Hypericum monogynum, and we sequenced its transcriptome to study the biosynthetic mechanism of hyperoside. After gene screening and functional verification, four kinds of key enzymes were identified. Specifically, HmF3Hs (flavanone 3-hydroxylases) and HmFLSs (flavonol synthases) could catalyze flavanones into dihydroflavonols, as well as catalyzing dihydroflavonols into flavonols. HmFLSs could also convert flavanones into flavonols and flavones with varying efficiencies. HmF3′H (flavonoid 3′-hydroxylase) was found to act broadly on 4′-hydroxyl flavonoids to produce 3′,4′-diydroxylated flavanones, dihydroflavonols, flavonols, and flavones. HmGAT  would transform flavonols into the corresponding 3-O-galactosides, including hyperoside. The parallel hyperoside biosynthesis routes were thus depicted, one of which was successfully reconstructed in Escherichia coli BL21(DE3) by feeding naringenin, resulting in a hyperoside yield of 25 mg/l. Overall, this research not only helped us understand the interior catalytic mechanism of hyperoside in H. monogynum concerning flower development and bioactivity, but also provided valuable insights into these enzyme families.Hyperoside is a bioactive flavonoid galactoside in both medicinal and edible plants. It plays an important physiological role in the growth of flower buds. However, the hyperoside biosynthesis pathway has not been systematically elucidated in plants, including its original source, Hypericaceae. Our group found abundant hyperoside in the flower buds of  O-galactoside, is abundant in many edible and medicinal plants, such as roses, dodders, ginkgo leaves, hawthorns, and Hypericum perforatum, contributing greatly to their nutritional and medicinal values [Hyperoside, namely quercetin 3-l values . It playl values , helps tl values , and enhl values . Moreovel values . These eHypericum monogynum, a self-pollinated dicotyledonous plant belonging to the Hypericaceae family, is widely distributed in middle and southern China, Australia, and South Africa [H. monogynum are mainly flavonoids, dianthrones, and phloroglucinol derivatives. A previous study reported the massive acquisition of hyperoside from the aerial parts of H. monogynum [H. monogynum [h Africa . As a wionogynum . In prevonogynum , 13, larp-coumaric acid:CoA ligase), CHS , CHI , F3H (flavanone 3-hydroxylase), FLS (flavonol synthase), F3′H (flavonoid 3′-hydroxylase), and GAT . Despite extensive studies of their genes in different plants [HaCHS [Natural hyperoside biosynthesis involves the participation of PAL , C4H (cinnamic acid 4-hydroxylase), 4CL , together with gene annotations from various public databases  and the conserved domain analysis. Genes with relatively high FPKM (fragments per kilobase per million mapped reads) levels (>30) were chosen for further analysis. They were HmF3H1–2, HmFLS1–3, HmF3′H, and HmGAT as shown  and specific tissues  from Lev. 4 flower buds were sampled for transcriptome sequencing and hyperoside content analysis . The canas shown S1.HmF3H1–2, HmFLS2–3, and HmF3′H had similar mRNA expression patterns  to characterize enzymatic functions  unless cofactors (ascorbate + α-glutaric acid) were added  family as flavanone 3-hydroxylases. They were respectively constructed into the pET28a(+) empty vector, and heterologously expressed in unctions S2 and S3re added S3B. In E enzymes , which cflavones  and S19.in vivo activities. With the addition of cofactors (ascorbate + α-glutaric acid), they could convert dihydroflavonols into flavonols smoothly -trans-DHQ and -cis-DHQ produced from eriodictyol . Both in vivo and in vitro assays demonstrated that HmF3′H could catalyze naringenin, DHK, kaempferol, and apigenin into eriodictyol, DHQ, quercetin, and luteolin, respectively . It was identified in E. coli to have strong 3-O-galactosyltransferase activities towards kaempferol and quercetin . The in vitro results , we observed a substantial decrease in hyperoside content over time (E. coli for toxicity abatement) mediated endogenous decomposition of flavonoids in E. coli BL21(DE3) and Rossetta(DE3), among which 3′,4′-dihydroxylated flavonoids were degraded most severely. Given that we failed in knocking out yhhW from E. coli BL21(DE3), we then attempted to inhibit its activity with different cofactors. It turned out that the cofactor combination (5 mM ascorbate +5 mM α-glutaric acid) was the most effective in inhibiting flavonoid decomposition .The high vigor of HmFLS1 in converting dihydroflavonols and flavanones into flavonols, together with its FPKM change similarity to HmF3H1, HmFLS1, trHmF3′H , AtCPR1 , BbGalE2, and HmGAT in E. coli BL21(DE3)  induction along with 5 mM cofactors (ascorbate + α-glutaric acid), followed by 6 h feeding of 0.3 mM naringenin plus 5 mM cofactors, was found to produce hyperoside mostly at 25 mg/l  (E. coli BL21(DE3) strain  , where eL21(DE3) . Consequformance S16. The formance S17. As activity) . In the ) strain , which pThe 2-ODD family is a large cytosol-soluble enzyme family catalyzing various oxidation reactions, which can be divided into three classes  in plants based on the sequence similarity and functions . DOXA clIn this study, though HmF3Hs belonged to the F3H family and HmFLSs belonged to the FLS family in the phylogenetic tree , they al2+ (as a cofactor), O2 and α-glutaric acid (as co-substrates) to exert functions [in vitro with all cofactors added. Ascorbate and α-glutaric acid were reported to be necessary and a low concentration of Fe2+ to be ancillary [E. coli cultures except for one that successfully characterized FLS activities in vivo by adding substrates together with cofactors [The 2-ODD enzymes typically require ascorbate (as an activator and iron-reducing agent), Feunctions , 27, 32.ncillary . Howeverofactors .in vivo verification of HmFLSs with no cofactors added. Later, with all cofactors  added, HmFLS-recombinant E. coli strains succeeded in synthesizing flavonols from dihydroflavonols. Furthermore, ascorbate and α-glutaric acid were found to be vital in producing flavonols in vivo while Fe2+ played a negative role in it (2+ (1–10 μM) for 2-ODD function [2+ into E. coli cultures would cause the formation of water-insoluble iron–flavonoid complexes as well as enhanced decomposition activity mediated by yhhW [E. coli. Therefore, the in vivo addition of extra Fe2+ was unnecessary and detrimental. The flavonols with 3′,4′-OH were also found to be unstable in the medium or solution [Ornithogalum caudatum) would exert different functions with or without Fe2+in vitro. In our study, Fe2+ played a positive but non-essential role during the in vitro verification of HmF3Hs and HmFLSs  could be selected, in which tobacco and A. thaliana were usually the first choice for plant-derived enzyme verification with much closer phylogenetic relationships. Previous studies reported that transient co-expression of most montbretin A biosynthesis genes in Nicotiana benthamiana resulted in the production of 2 mg/g fresh weight of myricetin 3-O-glucosyl rhamnoside [MrF3′5′H-transgenic Nicotiana tabacum could produce 0.15 mg/g fresh weight of myricetin rutinoside [Saccharomyces cerevisiae, the daidzin biosynthesis pathway was reconstructed with productivity of 73.2 mg/l [E. coli BL21(DE3), baicalein and scutellarein biosynthesis pathways were engineered with productivity of 23.6 mg/l and 106.5 mg/l, respectively [E. coli W led to the production of 1.12 g/l quercitrin [E. coli systems were much more direct, convenient, and less influenced by environmental factors, and pathway reconstruction was also much more adjustable and efficient in E. coli with short reproductive cycles, we decided to use E. coli BL21(DE3) for hyperoside biosynthesis pathway reconstruction.For the heterologous biosynthesis of hyperoside, three kinds of model system  strain through reported genome information  and PCRringenin , which wd before , with th batches , 48. One2.13 g/l . To our H. monogynum, which expanded our comprehension of the corresponding plant enzyme families significantly. Pathway reconstruction in E. coli not only provided a new way to obtain hyperoside but uncovered its remolding potential by knocking out yhhW. Furthermore, other than FNSIIs and FNSIs, the multifunctional role of HmFLS2–3 implied a third inconspicuous path towards flavone production and accumulation in plants.In conclusion, this research revealed the parallel hyperoside biosynthesis routes potentially existing in the flower buds of Hypericum monogynum plants were planted as ornamental shrubs at China Pharmaceutical University, Nanjing, People’s Republic of China. The species was identified by Professor Mian Zhang . . monogynumH flower buds at different stages  together with different tissues  of Lev. 4 were collected for transcriptome database construction . RNA was extracted using the E.Z.N.A. Plant RNA Kit , and first-strand cDNA synthesis was accomplished using the HiScript III 1st Strand cDNA Synthesis Kit .HmF3Hs, HmFLSs, HmF3′H, HmGAT) were obtained by local BLASTP in the H. monogynum transcriptome database (see Data availability section), combined with transcriptome annotations from various public databases  and the analysis of conserved amino acid residues. Primers with specific restriction sites and plasmid homologous arms (HmF3Hs and HmFLSs), pCDFDuet1-BbGalE2 (for HmGAT), or pYES2 (for HmF3′H) using the One Step Cloning Kit , followed by transformation into E. coli DH5α competent cells according to the protocol. Recombinant plasmids were extracted  for sequencing.Candidate genes (ous arms  were deswww.omicshare.com/tools) through Z-score normalization of FPKM values. Flavonoids were extracted according to reported procedures [2, followed by immediate lyophilization. Then, the lyophilized samples were immersed in 70% methanol (40 mg/ml) at 4°C for 24 h. After centrifugation at 12 000 rpm for 5 min, the supernatants were filtered through 0.22 μm organic filter membranes for HPLC analysis.Transcript heat maps were constructed using OmicShare tools (ocedures  with somE. coli BL21(DE3) competent cells. Positive colonies were selected and cultured in LB liquid medium with the corresponding antibiotic at 37°C, 180 rpm. When OD600 reached 0.5–0.7, IPTG  was added to induce protein expression . Proteins were purified and enriched using BeyoGold™ His-Tag Purification Resin  for subsequent SDS–PAGE analysis and in vitro enzyme assays. In vivo assays were conducted by 12 h feeding of 0.1 mM substrates plus cofactors (2.5 mM sodium ascorbate + 2.5 mM α-glutaric acid) after 5 h IPTG induction. The products from 2 ml E. coli cultures were extracted by adding the equivalent volume of ethyl acetate twice. The organic layers were collected and evaporated to dryness in vacuum, then 200 μl methyl alcohol was added to fully dissolve the solid mixture, which was followed by filtration through a 0.22 μm organic filter membrane and centrifugation at 12 000 rpm for 10 min. The supernatants were subjected to HPLC analysis.The successfully constructed recombinant vectors (pET28a(+)-HmF3Hs, pET28a(+)-HmFLSs, and pCDFDuet1-BbGalE2-HmGAT) were respectively transformed into WAT11 yeast strain harboring AtCPR1 was selected as the host of the recombinant vector pYES2-HmF3′H. Transformation was accomplished with the FROZEN-EZ Yeast Transformation II Kit . Positive colonies were further cultured in liquid SD-U medium (containing 2% glucose) at 30°C, 200 rpm. When OD600 reached 0.5–0.7, cells were centrifuged at 4000 rpm for 5 min and washed twice with ddH2O. Then the cell precipitates were resuspended in liquid induction medium  and protein expression was induced for 36 h at 28°C, 160 rpm. The microsomal proteins were extracted according to the reported protocol [in vitro enzyme assays. In vivo enzyme assays were conducted by feeding 0.1 mM substrates and culturing for 48 h after 5 h protein induction. The products from 2 ml S. cerevisiae cultures were extracted by adding the equivalent volume of ethyl acetate twice. The organic layers were collected and evaporated to dryness in vacuum, then 200 μl methyl alcohol was added to fully dissolve the solid mixture for HPLC analysis.The protocol  for subsin vitro reaction conditions (per 1 ml volume) were set as follows: 100 mM NaH2PO4 (pH 6.5), 10 mM α-glutaric acid, 10 mM sodium ascorbate, 0.25 mM FeSO4, 0.2 mM DMSO-dissolved substrates, and 300 μg purified protein, incubating at 30°C, 300 rpm for 2 h. For HmGAT, in vitro reaction conditions (per 1 mL volume) were as follows: 50 mM Tris–HCl buffer (pH 7.5), 0.5 mM sugar donor, 0.2 mM DMSO-dissolved substrates, and 600 μg crude protein, incubating at 30°C, 300 rpm for 2 h. For CYP450 enzyme HmF3′H, in vitro reaction conditions (per 1 mL volume) were set as follows: 50 mM Tris–HCl buffer (pH 7.5), 1 mM NADPH, 0.2 mM DMSO-dissolved substrates, and 1 mg microsomal protein, incubating at 30°C, 300 rpm for 6 h.For 2-ODD enzymes , Reactions were terminated by adding ethyl acetate . The organic layers were collected and evaporated to dryness in vacuum, then 200 μl methyl alcohol was added to fully dissolve the solid mixture for HPLC and LC–MS/MS analysis.HmF3H1 and HmFLS1 were sequentially cloned into the co-expression vector pETDuet1; the restriction sites were SacI/NotI and XhoI, respectively. HmGAT was cloned into the synthesized co-expression vector pCDFDuet1-BbGalE2; the restriction site for HmGAT was XhoI. Then, T7-RBS-trHmF3′H  competent cells in various combinations. Such culture conditions as IPTG concentration, protein-induction duration, naringenin concentration, substrate-feeding duration, cofactor kinds and concentration, and liquid medium kinds were explored to obtain the highest yield of hyperoside.https://www.expasy.org/resources/compute-pi-mw). Phylogenetic trees of plant F3Hs, FLSs, and GATs were respectively constructed by MEGA X (see Data availability section). The maximum likelihood method was applied and the bootstrap value was set as 1000.The protein sequences of plant GATs, HmF3Hs, and HmFLSs were subjected to multiple sequence alignment and analyzed by DNAMAN. The theoretical molecular weights of the corresponding proteins were calculated using the online analysis tool (https://swissmodel.expasy.org/) [The 3D protein models of HmF3Hs and HmFLSs were established by SWISS-MODEL (sy.org/)  based onsy.org/) . Moleculsy.org/) . The genE. coli DH5α for sequencing. Then, correct plasmids were extracted and transformed into E. coli BL21(DE3) to induce protein expression. When the OD600 of E. coli cultures reached around 0.5, a final concentration of 0.5 mM IPTG was added and E. coli cultures were cultivated at 150 rpm, 20°C for 17 h. Proteins were extracted by ultrasonication, then filtered through 0.45 μm membranes. The reaction conditions (per 1 ml volume) for each well were as follows: 40 mg crude protein, 15 mM sodium ascorbate, 15 mM α-glutaric acid, 0.25 mM FeSO4, 50 mM NaH2PO4 (pH 6.5), 0.2 mM substrate (naringenin or DHK), incubating at 30°C, 350 rpm for 1.5 h. Reactions were terminated by adding ethyl acetate . The organic layers were collected and evaporated to dryness in vacuum, then 200 μl methyl alcohol was added to fully dissolve the solid mixture for HPLC analysis.Site-directed mutagenesis of the three enzymes was conducted by using mutated primers for PCR cloning on the corresponding templates pET28a(+)-HmF3H1, pET28a(+)-HmFLS1, and pET28a(+)-HmFLS2. Primers designed for the experiment are listed in HPLC analysis was conducted on an Agilent 1290 Series HPLC System , and the mobile phases were as follows: 0.1% formic acid in ultrapure water as A, and chromatographically pure methanol as B. Elution conditions were as follows: 0–8 min, 50% B; 8–12 min, 50–98% B; 12–20 min, 98% B. The flow rate was 1 ml/min, column temperature