4, ShCOI1–5, and ShCOI1–6, the homologous genes of SsCOI1–4b, SsCOI1–1b, and SsCOI1–3b, respectively, were different in the interaction between the six different sugarcane cultivars and the smut pathogen , and the response time was earlier in the resistant cultivars than in the susceptible ones [TaCOI1 decreased significantly, and the rate of successful penetration by Bgt was higher than that of the control. This indicates that TaCOI1 may play a key role in wheat-Bgt interactions [A. thaliana, two mutant alleles of coil conferred hypersusceptibility to the necrotrophic pathogen Sclerotinia sclerotiorum than wild-type or heterozygous plants [ZmCOIs in the Arabidopsis coi1–1 mutant plants can cause the restoration of resistance to the leaf pathogen Botrytis cinerea and the soil-borne pathogen Pythium aristosporum [COI1 genes have multiple functions and participate in sugarcane defense against various environmental stresses via different regulatory mechanisms.It has been reported that JA and SA are mostly related to plant resistance to pathogen infection , 46. Thegen Fig. . Similarble ones . Using vractions . In A. ts plants . Furthertosporum . Taking cis-acting elements, tissue-specific expression, and expression profiles under various stresses suggests that COI1 genes participate in growth, development, and response to various stresses in sugarcane. Furthermore, three COI1 genes, ShCOI1–4, ShCOI1–5, and ShCOI1–6, were obtained by homologous cloning in the sugarcane cultivar ROC22 and could be induced by the stresses of drought, cold, ABA, SA, and S. scitamineum with divergent expression profiles. The results illustrate the fact that sugarcane COI1 genes may actively respond to biotic and abiotic stresses via different regulatory mechanisms. The present study laid a foundation for the functional identification of sugarcane COI1 genes and provided a theoretical basis for molecular breeding of sugarcane resistance.A total of 156 COI1s, including 21 SsCOI1s, seven SbCOI1s, and three ShCOI1s, were identified from 19 species and could be clustered into four groups. The analysis of Saccharum spp. hybrid cultivars  and smut whip were obtained from the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs .Eight + 1, bud, and epidermis tissues of nine consistent 10-month-old ROC22 plants  were collected [v/v), 100 μM MeJA , and 25% PEG 8000 at 28 °C with 16 h light and 8 h darkness [The root, stem pith, leaf ollected . Four-modarkness , 68. Thedarkness , 68. Fordarkness , 68.6 spores·mL− 1 S. scitamineum , and the control group was inoculated with sterile water  [S. scitamineum inoculation for RNA-seq [The stems of eight 10-month-old sugarcane cultivars, including smut-resistant cultivars YC05–179, YZ03–258, LC05–136, and YT96–86; and smut-susceptible cultivars GT02–467, ROC22, FN40, and YZ03–103, were cut into two-bud sets, immersed for 1 day in flowing water, and cultivated under a light–dark regime (16 h of light and 8 h of darkness) at 32 °C until the germinating seedlings with a bud height of about 2 cm , 69. The20, v/v) , 44. All20, v/v) , 44. Fiv RNA-seq .Each treatment included three biological replicates. All samples were immediately frozen in liquid nitrogen and stored at − 80 °C.Total RNA was extracted from the collected samples using TRIzol™ . RNA (1.0 μg) was reverse transcribed to the first-strand cDNA using a Prime-Script™ RT Reagent Kit  for RT-qPCR analysis. The cDNA used as cloning templates was synthesized from the RNA of ROC22 buds using a HiScript II 1st Strand cDNA Synthesis Kit .COI1 gene family, the genomic data of a total of 24 species were collected  were downloaded from Phytozome . The genomic data of C. crispus, C. merolae, and G. sulphuraria were downloaded from Ensembl (http://plants.ensembl.org/index.html). The genomic data of S. spontaneum was downloaded from the link of http://www.life.illinois.edu/ming/ downloads/Spontaneum_genome/ [http://sugarcane-genome.cirad.fr/) [http://pfam.xfam.org/search#tabview=tab1), were download from HMMER (https://www.ebi.ac.uk/Tools/hmmer) and used for the HMMER search [https://www.ncbi.nlm.nih.gov/cdd) to search the protein domain [COI1 gene family members were confirmed after removing incomplete sequences. Allele genes were designated as the same name followed by the letters “a,” “b,” “c,” and “d”, and duplicated genes were designated as the same name followed by the letter “e” in S. spontaneum. The COI1 genes in T. aestivum and Z. mays were named according the research of Bai et al. [To identify the _genome/ . The monrad.fr/) . Two HidR search . HmmsearR search . All obtn domain . The COIi et al. , respectCOI1 genes in S. bicolor, R570, and S. spontaneum were submitted to ExPASy (http://web.expasy.org/protparam/) to analyze their amino acid numbers, MW, theoretical pI, instability index, and GRAVY. All full-length proteins were submitted to SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) for secondary structure analysis. The predictions of signal peptides, transmembrane structures, and subcellular localizations were conducted by SignalP-5.0 , TMHMM (http://www.cbs.dtu.dk/services/TMHMM/), and Plant-mPloc (http://www.csbio.sjtu.edu.cn/bioinf/euk-multi-2/), respectively. In addition, the percent identity matrixes between COI1 proteins in S. bicolor, R570, S. spontaneum, and sugarcane hybrid cultivar ROC22 were calculated using DNAMAN.All the identified https://evolgenius.info/evolview-v2/#mytrees/SHOWCASES/showcase%2002) [Multiple sequence alignment (MSA) of COI1 proteins was conducted by ClustalW in MEGA 6.60 with default parameters. Three phylogenetic trees in this study, including one phylogenetic tree of the 156 COI1 proteins from 19 plant species, one phylogenetic tree of 21 SsCOI1, seven SbCOI1, and three ShCOI1 proteins, and one phylogenetic tree of 21 SsCOI1s, were constructed using the maximum likelihood method  based on the above alignments . Evolviese%2002)  was usedhttp://meme-suite.org/tools/meme) to identify the conserved motifs [http://gsds.gao-lab.org/). TBtools (Toolbox for Biologists) v1.09832 and Adobe Illustrator CS6 were used to display and edit the phylogenetic tree, conserved motifs, and gene structures [Amino acid sequences of SbCOI1s, ShCOI1s, and SsCOI1s were submitted to the Multiple Em for Motif Elicitation online program  software (http://mg2c.iask. in/mg2c_v2.1/). Multiple Collinearity Scan toolkit (MCScanX) and TBtools were used with the default parameters to analyze the synteny block and gene duplication pattern [COI1 gene family [The physical locations of  pattern , 77. Thee family .SbCOI1s, ShCOI1s, and SsCOI1s was retrieved from genomic data as the promoter sequence, and its cis-regulatory elements were predicted using the PlantCARE online program (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [A 2000 bp sequence upstream of the start site of gene translation of e/html/) . The rese/html/) .S. scitamineum for 0, 1, 2, and 5 d [S. spontaneum genome as the reference annotation library. The fragments per kilobase of transcript per million mapped (FPKM) was used as an indicator to measure the expression levels of transcripts or genes. For drought and cold treatments, the original data (PRJNA590595 and PRJNA636260) were downloaded from the Sequence Read Archive database (https://www.ncbi.nlm.nih.gov/sra/). The leaves of Saccharum hybrid cultivar Co 8021 under drought treatment were harvested at 0, 2, 6, and 10 d, and water-recovered at 10 d. The leaves of S. spontaneum under cold treatment were collected at 0, 0.5, 1, and 6 h. The sequence quality of these data was improved by Fastp [S. spontaneum genome [S. spontaneum, which was relevant to the related search of their homologous genes. The expression levels of SsCOI1s in different sugarcane tissues and in response to drought, cold, and S. scitamineum stresses were mined from these RNA-seq data. The heat map showing the log2 (FPKM or TMM) expression profiles was generated by TBtools [The RNA of the roots, stem piths, leaves, buds, and epidermis in ROC22 and the buds