However, the direct effects on antler growth and transcription, and the responses of the gut microbiota and metabolism of 25(OH)D supplementation in deer remain unclear. In this study, we demonstrated that 25(OH)D supplementation directly increased the growth and affected the gene expression profiles of sika deer antler. The bacteria community composition and metabolites in rumen were also changed following 25(OH)D supplementation, suggesting the enhanced metabolism of branch-amino acids. Our results confirmed the positive effect of 25(OH)D on antler growth and revealed the regulation of 25(OH)D supplementation on the host and microbes in sika deer.The antler is an important product of deer, which involves in the fast growth of bone. Previous study demonstrated that the production efficiency of 1,25(OH)n = 8) and a control group . 25(OH)D supplementation significantly increased the antler weight and growth rate. The levels of IGF-1,25(OH)D and 1,25-dihydroxyvitamin D were significantly higher in the 25(OH)D group than in the Ctrl group, while the levels of LDL-C were lower. The levels of valerate and branched-chain amino acids in the rumen fluid were significantly different between the 25(OH)D and Ctrl groups. The bacterial diversity indices were not significantly different between the two groups. However, the relative abundances of the butyrate-producing bacteria (families Lachnospiraceae and Succinivibrionaceae) and the pyruvate metabolism pathway were higher in the 25(OH)D group. The transcriptomic profile of the antler was significantly different between the 25(OH)D and Ctrl groups, with 356 up- and 668 down-regulated differentially expressed genes (DEGs) in the 25(OH)D group. The up-regulated DEGs were enriched in the proteinaceous extracellular matrix and collagen, while the down-regulated DEGs were enriched in the immune system and lipid metabolism pathways. Overall, these results provide novel insights into the effects of 25(OH)D supplementation on the host metabolism, rumen microbiota, and antler transcriptome of sika deer.The level of plasma 25-hydroxyvitamin D (25(OH)D) is associated with the growth of the antler, a fast-growing bone organ of Cervidae. However, the benefits of 25(OH)D supplementation on antler growth and the underlying mechanisms remain unclear. Here, the antler growth profile and transcriptome, plasma parameters, rumen bacteria, and metabolites (volatile fatty acids and amino acids) were determined in sika deer in a 25(OH)D supplementation group (25(OH)D,  Cervus nippon) is an important ruminant species that periodically grows velvet antlers, which is a completely regenerated organ with a fast growth rate in mammals [Sika deer , are essential for bone metabolism [2D indirectly stimulates the differentiation of precursor cells into osteoclasts by inducing the production of the receptor activator nuclear factor-κB ligand [2D [2D in white-tailed deer is associated with the antler growth rate [2D in antlers likely contributes to their bone growth in roe deer [2D production is also associated with the growth of reindeer antlers [Growing evidence has demonstrated that the active forms of vitamin D, 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D tabolism . 25(OH)DB ligand . The resgand [2D . For Cerwth rate . Sempereroe deer . Importa antlers . We thus antlers , an endo antlers . Vitamin antlers . Moreove antlers  were med antlers . Thus, t2D were associated with the abundances of specific butyrate producers from the Clostridiales order [It has also been reported that supplementation with 25(OH)D affects energy metabolism , promotees order . TherefoIn this study, we aimed to investigate (i) whether antler growth and plasma parameters are associated with 25(OH)D supplementation; (ii) whether rumen microbiota features and metabolites are affected by 25(OH)D supplementation using 16S rRNA gene sequencing and gas chromatography; and (iii) how the transcriptome in the AGC responds to 25(OH)D supplementation in sika deer.n = 8) or 3 mg/d 25(OH)D supplementation diet D group, n = 8). The sika deer in each group were raised in an individual pen, were fed twice daily at 07:00 a.m. and 16:00 p.m., and had free access to drinking water. The experiments were conducted for 8 weeks after the removal of the hard antler button, with 1 week for adaptation followed by 7 weeks of dietary treatment. All of the animal-specific procedures were approved and authorized by the Animal Ethics Committee of Jilin Agricultural University.A total of 16 four-year-old male sika deer (mean body weight = 109.5 ± 2.1 kg) with a similar casting time of the hard antler button (around 25 April 2020) were used in this study at the research farm of Jilin Agricultural University. The sika deer were randomly assigned to two groups, were fed a total mixed ration (TMR) based on roughage and concentrate , alanine aminotransferase (ALT), lactate dehydrogenase (LDH), total protein (TP), triglyceride (TG), total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glucose, and urea nitrogen using commercial kits . The levels of plasma IGF-1, 1,25(OH)2D, and 25(OH)D were determined using Enzyme-linked immunosorbent assay (ELISA) kits . The rumen liquid was centrifuged at 12,000× g for 5 min at 4 °C, the molar concentrations of volatile fatty acids (VFAs) were determined according to a previous study by gas chromatography  with a flame ionization detector and a DB-FFAP column [The blood samples were centrifuged at 3500× ® Fast DNA Stool Mini Kit , according to the manufacturer’s instructions. The primers 341F (5′-CCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the bacterial 16S rRNA gene in the V3-V4 region. Each primer pair contained the appropriate Illumina adapter sequence and an 8-bp barcode. The resulting amplicons were purified using the QIAquick PCR Purification Kit  and sequenced on the Illumina Novaseq 6000 to generate paired-end reads. The total RNA of the AGC tissue (five samples in each group) were isolated using Qiagen RNeasy Mini Kit , and checked for the RNA concentration and quality using a NanoDrop 2000 . RNA samples with an integrity number (RIN) greater than 7.0 were used for the library construction. A total of 1.5 μg RNA from each sample was used to construct the RNA-Seq library using NEBNext® UltraTM RNA Library Prep Kit . Each library was quantified using a Qubit 2.0 Fluorometer , and then sequenced on an Illumina HiSeq 4000 platform (150 bp paired-end sequencing).Microbial genomic DNA was extracted from the rumen liquid samples (five samples in each group) using a QIAampp values were determined based on 999 permutations. The phylogenetic investigation of communities by reconstruction of the unobserved states (PICRUSt) was applied to predict the functional profiles of the rumen microbiota resulting from reference-based OTU picking against the Greengenes database [For the 16S rRNA sequences, the paired-end sequences were first assembled into contigs using FLASH . The obtdatabase . The pre2(FC)| > 0.5 and with a false discovery rate (FDR < 0.05) based on the Benjamini and Hochberg correction. The principal component analysis (PCA) was applied to reveal the gene expression of AGC tissues between the two groups. The significantly enriched gene ontology (GO) term and KEGG pathways of DEGs were performed by KOBAS using a false discovery rate (FDR < 0.05) based on Benjamini and Hochberg correction [For the transcriptome analysis, Trimmomatic was used to remove the low quality and adapter sequence . After trrection . The genrrection .p values of the WRS tests were corrected using the false discovery rate of the Benjamini-Hochberg method. Linear regression analysis was used to estimate the association between the plasma 25(OH)D and IGF-1 concentrations. All p values ≤ 0.05 were considered to indicate statistical significance.The Wilcoxon rank sum (WRS) test was used to determine the statistical significance of the antler weight, antler growth, plasma parameter levels, volatile fatty acids (VFAs) and amino acid concentrations in the rumen fluid, alpha diversity indices, the relative abundance of bacteria according to taxonomy, and the enrichment of the KEGG pathways between the Ctrl and 25(OH)D groups. The p = 0.03, p = 0.01, p = 0.04), 25(OH)D (p < 0.01), and 1,25(OH)2D (p = 0.04) were significantly higher in the 25(OH)D group than in the Ctrl group. The level of LDL-C in the 25(OH)D group (p = 0.05) was lower than that in the Ctrl group  based on linear regression analysis  and the molar proportions of acetate (p = 0.77), propionate (p = 0.53), butyrate (p = 0.74), and isovalerate (p = 0.91) were not significantly different between the Ctrl and 25(OH)D groups (p = 0.01), indicating the alteration of the metabolism of branched-chain amino acids (BCAAs) in the rumen. Thus, we further compared the amino acid concentrations between the two groups. The results showed that a total of 11 amino acids, including aspartate, glutamate, glycine, alanine, valine, cysteine, methionine, leucine, tyrosine, lysine, and proline, as well as their related metabolites , were significantly enriched in the Ctrl group compared with those in the 25(OH)D group (p < 0.05); however, the concentrations of citrulline, phenylalanine, β-alanine, histidine, β-aminoisobutyric acid, and EOHNH2 were lower  was the most dominant bacteria, followed by bacteria belonging to Rikenellaceae RC9 (9.88%), Christensenellaceae R7 (5.35%), Prevotellaceae UCG 003 (3.20%), and Fibrobacter (3.04%), accounting for ~45.0% of the overall bacterial composition. In the 25(OH)D group, the genus Prevotella (22.76%) was predominant, followed by bacteria belonging to Succinivibrionaceae UCG 002 (7.80%), Rikenellaceae RC9 (7.56%), Christensenellaceae R7 (4.76%), and Ruminobacter (3.11%), accounting for 46.0% of the bacterial composition.Then, we examined the rumen bacterial community based on the 16S rRNA sequences. A total of 440,260 high-quality reads were obtained, with an average of 44,026 sequences for each sample . After rarefication to the minimum number, we obtained a total of 3464 OTUs based on 97% sequence identity. Based on these OTUs, we identified a total of 48 phyla in the rumen liquid from the two groups , dominatWe further revealed the difference in bacterial community composition between the Ctrl and the 25(OH)D groups. The PCoA results showed that the bacterial community composition in the rumen of the 25(OH)D group was distinct from that in the Ctrl group, based on Bray-Curtis dissimilarity matrices B, explaiAnaerosporobacter, Bauldia, Coprococcus, Iamia, Kribbella, Oribacterium, Ruminobacter, Succinimonas, Vibrio, and Virgisporangium, bacteria belonging to Lachnospiraceae NC2004, Lachnospiraceae NK4B4, and Succinivibrionaceae UCG 002, were significantly higher in the 25(OH)D group than in the Ctrl group D group D group separated from that in the Ctrl group E. MoreovMAMDC2, PTN, FN1, TNC, TNN, TGFBI, CDH2, TNFRSF11B, TCF7, and WINT10B, while the down-regulated DEGs included AFAP1L2, LCP1, IFI16, BCR, CYTIP, ALDH1A1, IL18, GPAT2, PTPRU, IF16, NPR3, IFITM1, SFMBT2, CXCR4, APCDD1L, and ARHGAP30 D supplementation. RNA sequencing was performed, and we confirmed that dietary 25(OH)D supplementation affected the gene expression of the AGC, as indicated by the PCA results, explaining approximately 46% of the total variation A. We ideARHGAP30 B. These ARHGAP30 C. For thARHGAP30 C.According to the KEGG analysis, the up-regulated DEGs were enriched in only a few pathways, including protein processing in the endoplasmic reticulum, focal adhesion, adherens junction, and ECM–receptor interaction D. In conELOVL5, ELOVL6, ELOVL7, PPARA, PPARG, ACSL1, ACSL3, ACSL5, ACADL, and FADS2. Moreover, the cytokine-cytokine receptor interaction, osteoclast differentiation, B cell receptor signaling pathway, PI3K-Akt signaling pathway, natural killer cell mediated cytotoxicity, and Rap1 signaling pathway, were also closely connected, involving genes such as NGF, NGFR, IL6R, EPOR, PRLR, IL3RA, IFNAR2, TNFRSF11B, IFNAR2, TGFBR2, CSF1, LCP2, SYK, and BLNK D supplementation can promote 1,25(OH)2D synthesis in weaning calves [2D concentration was significantly increased during antler growth in white-tailed deer [2D on antler growth. We also found a positive correlation between the plasma concentrations of 25(OH)D and IGF-1, an important antler-stimulating hormone [The results of this study indicate that 25(OH)D supplementation significantly increased the antler