was 30°C, and detection wavelengths were 270, 300, 330, and 360 nm.2) flow rate was 4.0 l/min; and the collision gas was ultrapure N2. Samples were introduced into the electrospray source at a flow rate of 4 μl/min. Targeted MS/MS mode (m/z 250 ± 200) was used to analyze the top two ions. The scan rate was three spectra per second in the range of m/z 100–1000 for MS and m/z 100–1000 for MS/MS.LC–MS/MS spectra were determined in a Bruker Esquire-LC quadrupole ion trap mass spectrometer (Germany). HPLC conditions were the same as mentioned above with detection wavelength at 270 nm. The ESI source operated in both positive and negative modes; capillary voltage was set at 4500 V; end plate offset voltage was set at 500 V; nebulizer pressure was 20.0 psi; drying gas temperature was 200°C; drying gas , and the ‘Double First-Class’ University project of China Pharmaceutical University (CPU2022QZ29). We appreciate Dr Hindra from the Department of Biology at McMaster University for helping us organize the manuscript and improve the language.J.L., Y.W., and L.K. designed the research; Y.W. performed the experiments, and Z.C., Q.L., S.Z., Y.L., and X.L. assisted with the experiments; Y.W. wrote the manuscript; J.L. and L.K. revised the manuscript. All authors read and approved the final version of the manuscript.All data related to this research are available in this paper and its supplementary materials published online.GenBank accession numbers involved in this article are as follows:Query proteins: OsFLS (XP_015624815.1); GbFLS (ACY00393.1); MtF3H (ACR15123.1); PhGAT (AAD55985.1); PhF3′H (AAD56282.1).Phylogenetic tree of plant GATs: PhGAT (AAD55985.10); VmGAT (BAA36972.1); AgGAT (AXU98426.1); DkGAT (BAI40148.1); DcGAT (AKI23632.1); EgGAT (BAF49284.1); EkGAT (QTU90759.1); AcGAT (BAD06514.1); AcGAT (A0A2R6Q8R5.1); SbGT (QBL54224.1); FaGLT (Q2V6K0.1); GmGLT (NP_001304377.2); VvGLT (AAB81682.1); FhFLT (ADK75021.1); EkGLT (QTU90760.1); PfGLT (BAA19659.1).Phylogenetic tree of plant F3Hs and FLSs: AbFLS1 (QBQ58058.1); AcFLS1 S(AQR58515.1); AtF3H (ABB60207.1); AcF3H (AXF74850.1); AtF3H (AAC49176.1); AtFLS1 (NP_196481.1); CcFLS (QCF41219.1); CmF3H (ARI70437.1); CsF3H (BAA36553.1); CsFLS1 (ASU87436.1); CsFLS3 (ASU87438.1); CsFLS2 (ASU87437.1); CuFLS (BAA36554.1); GbFLS (ACY00393.1); GbF3H (AAU93347.1); IbF3H (ANA53146.1); InF3H (BAA21897.1); LaF3H (ARA73611.1); OsF3H (XP_015634635.1); OsFLS (XP_015624815.1); PaF3H (QBI90546.1); PhFLS (CAA80264.1); PrF3H (AGY80772.1); PtF3H (QBI90549.1); PsF3H (ABK25766.1); PtFLS (XP_002325697.1); ZmF3H (AAA91227.1); ZmFLS (NP_001140915.1).OP585373, OP585374, OP585375, OP585376, OP585377, OP585378, and OP585379, respectively. The transcriptome database of H. monogynum has been submitted to NCBI under project ID PRJNA907155 [Sequence data of HmF3H1–2, HmFLS1–3, HmF3′H, and HmGAT identified in this study have been submitted to the GenBank database under accession numbers The authors declare no conflicts of interest.Horticulture Research online.Web_Material_uhad166Click here for additional data file.


Monochaetia is a pestalotioid genus that is morphologically characterized by 5-celled conidia with single apical and basal appendages. In the present study, fungal isolates were obtained from diseased leaves of Fagaceae hosts in China in 2016 to 2021 and identified based on morphology and phylogenetic analyses of the 5.8S nuclear ribosomal DNA gene with the two flanking internal transcribed spacer (ITS) regions, the nuclear ribosomal large subunit (LSU) region, the translation elongation factor 1-α (tef1) gene, and the β-tubulin (tub2) gene. As a result, five new species are proposed here, namely, Monochaetia hanzhongensis, Monochaetia lithocarpi, Monochaetia lithocarpicola, Monochaetia quercicola, and Monochaetia shaanxiensis. In addition, pathogenicity tests for these five species and Monochaetia castaneae from Castanea mollissima were conducted with detached leaves of Chinese chestnut. Results demonstrated that only M. castaneae successfully infected the host C. mollissima and caused brown lesions.Pestalotioid fungi have been frequently studied with respect to their morphology, molecular phylogeny, and pathogenicity. IMPORTANCEMonochaetia is a pestalotioid genus, with members that are commonly known as leaf pathogens or saprobes; some strains were isolated from air, in which case their natural substrate is so far unknown. Fagaceae represents an ecologically and economically important plant family that is widely distributed in the Northern Hemisphere, including an important tree crop species, Castanea mollissima, which is widely cultivated in China. In the present study, diseased leaves of Fagaceae in China were investigated, and five new Monochaetia species were introduced based on morphology and phylogeny of combined ITS, LSU, tef1, and tub2 loci. Additionally, six species of Monochaetia were inoculated onto healthy leaves of the crop host Castanea mollissima to test their pathogenicity. The present study provides significant data on the species diversity, taxonomy, and host range of Monochaetia and enhances our understanding of leaf diseases of Fagaceae hosts. Fagaceae is common and important, with 8 genera and about 927 species worldwide (Castanea mollissima), Quercus aliena, and Quercus variabilis. In China, C. mollissima is especially important for producing chestnut crops. Altogether, more than 320 members of Fagaceae are widely distributed in China.The plant family orldwide , 2. ManyGnomoniopsis, Neopestalotiopsis, Pestalotiopsis, Tubakia, and other genera , being characterized by the presence of a single apical appendage  from dead leaves of Ilex species, Monochaetia sinensis from dead leaves of Quercus species, Monochaetia castaneae from diseased leaves of Castanea mollissima, and Monochaetia schimae from diseased leaves of Schima superba  from 29 isolates, including two outgroup taxa, namely, Neopestalotiopsis cubana (CBS 600.96) and Pestalotiopsis australasiae (CBS 114126). Of the 2,735 characters included in the phylogenetic analyses, 642 were parsimony informative . The best maximum likelihood (ML) tree  revealed by RAxML is shown as a phylogram in Monochaetia, namely, Monochaetia hanzhongensis, Monochaetia lithocarpi, Monochaetia lithocarpicola, Monochaetia quercicola, and Monochaetia shaanxiensis.The combined sequence data set for the internal transcribed spacer (ITS), the large subunit (LSU), Monochaetia castaneae (CFCC 54354) produced brown lesions on the tested healthy leaves of Castanea mollissima. In contrast, there was no lesion development on the negative control or on leaves inoculated with M. hanzhongensis (CFCC 54451), M. lithocarpi (CFCC 54402), M. lithocarpicola (CFCC 54509), M. quercicola (CFCC 55515), or M. shaanxiensis (CFCC 54419)  . ReisolaMonochaetia hanzhongensis Ning Jiang sp. nov. . Description: conidiomata in culture sporodochial, aggregated or solitary, erumpent, pulvinate, black, 300 to 850 μm in diameter, exuding black conidial masses. Conidiophores indistinct, usually reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, cylindrical to subcylindrical, annelidic, 5 to 13 by 3 to 4 μm, mean ± standard deviation (SD) = 10 ± 3 by 3.7 ± 0.3 μm. Conidia fusoid, straight or slightly curved, 4-septate, smooth, slightly constricted at the septa, without appendages (17.5 to) 18 to 20 (to 21) by (5 to) 5.5 to 6 (to 6.5) μm, mean ± SD = 19 ± 1.2 by 5.7 ± 0.3 μm (n = 50), length/width ratio (L/W) = 2.9 to 3.8; basal cell obconic with a truncate base, thin walled, hyaline or pale brown, 3 to 4.5 (to 5) μm long (without appendage); median cells 3, trapezoid or subcylindrical, brown, thick walled, the first median cell from the base (3.5 to) 4 to 5 (to 5.5) μm long, the second cell (3 to) 3.5 to 4 μm long, the third cell 3.5 to 4 μm long, altogether (10 to) 10.5 to 12.5 (to 13) μm long; apical cell conic with an acute apex, thin