of YC05–179 and ROC22 inoculated with  and 5 d  were seqm genome , 82. The TBtools .SsCOI1s, the specific primers for ShCOI1–4, ShCOI1–5, and ShCOI1–6  system contained 1.0 μL cDNA template, 1.0 μL each of the forward and reverse primers (10 μM), 12.5 μL 2× Phanta Max buffer, 0.5 μL dNTPs (2.5 mM), and 0.5 μL Phanta Max Super-Fidelity DNA Polymerase , and 8.5 μL ddH2O. The PCR reaction conditions were as follows: 95 °C for 3 min; 35 cycles of 95 °C for 15 s, 56 °C for 15 s, and 72 °C for 2 min 30 s; and 72 °C for 5 min. PCR products were gel-purified, cloned into pMD19-T vector , and sequenced [ShCOI1–4, ShCOI1–5, ShCOI1–6, and ZmCOIs  was performed by NTI software [According to the sequences of equenced . Amino asoftware , 31, 45.SsCOI1 genes were designed by Beacon Designer 8.0  infected by smut pathogen and under hormones and abiotic stresses  were detected using RT-qPCR. The RT-qPCR was performed on Applied biosystems Q3  system using the SYBR-green dye method with the conditions of 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The total volume of the RT-qPCR reaction system was 20 μL, which included 10 μL of the 2 × ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of the primer (10 μM), 1.0 μL of the template (10 × cDNA diluted liquid), and 8.2 μL ddH2O. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)  and standard error (SE) were determined by the Duncan’s new multiple range test by using Data Processing System v9.50 software, and the histogram was graphed by Origin 9.0.Seven primer pairs of non-allelic lgorithm . SignifiAdditional file 1: Figure S1. Amino acid sequence alignment of ShCOI1s and ZmCOIs. Zea mays COI1s: ZmCOI1a (GRMZM2G125411), ZmCOI1b (GRMZM2G151536), ZmCOI1c (GRMZM2G353209), and ZmCOI2 (GRMZM2G079112). F-box domains were underlined with the black line. Transp_inhibit (transport inhibitor response 1 protein) domains were underlined with the red line. AMN1 domains (leucine-rich repeat (LRR) protein) were underlined with the blue line. Asterisks indicated the binding sites of coronatine/JA-Ile in the COI1-JAZ complex. Plus signs indicated conserved amino acid residues of F-box domains. Site 1, Site 2, Site 3, and Site 4 indicated four JAZ-binding sites involved in the COI1-JAZ interaction.Additional file 2: Figure S2. Chromosomal distribution of the COI1 gene family in Sorghum bicolor, R570, and Saccharum spontaneum. (a) The S. bicolor chromosome. (b) The S. spontaneum chromosome. (c) The Saccharum spp. hybrid cultivar R570 chromosome. SbCOI1, ShCOI1, and SsCOI1 represented the COI1 gene in S. bicolor, R570, and S. spontaneum. The scale bar on the left indicated the chromosome length . The name of each chromosome showed on the top of each chromosome.Additional file 3: Table S1. Identification of COI1 genes in different plants. Table S2. Conserved amino acid residues of COI1 proteins among different groups. Table S3. Characterization of SbCOI1, ShCOI1, and SsCOI1 genes. Table S4. Secondary structure analysis of SbCOI1, ShCOI1, and SsCOI1 proteins. Table S5. Percentage of identity between 7 SbCOI1s, 6 ShCOI1s, and 21 SsCOI1s was calculated using DNAMAN software. Table S6. Ten conserved motifs predicted in the COI1 proteins. Table S7. The gene type of COI1 genes in Sorghum bicolor, Saccharum spp. hybrid cultivar R570, and Saccharum spontaneum. Table S8. Syntenic relationships and selection pressure among Sorghum bicolor, Saccharum spp. hybrid cultivar R570, and Saccharum spontaneum. Table S9. Promoter cis-regulatory elements analysis of SbCOI1, ShCOI1, and SsCOI1 genes. Table S10. Sources of COI1 genes from sequenced species included in this study. Table S11 Primers used in this study.


Thermococcus, for which we propose the name Thermococcus bergensis sp. nov.Hyperthermophiles grow optimally above 80 °C and include mostly microorganisms belonging to the Archaea domain and are thriving in terrestrial and seafloor geothermal vents as well as in subsurface environments. From an anaerobic hyperthermophilic mixed culture obtained from water produced from a deep and hot oil reservoir we isolated and characterized a starch-degrading strain. Based on phylogenomic analysis, the strain represents a novel hyper-thermophilic species belonging to genus T, was isolated from a mixed sulfate-reducing consortium recovered from hot water produced from a deep North Sea oil reservoir. The isolate is a strict anaerobic chemo-organotroph able to utilize yeast extract or starch as a carbon source. The genes for a number of sugar degradation enzymes and glutamate dehydrogenase previously attributed to the sulfate reducing strain of the consortium (Archaeoglobus fulgidus strain 7324) were identified in the nearly completed genome sequence. Sequence analysis of the 16S rRNA gene placed the strain in the Thermococcus genus, but with an average nucleotide identity that is less than 90% to its closest relatives. Phylogenomic treeing reconstructions placed the strain on a distinct lineage clearly separated from other Thermococcus spp. The results indicate that the strain T7324T represents a novel species, for which the name Thermococcus bergensis sp. nov. is proposed. The type strain is T7324T (=DSM 27149T = KCTC 15808T).A novel hyperthermophilic archaeon, termed strain T7324 Thermococcus [Pyrococcus [Palaeococcus [http://lpsn.dsmz.de, accessed on 2 March 2021), the genus Thermococcus contains 33 validly described species, all being organotrophic and strictly anaerobic hyperthermophiles. They grow preferentially with proteinaceous compounds such as yeast extract or peptone and carbohydrates. Growth temperatures range from 50 to over 100 °C. Based on their DNA G + C content, Thermococcus spp. can be divided into a low G + C content group (38 to 47 mol%) and a high G + C group (50 to 58 mol%) [Thermococcus members have a marine origin, from deep hydrothermal vents or geothermally heated seashore environments. Two species, Thermococcus zilligii and Thermococcus waitotapuensis, originate from terrestrial fresh-water hot springs [Thermococcus sibiricus, originates from a high-temperature deep oil reservoir [The Euryarchaeota order  encompasses a diverse and widely distributed group of thermophilic and hyperthermophilic archaeal members isolated from geothermally heated environments . The ordmococcus , Pyrococrococcus  and Palaeococcus . Accordi springs ,7 while Thermovirga and Petrotoga [Archaeoglobus fulgidus, was initially isolated from a shallow hot vent at Vulcano island, Italy [Deep petroleum reservoirs represent extremophilic subsurface environments with a diversity of anaerobic thermophilic micro-organisms ,10,11 . Thed, Italy , and subd, Italy . Thermococcus strain, which apparently was responsible for the starch degradation [Thermococcus species that originated from the North Sea A. fulgidus culture, which was believed to be a pure culture at the time of deposition. The North Sea culture deposited at DSMZ (DSM 8774) in 1994 grew with lactate as a carbon and energy source under sulfate-reducing conditions but was later found to also grow on starch under sulfate-reducing conditions using a modified Embden–Meyerhof pathway . Howeverradation . Here weThermococcus strain, active cultures were transferred to an anaerobic lactate-free medium containing the following components (L−1 distilled water): 18 g NaCl, 2 g MgCl2∙6H2O, 0.32 g KCl, 0.14 g CaCl2∙2H2O, 0.11 g K2HPO4∙3H2O, 0.02 g KH2PO4, 0.5 mL 0.2% resazurin, 1 mL of a stock solution of 0.2% (NH4)2Fe(SO4)2∙6H2O and 10 mL of a stock solution containing 1.5 g Titriplex I, 0.5 g MnSO4∙2H2O, 0.1 g CoCl2∙6H2O, 0.1 g ZnSO4∙7H2O, 0.01 g CuSO4∙5H2O, 0.01 g H3BO3, 0.01 g Na2MoO4∙2H2O and 0.2 g NiSO4∙6H2O per litre. After autoclaving, the medium was flushed with N2/CO2 at a 9:1 ratio, 30 mL 1M NaHCO3, 4 mL 0.5 M Na2S and 10 mL vitamin solution [Thermococcus medium  under nitrogen at pH 7.0 and 80 °C. An active culture of DSM 8774 was obtained from DSMZ and cultivated anaerobically in 50 mL serum flasks under nitrogen with lactate and sulfate at 80 °C as described in . To isolFor initial phylogenetic characterization, genomic DNA was extracted from cells grown in the medium used for isolation as described above using a modification of the cetyl trimethylammonium bromide method . The parFor genome sequencing, DNA was isolated using a JetFlex Genomic DNA Isolation Kit (ThermoFisher Scientific). SMRTbell template library was prepared according to the instructions from PacificBiosciences, Menlo Park, CA, USA, following the procedure and checklist—20 kb Template Preparation using a BluePippin Size-Selection System. Briefly, for preparation of 15kb libraries 5µg genomic DNA were end-repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from Pacific Biosciences, Menlo Park, CA, USA. Reactions were carried out according to the manufacturer’s instructions. BluePippin Size-Selection was performed according to the manufacturer´s instructions . Conditions for the annealing of sequencing primers and the binding of polymerase to purified SMRTbell template were assessed with the calculator in RS Remote, PacificBiosciences, Menlo Park, CA, USA. SMRT sequencing was carried out on the PacBio RSII , taking a 240-min movie on two SMRTcells, which resulted in 101,716 post-filtered reads with a mean read length of 4947 bp.https://www.ncbi.nlm.nih.gov/genome/annotation_prok, accessed on 2 March 2021) and RAST . Genome completeness was assessed using CheckM [http://enve-omics.ce.gatech.edu/ani/, accessed on 2 March 2021) and the genome-to-genome distance calculator available at DSMZ .Long read genome assembly was performed with the “RS_HGAP_Assembly.3” protocol included in SMRTPortal version 2.3.0 using default parameters . Assembly resulted in 61 contigs, from which only five revealed the expected coverage values (>130×). The draft genome was annotated using the NCBI prokaryotic genome annotation pipeline (PGAP) . The isolate, designated strain T7324T, was unable to grow using lactate and sulfate as an electron donor and electron acceptor, respectively. PacBio sequencing and assembly yielded five contigs with a base coverage of 137× and a size range of 0.85 to 1.2 Mb  and T. kodakarensis ). The genome completeness was estimated to be 99.01%. Overall genome sequence comparisons revealed an ANI value between T. litoralis and strain T7324T of 87.9% and an in silico DNA-DNA hybridization value of 35.8%, which are significantly below the recommended species demarcation values of 98.7% [T therefore constitutes a separate species, for which we propose the name Thermococcus bergensis .-culture , the culobtained , with ceo 1.2 Mb . The totof 98.7%  and 70%,of 98.7% . Based oThermococcus litoralis strain oil (NZ_FJMQ00000000), was identified after further searches in the NCBI nucleotide database. This strain is the closest relative of strain T7324T based on overall genome similarity and also originates from a deep subsurface oil reservoir, but the two share an ANI value of only 89.85% and clearly represent different species. A phylogenomic tree including the closest relatives of strain T7324T shows a distinct branching of T. bergensis from all the other species/strains with 100% bootstrap support and highly significant pairwise ANI values , indicating an obligate syntrophic relationship between the two strains. Furthermore, although growing in isolation, T. bergensis grows much better as part of the co-culture, for reasons still not understood.  extract  or starcA. fulgidus DSM 8774 culture was found to be contaminated with a novel Thermococcus species, termed T. bergensis T7324, which was isolated and genome sequenced. The previously reported growth of the DSM 8874 culture on starch is now shown to be attributed to the T. bergensis partner of this consortium. There is an obligate syntrophic relationship between the two strains, as A. fulgidus 7324 has not been able to grow in pure culture. The molecular basis for this syntrophy has not been resolved.The


Metals, metallic compounds, and, recently, metallic nanoparticles appear in textiles due to impurities from raw materials, contamination during the manufacturing process, and/or their deliberate addition. However, the presence of lead, cadmium, chromium (VI), arsenic, mercury, and dioctyltin in textile products is regulated in Europe (Regulation 1907/2006). Metal determination in fabrics was performed by inductively coupled plasma-mass spectrometry (ICP-MS) after microwave-assisted acid digestion. The ICP-MS procedure has been successfully validated; relative standard deviations were up to 3% and analytical recoveries were within the 90–107% range. The developed method was applied to several commercial textiles, and special attention has been focused on textiles with nanofinishing . Arsenic content (in textile T4) and lead content  were found to exceed the maximum limits established by the European Regulation 1907/2006. Although impregnation of yarns with mercury compounds is not allowed, mercury was quantified in fabrics T1-2, T5, and T6. Further speciation studies for determining hexavalent chromium species in sample T9 are necessary . Some textile products commercialised in Europe included in this study do not comply with European regulation 1907/2006. Textile products can be contaminated with metals during both production and storage. In addition, manufacturers add intentionally metals or metallic compounds to textile products to achieve or improve specific functionalities. As example, approximately 80% of leathers are chrome-tanned ,2, while2NPs) is an example of nanofinishing to improve the existing textile manufacturing procedures, specifically the scouring and bleaching processes of fibres (nanoscouring and nanobleaching). The photocatalytic activity of TiO2NPs leads to a fast degradation  of natural colouring matter and pigments in fabrics . Another example is chromium, because several chromium-based dyes, such as chrome complexes with salicylic acid-based dyes (Mordant Yellow 1 and Mordant Yellow 5) give rise to mainly yellow and orange colours. In addition, Mordant Red 7, Mordant Red 19, Mordant Black 3, Mordant Black 9, Mordant Black 11, and Mordant Blue 13 dyestuffs, obtained by chroming aryl azo compounds, are also used in the textile industry [−1, except in textile coded as T9, which showed a very high chromium content (974 ± 6.01 µg g−1). Except for this sample (T9), similar concentrations were obtained by Menezes et al. [The presence of metals in textiles can be directly related to the dyeing process. That is the case of vanadium, since vanadium compounds are used as catalysts in the production of aniline black dye CI 50440) ,43. Vana0 ,43. Vas et al.  in texti−1) and T13 (0.758 ± 0.117 µg g−1) that contained the highest and lowest concentrations, respectively. Similarly, lead white and lead chromate pigments are also used in the textile industry [−1.In addition, barium sulphate (whitening colourant) and barium pigments can be used in the textile industry. Most of the analysed samples were found to contain barium, such as textiles T2-3  and textile T9 (1.26 ± 0.0630 µg g−1).Other metals such as copper, cobalt, and nickel are also used during the dyeing process as mordants . This isterature ,38,39. Sterature , and certerature . Sixteen−1). Furthermore, the presence of manganese in textiles is also related to the bleaching stage since several manganese complexes are used as catalysts to enhance this process [−1, respectively). The concentration of manganese in the remaining samples was in the range of 0.171–7.20 µg g−1.Iron can be used to form metal-complex dyes ,46,48 bu2NPs [2NPs provide self-cleaning [−1.Titanium dioxide is broadly used as delustering agent in synthetic fibres to reduce the lustre and transparency of yarns . Further2NPs ,52 can bcleaning ,54,55,56cleaning , and antcleaning ,58 to fa2NPs were added to these textile products (children’s T-shirt) to obtain protection against ultraviolet radiation  who specified that TiOadiation . It was 2NPs provide brightness and opacifying characteristics). Samples T10, T11, T12, T13, and