final weight and growth rate and 25(OH)D and 1,25(OH)g calves . Previouled deer  and fallled deer . These r hormone . Consist hormone . Moreove hormone . These f3 supplementation significantly lowers the circulating levels of BCAAs in type two diabetes mellitus [We further examined whether 25(OH)D supplementation affected rumen metabolism. The results showed that the molar proportion of valerate was significantly increased, and the concentrations of amino acids significantly differed in the 25(OH)D group. Valerate is an end-product of proline, lysine, and methionine degradation . Accordimellitus . These rPrevotella in the rumen of sika deer [2D [Ruminobacter) and Lachnospiraceae family  significantly increased in the 25(OH)D group. Members of the Succinivibrionaceae family mainly capture hydrogen to produce succinate, thereby promoting the production of VFAs [We further investigated how the rumen bacterial community was affected by 25(OH)D supplementation, due to its key role in rumen metabolism, and confirmed the dominance of ika deer . Howeverika deer , and thadeer [2D . The rel of VFAs , and are of VFAs . Members of VFAs . We alsoFN1, TNC, TNN, TGFBI, CDH2, TNFRSF11B, TCF7, WINT10B, and PTN. FN1 encodes the extracellular matrix (ECM) protein and is a vitamin D target [TNFRSF11B, encoding an osteoclastogenesis inhibitory factor, resulted in a strong upregulation of cartilage extracellular matrix components [CDH2, WNT10B, and PTN are highly expressed genes in the antler. The former mediates cell-cell interactions and enhances MSC aggregation, resulting in chondrogenic differentiation [TCF7 is considered a regulator of early osteoblast differentiation of MSCs [TGFBI is a core gene regulating antler growth and ossification [PTN has been identified as a critical factor promoting cartilage regeneration, angiogenesis, and chondrogenesis [2D regulated cell-to-cell communication by post-transcriptionally altering the expression of the genes involved in ECM organization and receptor interactions [We then examined whether and how the gene expression profiles in AGC responded to 25(OH)D supplementation. We identified 356 up-regulated DEGs in the 25(OH)D group in comparison with those in the Ctrl group, including D target , whose eD target , indicatmponents . CDH2, Wntiation , while t of MSCs ; TGFBI ification ; and PTNogenesis . In addiractions . Interes2D stimulates IL-10 release in dendritic cells, towards a less mature and more tolerogenic phenotype, resulting in a reduction in the anti-inflammatory activity of macrophages  [IL18 and IFI16 expression levels were significantly down-regulated in the 25(OH)D group. This finding is consistent with previous observations in mice, that 1,25(OH)2D markedly suppressed the expression of IL18 in primary keratinocytes and modulated the cutaneous inflammatory reactions [Notably, following 25(OH)D supplementation, the majority of changes (65.2%) in the gene expression reflected downregulation, and down-regulated genes were enriched in different pathways of the immune system, such as the B cell receptor signaling pathway, complement and coagulation cascades, and cytokine−cytokine receptor interaction. The immunomodulatory effects of vitamin D on both innate and adaptive immunity are well documented , indicatd TNF-α) . In our eactions . The DNAeactions . KeratinELOVL5, ELOVL6, ELOVL7, PPARA, and PPARG), including fatty acid elongation, biosynthesis of unsaturated fatty acids, the adipocytokine signaling pathway, and the PPAR signaling pathway. Moreover, the enriched biological process terms were the membrane lipid metabolic process and lipid catabolic process. This result suggests that 25(OH)D supplementation might result in the regulation of lipid metabolic regulatory pathways via target genes. Seven distinct elongation of very-long-chain fatty acid (FA) enzymes (ELOVL1–ELOVL7) play important roles in the elongation of medium to long-chain FAs into very-long-chain FAs [ELOVLs indicates a potential inhibitory effect of 25(OH)D supplementation on the synthesis of long-chain FAs in antlers. PPARα and PPARγ are key regulators of lipid homeostasis and are activated by a structurally diverse group of compounds, including long-chain arachidonic acid [2D, the results of the present study reveal the possibility of the interaction of vitamin D, long-chain FAs, and the PPAR signaling pathway in the regulation of metabolic homeostasis and antler growth.Another major finding of the transcriptome analysis was the enrichment of down-regulated DEGs in lipid metabolism pathways D supplementation (i) increases the antler weight and growth rate of sika deer; (ii) alters the rumen bacterial community composition and metabolic profiles, particularly regarding butyrate producers and the amino acid concentrations; and (iii) affects the genes expression of the immune response and lipid metabolism in antlers. Future studies are needed to elucidate the relationship between FA composition and antler growth. Taken together, these observations provide a better understanding of the effects of 25(OH)D supplementation on sika deer.


Cold stress is a major threat to the sustainability of rice yield. Brassinosteroids (BR) application can enhance cold tolerance in rice. However, the regulatory mechanism related to cold tolerance and the BR signaling pathway in rice has not been clarified. In the current study, the seedling shoot length (SSL), seedling root length (SRL), seedling dry weight (SDW), and seedling wet weight (SWW) were used as the indices for identifying cold tolerance under cold stress and BR-combined cold treatment in a backcross recombinant inbred lines (BRIL) population. According to the phenotypic characterization for cold tolerance and a high-resolution SNP genetic map obtained from the GBS technique, a total of 114 QTLs were identified, of which 27 QTLs were detected under cold stress and 87 QTLs under BR-combined cold treatment. Among them, the intervals of many QTLs were coincident under different treatments, as well as different traits. A total of 13 candidate genes associated with cold tolerance or BR pathway, such as BRASSINAZOLE RESISTANT1 (OsBZR1), OsWRKY77, AP2 domain-containing protein, zinc finger proteins, basic helix-loop-helix (bHLH) protein, and auxin-induced protein, were predicted. Among these, the expression levels of 10 candidate genes were identified under different treatments in the parents and representative BRIL individuals. These results were helpful in understanding the regulation relationship between cold tolerance and BR pathway in rice. Oryza sativa L.) is one of the most important food crops that grows in tropical to temperate regions worldwide [Rice , which is directly related to a large amount of physiological and biochemical processes and environmental factors ,4. To daOryza rufpogon Grif, DXWR) is a relative ancestor of the cultivated rice, which can be used as a donor of novel and favorable alleles for rice breeding [Dongxiang wild rice  are important steroid hormones, which play vital roles in the growth, development, and tolerance to various stresses in plants. The exogenous BR application can enhance low-temperature tolerance in plants ,21,22. Ie stress ,24,25,26e stress ,28,29,30In the current study, a backcross recombinant inbred lines (BRIL) population of 140 individuals, derived from a cross between the DXWR and a super rice SN265, was constructed. Based on this BRIL, a high-resolution genome-wide SNP genetic map was developed using the GBS technique. Furthermore, we identified some QTLs and candidate genes under cold stress and BR-combined cold treatment at the seedling stage. The results have an important significance for the discovery of the genes involved in cold tolerance and for understanding the molecular mechanism that regulates the cold tolerance related to the BR signaling pathway in rice.Cold tolerance of each BRIL individual was evaluated under cold stress and BR-combined cold treatment at the seedling stage. The BRIL population showed varying levels of cold tolerance and significant contrasting response in SSL, SRL, SDW, and SWW . SSL wasFor SDW and SWW, both cold stress and BR-combined cold treatment resulted in a significant decrease in SDW and SWW of the majority of BRIL individuals. The change in the number of BRILs with SDW in the range of 0.01–0.03 g and SWW in the range of 0.1–0.2 g were remarkable, respectively. Compared to cold stress, the number of BRILs with SDW in the range of 0.02–0.03 g and SWW in the range of 0.15–0.2 g increased under the BR-combined cold treatment, respectively d,e.p ≤ 0.01), SSL and SDW , SSL and SWW , SRL and SDW , SRL and SWW , and SDW and SWW  under cold stress, respectively. Under BR-combined cold treatment, the positive correlations between SSL and SRL  as well as between SDW and SWW  were shown similar to under cold stress. Additionally, under both cold stress and BR-combined cold treatment, all of the SSL, SRL, SDW, and SWW showed highly significant positive correlations with r = 0.19 (p ≤ 0.1), r = 0.19 (p ≤ 0.1), r = 0.43 (p ≤ 0.01), and r = 0.76 (p ≤ 0.01), respectively. These results suggested that all four traits showed continuous single-peak pattern distributions, which met the requirements for QTL mapping. Moreover, the treatment of BR improved the SSL, SDW, and SWW of some BRILs under cold stress.The correlation among all four traits with different treatments in the BRIL population was analyzed. The results revealed that all traits showed continuous and approximately normal distributions. Most of the traits showed a significant positive correlation regardless of cold stress or BR-combined cold treatment . For exaThrough the GBS approach, a total of 64.48 Gb high-quality filtered reads were filtered and 96.19% of these reads were mapped to the rice reference genome. Furthermore, a total of 10,836 SNPs were validated for the determination of recombinant events. The ratio of Q20 for BRILs was above 90% and the guanine-cytosine (GC) content was 43.55%, thus the quality of the data met the requirements for further analysis. A total of 10,836 unfiltered SNPs were validated for the determination of recombinant events. Finally, after filtration, followed by the ABH plugin, a total of 1145 bin markers were retained and used in the linkage map construction using the polymorphic markers to map all 12 rice chromosomes. All 12 linkage groups varied in the number and density of markers, with a total of 3188.33 cM in genetic distance and the individual linkage groups ranged from 186.74 to 346.46 cM in length. The interval of genetic distance of each polymorphic marker ranged from 1.9 to 146.3 cM and the average genetic distance between markers was 0.3 cM. The highest number of markers was 117 on chromosome 4, while the lowest number was 77 on chromosome 7 .The genetic linkage map was used to map the QTLs related to cold tolerance based on SSL, SRL, SDW, and SWW, respectively. A total of 114 QTLs were identified, of which 27 QTLs were detected under cold stress and 87 QTLs under BR-combined cold treatment. The maximum LOD score was 11.4737, while the maximum phenotypic variation explained (PVE) value was 18.2439% .qSSL1-2, qSSL3-1, and qSSL11-1) were localized in cold stress. Under BR-combined cold treatment, 4 QTLs  were identified using SSL under normal temperature condition as control, while 2 QTLs (qSSL2-2 and qSSL7-1) were localized using SSL under cold stress as control  were identified on chromosome 10 and 11 under cold stress. Under BR-combined cold treatment, only 2 QTLs (qSRL1-3 and qSRL8-1) were localized on chromosomes 1 and 8 using SRL under normal temperature condition as control, while 29 QTLs were localized on all chromosomes except for chromosome 8 with the control of SRL under cold stress. The LOD scores of all QTLs ranged from 3.0047 to 4.3086 and the PVE values ranged from 0.7550% to 9.4108%  were localized under cold stress. Under BR-combined cold treatment, 5 QTLs were localized with the control of SWW under normal temperature condition, while 15 QTLs were localized with the control of SWW under cold stress. Similar to SDW, the intervals between several pairs of QTLs under cold stress and BR-combined cold treatment were also coincident, including qSWW6-1 and qSWW6-2, qSWW6-3 and qSWW6-4, and qSWW6-6 and qSWW6-7, respectively  were used to predict candidate genes based on the Rice Genome Annotation Project website. A total of 156 candidate genes were predicted, of