walled, hyaline, 2.5 to 4 μm long (without appendage); basal appendage single, unbranched, tubular, centric, straight or bent, (3 to) 3.5 to 9.5 (to 12) μm long, mean ± SD = 6.3 ± 3.4 μm; apical appendage single, unbranched, tubular, centric, straight or bent, (6.5 to) 7 to 18.5 (to 23) μm long, mean ± SD = 12.9 ± 5.7 μm. Sexual morph unknown. Culture characteristics: colonies on malt extract agar (MEA) flat, spreading, with flocculent aerial mycelium, folded surface, and diffuse edge, white to fawn, reaching 50 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses; colonies on potato dextrose agar (PDA) flat, spreading, with flocculent aerial mycelium and undulate margin, white to pale luteous, reaching 40 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses.sp. nov. . MycoBanMonochaetia hanzhongensis is phylogenetically close to Monochaetia dimorphospora, Monochaetia kansensis, M. lithocarpi, Monochaetia quercus, and M. shaanxiensis  . M. hanzhongensis differs from M. dimorphospora, M. kansensis, and M. shaanxiensis in conidial width  and has shorter conidia than M. quercus (17.5 to 21 μm versus 22.5 to 29 μm in M. quercus) (Notes: nxiensis . Morpholhocarpi)  but can quercus)  17)..MonochaeMonochaetia lithocarpi Ning Jiang sp. nov. . Description: conidiomata in culture sporodochial, solitary, erumpent, pulvinate, dark brown, 250 to 600 μm in diameter, exuding dark brown conidial masses. Conidiophores septate and branched, hyaline or pale brown, thin walled. Conidiogenous cells hyaline or pale brown, smooth, cylindrical to subcylindrical, annelidic, 4.5 to 17 by 2 to 4 μm, mean ± SD = 10.3 ± 3.4 by 3 ± 0.6 μm. Conidia fusoid, straight or slightly curved, mostly 4-septate, occasionally 3-septate, wall smooth or undulate, not constricted at the septa but commonly collapsed at the septa, without appendages (15 to) 17 to 22 (to 24.5) by (4.5 to) 5 to 6.5 (to 7) μm, mean ± SD = 19.4 ± 2.6 by 5.8 ± 1.6 μm (n = 50), L/W = 2.6 to 4.6; basal cell obconic with a truncate base, thin walled, hyaline or pale brown, (2.5 to) 3 to 4.5 (to 5) μm long (without appendage); median cells 3 (rarely 2), trapezoid or subcylindrical, brown, thick walled, the first median cell from the base 4 to 5 μm long, the second cell (2.5 to) 3 to 4.5 (to 5) μm long, the third cell 3 to 4 μm long, altogether (10 to) 10.5 to 12.5 (to 13) μm long; apical cell conic with an acute apex, thin walled, hyaline or pale brown, 3 to 4 μm long (without appendage); basal appendage single, unbranched, tubular, centric, straight or bent, 4 to 13 (to 17) μm long, mean ± SD = 8.5 ± 3.3 μm; apical appendage single, unbranched, tubular, centric, variously bent, (13 to) 14.5 to 21.5 (to 23.5) μm long, mean ± SD = 17.8 ± 3.5 μm. Sexual morph unknown. Culture characteristics: colonies on MEA flat, spreading, with radially folded surface and undulate edge, olivaceous gray, reaching 35 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses; colonies on PDA flat, with moderate aerial mycelium and entire edge, white to pale luteous, reaching 40 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses. Additional materials examined: China, Guangdong Province, Qingyuan City, Yangshan County, Guangdong Nanling Nature Reserve, on diseased leaves of Lithocarpus glaber, 4 December 2019, Shang Sun (cultures CFCC 54379 and CFCC 54500).sp. nov. . MycoBanLithocarpus glaber clustered into a distinct and well-supported clade in the phylogram, which is described here as the new species Monochaetia lithocarpi  (M. quercus in the length of three median cells of conidia (10 to 13 μm versus 15 to 20 μm in M. quercus)  .Notes: Three isolates from leaf spots of thocarpi . Monochanxiensis . Morpholhospora)  and diffxiensis) . HoweverMonochaetia lithocarpicola Ning Jiang sp. nov. . Description: conidiomata in culture sporodochial, aggregated or solitary, erumpent, pulvinate, brown, 300 to 700 μm in diameter, exuding brown conidial masses. Conidiophores septate and branched, hyaline, thin walled. Conidiogenous cells hyaline, smooth, cylindrical to subcylindrical, annelidic, 5 to 22.5 by 2 to 3.5 μm, mean ± SD = 15.8 ± 4.5 by 2.9 ± 0.4 μm. Conidia fusoid, straight or slightly curved, 4-septate, smooth, constricted at the septa, without appendages (31 to) 31.5 to 34 (to 35) by (7.5 to) 8 to 8.5 (to 9) μm, mean ± SD = 33 ± 1.2 by 8.3 ± 0.4 μm (n = 50), L/W = 3.5 to 4.5; basal cell obconic with a truncate base, thin walled, hyaline, (5 to) 5.5 to 7.5 (to 8) μm long (without appendage); median cells 3, trapezoid or subcylindrical, pale brown, thick walled, the first median cell from the base (5.5 to) 6 to 7 (to 7.5) μm long, the second cell (6 to) 6.5 to 8.5 μm long, the third cell (6 to) 6.5 to 8.5 (to 9) μm long, altogether (18 to) 19 to 22.5 (to 23) μm long; apical cell conic with an acute apex, thin walled, hyaline, 5.5 to 6.5 μm long (without appendage); basal appendage single, unbranched, tubular, centric, straight or slightly bent, (6.5 to) 7 to 12.5 (to 13.5) μm long, mean ± SD = 9.7 ± 2.6 μm; apical appendage single, unbranched, tubular, centric, bent, (12 to) 13 to 23 (to 24) μm long, mean ± SD = 18 ± 4.8 μm. Sexual morph unknown. Culture characteristics: colonies on MEA flat, with ruffle sag on the surface and undulate edge, white to pale luteous, reaching 25 mm in diameter after 10 days at 25°C, forming brown conidiomata with brown conidial masses; colonies on PDA flat, spreading, with entire edge, white to pale luteous, reaching 25 mm in diameter after 10 days at 25°C, forming brown conidiomata with brown conidial masses. Additional material examined: China, Guangdong Province, Qingyuan City, Yangshan County, Guangdong Nanling Nature Reserve, on diseased leaves of Lithocarpus glaber, 4 December 2019, Shang Sun (culture CFCC 54448).sp. nov. . MycoBanM. lithocarpicola is the closest relative of Monochaetia massachusettsianum, which was isolated from air in the United States and for which the natural host is therefore unknown. However, M. lithocarpicola is distinguished from M. massachusettsianum by obviously longer conidia (31 to 35 μm versus 23 to 30 μm in M. massachusettsianum)  .Monochaetia quercicola Ning Jiang sp. nov. . Description: conidiomata in culture sporodochial, aggregated or solitary, erumpent, pulvinate, dark brown, 50 to 350 μm in diameter, exuding dark brown conidial masses. Conidiophores septate and branched, hyaline, thin walled. Conidiogenous cells hyaline, smooth, cylindrical to subcylindrical, annelidic, 8.5 to 21 by 2.5 to 3 μm, mean ± SD = 14.7 ± 3.6 by 2.8 ± 0.3 μm. Conidia fusoid, slightly curved, 4-septate, smooth, constricted at the septa, without appendages (19.5 to) 20.5 to 23 (to 25) by (7 to) 7.5 to 9 (to 9.5) μm, mean ± SD = 21.8 ± 1.4 by 8.2 ± 0.6 μm (n = 50), L/W = 2.3 to 3.3; basal cell obconic with a truncate base, thin walled, hyaline, 3 to 4.5 μm long (without appendage); median cells 3, trapezoid or subcylindrical, pale brown to brown, thick walled, the first median cell from the base (4 to) 4.5 to 6 μm long, the second cell 4.5 to 5 (to 5.5) μm long, the third cell (4 to) 4.5 to 5 (to 6) μm long, altogether (13 to) 13.5 to 15.5 μm long; apical cell conic with an acute apex, thin walled, hyaline, 3 to 4 (to 4.5) μm long (without appendage); basal appendage single, unbranched, tubular, centric, straight or slightly bent, (5 to) 6 to 11.5 μm long, mean ± SD = 8.7 ± 2.6 μm; apical appendage single, unbranched, tubular, centric, bent, (5 to) 9.5 to 17 (to 17.5) μm long, mean ± SD = 13.3 ± 3.8 μm. Sexual morph unknown. Culture characteristics: colonies on MEA flat, spreading, with radially folded surface and entire edge, sienna to hazel, reaching 60 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses; colonies on PDA flat with flocculent mycelium and undulate edge, white to luteous, reaching 60 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses. Additional material examined: China, Henan