T14  were all-white clothes. Sample T6 was the sample with highest concentration of titanium, and it was half white and half black (one colour in each side of the piece of garment). In addition, titanium was present in T1-2, T2-2, T2-3, T3-2, and T3-3 which were subsamples that contain white fibres mixed with coloured fibres.The manufacturers of the remaining samples did not specify the addition of titanium, which could mean that titanium was used to enhance fire retardation or opacity of fibres. Moreover, titanium could be present in these samples since they contain totally or partially white fibres . In addition, another two samples (T1-1 and T2-1) were found to contain silver, although silver ions/NPs were not listed in the textile composition.Finally, several manufacturers of the studied textiles have specified the presence of silver ions or AgNPs in the garments since silver compounds offer biocide properties to the textiles . In acco−1. The levels of Mo in the samples were lower than those reported by Rezyć et al. [−1 in textile samples. However, antimony was present at high concentrations (0.301 to 218 µg g−1). As previously commented, high antimony concentrations in polyester fibres can be due to its use in the fabrication of polyethylene terephthalate (the most common type of polyester used in garments) [−1, samples T2-2, T2-3, T3-2, T3-3, T5, T6, T11, and T13).The presence of metals such as molybdenum and antimony is related to their use in flame retardant formulations ,8,61. Moć et al.  whose foarments) . A direc−1, except for sample T4 (15.8 ± 0.920 µg g−1), where high arsenic levels could be associated with high cotton percentage (80%) in the garment.Arsenic compounds are effective insecticides and herbicides and the use of organoarsenicals is a common practice to control weeds in cotton fields and as defoliants of cotton plants . ArsenicA microwave-assisted acid digestion procedure using a mixture of nitric acid, hydrogen peroxide, hydrochloric acid, and hydrofluoric acid has found adequate for decomposing a vast variety of textiles and (nano)textiles. ICP-MS multi-element determinations were found to be accurate  and precise (RSDs varied between 1 and 3%).−1) exceeded the maximum legal concentration of arsenic in textile products manufactured in Europe (1 µg g−1). Subsamples T1-1, T1-2, and T3-3 showed concentrations of lead which were close to the maximum legal limit or over it, 1 µg g−1 . Impregnation of yarns with mercury compounds is not allowed, but mercury was found in fabrics T1-2 (0.375 ± 0.0462 µg g−1), T5 (0.0818 ± 0.00544 µg g−1), and T6 (0.157 ± 0.00597 µg g−1).The content of cadmium, lead, chromium (VI), arsenic, mercury, and dioctyltin in fabrics is regulated in Europe (Regulation 1907/2006). The concentration of arsenic found in textile T4 .On the other hand, speciation studies are necessary in the analysis of chromium. Hexavalent chromium is the only species of chromium regulated in textile products, and its maximum allowable concentration is 1 µg g2NPs, and ZnONPs) in functionalised (nano)textiles, high levels have been found in some garments which were reported to contain NPs by the manufacturers. Further studies would be welcome in order to characterise the particle size distribution as well as the release of these metallic NPs under conventional washing (laundry) conditions.Regarding metals such as silver, titanium, and zinc, which are mainly added as NPs (AgNPs, TiO


Coxiella burnetii, the causative agent of query (Q) fever in humans, is an obligate intracellular bacterium. C. burnetii can naturally infect a broad range of host organisms  and cell types. This amphotropic nature of C. burnetii, in combination with its ability to utilize both glycolytic and gluconeogenic carbon sources, suggests that the pathogen relies on metabolic plasticity to replicate in nutritionally diverse intracellular environments. To test the significance of metabolic plasticity in C. burnetii host cell colonization, C. burnetii intracellular replication in seven distinct cell lines was compared between a metabolically competent parental strain and a mutant, CbΔpckA, unable to undergo gluconeogenesis. Both the parental strain and CbΔpckA mutant exhibited host cell-dependent infection phenotypes, which were influenced by alterations to host glycolytic or gluconeogenic substrate availability. Because the nutritional environment directly impacts host cell physiology, our analysis was extended to investigate the response of C. burnetii replication in mammalian host cells cultivated in a novel physiological medium based on the nutrient composition of mammalian interstitial fluid, interstitial fluid-modeled medium (IFmM). An infection model based on IFmM resulted in exacerbation of a replication defect exhibited by the CbΔpckA mutant in specific cell lines. The CbΔpckA mutant was also attenuated during infection of an animal host. Overall, the study underscores that gluconeogenic capacity aids C. burnetii amphotropism and that the amphotropic nature of C. burnetii should be considered when resolving virulence mechanisms in this pathogen. Coxiella burnetii, the causative agent of query (Q) fever in humans and coxiellosis in other species , is a highly infectious obligate intracellular bacterium   , 15.C. burnetii has undergone reductive evolution, resulting in a relatively small (∼2 Mb) genome  unable to undergo gluconeogenesis demonstrated that C. burnetii exhibits metabolic plasticity in carbon metabolism via the use of glucose for biomass production  genome , yet enccal data , a correcal data , 18, andcal data –22, withal data –. Recentloduction . Becauseoduction , composiC. burnetii was assessed. Intracellular replication within four distinct mammalian- and three arthropod-derived cell lines was compared between a metabolically competent parental strain and the CbΔpckA mutant. Additionally, because the nutritional environment directly impacts host cell physiology—upon which pathogen fitness is intimately dependent—the analysis was extended to investigate C. burnetii intracellular replication within mammalian cells cultivated in a novel physiological medium that resembles the nutrient composition of mammalian interstitial fluid (IF), interstitial fluid-modeled medium (IFmM).In the present study, the role of metabolic plasticity in supporting amphotropism  of C. burnetii intracellular replication under conditions more reflective of nutrient availability within host tissues, a novel cell culture medium modeled on mammalian IF, IFmM, was designed. Mammalian IF serves as a conduit for supply of nutrients to tissues and thus is a physiologically relevant source of nutrients for most Coxiella-mammalian host interactions. In IFmM, concentrations of the principal ions sodium (Na+), potassium (K+), and chloride (Cl−) were based on those used in the commercially available medium RPMI 1640 (RPMI)   (see Tab0 (RPMI) , 28 (see0 (RPMI) , 30 (Tab0 (RPMI) ; however0 (RPMI) , Dulbecc0 (RPMI) , 32 form0 (RPMI) . The comC. burnetii strains exhibit unique replication potentials within a single cell type, including human alveolar macrophages   of 5, and bacterial growth was determined by quantifying GE every 2 days for a total of 8 days. Because host nutrient availability is expected to influence intracellular replication of C. burnetii, replication potentials were compared between C. burnetii-infected host cells cultured in RPMI and our novel IFmM. C. burnetii replication in J774A.1 cells was indistinguishable between conditions for the first 2 days and then diversified to result in final bacterial burdens ∼4 times greater in cells maintained in IFmM  and the influence of key nutrients on pathogen intracellular replication. Our results indicate that C. burnetii GE are comparable on day 0 and day 2 postinfection (p.i.) for naturally phagocytic cell lines , regardless of cell culture medium used  and cultured in IFmM, where (i) the glucose concentration was adjusted from 2.5 mM (control) to 5.5 or 11 mM , or (ii) amino acids were