which 65 were annotated with known function, while the other genes were identified as retrotransposon and transposon proteins (40), expressed proteins (44), and hypothetical proteins (7), respectively .Among the 65 annotated genes, 13 were possibly involved in cold resistance or BR signal transduction pathway . They inTo further validate functions of the candidate genes, qRT-PCR was used to determine the expression levels of the 10 candidate genes under cold stress and BR-combined cold treatment in the parents and representative BRIL individuals (three excellent cold tolerance and three non-excellent cold tolerance).The expression levels of all transcription factor genes, including LOC_Os01g40260 (OsWRKY77), LOC_Os11g45740 (MYB), LOC_Os07g05805 (OsBZR1), LOC_Os07g08440 (bHLH), LOC_Os06g44750 (AP2), and LOC_Os05g39380 (zinc finger), were higher under both cold stress and BR-combined cold treatment than those under normal temperature in all of the materials mentioned above. For different treatments, the expression levels of LOC_Os07g05805 (OsBZR1) and LOC_Os07g08440 (bHLH) under BR-combined cold treatment were higher than those under cold stress. For the maternal and paternal parents, the expression levels of LOC_Os11g45740 (MYB) and LOC_Os07g08440 (bHLH) were higher in DXWR than in SN265 under both cold stress and BR-combined cold treatment, whereas the opposite expression pattern was monitored for LOC_Os07g05805 (OsBZR1). For the representative BRIL individuals, the expression levels of LOC_Os11g45740 (MYB), LOC_Os07g05805 (OsBZR1), and LOC_Os07g08440 (bHLH) in three excellent BRIL individuals were higher than those in three non-excellent BRIL individuals under cold stress as well as under BR-combined cold treatment, whereas the opposite expression pattern was detected for LOC_Os01g40260 (OsWRKY77) . In addiO. rufipogon) was discovered in Dongxiang County, Jiangxi Province, China, which has the northernmost distribution in all species of wild rice (28°14′ N). Since DWXR plays important roles in basic rice research and industrial development, it is known as “the panda of wild plants” [The rich genetic diversity is an important basis for improving the agronomic trait and stresses tolerance in plants. Previous studies regarding evolution and genetic divergence showed that cultivated rice was domesticated from wild rice species. However, during the domestication process with thousands of years of evolution and natural selection, the diversity of many morphological traits was reduced, and many gene resources were lost in cultivated rice ,31,32,33 plants” ,18,37. D2 or BC1 populations and the permanent primary genetic populations, such as recombinant inbred line were constructed and used for QTL mapping, they could show not only a low QTL detection efficiency, but also poor stability. Therefore, a stable and reliable mapping population is essential for utilizing wild rice [4F1). Furthermore, the BC4F1 population was self-crossed for eight times to obtain advanced BRIL populations (BC4F8). The back- and self-crossing for multiple generations reduced the genetic differences caused by different genetic bases between cultivated rice and wild rice, which made the result of QTL mapping more stable and reliable.Although wild rice and cultivated rice have the same genome type, they exhibit many differences in their genome sequences. If the temporary genetic populations, such as Fild rice ,33,38. Tild rice ,18,19,30DREB (C-repeat binding factors/dehydration-responsive element-binding proteins) transcriptional factor, ICE (inducer of CBF expression) activated factor, and diverse downstream functional proteins called cold-regulated (COR) proteins. The CBFs belong to the AP2/ERF  family of transcription factors, which are the most important in the ICE–CBF–COR pathway. The CBFs control the expression of COR genes in response to cold stress, while the ICE acts as a positive upstream regulator of CBF genes [It is well known that the low-temperature tolerance of plants is a complicated quantitative trait controlled by polygenes. To cope with low-temperature stress, plants modify their physiology, metabolism, and growth by means of signal transduction and expression regulation of many genes associated with cold tolerance when plants are exposed to low-temperature stress . A substBF genes ,40,41.CBF/DREB homologous genes (OsDREB1A-OsDREB1J) have been identified. Among them, OsDREB1A, OsDREB1B, OsDREB1D, OsDREB1F, and OsDREB1G enhanced cold tolerance in Arabidopsis or rice [DREB1G and OsDREB1J were identified as the candidate genes related to cold tolerance for three QTLs, qLOP2, qPSR2-1 and qSR8-3, using the DXWR populations as the donor parent, respectively [OrbHLH001, was isolated from DXWR, which enhanced cold tolerance when expressed in transgenic Arabidopsis [OsbHLH002, a homolog of ICE1 in rice, positively regulated cold tolerance by promoting the expression of OsTPP1, which encoded a key enzyme for trehalose biosynthesis [CBF genes by regulating the degradation of EIN3 and PIF3 [In rice, 10  or rice ,43. In tectively ,30. In abidopsis . Apart fbidopsis ,45. OsbHynthesis . Two F-band PIF3 ,48. In tqSSL7-1 under BR-combined cold treatment in the current study. The expression level of OsBZR1 was upregulated both under cold stress and BR-combined cold treatment, and it showed higher transcript levels in the excellent BRILs than the non-excellent BRILs under cold stress and BR-combined cold treatment. Moreover, BZR1 regulated other genes uncoupled with CBFs, such as WKRY6, PYL6, and SOC1, etc., to improve cold tolerance in Arabidopsis [CBF genes were attenuated [OsWRKY77 was upregulated in the sensitive genotype, suggesting its negative role in cold tolerance at the germination stage in rice [OsWRKY77 were upregulated both under cold stress and BR-combined cold treatment, and its expression levels in the excellent BRILs were lower than the non-excellent BRILs under cold stress and BR-combined cold treatment. In addition to the transcription factors and genes related to cold tolerance and BR signaling pathway mentioned above, some other genes related to cold tolerance were identified, such as zinc finger domain containing protein, auxin-induced protein, oxygen evolving enhancer protein, heat shock protein, etc. The precise function and regulatory mechanism of these genes still needs further investigation.Previous studies have shown that the exogenous application of BR can improve low-temperature tolerance at different growth stages in various plants, including rice ,28, maizbidopsis ,26. As a in rice . The expIn conclusion, many QTLs and candidate genes related to cold tolerance or BR pathway were identified by mapping analysis and qRT-PCR. This study provided a basis for further mining the genes involved in low-temperature tolerance or BR signaling pathway and investigating the mechanism regulating low-temperature tolerance in rice. Further studies were needed to thoroughly investigate whether these genes are associated with both cold tolerance and BR signaling pathway in rice.1 plants were backcrossed with the recipient parent SN265 for 4 times to construct the BC4F1 generation. Then, the BC4F1 population was used to develop a BRIL population with 140 individuals in F8 generation (BC4F8).A super rice variety SN265 was used as the recipient parent to cross with the donor parent DXWR. FEvaluation of cold tolerance at seedling stage was performed according to the previous studies with minor changes ,29,30. Fwww.R-project.org, accessed on 1 July 2022).All of the experiments for evaluating cold tolerance were repeated three times under the same conditions, and the average cold tolerance scores from three replicates were used for the QTL mapping analysis. R software 3.5.1  was used to analyze the frequency distributions and correlations of all 4 phenotypic traits . The Tassel software was used to analyze the raw data and high-quality SNPs between parents were termed by alignment with Nipponbare reference genome [Young leaves of 140 BRIL individuals and their parental lines were collected separately for genomic DNA extraction using a DNA extraction kit . DNA libraries from BRILs were constructed using the GBS technology. The restriction enzymes e genome .The Illumina data of BRIL population was used to construct a linkage map using the QTL IciMapping sofware v4.163  . The genp = 0.05. The threshold for the logarithm of odds (LOD) scores was set to 3.0. QTL nomenclature was followed by the method of McCouch et al. (2008) [QTLs were mapped using the inclusive composite interval mapping of the additive (ICIM-ADD) mapping method. QTLs were computed by a permutation test involving 1000 runs at a significance level of . (2008) .http://rice.plantbiology.msu.edu, accessed on 1 July 2022). The seedlings at the third leaf stage from 2 parental lines, 3 excellent cold tolerance BRIL individuals, and 3 non-excellent cold tolerance BRIL individuals at normal temperature (25 °C), for 12 h under cold stress (4 °C), and for 12 h under BR-combined cold treatment  were collected to extract total RNA using the Trizol reagent. Quantitative real-time PCR (qRT-PCR) was used to investigate the expression patterns of 10 representative candidate genes. Moreover, qRT-PCR was carried out using a q225 Real-Time PCR System  under the following conditions: 95 °C for 5 min, 30 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min, and 1 cycle of 72 °C for 15 min. Each sample was analyzed in three biological and three technical replicates. The relative expression levels were calculated via the ∆Ct method [To predict potential candidate genes within QTL intervals, the physical positions of SNP markers flanking the QTLs were searched in the Rice Genome Annotation Project website (t method . The act


Mycotoxin exposure in humans is primarily assessed through its occurrence in external sources, such as food commodities. Herein, we have developed a direct competitive ELISA to facilitate the detection of aflatoxin B1 (AFB1), deoxynivalenol (DON), fumonisin (FUM B1/B2), ochratoxin A (OTA), and zearalenone (ZEA) in human serum. The analytical validation of the assay followed practices endorsed by the international research community and the EU directive 96/23/EC in order to examine detection capability, recovery, and cross-reactivity. The assay demonstrated a lower limit of quantitation (LLOQ) for AFB1 [0.61 ng/mL (hereon ng/mL = ppb)], DON (19.53 ppb), FUM (4.88 ppb), OTA (19.53 ppb), and ZEA (0.15 ppb). Recovery from human serum for all mycotoxins spanned from 73% to 106%. Likewise, the specificity for monoclonal antibodies against cross-reactant mycotoxins ranged from 2% to 11%. This study compares the LLOQ and recovery values with commercial and emerging immuno-based methods for detecting mycotoxins in foodstuffs. The LLOQ values from the present study were among the lowest in commercial or emerging methods. Despite the differences in the extraction protocols and matrices, the recovery range in this study, commercial tests, and other procedures were similar for all mycotoxins. Overall, the assay detected AFB1, DON, FUM, OTA, and ZEA in human serum with excellent accuracy, precision, and specificity. Many fungal species like Penicillium, Fusarium, Aspergillus, and Alternaria produce secondary metabolites known as mycotoxins . MycotoxHuman biological monitoring (HBM) for mycotoxins is becoming essential due to the presence of mycotoxins in our food  and expoThe heterogeneous distribution of mycotoxin in food and the matrix effect during analytical detection can result in an underestimated exposure level ,22.Accurate data on food consumption and source for every individual is not always available to trace the origin and levels of exposure ,22.Mycotoxin in food does not always imply exposure as their bioavailability, food treatment or composition, and individual-to-individual differences are diverse .Climate change will present favorable growing conditions for fungi to promote AFB1 and DON contamination in maize and wheat in Europe . Thus, mIndividual HBM can help to circumvent the abovementioned challenges by directly measuring mycotoxins in human matrices ,24. AddiLC/MS/MS can only aid in directly determining unmodified mycotoxins that are available as reference substances and indirectly detecting metabolized mycotoxins that have been reduced to their free form . For theHerein, we propose using the direct enzyme-linked immunosorbent assay (ELISA) format to produce an accurate, sensitive, and selective multi-mycotoxin detection tool for human serum. The present study is critical because LC machines are expensive and not easily accessible  in univeThe results presented below provide an analytical proof-of-concept for using the direct competitive ELISA format for measuring AFB1, DON, FUM, OTA, and ZEA in human serum. The log10 concentrations were plotted on a logarithmic x-axis in The recovery percentages in Lastly, the cross-reactivity was 100% between the antibodies and their matching pair of mycotoxin standards , demonstThe risk of mycotoxin exposure in humans is predominantly assessed through its occurrence in external sources, such as food commodities ,29. HoweGuidelines for validating analytical methods that aim to detect mycotoxins in human body fluids do not exist. We have adapted analytical validation practices endorsed by the international research community  and the TM [® [The monitoring of mycotoxins in humans through ELISA is limited to AFB1 adducts with albumin or lysine ,32. As aTM ,47,48,49TM [® ,52,53,54TM [® ,57,58,59TM [® ,35,36,37® [The recovery percentages were unavailable or not indicated for the Cusabio , AgraQua® ,52,53,54® ,57,58,59® , and Jos®  assays d® ,47,48,49® ,47,48,49In In LC/MS/MS, which is a prerequisite for detecting multiple mycotoxins in humans , will imAFB1, DON, FUM B1, FUM B2, OTA, and ZEA reference materials were purchased from LGC standards GmBH, Germany, with a certificate of analysis confirming the purity of said mycotoxins using liquid chromatography with ultraviolet, fluorescence, or diode-array detectors. Nunc MaxiSorp™ ELISA plates with a plate shaker, recombinant protein G, methanol, acetonitrile, formic acid, and the Guardian™ peroxidase conjugate diluent were procured from Fisher Scientific, Vantaa, Finland. At 20 mg/mL concentration, FUM B1 and B2 were stored in 50% acetonitrile:water at 4 °C, and the remaining mycotoxins were stored in 100% methanol at −20 °C. Monoclonal antibodies against AFB1, DON, FUM B1/B2, OTA, ZEA, and their conjugates with horseradish peroxidase (HRP) were obtained for direct competitive ELISA from Creative Diagnostics, USA.SeramunBlock NP and 3,3′,5,5′ tetramethylbenzidine substrate (TMB) were acquired from Seramun GmBH in Germany and Neogen Corporation in the UK, respectively. Phosphate buffer saline  was prepared in-house using sodium chloride (137 mM), potassium phosphate monobasic (2 mM), disodium hydrogen phosphate (8 mM), and potassium chloride (3 mM) from Merck, Finland. To coat the recombinant protein G on the Nunc MaxiSorp™ ELISA plate, 100 mM carbonate buffer (pH 9.5) was prepared from sodium carbonate  and bicarbonate . Sera from a healthy human donor  was used to scrutinize mycotoxin recovery and the matrix effects. The study employed EL406 and Synergy H1 with Gen5 software  to wash and read the ELISA microplate.The optimal amounts of recombinant protein G, monoclonal antibodies, and HRP conjugated mycotoxins were determined through checkerboard titrations . The ideA 2% BSA  in 1× PBS functioned as the diluent for all mycotoxin standards and as a surrogate to the human matrix ,70,71. Mv/v, 1.2 mL) was added to 0.4 mL of spiked and blank human sera, vortexed for 10 sec, mixed through shaking at RT for 5 min, and centrifuged at 12,000× g 4 °C for 10 min [Acetonitrile with 1% formic acid  and www.mycurvefit.com (accessed on 29 September 2022) to fit regression curves. The four-parameter logistic (4PL) and Gaussian fits were employed to draw a non-linear relation between the mycotoxin concentrations in 2% BSA/PBS  and OD at 450 nm  [The study includes calibration curve data performed on three different days in duplicates and the use of  y-axis) ,76,77. T y-axis) ,34,35. I y-axis) ,76,77. IThe ‘My Assay’ program was used to obtain the coefficient of determination (R²), the LLOQ, and accuracy percentages at said LLOQs achieved through back-calculation. The coefficient of determination (R²) is the ratio of variation that the 4PL curve-fitting model explains to the total variation in the model ,78. The In Equation (1), a = curve peak height, e = Euler’s number, x = integer, b = position of the peak’s center, and c = standard deviation.In Equations (2)–(4), concentration refers to the log10 values predicted using the Gaussian curve and the associated coefficient values in Experiments for recovery and matrix effect were repeated on two different dates in duplicates for all the mycotoxins. Equation 2 and the coefficient of variation (%CV) were used to identify the accuracy and precision of the assay, which are expressed as average recovery (%) and repeatability (%CV). For matrix interference (Equations (3) and (4)), MF values < 1 and negative %ME were interpreted as matrix suppression, whereas MF values > 1 and positive %ME were interpreted as an enhancement. The assay was also performed to ascertain specificity and create a heatmap to demonstrate the cross-reactivity percentage using Equation (5), adapted from a previous publication .Cross-rIn Equation (5), OD refers to the optical density value at 450 nm with target or cross-reactant mycotoxins at a concentration that reduces the detection signal by at least 50%.


Systemic sclerosis (SSc) is an autoimmune rheumatic disease with high mortality, which is featured by inflammation, vascular damage, and aggressive fibrosis. To date, the pathogenesis of SSc remains unclear and effective treatments are still under research. Active NLRP3 recruits downstream proteins such as ASC and caspase-1 and assembles into inflammasome, resulting in excretion of inflammatory cytokines including IL-1β and IL-18, as well as in pyroptosis mediated by gasdermin D. Various studies demonstrated that NLRP3 inflammasome might be involved in the mechanism of tenosynovitis, arthritis, fibrosis, and vascular damage. The pathophysiological changes might be due to the activation of proinflammatory Th2 cells, profibrotic M2 macrophages, B cells, fibroblasts, and endothelial cells. Here, we review the studies focused on NLRP3 inflammasome activation, its association with innate and adaptive immune cells, endothelium injury, and differentiation of fibroblasts in SSc. Furthermore, we summarize the prospect of therapy targeting NLRP3 pathway. Systemic sclerosis (SSc) is an autoimmune disease characterized with inflammation, vascular damage, and fibrosis, in which the progressive fibrosis of skin and lungs is the most characteristic . EpigeneAs the first line against invading microbes and cellular damage, innate immune cells could be activated by pathogens or defective cells through various PRRs expressed on membranes or in cytoplasm. PRRs expressed on membranes are transmembrane proteins, including Toll-like receptors (TLRs) and C-type lectin receptors (CLRs). The cytoplasmic PRRs consist of retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), and NLRs . The hetSSc, as an autoimmune disease featured by inflammation, vascular damage, and fibrosis, involves immune cells, endothelial cells (ECs), and fibroblasts . StudiesThe activation of NLRP3 requires two signals. The first one is the priming signal initiated by TLRs, regulating gene expression and posttranslational modification . Once acNterm), central linker region, and C-terminal self-inhibition domain [Pyroptosis is a recently discovered type of programmed cell death which could be conducted by activated NLRP3 inflammasome . Differen domain , 27. Oncn domain .Besides the canonical NLRP3 activation which promotes caspase-1 activation, the non-canonical NLRP3 activation contributes to activation of caspase-4 (or caspase-11 in mice) and caspase-5 . Caspase+ efflux appears to be necessary for never in mitosis A-related kinase 7 (NEK7) activation and is confirmed to be an upstream event of NLRP3 activation [+ efflux before inflammasome activation, and the activation could be induced by low potassium independent of other triggers [+ concentration remains unchanged while NLRP3 activating in some research [2+ influx and Cl− efflux may also play a role in NLRP3 activation [2+ influx or Cl− efflux [Different from typical PRRs, NLRP3 appears not to directly sense DAMP or PAMP. Although the mechanism remains to be fully elucidated, there are some studies trying to illuminate how NLRP3 inflammasome gets activated. Transmembrane pore proteins are supposed to play a crucial part in initiating inflammasome assembly. Stimuli like nigericin, mostly bacterial toxins, are pore-forming compounds, which might permeabilize cell membrane for ion flows while ion redistribution seems to be the key to inflammasome activation . K+ effltivation , 38. Thetivation , 39, 40.triggers –43. Howeresearch . Other itivation , 46. But− efflux , 47. It Research indicates mitochondria damage may initiate NLRP3 activation by mitochondrial DNA (mtDNA) release and cardiolipin externalization . MitophaAlthough what ignites the onset of disease remains an issue, immunology dysfunction and vascular damage seem to be early pathological changes in SSc, as perivascular edema and infiltration of immune cells are seen ahead of fibrotic involvement , 54. ConCirculating monocytes could differentiate into fibroblasts and macrophages , and bot+ efflux [Th1 and Th2 are two classical subtypes of T helper (Th) cell. Th1 secretes IFN-γ and tumor necrosis factor (TNF)-α, showing proinflammatory function , while T+ efflux . NLRP3 d+ efflux , 80. TheMicrovascular dysfunction appears to play a prominent role at the early stage in SSc . An incrECs or endothelium injury gives rise to increased permeability as well as persistent vascular leakage and triggers the vascular microenvironment ischemia along with activating inflammation , 89. ECsExcessive fibrosis is the most characteristic pathological feature in SSc, which results from excessive synthesis and deposition of ECM. The fibroblast-to-myofibroblast transition (FMT) after activation by inflammatory factors and mechanical damage is a crucial procedure in fibrosis. Myofibroblasts feature increased excretion of inflammatory factors and ECM . ECM conRecently, the role of NLRP3 inflammasome in SSc fibrosis has attached great concern. High expression of NLRP3, caspase-1, IL-1β, and IL-18 and over-activated NLRP3 inflammasome were found in skin biopsy specimen from SSc patients and showed a positive correlation with modified Rodnan skin thickness score (mRSS), a measure of skin involvement, in which higher scores represent severe skin fibrosis . NLRP3 s2+ permeability responding to P2X7R activating, which might give rise to NLRP3 activation in SSc [Recently, studies showed more interesting mechanisms of SSc onset. Fibroblasts from SSc patients exhibited higher Can in SSc . MiR-155n in SSc , 98, andn in SSc .Musculoskeletal symptoms, involving joints, tendon, and muscle are quite common in SSc patients and contribute greatly in reducing quality of life , 101. ArRecently, research of treatments aiming at NLRP3 inflammasome signaling cascade shows a promising target for inflammatory diseases including SSc. MCC950, also known as CP-456,773, is a highly specific NLRP3 inhibitor which declines maturation and excretion of IL-1β through suppressing assembly of NLRP3 inflammasome , 108. Va+ efflux and ASC speck formation [Addition to small-molecule inhibitors, several endogenous molecules targeting NLRP3 are also of note. A20, an anti-inflammatory protein which is also known as TNF-α-induced protein 3 (TNFAIP3), shows a protective role in pulmonary fibrosis and it was reported to reduce NLRP3-mediated cytokine secretion and pyroptosis in RA , 118. Whormation . Intriguormation .Caspase-1 as the executor in NLRP3 inflammasome is also a remarkable target for NLRP3 block. YVAD-CHO is a reversible caspase-1 inhibitor, which reduces IL-1β excretion in SSc monocytes . TreatmePossible side effects should be of note as NLRP3 plays an important role in immune defense system. Treatment with MCC950 weakens early protective immune response towards influenza A virus . InfectiAs its function on mediating proinflammatory cytokines such as IL-1β and IL-18, NLRP3 is involved in many inflammatory immune-relevant diseases, including SSc. This review elaborate NLRP3 and its potential association with the autoimmune dysfunction, vascular damage, fibrosis of skin, and visceral organ in SSc. Despite contradictory conclusions, NLRP3 might play a potential role in SSc pathogenesis and offers promising insight into SSc treatment.