Province, Xinyang City, Shihe District, Jigong Mountain, on diseased leaves of Quercus acutissima, 7 August 2019, Yong Li (culture CFCC 55248).sp. nov. . MycoBanMonochaetia quercicola, from leaf spots of Quercus acutissima and Q. aliena, formed a distinct clade in the phylogram close to M. ilicis and M. schimae (Monochaetia quercicola can be distinguished from M. ilicis and M. schimae by wider conidia (7 to 9.5 μm versus 3 to 5 μm in M. ilicis versus 4.5 to 6 μm in M. schimae) (Notes: Two isolates of  schimae . Howeverschimae) , 16.Monochaetia shaanxiensis Ning Jiang sp. nov. . Description: conidiomata in culture sporodochial, aggregated or solitary, erumpent, pulvinate, dark brown, 150 to 450 μm in diameter, exuding dark brown conidial masses. Conidiophores septate and branched, hyaline, thin walled. Conidiogenous cells hyaline, smooth, cylindrical to subcylindrical, annelidic, 11.5 to 20.5 by 2 to 3 μm, mean ± SD = 15.7 ± 2.7 by 2.4 ± 0.2 μm. Conidia fusoid, straight or slightly curved, 4-septate, smooth, slightly constricted at the septa, without appendages, (19 to) 19.5 to 21.5 (to 22) by 6.5 to 7.5 (to 8.5) μm, mean ± SD = 20.6 ± 0.9 by 7.1 ± 0.5 μm (n = 50), L/W = 2.5 to 3.2; basal cell obconic with a truncate base, thin walled, hyaline or pale brown, 3 to 4 (to 4.5) μm long (without appendage); median cells 3, trapezoid or subcylindrical, brown, thick walled, the first median cell from the base 4.5 to 5 (to 5.5) μm long, the second cell 3.5 to 4.5 μm long, the third cell (3 to) 3.5 to 4.5 μm long, altogether 11.5 to 13 μm long; apical cell conic with an acute apex, thin walled, hyaline, (4 to) 4.5 to 5.5 μm long (without appendage); basal appendage single, unbranched, tubular, centric, straight, 2 to 3 μm long, mean ± SD = 2.5 ± 0.3 μm; apical appendage single, unbranched, tubular, centric, straight, (3.5 to) 4 to 6.5 (to 8) μm long, mean ± SD = 5.1 ± 1.4 μm. Sexual morph unknown. Culture characteristics: Colonies on MEA flat, spreading, with radially folded surface and entire edge, luteous to olivaceous gray, reaching 35 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses; colonies on PDA flat, spreading, with flocculent aerial mycelium and undulate edge, white to pale luteous, reaching 45 mm in diameter after 10 days at 25°C, forming black conidiomata with black conidial masses.sp. nov. . MycoBanMonochaetia shaanxiensis is phylogenetically close to M. hanzhongensis, M. kansensis, and M. lithocarpi , a shorter basal appendage than M. kansensis (2 to 3 μm versus 3 to 15 μm in M. kansensis), and wider conidia than M. hanzhongensis (6.5 to 8.5 μm versus 5 to 6.5 μm in M. hanzhongensis) (Notes: thocarpi . Howeverngensis) .Fagaceae leaf spots in China were identified as 5 new species of Monochaetia. However, we investigated only 5 Fagaceae hosts of more than 320 reported species in China, indicating that many hidden Monochaetia species may remain to be discovered from Fagaceae in the future.In the present study, 9 isolates from Monochaetia are commonly known as leaf pathogens or saprobes and are sometimes isolated from air  have not yet been sequenced. In the present study, 5 additional new species were revealed from Fagaceae based on conidial characteristics and molecular phylogenetic evidence , M. hanzhongensis (from Quercus variabilis), M. lithocarpi (from Lithocarpus glaber), M. lithocarpicola (from L. glaber), M. quercicola  and M. shaanxiensis (from Quercus baronii) to healthy leaves of C. mollissima. Only M. castaneae successfully infected C. mollissima and caused brown lesions, which implied that the other five fungal species are not pathogenic to the important crop fagaceous species C. mollissima.This study revealed five new in China , we testFagaceae, namely, Lithocarpus glaber, Q. variabilis, Q. aliena, Q. acutissima, and Q. baronii, were investigated in the present study. The leaf samples were packed in paper bags and transferred to the laboratory for fungal isolation.Investigations were conducted in Guangdong, Henan, and Shaanxi Provinces in China from 2016 to 2021 to collect diseased fagaceous leaf samples. A total of two host genera and five species in http://cfcc.caf.ac.cn/) and the specimens in the herbarium of the Chinese Academy of Forestry (CAF) (http://museum.caf.ac.cn).The diseased leaf samples were surface sterilized for 1 min in 75% ethanol, 3 min in 1.25% sodium hypochlorite, and 1 min in 75% ethanol, rinsed for 2 min in distilled water, and blotted on dry sterile filter paper. Then the diseased areas of the leaves were cut into pieces (0.5 by 0.5 cm) with an aseptic razor blade, transferred onto the surface of both PDA  and MEA  plates, and incubated at 25°C to obtain fungal hyphae. Hyphal tips were then removed to new PDA and MEA plates to obtain pure cultures. The cultures were deposited in the China Forestry Culture Collection Center (CFCC) (tef1) gene (tub2) gene (tef1 and tub2), and 1 min at 72°C, and a final elongation step of 7 min at 72°C. PCR amplification products were checked via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI Prism 3730XL DNA Analyzer with a BigDye Terminator kit v.3.1  at the Shanghai Invitrogen Biological Technology Company Ltd. .Genomic DNA was extracted from colonies grown on cellophane-covered PDA plates using a cetyltrimethylammonium bromide (CTAB) method . DNA quaf1) gene , 25, andb2) gene . The PCRhttp://mafft.cbrc.jp/alignment/server) (https://www.phylo.org) using RAxML-HPC BlackBox v.8.2.10  database. Sequences were aligned using MAFFT v.7 (/server)  and corr/server) . The nucv.8.2.10 , employiv.8.2.10 . Two MCMtef1, and tub2 sequences of the species were pairwise compared. For this, the sequences of species pairs were aligned, and the parts containing leading/trailing gaps were removed. Sequence differences of this alignment are recorded in the following way: number of nucleotide substitutions (excluding insertions and gaps)/total number of nucleotide characters, percentage of sequence substitutions, and numbers of insertions and gaps.For closely related species with similar morphology, ITS, LSU, The morphological data for the isolates collected in the present study were obtained from sporulating pure cultures grown on PDA or MEA plates in the dark at 25°C. The conidiomata were observed and photographed using a dissecting microscope . Microscope slides of conidiogenous cells and conidia were prepared in tap water, and the slides were examined and photographed with an Axio Imager 2 microscope  equipped with an Axiocam 506 color camera or an Eclipse 80i microscope  equipped with a Nikon digital sight DS-Ri2 camera, using differential interference contrast (DIC) illumination. For measurements, 50 conidia were randomly selected. Measurements of the conidia are reported as maximum and minimum (in parentheses) and the range representing the mean ± SD of the number of measurements indicated. Culture characteristics were recorded from 9-cm PDA or MEA plates after 10 days of incubation at 25°C in the dark. To enable comparison of species growing on fagaceous hosts, measurement data and sequence data available are summarized in Monochaetia castaneae (CFCC 54354), M. hanzhongensis (CFCC 54451), M. lithocarpi (CFCC 54402), M. lithocarpicola (CFCC 54509), M. quercicola (CFCC 55515), and M. shaanxiensis (CFCC 54419) were selected for inoculations. Healthy leaves of Castanea mollissima were washed in distilled water, surface sterilized in 75% ethanol for 1 min, rinsed in distilled water, and then surface wounded with a sterile needle in both the left and right portions of the leaves. Conidia of the six Monochaetia species were harvested from 4-week-old PDA plates with 10 mL of sterilized water, and the conidial suspension was filtered through two layers of cheesecloth to eliminate debris and mycelia. The conidial suspension was adjusted to a final inoculum concentration of 1 × 106 conidia/mL with sterile deionized water. Then, 10 μL of conidial suspension was placed in the left portion of the leaves; sterile water inoculated in the right portion served as the negative control. Each treatment had five replicates, and the experiment was carried out twice. The inoculated leaves were placed in transparent plastic bags at 25°C and >90% humidity in the dark for 10 days. After the appearance of symptoms, fungal isolates were reisolated from the infected leaves and identified based on the morphological and phylogenetic analyses to fulfill Koch’s postulates.The isolates representing


The SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages.ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing.The presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages.All genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the TThe developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.The online version contains supplementary material available at 10.1186/s12985-023-02137-5. The global coronavirus disease 2019 (COVID-19) pandemic was caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) virus, and Omicron, the fifth variant of concern (VOC) designated by the World Health Organization (WHO), has been the prevalent strain of SARS-CoV-2 virus in circulation since November, 2021, causing major outbreaks around the world. Recent computational and whole-genome sequencing analyses have identified five main lineages of the Omicron variant descending from an original Omicron ancestor, which are designated BA.1, BA.2, BA.3, BA.4 and BA.5, and several sub-lineages, including BA.1.1, BA.2.12.1, BA.2.11, BA.2.75, BA.4.6 [Vaccination has been proven to be the most effective way to prevent and control COVID-19 , 5. The Compared to previous SARS-CoV-2 VOC strains , Omicron demonstrates enhanced transmissibility and can evade RBD-targeted neutralizing antibodies, due to RBD mutations promoting contagiousness and vaccine escape . UnpreceAt present, identification of SARS-CoV-2 variants and lineages mainly relies on viral whole-genome sequencing (WGS) by next-generation sequencing (NGS) . HoweverIn the present study, we developed a two-tube, four-color assay that uses asymmetric PCR-based melting curve analysis to detect seven immune-escape RBD mutations of Omicron, in order to provide an accurate, stable, rapid, simple, inexpensive, and open-source method for the preliminary identification of the Omicron variant and discrimination of the BA.1, BA.2, BA.4/5, and BA.2.75 lineages, which have been prevalent in the mainland of China. In the future, this technique can be easily applied and generalized as a public health strategy for managing COVID-19. The ability to identify immune-escape RBD mutations also may be useful for guiding precision therapy and predicting prognosis.RNA was extracted from the clinical nasopharyngeal swabs using an RNA extraction kit  according to the manufacturer’s instruction. The sample input and output volumes were 200 μL and 80 μL, respectively. RNA extracts were tested for SARS-CoV-2 by quantitative reverse transcription-PCR (RT-PCR). Then, we obtained the remnant RNA extracts which had been tested positive (n = 40) and negative (n = 20) for SARS-CoV-2 for clinical validation of the current methodology. The RNA extracts were stored at –80 °C before use. The study protocol was implemented in accordance with the Declaration of Helsinki II and with approval from the ethics committee of China-Japan Friendship Hospital .N gene and 12,961–13,310 coding sequence in the ORF1ab gene were inserted into the plasmid pUC57, separately. The sequences inserted in the plasmids are presented in Additional file Plasmids with genome fragments of SARS-CoV-2 were obtained from Tsingke Co. Ltd . Fragments containing the 1129 ~ 1584 or 1212 ~ 1584 coding sequence of the spike genes of the original wild-type virus (Wuhan) and the BA.1, BA.2, BA.4/5, and BA.2.75 lineages of the Omicron variant, as well as the 481 ~ 830 coding sequence in the ORF1ab plasmid and N plasmid equally, and diluted to a final concentration of 10,000 copies/μL of each plasmid. The plasmid mixtures were stored at –20 °C.The plasmids were dissolved in TE buffer  to 2 μg/mL. They were further diluted using nuclease-free water  to 40,000 copies/μL. Viral lineages were mimicked by mixing wild-type or mutant spike gene plasmids with Pseudotyped lentiviruses with SARS-CoV-2 wild-type virus  and Omicron BA.1 virus  were obtained from Guangzhou BDS Biological Technology Co., Ltd. . Pseudotyped lentiviruses with influenza virus A (Flu A), influenza virus B (Flu B) and respiratory syncytial virus (RSV) were obtained from American Type Culture Collection  for analysis of the analytical specificity of the developed method. Viral RNA was extracted using an RNA extraction kit  with an input volume of 200 μL and output volume of 80 μL.The extracted RNA was used to synthesize random hexamer-primed cDNA with M-MuLV reverse transcriptase  according to the manufacturer’s instruction. The cDNA was then used for PCR experiments.ORF1ab or N gene was detected in each tube as an internal control. Three pairs of primers and eight dual-labeled self-quenched fluorescent probes were designed to detect spike mutations L452R (c.1355 T > G), N460K (c.1380 T > G), E484A (c.1451A > C), F486V (c.1456 T > G), Q493R + Q498R (c.1478A > G + c.1493A > G), and Y505H (c.1513 T > C), along with ORF1ab and N, respectively  as an internal indicator for SARS-CoV-2 virus. The second reaction (reaction 2) contained probes targeting the spike E484A , Q493R + Q498R , and L452R , with a probe targeting the N gene (in Cy5 channel) as an internal indicator. Each 25-μL PCR mixture contained TaKaRa Ex Taq® Polymerase (0.125 U/μL) supplied in 10 × Ex Taq Buffer (pH 8.5), dNTP (200 μM each) and 2 mM MgCl2 , with 0.1–0.3 μM limiting primers, 0.8–2.4 μM excess primers, 0.1–0.4 μM probes, and 5 μL plasmids or cDNA template. The concentration details for the primers and probes are presented in Additional file ORF1ab gene, or the N gene, 0.1 μM restriction primer, 0.8 μM excess primer, and 0.2 μM probe were used in the reaction.The method was then optimized and performed using a SLAN®-96P fluorescent quantitative PCR system . The first reaction (reaction 1) contained probes targeting N460K , F486V , and Y505H , with a probe targeting the ORF1ab and N gene were 343 bp, 119 bp, and 99 bp, respectively. After the amplification procedure, the melting curve program included three steps: denaturation at 95 °C for 2 min, renaturation at 40 °C for 2 min, and subsequent melting with continuous acquisition of fluorescence from 40 to 80 °C at a ramp rate of 0.08 °C/s.The PCR cycling program consisted of initial denaturation at 95 °C for 5 min followed by 50 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 1 min, and extension at 72°C for 30 s for amplification. The amplified products for spike gene, ORF1ab gene (Cy5 channel in reaction 1) and N gene (Cy5 channel in reaction 2) suggested the existence of SARS-CoV-2 virus, and the Tm of each melting curve in the FAM, HEX, and ROX channels indicated the genotype of the targeted mutations.The results were then interpreted via combinatory analysis of the four fluorescence signals of each reaction  of each reaction was defined as the copy number of the input template that provided the appearance of a melting peak for all targets in > 95% technical repeats.ms. Inter-assay precision of Tms was assessed based on the results for five replicates of each plasmid mixture tested on four independent days by different operators.Intra-assay precision was assessed for the plasmid mixtures representing each SARS-CoV-2 virus lineage with an amount of 312.5 copies per reaction in five replicates to determinate the percent coefficient of variance (CV) of TThe presented method was tested using RNA extracts of pseudotyped lentiviruses with