diluted 10-fold  or increased 5-fold . At 5 days p.i., cultures were processed for quantification of GE and imaged for qualitative assessment of CCV size. During infection of J774A.1 cells, increasing concentrations of glucose to 11 mM or amino acids 5-fold resulted in significantly reduced yields compared to those of complete IFmM   . When me reduced . In VEROteration . Despiteteration . In JEG-i yields . Increasent foci . These dC. burnetii metabolic fitness is expected to be directly impacted by access to specific nutrients that are mainly acquired or produced by the host cell. Based on documented roles in C. burnetii physiology and data presented in J774A.1 = 13.32 ± 2.55 ng μl−1; glutamateVERO = 16.17 ± 2.23 ng μl−1) and significantly lower than the concentration measured in JEG-3 cells (36.19 ± 6.15 ng μl−1) (−1) remained lower than those measured for JEG-3 (39.38 ± 8.87 ng μl−1) and VERO cells (36.09 ± 8.72 ng μl−1). Moreover, VERO cells cultured in IFmM contained an ∼2-fold higher concentration of glutamate than cells cultured in RPMI (P = 0.0513) (−1 glucose measured regardless of cell type or culture medium (C. burnetii—are host-cell specific and can change based on medium nutrient composition.ng μl−1) . In comp 0.0513) . Measuree medium . OverallpckA, encoding phosphoenolpyruvate carboxykinase, prevents gluconeogenic activity in C. burnetii. Previously, the pckA deletion mutant CbΔpckA was determined to have a moderate (glucose-dependent) growth defect in VERO cells cultured in RPMI  was compared to that of the CbΔpckA mutant and its functional complement CbΔpckA::pckA in J774A.1, JEG-3, THP-1, and VERO cells. In J774A.1 cells maintained in IFmM, the CbΔpckA mutant had a severe (∼1.5 to 2 log) growth defect from 2 to 8 days p.i. compared to the Pt strain (CbΔpckA mutant tended to exhibit reduced growth from 4 to 8 days p.i. compared to the Pt strain (CbΔpckA mutant with pckA (CbΔpckA::pckA) did not restore growth within the JEG-3 cell line , JEG-3 , and VERO . The use of IFmM did not affect replication of either the Pt or CbΔpckA strain in THP-1 cells  (mock). Both the Pt and CbΔpckA::pckA strains were equally virulent with ≤4% of the infected flies surviving relative to mock infected flies at 40 days p.i. . Therefore, gluconeogenic capacity is required for optimal virulence via a mechanism that does not depend on overall fly colonization.The growth defect displayed by the al model  was emplays p.i. . In contA strain . To deteCbΔpckA mutant to replicate in flies and reach overall burdens similar to the Pt strain, yet exhibit a defect in virulence, suggested that the mutant was incapable of colonizing specific cells critical for fly viability. Because D. melanogaster-derived S2 hemocytes  were equivalent, the CbΔpckA strain replicated less effectively than the Pt strain in S2 and ISE6 cells, resulting in significantly reduced bacterial loads at one or more time point from day 4 to 10  compared to that measured for DAE100 cells (423.57 ± 112.55 ng μl−1)  . In compnificant . These dC. burnetii is a zoonotic pathogen with an extensive host range, including humans, domestic animals, wildlife, and arthropods. While determinants for species-specific colonization are poorly defined, carriage of the plasmid QpH1 has been linked to C. burnetii colonization of murine macrophages —a conduit for nutrients that connects blood plasma with the cellular constituents of tissues—is a physiologically relevant nutritional environment supporting most Coxiella-mammalian host interactions. Accordingly, we designed a physiological medium, IFmM, which better reflects the nutrient composition of IF to test C. burnetii replication phenotypes in different cell types  were compared between a metabolically competent parental strain and the gluconeogenesis-deficient CbΔpckA strain. As expected, both the parental strain and mutant exhibited host cell-dependent intracellular replication phenotypes. Moreover, different intracellular levels of glycolytic  and gluconeogenic  substrates in different cell types are consistent with a requirement for metabolic plasticity to facilitate C. burnetii amphotropism.rophages . Upon inll types . The sigSalmonella enterica, medium composition  can influence pathogen intracellular replication and mask the metabolic state of the bacterium during infection . Beyond applicability in the analysis of C. burnetii physiology and virulence, IFmM may prove useful in the study of other host and host-pathogen systems for which IF serves as the natural conduit of nutrients. For instance, the obligate intracellular bacterium Chlamydia trachomatis, which infects mucosal epithelial cells and is dependent on scavenging a wide range of nutrients from the host . We also show that the use of an IF-based physiological medium facilitates resolution of C. burnetii intracellular replication phenotypes. Overall, our results indicate that consideration of C. burnetii amphotropism is critical when attempting to resolve virulence mechanisms in this pathogen.In conclusion, we have demonstrated a requirement of C. burnetii Nine Mile phase II (NMII)  was propagated in ACCM-2 and prepared for long-term storage as described  supplemented with GlutaMAX  and 5% (vol/vol) hiFetalPlex at 37°C and 5% CO2. Human monocyte-derived THP-1  cells were maintained in RPMI 1640 medium containing 2 mM l-glutamine , 10 mM HEPES , 1.0 mM sodium pyruvate and 10% hiFetalPlex at 37°C and 5% CO2. Unless otherwise stated, host cells were seeded in 6-well or 12-well plates at a density of 2 × 105 cells per 3 or 1.5 ml, respectively, and maintained in complete RPMI 1640 at 37°C and 5% CO2 for 24 h. Subsequently, host cells were infected at an MOI of 5 in 1 ml of plain RPMI 1640. Infections were facilitated by centrifugation at 400 × g for 30 min at room temperature (RT). Following infection, host cells were washed 3 times with IFmM inorganic salts solution (pH 7.2) to remove unattached and noninternalized bacteria before complete IFmM or RPMI 1640 infection medium  was added to each well. Media were replenished every 48 h for the duration of the 8-day infection cycle. At the time of sampling, host cells from duplicate wells were detached via gentle scraping or use of trypsin and transferred to 1.5-ml tubes for processing and quantification of bacterial GE. Samples for the zero hour time point were collected ∼2 h postinfection.Mouse macrophage-like J774A.1 , African green monkey kidney epithelial VERO , and human placental trophoblast-derived epithelial JEG-3  cells were maintained in complete RPMI 1640 medium  cells  were maintained in Schneider’s insect medium  containing 10% (vol/vol) hiFetalPlex at 28°C. Dermacentor andersoni DAE100 and Ixodes scapularis ISE6 cells were maintained in complete L15B medium  hiFetalPlex or complete L15B was added to each well for the S2 and DAE100/ISE6 cells, respectively. Arthropod infections were allowed to progress for 10 days, with samples taken every 48 h. At time of sampling, cultures from duplicate wells were transferred to 1.5-ml tubes and host cells pelleted via centrifugation for 10 min at 9,000 × g. Supernatants were removed and pellets stored at −20°C until GE quantification.B medium  at 34°C.g (RT). The resulting supernatants were assayed directly.Intracellular glucose and glutamate pools were quantified using commercially available assay kits  according to manufacturer’s instructions. For J774A.1, JEG-3, VERO, and S2 cell lines, 2 to 4 confluent T-75 flasks were used for the analyses. JEG-3 and VERO cultures were harvested via trypsinization, while J774A.1 cultures were harvested by gentle scraping. For the DAE100 and ISE6 cell lines, 2 or 3 confluent T-25 flasks were harvested via repeated pipetting using a serological pipette. Cells were washed twice with PBS (pH 7.2) before cell numbers were determined via direct cell counts for normalization purposes. Finally, cells were pelleted and resuspended in 0.25 ml of PBS containing 0.1% (wt/vol) SDS, heated for 10 min at 95 to 100°C to inactivate host enzymes, and