Origanum majoranum L. is a Lamiaceae medicinal plant with culinary and ethnomedical applications. Its biological and phytochemical profiles have been extensively researched. Accordingly, this study aimed to investigate the chemical composition and the antibacterial and antioxidant properties of O. majoranum high features, as well as to search for techniques for activity optimization. A metabolomics study of the crude extract of O. majoranum using liquid chromatography-high-resolution electrospray ionization mass spectrometry (LC ± HR ± ESI ± MS) was conducted. Five fractions  were derived from the total extract of the aerial parts. Different chromatographic methods and NMR analysis were utilized to purify and identify the isolated phenolics (high features). Moreover, the antimicrobial, antibiofilm, and antioxidant activity of phenolics were performed. Results showed that metabolomic profiling of the crude extract of O. majoranum aerial parts revealed the presence of a variety of phytochemicals, predominantly phenolics, resulting in the isolation and identification of seven high-feature compounds comprising two phenolic acids, rosmarinic and caffeic acids, one phenolic diterpene, 7-methoxyepirosmanol, in addition to four flavonoids, quercetin, hesperitin, hesperidin, and luteolin. On the other hand, 7-methoxyepirosmanol (OM1) displayed the most antimicrobial and antioxidant potential. Such a phenolic principal activity improvement seems to be established after loading on gold nanoparticles. Origanum is one of 200 genera of the Lamiaceae family, containing 3500 species worldwide. The vast majority of species are fragrant and grow naturally in the Mediterranean region [Origanum majoranum L., also known as Majorana hortensis Moench, is a tender perennial herb in the genus “Origanum” [p-cymene, sabinene, and trans-sabinene hydrate [n region ,2,3,4. Tn region ,7. Origariganum” . It is sriganum” . It is griganum” ,11,12. Priganum” ,15,16,17 hydrate . Various hydrate ,19, flav hydrate ,22.In natural product research, dereplication has been widespread, allowing for rapidly identifying known metabolites in complex combinations. ,24. It iOriganum majoranum will be initially assessed and dereplicated utilizing metabolomic analysis via liquid chromatography combined with high-resolution electrospray ionization mass spectrometry (LC-HRESIMS). Subsequently, we assess datasets for correlations between its previously reported antioxidant, antibacterial, and anti-biofilm efficacy, and the related chemical profile, as well as purification of its high features. Afterwards, in vitro activities will be investigated to identify the most promising metabolite(s) and how to optimize their efficacy via nanotechnology.This study intends to investigate the chemical and biological profiles of the plant as mentioned above as part of our ongoing research on it. In this approach, the secondary metabolites of The mass resolution in this current study was 50,000 (atm/z 400), which is sufficient to differentiate closely related metabolites. The total number of features found by LC-HRMS in OM extract is documented in In a target-based functional assay, crude hydromethanolic extracts of OM were active as antimicrobials and antioxidants ,30. Most1H and 13C NMR spectral analysis of purified metabolites are represented in All physical characteristics and O. majoranum purified metabolites (high features) were investigated against E. coli and S. aureus using the MTP assay. Results showed that all purified metabolites displayed low to moderate antimicrobial properties against all tested bacteria, with inhibition ratios ranging from 13.720% to 63.160%. In addition, compound OM1 exhibited the highest antibacterial activity with an inhibition ratio of 63.160%. To compare the inhibitory effects of OM1 and AuNPs-OM1 on the growth of microbes, different bacterial and fungal species were tested. The OM1 compound exhibited low to moderate antimicrobial activity toward all tested bacterial and fungal strains, with inhibition ratios ranging from 12.512% and 49.377%. Additionally, the antibacterial activity of OM1 was elevated after loading on gold nanoparticles (AuNPs), which caused the increase in the inhibition ratios against P. vulgaris and E. coli to be 24.419% and 50.658%, respectively. Moreover, AuNPs-OM1 exhibited better inhibitory activity against A. niger and C. albicans fungal strains than OM1, individually with inhibition ratios of 73.150% and 65.200%, respectively , and the biofilms of each of these bacteria were compared to the control (untreated biofilms). In preliminary antibiofilm experiments, the phenolic OM1 demonstrated limited antibiofilm activity against all tested bacteria with biofilm inhibitory activity up to 10.552%. Additionally, the AuNPs-OM1 reduced the biofilm formation of all strains, especially E. coli, by 30.02% (see Biofilm inhibition activity was examined using microtiter plates. The biofilm inhibition efficiency of the substances OM.02% see .1 has the highest DPPH scavenging activity (IC50 = 2.41 µg). In contrast, the lowest DPPH depletion was found in the OM5, revealing a low antioxidant “power” of this compound. The other phenolics, namely quercetin, rosmarinic acid, caffeic acid, luteolin, and hesperidin, showed similar scavenging activities to OM1, ranging from 65.63% to 89.38%. The AuNPs-OM1 did not potentiate the antioxidant activity of OM1 compared to antimicrobial and antibiofilm activities (scavenging activity = 55.50%)  see ; Table 51 was selected for loading on gold nanoparticles. Gold nanoparticles (AuNPs) were synthesized utilizing GSH in this study. Creating a covalent bond between the cysteine thiolate of GSH and the gold nanoparticles’ surface in the HAuCl4.3H2O mediates the synthesis. This interaction caused AuNPs to cluster together on GSH molecules, and the addition of NaBH4 at pH 8 resulted in the production of ruby-red AuNPs. The prepared AuNPs using GSH and NaBH4 exhibited a characteristic surface plasmon band (SPR) at 520 nm, and the surface plasmon resonance (SPR) absorption spectral range was from 385 to 540 nm. On the other hand, the plasmon band of the conjugate was also measured  and field emission scanning electron microscopy (FESEM). According to the TEM micrograph, the produced AuNPs had an average particle size of approximately 5.02 to 30.20 ± 25 nm, with a spherical shape , (2 0 0), and (2 2 0), respectively.XRD is considered the most important technique to study the structural properties of the prepared nanomaterials. Therefore, the prepared AuNPs were examined via the XRD diffraction pattern. 1, FTIR analysis is required. The FTIR spectra of the compound alone, OM1, and AuNPs-OM1 were recorded in the spectral region of 4000–400 cm−1 and are exhibited in For the characterization of functional groups presenting AuNPs and AuNPs-OMThe topic of oxidative stress and its control by antioxidants is receiving more attention than ever. In nutrition, many consumers and healthcare practitioners closely examine the antioxidant content of typical diet components . The pheOriganum majorana L. was previously tested against diverse fungi such as Aspergillus niger, Fusarium solani, Candida albicans, and A. parasiticus, and different bacteria such as Bacillus subtilis, B. megaterium, Escherichia coli, and Proteus vulgaris, as well as in vitro antioxidant activity against reactive oxygen species was evaluated. As a result, both antimicrobial and antioxidant assays suggested that the alcoholic extract of O. majorana can be used as an effective herbal protectant against different pathogenic bacteria and fungi and has a powerful antioxidant capacity toward various free radicals [The in vitro microbicidal activity of the alcoholic extracts of radicals ,38,39,40O. majorana extract were performed to identify various compounds and understand the leading causes of the previously reported antimicrobial and antioxidant potential. According to metabolomics and dereplication, it was clear that O. majorana extract possesses a high chemical diversity. In particular, there were eight phenolic compounds identified as high features (high intensity), namely 7-methoxyepirosmanol, rosmarinic acid, quercetin, caffeic acid, hesperitin, luteolin, apigenin, and hesperidin. Most of these metabolites were previously reported in O. majorana alcoholic extracts [To establish a reason for this result, metabolomics utilizing LC-HRMS and dereplication of extracts ,35. An eextracts ,42,43, wextracts ,45,46, wPolyphenolic-nanoparticle conjugates have recently been investigated for targeted medication activity augmentation. Nanoparticle-based drug delivery techniques such as vesicular drug delivery (liposomes), nanocrystals, nanoparticles, solid dispersion, and phospholipid complexes have been used to solve the challenges of poor solubility and low bioavailability ,49,50,51Gold nanoparticles (AuNPs) have been employed in a wide range of applications due to their highly tunable physicochemical features . Surface1 against several pathogens was substantially (p < 0.05) higher than that of free OM1. The bactericidal activities of AuNPs-OM1 against microorganisms were consistent with the findings of other researchers. In one investigation, azithromycin-loaded nanoparticles outperformed free azithromycin against S. Typhimurium [Escherichia aerogenes, M. luteus, P. aeruginosa, S. enterica, and for 20 days, compared to free nisin, which had antibacterial action for 6 days [Loading 7-methoxyepirosmanol on nanoparticles exhibited an optimization result for both antimicrobial and antibiofilm activities but not for antioxidant scavenging activity. To our knowledge, gold nanoparticles have not exerted any role in the alteration of the antioxidant capacity of various reducing agents but have been considered analytical tools for antioxidant capacity assessment . Moreovehimurium . Nisin-lr 6 days . That efr 6 days .O. majoranum (OM) plant were obtained in March 2021 from a field near Elwasta Capital, Beni Suif, Egypt. Professor Abdel-Halim A. Mohammed, Horticultural Research Institute, Department of Flora and Phytotaxonomy Research, Dokki, Cairo, Egypt, certified the plant’s authenticity. A voucher specimen (2021-BuPD 57) was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Beni Suif University.The leaves of O. majoranum powdered plant (1 kg) was macerated with 80% MeOH at room temperature and then concentrated under reduced pressure using a rotary evaporator  to a syrupy consistency. The concentrated methanolic extract yielded 60 g, and the dried extract was stored at 4 °C for in vitro and metabolomic studies.O. majoranum powder was subjected to metabolomic analysis using the LC-HRESIMS technique, detailed in According to Hifnawy et al. , the extThe concentrated methanolic extract of OM (170 gm) was suspended in distilled water (500 mL) and extracted with petroleum ether, DCM, EtOAc, and BuOH, in that order. Under reduced pressure, the organic phase of each step was evaporated individually to afford the corresponding fractions OM-I (0.6 g), OM-II, (35 g) OM-III (18 g), and OM-IV (60 g), respectively. The resulting EtOAc (OM-III) and BuOH (OM-IV) fractions were kept at 4 °C for the phytochemical investigation.1–OMI10). Sub-fraction OMI5 was washed several times with chloroform to afford compound OM1 (96 mg).On a