Flu A, Flu B, and RSV as well as human genomic DNA, to verify that it did not detect either viral nucleic acid from these other widespread human viral pathogens causing respiratory infections or human genomic DNA.To assess the accuracy, the presented method was used to test clinical samples for which the status was known, as well as SARS-CoV-2 wild-type RNA weakly positive quality control reference material and Omicron BA.1 quality control reference material. The mutation genotyping results for the positive samples were compared with Sanger sequencing results.2 , 0.5 μM forward primer, and 0.5 μM reverse primer. The PCR cycling program consisted of initial denaturation at 95 °C for 5 min followed by 50 cycles of 95 °C for 20 s, 60 °C for 1 min and 72 °C for 30 s, with a final extension at 72 °C for 10 min . The amplicons were 343 bp and were sent to Tsingke Biotechnology Co., Ltd.  for unidirectional sequencing using an ABI 3730xl DNA Analyzer .For Sanger sequencing, the primers used to amplify the targeted region of the spike gene in SARS-CoV-2 virus were designed as follows: 5’-AATCGCTCCAGGGCAAACTG-3’ (forward primer), and 5’-AGTTGCTGGTGCATGTAGAA-3’ . PCR was carried out in a 50-μL reaction mixture containing TaKaRa Ex Taq® Polymerase (0.125 U) in 10 × Ex Taq Buffer (pH 8.5) with dNTP (200 μM each), 2 mM MgClThe data are presented as mean ± standard deviation (SD), and CVs were calculated using SAS .m values are automatically generated.Figure ORF1ab gene as the internal control, while reaction 2 detects E484A, Q493R + Q498R and L452R with the N gene as the internal control. As expected, Tms of probes in different combinations were usually slightly changed compared with those in single tests . Since an additional mutation F486V in BA.4/5 is covered by the probe targeting E484A, its Tms was shifted to 52.71 °C. The probe targeting Q493R + Q498R was designed to be completely matched with the haplotype of BA.2  with a Tm of 61.12 °C. The mean Tm for wild-type virus was significantly decreased to 49.34 °C because of the 2-bp mismatches . The Tm for BA.1  was lower (54.78 °C) than that for BA.2 because of the 1-bp mismatch. The mean Tms for BA.4/5 and BA.2.75 were shifted to 55.50 °C and 54.94 °C, respectively, because of the existence of the R493Q reversion mutation. The Tms for the BA.1, BA.4/5, and BA.2.75 did not differ significantly, but the respective melting curves had slightly different shapes .The genotypes of mutations could be further identified by their corresponding Tms  for BA.4/5 than for wild-type virus, Omicron BA.1, BA.2, and BA.2.75 .The BA.4 and BA.5 lineages have identical spike mutations. Relative to BA.2, BA.4/5 have additional RBD mutations, L452R and F486V. The probes targeting L452R and F486V showed significantly higher Tm of 63.56 °C, which was obviously higher than the Tms for wild-type virus, Omicron BA.1, BA.2, and BA.4/5 (58.89–58.93 °C).The BA.2.75 lineage has an additional RBD mutation N460K. The probe matching N460K showed a mean TWe use plasmid DNA as the amplification template with varying amounts at 1250, 625, 312.5, 156.3, 78.1, 39.1, 19.5, and 9.8 copies per reaction to assess the analytical sensitivity. The LOD of the present method reached 39.1 copies per reaction for all lineage types, whereas the LODs for each probe in a single test were even lower  with a SD ≤ 0.38 °C  and an inter-run Tm CV ≤ 0.84%  with a SD ≤ 0.41 °C . The Tm values had three times standard deviation (3SD) values ≤ 1.17 °C. All the other genotypes could be successfully resolved with a Tm difference larger than 4 °C, which is much larger than the 3SD values, except that the haplotype in BA.1  versus the haplotype of BA.4/5 and BA.2.75  could not be distinguished by the 493R + 498R probe.Five replicates of each genotype were tested on four independent days by different operators to determine the intra-run and inter-run precision. As shown in Table To assess the specificity of this method, cDNA generated from RNA extracts of pseudotyped lentiviruses of common human respiratory pathogens, including Flu A, Flu B, and RSV, were tested. No cross-reactivity was seen for any of these pathogens in this assay. Moreover, no signal was detected after addition of human genomic DNA.ORF1ab and N gene peaks in the Cy5 channel of all SARS-CoV-2–positive samples with Tms ranging from 63.59 to 65.01 °C and from 63.75 to 64.40 °C, respectively. Furthermore, our method accurately identified the wild-type quality control reference material and BA.1 quality control reference material. The current method also correctly classifies all of the BA.2 (n = 36) and BA.4/5 (n = 4) among the 40 positive clinical samples. Complete concordance between the melting curve method and Sanger sequencing results was obtained, demonstrating that the developed method is able to preliminarily distinguish Omicron variants and its lineages  and –negative (n = 20) nasopharyngeal swabs, as well as weakly positive quality control RNA reference material of SARS-CoV-2 wild-type virus and a quality control reference material of Omicron BA.1. No signal was detected from cDNA sample generated from SARS-CoV-2–negative swabs. The results for positive samples were compared with Sanger sequencing results. The developed method detected the Since its discovery in December of 2019, the SARS-CoV-2 virus has continuously gained evolutionary advantages in the RBD of its spike protein either by mutations to strengthen the ACE2–RBD binding affinity or by mutations to escape antibody protection, due to natural selection . For theIn the present study, we developed an asymmetric PCR-based melting curve assay to rapidly identify the key immune-escape mutations in SARS-CoV-2 and demonstrated that this assay can successfully identify wild-type virus, Omicron BA.1, BA.2, BA.4/5, and BA.2.75 that were once prevalent in the mainland of China. We selected spike RBD mutations in the receptor-binding motif (amino acids 438–506). In this motif, six identical mutations  are present in all five Omicron main lineages. Among them, E484A, Q498R and Y505H are Omicron-specific mutations. Furthermore, both BA.1 and BA.2 have Q493R, whereas BA.1 has an additional G496S mutation compared to BA.2. BA.4/5 also have L452R, F486V, and a reversion mutation R493Q compared with BA.2 in this motif . NotablyCurrently, WGS of viruses are routinely used to identify SARS-CoV-2 variants and lineages. However, this is a comprehensive approach for variant detection. The sequencing platform, which requires routine, time-consuming and labor-intensive maintenance, is not available in most clinical laboratories outside of genomic centers. Data interpretation is also challenging and requires experienced technicians. As an alternative to NGS or Sanger sequencing, several groups have attempted to develop PCR-based preliminary methods for variant determination via detection of specific mutations –29. PCR-m difference larger than 4 °C. Although the haplotype of BA.1  versus the haplotype of BA.4/5 and BA.2.75  cannot be distinguished from each other by the Tms of the 493R + 498R probe in this assay because both genotypes have a 1-bp mismatch compared with the probe, these lineages can be successfully identified based on the results for the other mutations or by applying single probe tests (mean Tms of 493R + 498R probe: 53.57 °C for BA.1 and 55.47 °C and 55.35 °C for BA.4/5 and BA.2.75). Thus, the results can be easily determined by both experienced and inexperienced technicians. The clinical validation data further demonstrated that the developed method can accurately genotyped wild-type, BA.1, BA.2, BA.4/5 viruses with 100% accuracy. The assay can detect each genotype with a sensitivity as low as 39.1 copies per reaction. The intra- and inter-run precision are also satisfactory with CVs for the Tms ≤ 0.69% and ≤ 0.84%, as well as SDs ≤ 0.38 °C and ≤ 0.41 °C. The internal