then subsequently centrifuged for 10 min at 10,000 × D. melanogaster w1118 flies were infected and survival monitored as described . Following injection, fly mortality was determined every 24 h over 40 days with flies collected at 15 and 30 days p.i. to determine relative bacterial loads by measuring GE.Male escribed . BrieflyC. burnetii NMII cultures was added to a 1.5-ml screw-cap tube containing 0.1-mm zirconia beads  and subjected to mechanical lysis  via three, 30-s pulses at 5.0 m s−1. Samples were serially diluted and GE quantified via detection of the C. burnetii gene CBU1206  containing 0.3 ml of molecular grade water. Tubes were subjected to an initial round of mechanical homogenization at 6.0 m sC. burnetii-infected S2, ISE6, and DAE100 cells using the Nucleospin tissue kit . Briefly, cell pellets were resuspended in 0.18 ml T1 buffer and supplemented with 0.025 ml of proteinase K. Cells suspensions were incubated overnight at 56°C, and subsequent DNA extraction followed manufacturer’s instructions. Total DNA was eluted in 0.1 ml of 70°C molecular grade water. For GE quantification, 10 to 100 ng total DNA was used as template.Total DNA was extracted from C. burnetii infection of D. melanogaster, depicted data show the results from at least 3 independent experiments (n).Statistical analysis was done using Prism software . Except for


A popular food in China, soybean seeds and sprouts contained many biologically active substances which are beneficial to the human body, such as flavonoids. Northeast of China is the main producing area of soybean. The experimental materials came from the main soybean producing areas in Northeast China, this study compared flavonoids of two China cultivars of soybeans, Heinong52(HN52) and Heinong71(HN71). Here, we also considered the effects of germination on the chemical profile of flavonoids. Using a LC–ESI–MS/MS system, 114 differential flavonoid metabolites were identified. A total of 18 metabolites were significantly different between the two soybean varieties before germination, of which 14 were up-regulated and 4 were down-regulated. After germination, 33 significantly different metabolites were found in the two soybean sprouts, of which 19 were up-regulated and 14 were down-regulated. These experimental results revealed significant up-regulation of metabolites in soybean sprouts compared with soybean seeds, thus suggesting that soybean germination may increase content of flavonoid metabolites. There are six main pathways for the synthesis of flavonoids: isoflavonoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, biosynthesis of secondary metabolites, and biosynthesis of phenylpropanoids. Soybean seeds lack flavone and flavanol biosynthesis and develop the capacity for this biosynthetic pathway after germination as sprouts. Isoflavonoid biosynthesis is the most abundantly utilized pathway. Glycine max(L.) Merr.), with a cultivation history of at least 4,000 years. Nowadays, soybean is gaining popularity in many countries largely as a vegetable protein and oil source, owing to its bean composition of approximately 40% protein and 20% oil1. Soybean is an important nutritional component of diets and is used in many foods, such as soybean oil, soybean sprout, paste, soymilk, and tofu2. Mostly present in plant leaves and fruits, flavonoids primarily exist as glycosides combining with sugars. An important phenolic secondary metabolite in plants, flavonoids can support plant disease resistance, and in the human body have roles such as anti-oxidation, anti-cancer, and anti-aging4. Certain major flavonoids, such as flavonols, flavonol glycosides, isoflavones, chalcones, anthocyanins and procyanidins have been previously isolated and identified from soybeans6. Additionally, recent research showed that pinto and black beans as excellent dietary sources of natural antioxidants and thus may serve as disease preventative and healthy foods7. Moreover, a recent review analyzing epidemiological studies in the last ten years and the flavonoid content of diets concluded that dietary flavonoids may prevent a range of different types of cancer8. In fact, free phenolic compounds, such as flavonols and isoflavones, have been found to have with immunomodulatory and cellular antiproliferative functions9.China is the first country to grow and domesticate the soybean and mass spectrometry, for comprehensive metabolic analysis of plant substrates23. Recent technological developments in the field of metabolomics, especially as a result of the widespread use of LC-high resolution MS have considerably improved accuracy and sensitivity of metabolite detection24. For example, non-target LC–MS-Orbitrap metabolomics methods were used to compare and analyze the biologically active metabolites of frozen, boiled, and canned chickpeas, lentils, and white beans25. The main purposes of this study were to: (1) compare the flavonoid contents in soybean seeds and sprouts grown under dark conditions for 3 days at 24 °C (2) analyze the metabolism of flavonoids in soybean sprouts.The sprouting process induces a variety of biochemical changes in soybean seeds, leading to the accumulation of primary and secondary metabolites26. The two soybean cultivars Heinong52 (HN52), and Heinong71(HN71) used in this experiment were cultivated at the Harbin Soybean Research Institute , Heilongjiang Academy of Agricultural Sciences, Harbin, China(statement as follow). Soybean seeds were harvested from three replicates of each cultivar for a cropping year and stored at room temperature until analysis of flavonoids compounds. Briefly, high-quality soybean grains were selected, rinsed with clean water, soaked in distilled water at 24 °C for 4 h, spread in a single layer on a tray, covered with gauze, and placed in an artificial climate box  at about 24 °C and 80% humidity to germinate for 3 days in the dark. The growth chamber was set up to spray water for 3 min every 1 h. After germination, approximately 20 g FW(fresh weight) of soybean sprouts  per cultivar with three replicates every sampling day were randomly collected from containers, and freeze-dried at − 50 °C for 20 h. The freeze-dried sprouts and seeds were crushed using a mixer mill  with a zirconia bead for 1.5 min at 30 Hz. 100 mg powder was weighed and extracted overnight at 4 °C with 1.0 mL 70% aqueous methanol. Following centrifugation at 10,000 g for 10 min, the extracts were absorbed  and filtrated  LC–MS analysis(contained “2.2 HPLC Conditions” and “2.3 ESI-Q TRAP-MS/MS”) .According to our previous detection of isoflavones in 12 main soybean varieties grown in Heilongjiang, China, two varieties with high isoflavone content were selected for experimentationwww.shimadzu.com.cn/; MS, Applied Biosystems 4500 Q TRAP, www.appliedbiosystems.com.cn/). We used the following steps to analyze, HPLC: column, Waters ACQUITY UPLC HSS T3 C18 ; solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program,100:0 V/V at 0 min, 5:95 V/V at 11.0 min, 5:95 V/V at 12.0 min, 95:5 V/V at 12.1 min, 95:5 V/V at 15.0 min; flow rate, 0.40 mL/min; temperature, 40 °C; injection volume: 5μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS27.The sample extracts were analyzed using a LC–ESI–MS/MS system  using HPLC–DAD-MS/MS-based methods and found the existence of 105 phenolic compounds, alkaloids, jasmonic acids, and organic acids32.LIT and triple quadrupole (QQQ) scans were acquired using a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 4500 Q TRAP LC/MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in a positive ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The parameters of ESI source operation were as follows: ion source, turbo spray; source temperature 550 °C; ion spray voltage (IS)5500 V; ion source gas I (GSI), gas II(GSII), curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively; the collision gas(CAD) was high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this periodP < 0.05. Data analysis was carried out with SPSS . All data are presented as mean ± standard error of means.For all experiments, three independent assays were carried out. One-way analysis of variance was used to study the differences between means, with a significant level at Principal component analysis (PCA) was performed on metabolic data to visualize the biological variability of soybean seeds and sprouts. These results indicated that the dominating source of variance was the differential flavonoid content in the two soybean varieties across all germination stages PC1). From Fig. . From Fi30. These were C13H16O10 , C13H16O10,C13H16O10 , C27H30O16(Kaempferol-3-O-neohesperidoside), C27H30O16 -glucose), C21H22O11 (Aromadendrin-7-O-glucoside), C27H30O16 (Quercetin-3-O-robinobioside), C27H30O17 (Quercetin-3-O-sophoroside (Baimaside)), C33H40O21 (Kaempferol-3-O-sophoroside -7-O- glucoside) , C27H32O15 (Eriodictyol-7-O-Rutinoside (Eriocitrin)), C27H30O16 (Quercetin-3-O-rutinoside (Rutin)), C28H32O17 , C27H30O15 (Eriodictyol-7-O-Rutinoside (Eriocitrin)), respectively. This indicated that these flavonoids may be universal in beans.From Table 37.Metabolomic data is massive and multidimensional, so it is imperative to combine univariate and multivariate statistical analyses, to analyze the data from a multivariate perspective according to the characteristics of the data, and to accuratelymine differential metabolites. This study has three biological replicates. The method of combining fold change ≥ 2 and OPLS-DA model VIP  ≥ 1 was used to screen for differential metabolites. Shown in Fig. Each point in the volcano map represents a metabolite. The abscissa represents the logarithm of the quantitative difference of a certain metabolite in the two samples; the ordinate represents the VIP value. The larger the absolute value of the abscissa, the greater the multiple difference in the expression amount between the two samples; the larger the ordinate value, the more significant the differential expression, and the more reliable the differentially expressed metabolites screened. The green dots in the figure represent down-regulated differentially expressed metabolites, the red dots represent up-regulated differentially expressed metabolites, and black represents detected but not significantly different metabolites.40.As shown in Fig. 41. Additionally, Ayet et al. revealed that germination increased the content of soya saponins in lentils43. Moreover, Mekky et al. revealed that germination increased the quantities of flavonoids, phenolic acids and saponins in fava beans30.As shown in Fig. 44 to annotate and display the differential metabolites identified in these samples, 6 metabolite were significantly up-regulated in the isoflavone synthesis pathway, 1 metabolite was significantly down-regulated, and 13 metabolites did not change significantly (Fig. In the homepage of the KEGG website, we directly enter the keyword "Flavonoids" and the top 4 pathways are all pathways involving flavonoid metabolites, including:flavonoid synthesis, anthocyanin synthesis, isoflavone synthesis, flavonoids and flavonols synthesis. Using the KEGG databasetly Fig. .Figure 5There are six primary metabolic pathways for differential metabolism of flavonoids; isoflavonoid biosynthesis, flavonoid biosynthesis, flavone and flavonol biosynthesis, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis Fig. . Unlike As shown in Fig. In this study, two soybean varieties with high isoflavone content and their soybean sprouts in Heilongjiang Province, China were analyzed with flavonoid metabolome analysis. 114 differential metabolites were detected which contained 41 isoflavones and 26 flavonoids. These experimental results revealed that the differentially expressed and up-regulated metabolites in soybean sprouts increased compared to soybean seeds. The flavonoid metabolites in the same variety of soybean seeds and sprouts differ greatly in multiples, indicating that flavonoid metabolism is very active during soybean germination, and germination may be a way to accumulate flavonoids. Metabolic pathway data explained why isoflavones were higher than different metabolites. This study detected and analyzed the changes of flavonoid metabolites after soybean germination, providing data reference for the research of flavonoids, and providing ideas for future research on plant secondary metabolism.


We report here the nearly complete genome sequences of two human BK polyomavirus (BKV) strains recovered from two Vietnamese renal allograft recipients and belonging to subtypes IVc1 (strain VN_PBK185) and Ib1 (strain VN_PBK212). The genome sequences of VN_PBK185 and VN_PBK212 were highly similar (99.9% nucleotide identity) to the reference BKV strains VNM-1 and VNM-9, respectively. Polyomaviridae, genus Betapolyomavirus, and has a non-enveloped capsid. The genome consists of a circular double-stranded DNA molecule about 5.1 kb long. Based on either genotyping or serotyping methods, BKV is classified into four genotypes (I to IV), with genotype I predominant worldwide, followed by genotype IV. Meanwhile, genotypes II and III are found at lower rates (Human BK polyomavirus (BKV) is a ubiquitous pathogen with worldwide distribution among humans. In the majority of cases, this virus does not cause any symptoms but only becomes an emerging opportunistic infectious pathogen in immunocompromised patients, especially individuals with kidney transplantation . Taxonomer rates , 3. Phyler rates . Chen et4 copies/ml and 109 copies/ml (patient 1) and >103 copies/ml and 108 copies/ml (patient 2), respectively, as quantified using the RealStar BKV PCR 1.0 kit . Next, DNA was extracted from the urine specimens, and a PCR amplicon of 5.1 kb was generated using the following forward and reverse primers: wBK_aF (672 to 700), 5′GAG GCT GCT GCT GCC ACA GGA TTT TCA GT 3′, and wBK_aR (680 to 651), 5′ AGC AGC CTC AGA TAC ACT GGC AAC TAG GTC 3′. The reaction mixture (50 μl) contained 5 μl DNA template, 1× Phusion master mix with high-fidelity (HF) buffer , 0.5 μM primer wBK_aF/R, and deionized water. The PCR conditions were 98°C for 2 min, followed by 35 cycles of 98°C for 20 s, 58°C for 20 s, and 72°C for 4 min, with a final extension at 72°C for 4 min. The PCR products were gel extracted , purified, and subsequently used for direct Sanger sequencing with 12 primers . These clinical samples were sent to our laboratory to measure the BKV DNA loads and for subsequent BKV genotyping. The purpose of the study was explained to each patient, and informed consent was obtained, after the study protocol was approved by the local ethics committee. Briefly, the BKV DNA loads in plasma and urine were >10 primers  using thNC_001538), a contiguous sequence represented by 5,145 nucleotides  contained a total GC content of 39.44% (patient 1), and a contiguous sequence represented by 5,139 nucleotides  contained a total GC content of 39.37% (patient 2). Phylogenetic analysis showed that these BKV sequences belong to subtypes IVc1 (patient 1) and Ib1 (patient 2), with both sequences having 99.9% nucleotide sequence similarity to the reference strains VNM-1 and VNM-9   . In concMW678842 and MW678843, respectively.The complete genome sequences of BKV strains VN_PBK185 and VN_PBK212 have been deposited in the GenBank database under the accession numbers