silica gel column , a portion of fraction OM-I (600 mg) was fractionated. First, elution was carried out utilizing a petroleum ether-EtOAc gradient mixture in order of increasing polarity , then EtOAc-MeOH (80:20), (50–50), and finally MeOH 100%. The effluents were then collected in test tubes (20 mL), concerning each fraction of 200 mL. Afterwards, each resulted fraction was concentrated and visualized by TLC. Similar fractions were grouped and concentrated under reduced pressure to provide 10 sub-fractions , then with EtOAc-MeOH (80:20), (50–50), and finally with MeOH 100%. The effluents were separated into fractions of 400 mL each, which were then divided into test tubes (20 mL), and each fraction was concentrated and monitored using TLC. Six sub-fractions (OMIII1–OMIII6) were created by grouping similar fractions together and concentrating them under reduced pressure. Next, a portion of subfraction OMIII4 (2 g) was fractionated under the same conditions as fraction OM-III, yielding OMIII4-F4 and OMIII4-F5, the latter of which is pure compound OM2 (105 mg). OMIII4-F4 was then subjected to a Sephadex LH-20 column  using MeOH-H2O (8:2) to afford compounds OM3 (32 mg), OM4 (43 mg), OM5 (54 mg), and OM6 (102 mg).On a silica gel column , a portion of fraction OM-III (15 g) was fractionated. First, elution was carried out utilizing CHCl2O gradient mixtures in order of decreasing polarity . The effluents were collected in various fractions (600 mL each), and each resulted fraction was concentrated and visualized by TLC. To create 16 sub-fractions (OMIV1–OMIV16), similar fractions were clustered together and concentrated at reduced pressure. OMIV6 (200 mg) was then subjected to chromatographic separation using a Sephadex LH-20 column  using MeOH-H2O (8:2) to yield OMIV6-F3, which was afforded to be compound OM7 (24 mg). All chemicals, reagents, and apparatus are mentioned in On a polyamide-6 column , a portion of fraction OM-IV (10 g) was fractionated. Afterwards, elution was carried out utilizing MeOH-HProteus vulgaris, Salmonella typhimurium, and Escherichia coli ATCC 25955), one Gram-positive bacteria (Staphylococcus aureus NRRL B-767), and two yeasts  were used as test organisms and antibacterial tests were performed [600 in a Spectrostar Nano Microplate Reader .To test pure compounds for antibacterial activity, three Gram-negative bacteria (erformed . The expStaphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (Pseudomonas areuginosa and Escherichia coli) [w/v) was applied to each well for 1 h, then the surplus stain was removed, and the plates were retained for drying. Furthermore, dried plates were washed with 95% ethanol, and then optical density was evaluated at an optical density of 570 nm using a Spectrostar Nano Microplate Reader .The 96-well flat polystyrene plates were used to test the biofilm inhibitory activity of compound OM1 and AuNPs-OM1 against four clinical microorganisms, including Gram-positive bacteria (ia coli) . In brieThe DPPH free radical scavenging experiment was used to assess the antioxidant activity of various metabolites . A fresh2 = 0.999) suggested that the model was well-fitting. The kinetic measurements for each antioxidant investigated were performed using the spectrophotometer model Cary Bio 100 . Moreover, the sample chamber’s temperature was kept under control using a Peltier device incorporated into the chamber. In the literature, DPPH radical scavenging by H atom-donating antioxidants has been described utilizing at least two methods: (a) the fixed reaction time approach and (b) the steady-state saturation method. We tested both strategies to compare their outcomes.The linear regression (r4 (2 mg/mL) under strong stirring until the ruby-red color formed. The supernatant was declined after centrifugation, and the gold nanoparticles were distributed in water before centrifugation was performed again to obtain clean AuNPs.Gold nanoparticles were prepared as described by Wu et al., .50 mL of3+ to Au0 was tracked by regularly sampling aliquots (1 mL) of the mixture and analyzing the UV-vis spectra of the solutions with a SPECTROstar Nano Absorbance Plate Reader (BMG LABTECH). Finally, the gold nanoparticle solution was drop-coated onto a glass substrate, and the X-ray diffraction patterns were recorded using a PANalytical X’pert PRO X-ray diffractometer (The Netherlands) with Cu Ka1 radiation at 40 kV and 30 mA, respectively. Further, the diffracted patterns were captured at 2θ with the scanning speed of 0.02°/min from 10° to 80°. According to Brock-Neely, the ATR-FTIR spectra (Thermo Nicolet 380) of gold nanoparticles were obtained utilizing Broker vertex 80 v in the range of 4000–400 cm−1 with a resolution of 4 cm−1 (1957) [The formation of AuNPs was initially monitored by a color change of the solution. Then, the transition of Au1 (1957) .Transmission electron microscopy (TEM) and scanning electron microscopy were used for analysis of the size and morphology of the produced gold nanoparticles and conjugate. First, 2–4 µL of gold nanoparticle solution was placed on carbon-coated copper grids for sample preparation. Next, the thin film was formed and air-dried under ambient circumstances and detected using Philips 10 Technai with an accelerating voltage of around 180 keV with a wavelength (λ) of 0.0251 Å. Next, scanning electron microscopy (SEM) was used to detect the elemental analysis, with a Field Emission Scanning Electron Microscope (FE-SEM)  acceleration voltage of 20 kV, attached with EDAX .1 and the prepared AuNPs was carried out according to Sulaiman et al. [1 (500 μg mL−1) and stirred at room temperature overnight. The conjugated AuNP-OM1 was centrifuged for 1 h at 10,000 rpm after preparation to eliminate excess OM1.Conjugation of compound OMn et al. , wherebyO. Majoranum, particularly of its metabolites purified from ethyl acetate and butanol fractions. Furthermore, metabolomic and phytochemical investigations of the plant revealed its ability to accumulate and biosynthesize several secondary metabolites, and primarily phenolics, implying their involvement in O. majoranum’s previously reported antibacterial and antioxidant activities. As a result, O. majoranum’s previously noted antibacterial ability may be partly attributed to the combined effects of these phytochemicals and/or their synergistic interactions. The antibacterial study confirms that the AuNPs-OM1 is more effective at controlling the development of the microorganisms tested and in bacterial biofilm inhibition compared with free OM1 (the most active compound). After loading the 7-methoxyepirosmanol in gold nanoparticles, the higher antibacterial activity could be attributed to increased cell penetration and uptake. These discoveries may assist in broadening the potential of this plant in future phytotherapy. Given its dietary supplementation and reported edibility, O. majoranum may be considered to protect against a variety of disorders. In the near future, more research into the cellular mechanisms and molecular aspects of O. majoranum’s antibacterial and antioxidant properties, as well as its phenolic metabolites, is required.The present work revealed the antimicrobial and antioxidant effects of the aerial parts of


Siniperca chuatsi rhabdovirus (SCRV), are epidemic pathogens that harm fish aquaculture. To clarify the interactions between SCRV and its host and explore antiviral targets, the present study performed transcriptome analysis in a cultured S. chuatsi skin cell line (SCSC) after SCRV infection at 3, 12, 24, and 36 h post-infection (hpi). Comparison with control obtained 38, 353, 896, and 1452 differentially expressed genes (DEGs) in the detected time points, respectively. Further analysis of the Go terms and KEGG pathways revealed the key pathways “Cytokine-cytokine receptor interaction” and “interferon related pathways” in SCSC cells responding to SCRV infection. The significantly up-regulated genes in the pathways were also verified by qPCR. Furthermore, gene cloning and overexpression revealed that five interferon-stimulated genes (ISGs) IFI4407, IFI35, Viperin, IFIT1, and IFIT5 had the ability to inhibit SCRV replication in FHM (Fathead minnow) cells, especially an inhibition efficiency more than 50% was observed in IFI35 overexpressed cells. In summary, current study revealed the main innate immune pathways in S. chuatsi cells induced by SCRV infection and the major ISGs of S. chuatsi in controlling SCRV replication.Fish rhabdoviruses, including  Siniperca chuatsi  is an economically important fish cultured in China with high market value, and it belongs to the family Serranidae in the order Perciformes [S. chuatsi aquaculture industry has been hampered by diseases caused by viruses such as rhabdoviruses [Aquaculture has become one of the fastest-growing agricultural and food production industries in the world . The culciformes ,4. Howevoviruses .Rhabdoviridae contains viruses with a bullet-shaped virion and negative-sense single-stranded RNA genome [S. chuatsi rhabdovirus (SCRV) was first isolated from diseased mandarin fish in 1999 in China [S. chuatsi. For example, Huang et al. investigated the transcriptome responses of S. chuatsi at 24 h (h) and 60 h post-infection (hpi) with SCRV [S. chuatsi cell line during SCRV infection are still lacking. More research on the genes of S. chuatsi that function against SCRV infection is still needed.The family A genome . S. chuain China , which hin China . Isolatein China . Knowledith SCRV , which rith SCRV . HoweverS. chuatsi skin cell line (SCSC), which showed high sensitivity to SCRV infection [Recently, we established a nfection . In the S. chuatsi [The SCSC cell line and the FHM (Fathead minnow) cell line have been preserved in our laboratory ,14. SCRV chuatsi . In the For preparing virus stocks, SCSC cells were infected by SCRV at a MOI (multiplicity of infection) of 0.1. The cells were harvested when about 80% of cells had a cytopathic effect (CPE). After freezing and thawing three times, they were divided into small pieces and incubated at −80 °C until use. For preparing the infected cells used in RNA-seq, SCSC cells were infected by SCRV prepared above at a MOI of 1, and then were collected at 0, 3, 12, 24, and 36 h post-infection (hpi) and subjected to RNA extraction.n = 3) was extracted by using TRIzol reagent . RNA quality, including purity, integrity, and concentration, was examined by using a NanoDrop 2000 spectrophotometer  and electrophoresis. The TruseqTM RNA sample prep kit  was used to construct libraries with the RNA samples with high quality. In brief, mRNA was enriched with poly-T magnetic beads and then fragmented. cDNA was synthesized, and their cohesive ends were repaired. Adapters were ligated to the 3′ ends after the addition of adenosines. PCR was used to enrich the cDNA fragments, and the products were purified by electrophoresis and quantified using Qubit 2.0 and Agilent 2100 bioanalyzer. Then, the libraries were sequenced on MGI DNBseq-T7 in 150 bp paired-end mode, and raw data reads were generated.Total RNA of the collected cells  was performed with HISAT2 (v2.1.0) software. Expression analysis was performed with RSEM software, and the number of reads mapped to each transcript of each sample was obtained, then, FPKM (Fragments Per Kilobase Per Million bases) conversion was performed.Poor-quality reads, adapters, and poly-N were deleted from the raw reads by using software Trimmomatic to obtain clean reads. Data quality was evaluated by calculating the Q20, Q30, and GC contents. Aligning the clean reads to the reference genome of p-values were corrected by using multiple corrections, including Bonferroni, false discovery rate, Holm, and Sidak. DEGs with a corrected p-value ≤ 0.05 were considered significantly enriched.DESeq2 package was used to perform the differential expression analysis based on aligned transcripts (unigenes) obtained above and expression levels obtained from RSEM software. The unigenes with |log2Fold Change| (|log2FC|) > 1 and FDR < 0.05 were assigned as differentially expressed genes (DEGs). Gene ontology (Go) annotation and pathway analysis were performed using blast2go software and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, respectively. Goatools and KOBAS were used to perform enrichment analyses of Go terms and KEGG pathways based on Fisher’s exact test, respectively. S. chuatsi was used as an internal control. The relative expression ratios of the genes were calculated by the 2−ΔΔCT method. The genes and primers used in RT-qPCR were collected in The genes involved in chemokine or interferon (IFN) signaling pathways were selected for real-time quantitative PCR (RT-qPCR) analysis. The samples used for RNA extraction were collected as described above. The HiScript III RT SuperMix for qPCR (+gDNA wiper)  was used to perform first-strand cDNA synthesis. RT-qPCR was performed in a Quantagene q225MX Real-Time PCR Instrument . Each qPCR mixture contained 1 μL of cDNA, 5 μL of SYBR Premix (2×), 1 μL of primers (0.5 μL for each primer), and 3 μL of ultrapure water. The qPCR conditions were conducted as described previously . The β-a07, IFI4408, IFI35, Viperin, IFIT1, and IFIT5) were amplified with Pfu DNA polymerase  using the cDNAs as a template. We noted that there were two genes (LOC122878107 and LOC122878108) both encoding for IFI44, and we named the two ISGs IFI4407 and IFI4408 based on the last numbers of the two genes. PCR products were ligated into pEASY-Blunt cloning vector  and sequenced. The verified products were then amplified and ligated into pcDNA3.1-2 × HA vector by infusion cloning. The vector gives a 2 × HA tag on the N-terminal of the cloned ISGs, which makes it easy for expression detection by Western blot. The finally obtained plasmids were sequenced to ensure they were correct.For cloning of the genes encoding ISGs, cDNA was synthesized by using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper)  using the RNA described above. The coding sequences of the ISGs . For most of the samples, more than 91% of the reads could be mapped to the genome, except S12-3, which had a map rate of 84.57%. In addition, more than 96% of the mapped reads were located on the exons region of the genome. The mapped results showed that a total of 26,250 genes was expressed in SCSC cells under SCRV infection, with about 20,000 genes expressed in each sample. Subsequent analysis was based on the expressed genes.To further investigate the response of the SCSC cells under SCRV infection, we performed transcriptome analysis with SCRV-infected SCSC cells. The time points 0, 3, 12, 24, and 36 hpi were selected to reveal the responses. A total of 15 libraries were constructed and sequenced, which generated 151.23 Gb of clean data with more than 8.06 Gb for each sample . The Q30The DEGs at different time points  were analyzed by comparison with the samples at 0 hpi. The results obtained 38 (13 up-regulated), 353 (168 up-regulated), 896 (634 up-regulated), and 1452 (997 up-regulated) DEGs in the 3 (S3 vs. S0), 12 (S12 vs. S0), 24 (S24 vs. S0), and 36 (S36 vs. S0) hpi groups .Go and KEGG pathway enrichment were further performed to analyze host responses based on the DEGs. There were 25, 20, 30, and 32 significantly enriched Go terms in the groups of S3 vs. S0, S12 vs. S0, S24 vs. S0, and S36 vs. S0, respectively . The topFor KEGG pathway enrichment , only fobmp6 (encoding bone morphogenetic protein 6) and tgfb3 (encoding transforming growth factor beta-3 proprotein) were significantly down-regulated from 12 hpi to 36 hpi. Expression of il11a (encoding interleukin 11a) were significantly down-regulated at the four examined time points. Another 19 cytokine and cytokine-receptor-related genes were up-regulated, especially at 36 hpi. The top up-regulated cytokine genes included LOC122880389 , LOC122888056 , LOC122875308 , LOC122875929 , LOC122884604 , and LOC122873767 , while the top up-regulated cytokine receptor genes included LOC122881975 , LOC122881976 , and LOC122885641 .The KEGG pathway “Cytokine-cytokine receptor interaction” was significantly enriched in the groups of S12 vs. S0, S24 vs. S0, and S36 vs. S0. The DEGs in the pathway were further collected in IL12ba was not obvious until 36 hpi. The other two examined genes, IL11a and IL15, were down-regulatory expressed after SCRV infection. Although the elevated levels of several genes, such as CXCL11, were higher than those detected in transcriptome analysis, the changing trend of the genes between RT-qPCR and transcriptome is consistent. Different expression levels detected between RT-qPCR and transcriptome could be caused by sample collection.Expression of several cytokine and cytokine-receptor-related genes, encoding for CXCL11, CXCR2, CXCL6, IL11a, IL8, CXCL10, CXCL9, IL12ba, and IL15, were further examined by RT-qPCR in SCRV-infected cells . The gendhx58, encoding LGP2; ifih1, encoding MDA5; nlrc5; ptx3a), interferon-regulatory factors (IRFs) , and ISGs. In the transcriptome analysis, these genes were not significantly up-regulated at 3 hpi, and most of these were significantly up-regulated at 24 and 36 hpi.Considering the important roles of IFN in antiviral responses, the significantly enriched DEGs on PRRs (Pattern Recognition Receptors), IFN, and IFN stimulated genes (ISGs) were collected in irf9 and irf10 were up-regulated from 12 hpi, and irf7 was up-regulated from 24 hpi. For the ISGs, most of them were significantly up-regulated from 12 hpi, while the genes encoding for viperin and IFIT1 were up-regulated from 3 hpi. The two ISGs also had the highest elevation during SCRV infection.Expression of the IRFs and some ISGs was further examined by RT-qPCR in SCRV infected cells. As shown in 07, IFI4408, IFI35, Viperin, IFIT1, and IFIT5) were cloned successfully from SCRV-infected SCSC cells. A 2 × HA tag was fused on the N-terminal of these ISGs in favor of the expression detection. FHM cells were used in the overexpression assay for its high transfection efficiency. As shown in 07. SCRV replication was detected using RT-qPCR in the overexpressed cells. As shown in 07-, IFI35-, Viperin-, IFIT1-, and IFIT5-overexpressed FHM cells. Among them, IFI35 had the highest inhibition efficiency (more than 50%), although Western blotting showed that the expression level of IFI35 was the lowest. In general, the overexpression of several ISGs coming sfrom SCSC inhibited SCRV replication in FHM cells.To investigate the effect of these significantly up-regulated ISGs on SCRV infection, the ORFs encoding six ISGs . NLRC5 has been reported to be a negative regulator in NF-κB signaling pathway and activator of MHC class I genes [Innate immune responses rely on the recognition of pathogen-associated molecular patterns (PAMPs) by the PRRs . It has  I genes .IFNs are a family of cytokines that play critical roles against virus invading in innate immunity, whose expression is regulated by IRFs ,28. The 07, IFI4408, IFI35, Viperin, IFIT1, and IFIT5) from SCSC cells and examined the antiviral effect in overexpressed FHM cells. The function of the IFI44 in mammalian cells is divergent. It has been reported that IFI44 restricted the replication of respiratory syncytial virus but supported influenza A virus replication [Carassius auratus herpesvirus-infected gibel carp [07) showed antiviral capacity in FHM cells in our overexpression assay. It should be noted that there were two protein bands in IFI4407 over-expressed cells. Because the 2 × HA tag was fused on the N-terminal of the ISG, the two protein bands could not be fround from a different translation start site. We speculated that the two bands might be caused by the processing of mRNA or translated protein, but it needs to be proven in future studies, as do the functions of the two products.ISGs are genes induced during IFN response, which have functions in antiviral defense, antiproliferative activities, and stimulation of adaptive immunity . In the lication ,37. The bel carp . The preS. chuatsi IFI35 inhibited the replication of SCRV in SCSC cells.IFI35  has been reported as a proinflammatory factor during cellular infection . For fisS. chuatsi. The IFN-induced protein with the tetratricopeptide repeats (IFITs) family contains several members including IFIT1, IFIT2, IFIT3, IFIT4, and IFIT5 [It has been reported that viperin has the ability to act against various viruses via multiple mechanisms . The antnd IFIT5 . IFIT1 (nd IFIT5 ,49,50,51nd IFIT5 .07, IFI35, Viperin, IFIT1, and IFIT5 in anti-SCRV. Thus, these results provide new information in the research on SCRV infection and can be beneficial to the antiviral exploration in the aquaculture of Mandarin fish.In summary, the present study analyzes the important cellular responses and antiviral genes of a new cell line SCSC under SCRV infection by transcriptome and RT-qPCR, which revealed several key pathways in SCRV infection, such as the pathways of “Cytokine-cytokine receptor interaction” and “interferon related pathways”. Further functional analysis verified the major ISGs including IFI44