More importantly, the expression levels of BRD4 and pyroptosis indicators increased significantly in ALD patients. Molecule docking analysis revealed a potent binding of SA with BRD4. In conclusion, this study demonstrates that SA reduces ALD through BRD4, which is a valuable lead compound that prevents the ALD process.Alcoholic liver disease (ALD) is one of the main causes of death in chronic liver disease. Oxidative stress and pyroptosis are important factors leading to ALD. Bromodomain-containing protein 4 (BRD4) is a factor that we have confirmed to regulate ALD. As a phenolic acid compound, sinapic acid (SA) has significant effects in antioxidant, anti-inflammatory and liver protection. In this study, we explored whether SA regulates oxidative stress and pyroptosis through BRD4 to play a protective effect in ALD. Male C57BL/6 mice and AML-12 cells were used for experiments. We found that SA treatment largely abolished the up-regulation of BRD4 and key proteins of the canonical pyroptosis signalling in the liver of mice fed with alcohol, while conversely enhanced the antioxidant response. Consistantly, both SA pretreatment and BRD4 knockdown inhibited oxidative stress, pyroptosis, and liver cell damage  Long-term and excessive alcohol consumption can lead to progressive steatohepatitis and fibrosis, which further develops into liver cirrhosis and hepatocellular carcinoma. Among men, the proportion of global deaths caused by alcohol is 7.6%, among women it is 4.0%, and 139 million disability-adjusted life-years are lost due to drinking, accounting for 5.1% of the total global disease burden . AlcoholSinapic acid , one of the phenolic acid compounds, is the effective ingredients of traditional Chinese medicine. It is widely found in various oil crops, grains, vegetables, and berries. SA has anti-inflammatory, antioxidant, and antibacterial effects . It has Oxidative stress has been identified as a potential mechanism of ALD, which is caused by the imbalance between antioxidant capacity and reactive oxygen species (ROS) . The nucBromodomain-containing protein 4 (BRD4) is the most widely studied member of the bromodomain and extra terminal domain-containing (BET) protein family. Recently, we first determined that BRD4-induced inflammatory response is a split-new access for ALD . StudiesIn this study, we first explored whether SA can protect against ALD. Subsequently, molecular docking and biochemical evaluation were applied to verify the binding mode of SA and BRD4. It is revealed that SA targets BRD4 to alleviate the oxidative stress and pyroptosis of ALD.−1 per day) group, EtOH group, and two groups of SA-treated EtOH mice that were respectively administrated (i.g.) with SA at the dosages of 10 and 20 mg kg−1 per day. Subsequently, blood was gathered from the abdominal aorta and liver tissues were collected for further analysis.Male C57BL/6 mice (8 weeks old) were obtained from the Animal Center of Dalian Medical University . All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of Dalian Medical University . SA (>98% purity) and sodium carboxy methyl cellulose (CMC-Na) were separately purchased from Sigma-Aldrich  and Solarbio . SA is suspended in 0.1% CMC-Na solution before use. The mice were adapted for one week before the experiment and then fed either a normal diet (control) or a Lieber-DeCarli liquid diet containing 5% ethanol (v/v) (EtOH) for 8 weeks. Liquid diets were freshly prepared before distribution. Sixty mice were equally divided into five groups, including control group, control + SA , and were handled under the approval of the Ethics Committee of Dalian Medical University. All patients signed informed consent forms. The basic biological information of controls and patients were presented in 2, and cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium  containing 40 ng/ml dexamethasone, 5 μg/ml Insulin-Transferrin-Selenium , and 10% fetal bovine serum .Mouse hepatocyte cell line AML-12 cells (American Type Culture Collection (ATCC), Manassas, VA, United States) were maintained at 37 °C in a humidified atmosphere with 5% COBefore protein extraction, cells were pretreated with 20 μM SA for 6 h, 100 mM ethanol, or neither (control) for 24 h. Small interfering RNA (siRNA)was designed and synthesized by GenePharma . Lipofectamine 3,000 (Invitrogen) was used for transfection. The sequences of the BRD4 siRNA were as follows: sense 5′- GCCUGAGAUGAAGCCUGUATT -3′, antisense 5′- UACAGGCUUCAUCUCAGGCTT -3′. After transfection for 24 h, the cells were incubated either with or without 20 μM SA for 6 h and either with or without ethanol for 24 h. Finally, the cells were harvested and processed for protein extraction.Cell viability was quantitatively analyzed with the Cell Counting Kit-8 . The experiments were carried out in 96-well plates, incubated at 37 °C for 2 h, and then optical density (OD) values were measured with Thermo Multiskan FC microplate luminometerat 450 nm.Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG) and alcohol dehydrogenase (ADH) levels were assayed in serum, while malondialdehyde (MDA) and glutathione (GSH) levels were determined in liver tissues by commercial kits according to the manufacturer’s instructions .Isolated liver lobes were fixed in 4% paraformaldehyde at room temperature at least overnight and then embedded in paraffin for hematoxylin-eosin (H&E) or used to prepare frozen sections for Oil Red O staining. The sections were examined by light microscopy.For immunohistochemistry, antigen retrieval was performed in a Tris/EDTA buffer (pH 9). Then, the sections were incubated with F4/80, NLRP3 or caspase-1 primary antibody , followed by the corresponding secondary antibody. After visualizing with 3,3-diaminobenzidine (DAB) colour-rendering solution, the sections were counterstained with hematoxylin.Liver sections from ALD patients were incubated with anti-BRD4 primary antibody  overnight at 4 °C, followed by incubation with secondary antibody for 2 h at room temperature. Next, DAPI  was used to stain nuclei.β-actin .The tissues and cells were lysed in Lysis Buffer for Western with 1 mM PMSF according to the manufacturer’s instructions . Total protein solution was separated by centrifuge at 4 °C for 10 min, and the supernatant was quantified with a BCA kit . Then, the supernatant was boiled for 10 min by adding loading buffer and separated by SDS-PAGE. The primary antibodies used for western blotting were as follows: BRD4, GSDMD, IL-18, Nrf2, HO-1 , caspase-1, IL-1β , NLRP3, SOD2, and http://www.rcsb.org/pdb). The structure sequence was analyzed by PyMOL software to remove the original ligands and water molecules. Coordinates for both the ligand (SA) and target protein (BRD4) were prepared with AutoDock Tools-1.5.6, and molecular docking analysis of ligand-protein complexes was conducted using the AutoDock4.2. The docking results are presented after mapping with Discovery Studio version4.5.The BRD4 protein structure (PDB: 3P5O) was taken from the Protein Data Bank . The images were observed with a fluorescence microscope.AML-12 cells were stained with 10 μM DCFH-DA staining  for 30 min at 37 °C away from light. DCFH-DA fluorescence results were viewed using a fluorescence microscope.t-test (two-group comparisons) and one-way ANOVA (multigroup comparisons) were executed. p < 0.05 was considered statistically significant.The results are expressed as the mean ± SD. GraphPad Prism  was used to analyze the data. Student’s unpaired We first explored whether SA exerts a protective effect in alcohol-induced liver injury. Compared to the control, serum ALT, AST , TC, TG Oxidative stress is a main contributor to the pathogenesis of liver damage caused by alcohol. Indeed, mice that consumed alcohol showed decreased levels of antioxidant genes including Nrf2, HO-1, and SOD2  and hepaIn recent years, the key role of pyroptosis and inflammasome activation in the development of ALD has been shown by substantial evidence . Based o. Based on the above findings, AML-12 cells were induced by EtOH for 24 h to establish the ALD model in vitro. AML-12 cells transfected with BRD4 siRNA, and then different concentrations of SA pretreated the cells. In vitro we got the same trend, 20 μM SA was determined for the following experiments  is downregulated in idiopathic pulmonary fibrosis myofibroblasts by Brd4 inhibition, (Oxidative stress is a process in which organisms or cells produce excessive ROS and then destroy proteins and DNA . Studiesibition, . In addiibition, . HoweverIn aggregate, our results demonstrated for the first time that SA could be a promising therapeutic agent for ALD via BRD4, which reduces oxidative stress and pyroptosis. This study may provide a new strategy for the treatment of alcohol-associated liver disease.