SO2 can be endogenously produced and rapidly transformed into sulfur-containing compounds  to maintain its homeostasis in plant cells. Exogenous application of SO2 in the form of gas or solution can trigger the expression of thousands of genes. The physiological functions of these genes are involved in the antioxidant defense, osmotic adjustment, and synthesis of stress proteins, secondary metabolites, and plant hormones, thus modulating numerous plant physiological processes. The modulated physiological processes by SO2 are implicated in seed germination, stomatal action, postharvest physiology, and plant response to environmental stresses. However, the review on the signaling role of SO2 in plants is little. In this review, the anabolism and catabolism of SO2 in plants were summarized. In addition, the signaling role of SO2 in seed germination, stomatal movement, fruit fresh-keeping, and plant response to environmental stresses  was discussed. Finally, the research direction of SO2 in plants is also proposed.Sulfur dioxide (SO SO2 can dissolve in water and form sulfurous acid (H2SO3), which in turn dissociates into sulfite (+). In neutral solution, the ratio of 2 mainly roots in three derivants, that is, +. + can lower the pH value of the cells and their compartmentations, followed by affecting the activities of the enzymes and interfering with the cellular metabolism (2 can induce the accumulation of reactive oxygen species (ROS) by oxidizing sulfite into sulfate and/or activating NADPH oxidase (NOX), which in turn cause biomembrane damage, protein disintegration, DNA damage, chromosome aberration, gene mutation, Golgi body destruction, and programmed cell death (PCD) , growth inhibition, and even plant death  . Therefont death .2, as a signaling molecule, can endogenously produce by the catalysis of aspartate aminotransferase (AAT) and NOX using hydrogen sulfide (H2S) as substrate in animal system . Therefore, the abnormal production of endogenous SO2 commonly leads to colitis, hypertension, atherosclerosis, neuronal damage, vascular calcification, myocardial hypertrophy, myocardial injury, pulmonary hypertension, and acute lung injury , ROS, nitric oxide (NO), and H2S, exhibit the common characteristics: small molecule, fast dispersal, dual effects, controllability of generation and elimination, biological activity, reprogramming gene expression, and so forth  or solution , at low concentrations, has been found to exert a positive role in seed germination  were concluded. The aim of this paper highlighted that SO2 is an emerging signaling molecule in plants.In plants, SOeduction . Recentlmination , stomatamination , fruit fmination , and plamination . These s2 homeostasis in plants is maintained by its production  and elimination  by APS reductase (APR) using glutathione (GSH) as reducing agent  and its derivant thiosulfate (TS) under the catalysis of NOX, sulfide oxidase (SO), and TS sulfurtransferase (TST), respectively  or solution  form could reprogramme the expression of thousands of genes. The physiological functions of these genes include antioxidant defense, osmotic adjustment, cell wall modification, and the synthesis of stress proteins , secondary metabolites, and plant hormones , and plant hormones  facilitated seed germination and increased seed vigor. In addition, the SO2-pretreated germinating seeds had higher NOX activity and ROS level, while NOX inhibitor, diphenyleneiodinium, inhibited ROS accumulation and germination and vigor of maize seeds pretreated with SO2  postponed PCD, restrained the coalescence of small protein storage vacuoles, and reduced the accumulation of ROS  in aleurone cells pretreated with gibberellin , guaiacol peroxidase (POD), ascorbate peroxidase (APX), and superoxide dismutase (SOD), while had lower activities of lipoxygenase (LOX) and polyphenol oxidase  accelerated PCD in both SO2– and H2S-pretreated aleurone cells  seeds, pretreatment with SO2 donor  alleviated PCD induced by gibberelin in aleurone layers in a concentration-dependent fashion , while weakened that of LOX, reduced the levels of H2O2, O2–, and malondialdehyde (MDA) in aleurone layers , H2S, and plant hormones , treatment of epidermal strips with the different concentrations (0 ~ 5 mM) of Na2SO3/NaHSO3 solutions (SO2 donor) rapidly increased the levels of endogenous H2S and NO, and then induced stomatal closure in a dose-dependent manner (2 was reversed by hypotaurine (H2S scavenger) and cPTIO , low concentrations (0.0001 ~ 0.1 μM) of sulfurous acid  facilitated stomatal opening by reducing the level of endogenous ABA to antagonize its action  influences photosynthesis, respiration, and transpiration, as well as plant resistance to environmental stress, especially drought stress . Stomatat manner . In addis action . Adversenous ABA .Hemerocallis fulva, the epidermal strips treated with the different concentrations (1 ~ 5 mM) of SO2 donor (Na2SO3/NaHSO3) were found to reduce the guard cells vigor and induce cell death in a concentration-dependent manner , indicating SO2 could trigger PCD in guard cells. Additionally, the levels of endogenous NO, ROS, and Ca2+ were increased by SO2, while reduced by NO scavenger (cPTIO), nitrate reductase (NO-producing enzyme) inhibitor (NaN3), ROS scavengers , Ca2+ chelating agent (EGTA), and plasma membrane Ca2+ channel blocker (LaCl3), thus decreasing cell death  induced guard cell death in a dose-dependent fashion  and synthesis inhibitors (L-NAME and tungstate) weakened SO2 toxicity , Ca2+ chelating agent (EGTA), and Ca2+ channel inhibitor , stomatal index (SI), and SD/SI ratio in Lepidozamia hopei, Lepidozamia peroffskyana, Ginkgo biloba, Nageia nagi, Podocarpus macrophyllus, Araucaria bidwillii, and Aagathis australis.Similarly, in toxicity . Likewisr LaCl3; . Also, tmulation . These dDimocarpus longan Lour. cv. Daw) fruits, SO2 fumigation  reduced pericarp browning, lengthened shelf-life, and improved the quality of fruits by enhancing antioxidant capacity compared with the control without SO2 fumigation  inhibited fruit decay and reduced the levels of H2O2 and MDA compared to the control without SO2 treatment -lyase, as well as the content of Cys  was up-regulated by SO2 treatment in table grapes, while the transcription level of VvHPCA1-4 and VvHPCA3  was down-regulated  improved the survival percentage and relative water content (2 could increase drought tolerance. The further experiments showed that SO2 pretreatment improved the content of proline (Pro) and activities of SOD and POD, which in turn reduced the accumulation of H2O2 and MDA in drought-treated wheat seedlings. In addition, SO2 pretreatment down-regulated the expression of TaNAC69 (transcription factor gene), while the expression of other transcription factor genes (TaERF1 and TaMYB30) insignificantly changed but maintained a higher levels in wheat seedlings under drought stress conditions  decreased stomatal apertures and leaf transpiration rate, which in turn improved the relative water content in the leaves of drought-stressed seedlings (2 pretreatment increased the activity of Δ1-pyrroline-5-carboxylate synthetase (P5CS), reduced that of Pro dehydrogenase (ProDH), and corresponding gene expression, followed by accumulating Pro in the leaves of drought-stressed seedlings  by scavenging H2O2  increased SOD, POD, and O-acetylserine(thio)lyase (OASTL) activities and GSH, Cys, and nonprotein thiol (NPT) contents in Arabidopsis plants, which in turn alleviated oxidative stress , followed by promoting the synthesis of anthocyanin. Interestingly, SO2 pretreatment also activated the expression of genes  involved in C-repeated binding factor (CBF) signaling pathways, mainly cold-response signaling pathways  and freezing stress . Both chilling and freezing stress can trigger osmotic and oxidative stresses . Therefofficient . Similarous H2O2 . Meanwhipathways .2 (30 mg m−3) exposure reduced stomatal aperture and ROS accumulation by enhancing POD activity under cold stress  to the different concentrations of SO2 increased O2– generation rate and H2O2 content in Arabidopsis shoots, and up-regulated the gene expression of POD, SOD, and glutathione peroxidase (GPX), as well as increased the activity of corresponding enzymes and the content of GSH. Additionally, SO2 exposure increased isoenzymatic isoforms of SOD (FeSOD and Cu/ZnSOD) and POD, while decreased CAT isoforms  down-regulated the expression of miR398, which in turn increased the transcript level of its target genes Cu/Zn-SOD (CSD1 and CSD2) and increased the activity of SOD  and sulfate transporter , implying miR398 and miR395 participate in the resistance of Arabidopsis plants to oxidative stress induced by SO2 exposure. These data suggest that SO2-induced ROS act as a signal to trigger plant defense response.Parallelly, SOd stress . Also, Spathways , thus imy of SOD . Similar2 (Na2SO3/NaHSO3 (3/1) solution as SO2 donor) in a concentration-dependent manner and the optimal concentration was between 1 and 2 mM  and total soluble protein (TSP) in germinating seeds under Cd stress , mercury, arsenic, and aluminum, have become the major pollutants with the development of modern agriculture and industry. Heavy metals can disturb cellular metabolism, inhibit seed germination and plant growth, and even influence on human health by entering into food chain . Therefoand 2 mM . Additiod stress . Also, Sd stress . Furtherat seeds . Interesat seeds . These d2 derivatives (0.5 mM) reduced the Cd-inhibited seedling growth and Cd-induced oxidative damage  in the leaves of seedlings  by promoting sulfur assimilation, followed by enhancing Cd-detoxification capacity in seedlings. Further experiments showed that SO2 derivatives down-regulated the expression of genes related to Cd uptake and translocation  at 1.2 mM increased the activities of POD, CAT, and APX, and lowered that of LOX, and reduced the accumulation of O2–, H2O2, and MDA in germinating seeds under aluminum stress  in seedling radicles under aluminum stress  increased the resistance to Botrytis cinerea , which encode phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), β-1,3-glucanase (BGL), and chitinase (CHI), and increased the activities of PAL, PPO, BGL, and CHI , correspondingly decreased the gene expression of their targets involved in the auxin signaling pathway. Adversely, the expression levels of the primary auxin-response genes  were down-regulated by SO2 pre-exposure in Arabidopsis plants  led to the change in the expression of 2,780 genes, the genes involved in biotic stress resistance, ROS production and scavenging, and sulfur assimilation were up-regulated (2 (30 mg m−3) treatment triggered the different expression of 2,780 genes, the functions of which were mainly involved in signal transduction, transcription regulation, molecular structure, transport, binding, and metabolism. Many different expression genes encoding antioxidant enzymes , heat shock proteins (HSP), pathogenesis-related (PR) proteins, and cytochrome P450 were up-regulated by SO2 in Arabidopsis shoots (2 exposure (30 mg m−3), they encoded antioxidant enzymes , HSP, and PR, as well as were involved in the ETH signaling pathway, phenylpropanoid pathway, and cell wall modification. Similarly, SO2 exposure enhanced the production of ROS, as signaling molecule, increased the activities of SOD, POD, glutathione peroxidase (GPX), and GST in Arabidopsis plants (V. vinifera L. “Crimson Seedless”), transcriptomics approaches also found that SO2 treatment (140 μl L−1) up-regulated the expression of enzymes genes related to sulfur-metabolizing enzymes , redox homeostasis, and plant hormones  signaling pathways (snc1 mutants (with high SA content) had higher tolerance to SO2 than nahG plants (with low SA content), implying that endogenous SA and signaling might play an essential role in plant responses to SO2 stress. These studies further support the fact that SO2 increases disease resistance in Arabidopsis plants.In addition, Arabidopsis plants, the transcriptome analysis identified that SOegulated . Likewiss shoots . In Arabs plants . Also, ipathways . General2 can not only regulate seed germination, stomatal movement, and postharvest physiology, but also plants respond to environmental stresses, such as drought, cold, and heavy metal, and pathogen stresses. Numerous studies identified that SO2 meets the requirements of signaling molecules in plants, emerging as a novel signaling role in many plant physiological processes. SO2 as a novel signaling molecule can exert its physiological functions either alone or interaction with other signaling molecules, such as Ca2+, H2O2, NO, H2S, cGMP, and plant hormones  and osmoregulation system , drive secondary metabolism , synthesize stress proteins , and modulate epigenetic modifications  and miRNAs), thus regulating several plant physiological functions .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.