control (ORF1ab or N) in each reaction is detected to ensure reliability. The amplification products are directly analyzed in a closed-tube, which can avoid contamination caused by open tube. The design of probes and primers of this method are conventional and feasible, which can be generalized to novel mutations in the future. Also, this method has high-throughput potential for performance in a 96-well or 384-well PCR analyzer.The presented method developed by our group is a two-tube, four-color assay based on a fluorescent PCR platform. This assay requires simple and labor-saving operation and can be easily performed locally in most clinical laboratories. It is basically a reverse transcription and multiple asymmetric PCR analysis. All mutations recognized by 484A, 505H, 486V, 452R, and 460K probes can be successfully resolved with a TMoreover, the current melting curve method requires only one traditional probe for each mutation, which is cost-efficient compared to the other TaqMan-based real time PCR assays. TaqMan-MGB (minor groove binder)-PCR  and ARMSORF1ab, and N fragments to ensure appropriate amplification efficiency for all products, while the combinations and concentrations of probes were precisely moderated. Finally, the PCR conditions were optimized to ensure the amplification of all fragments and that enough single-chain products were obtained efficiently, with PCR analysis done within 2.5 h.For a multi-PCR assay, due to unequal reaction efficiencies for different amplicons or the interactions between oligonucleotide primers and probes, simultaneous amplification of multiple amplicons can be hampered, resulting in experimental failure and reduced analytical sensitivity , 42. Thim is observed, it may prompt the occurrence of novel mutation in the probe targeted sequence, and sequencing methods should be then applied to clarify the exact mutation. Also, when there’s a need to apply this method to novel mutations, probes and primers should be reevaluated and usually redesigned. For assay design, to introduce as few amplification products as possible in the multiplex PCR is recommended, in order to reduce the risk of interference between the primer pairs. Longer amplicons can be employed when necessary, and the elongation process of each cycle should be extended accordingly. Notably, since the efficiency of asymmetric PCR is largely depended on the accurate concentration ratio of restriction primer and excessive primer, quantification of primers using a nucleic acid quantizer before experiment is recommended.As an PCR-based method, the current method cannot detect novel mutations, which is its major limitation. However, if an unexpected change of Tm for each melting peak may change slightly and should be determined preliminarily before use.In addition, the presented method can be applied in a one-step RT-PCR system using viral RNA via adding random primer for reverse transcription, gene-specific primer pairs and probes in a commercial one-step PCR mixture and amplified directly. For the PCR procedure, a reverse transcription step (commonly 30 min) should be added before the initial denaturation step. With differences in components and their concentrations in the reaction buffer, the reference ranges of TIn conclusion, we described an asymmetric PCR-based melting curve analysis method for the rapid identification of immune-escape mutations located in the spike RBD of the SARS-CoV-2 Omicron strain. This assay can provide accurate, reliable, rapid, simple and low-cost preliminary detection of Omicron and its lineages in two PCR wells. We expect its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.Additional file 1: Table S1. Insert sequence of plasmids.Additional file 2: Table S2. Reaction mix composition for the asymmetric PCR melting curve analysis-based method.Additional file 3: Table S3. Mean±SD values of Tms in single probe tests.Additional file 4: Fig. S1. Performance of single probe tests. The performance of each single probe test for wild-type, Omicron BA.1, BA.2, BA.4/5, and BA2.75 with 312.5 template copies per reaction.Additional file 5: Fig. S2. Limit of detection of single probe tests. The performance of each single test with varying amounts of plasmid templates at 1250, 625, 312.5, 156.3, 78.1, 39.1, 19.5, and 9.8 copies per reaction.Additional file 6: Fig. S3. The asymmetric PCR melting curve analysis-based method clearly distinguishes Omicron variant and lineages in SARS-CoV-2-positive samples. The asymmetric PCR melting curve analysis-based method were performed in 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials. (A) In reaction 1, four colored lines of each sample indicate the melting curves of RBD mutation site 460 (blue), 486 (green), 505 (orange) and the ORF1ab gene (red). (B) In reaction 2, the colored lines indicate the melting curves of RBD mutation site 484 (blue), 493+498 (green), 452 (orange) and the N gene (red).Additional file 7: Table S4. Values of Tms in SARS-CoV-2-positive samples validation using the asymmetric PCR melting curve analysis-based method.Additional file 8: Table S5. Sequence of the targeted spike gene region in SARS-CoV-2 positive clinical samples using Sanger sequencing.


Background: Plant cell culture technology is a potential way to produce polyphenols, however, this way is still trapped in the dilemma of low content and yield. Elicitation is regarded as one of the most effective ways to improve the output of the secondary metabolites, and therefore has attracted extensive attention.Methods: Five elicitors including 5-aminolevulinic acid (5-ALA), salicylic acid (SA), methyl jasmonate (MeJA), sodium nitroprusside (SNP) and Rhizopus Oryzae Elicitor (ROE) were used to improve the content and yield of polyphenols in the cultured Cyclocarya paliurus  cells, and a co-induction technology of 5-ALA and SA was developed as a result. Meanwhile, the integrated analysis of transcriptome and metabolome was adopted to interpret the stimulation mechanism of co-induction with 5-ALA and SA.Results: Under the co-induction of 50 μM 5-ALA and SA, the content and yield of total polyphenols of the cultured cells reached 8.0 mg/g and 147.12 mg/L, respectively. The yields of cyanidin-3-O-galactoside, procyanidin B1 and catechin reached 28.83, 4.33 and 2.88 times that of the control group, respectively. It was found that expressions of TFs such as CpERF105, CpMYB10 and CpWRKY28 increased significantly, while CpMYB44 and CpTGA2 decreased. These great changes might further make the expression of CpF3′H (flavonoid 3′-monooxygenase), CpFLS (flavonol synthase), CpLAR (leucoanthocyanidin reductase), CpANS (anthocyanidin synthase) and Cp4CL (4-coumarate coenzyme A ligase) increase while CpANR (anthocyanidin reductase) and CpF3′5′H  reduce, ultimately enhancing the polyphenols accumulationConclusion: The co-induction of 5-ALA and SA can significantly promote polyphenol biosynthesis in the cultured C. paliurus cells by regulating the expression of key transcription factors and structural genes associated with polyphenol synthesis, and thus has a promising application. Polyphenols are one of the most diverse and widely distributed secondary metabolites in the plants . CatechiAt present, polyphenols are mainly obtained from plant materials such as leaves, stems, flowers and fruits by extraction and purification . There aetc. Other species, however, cannot be utilized on a large scale probably due to the low contents and yields of active compounds. For instance, the yields of catechin, caffeic acid, kaempferol and apigenin in the cultured date palm cells were only 155.9, 162.7, 89.7, and 242.7 μg/L, respectively , compact green callus (CG), wet grayish brown callus (WGB), crisp white callus (CW), crisp light pink callus (CPC), and red callus (CR), among which CYG was the most common and typical line, while CR was extremely rare . ROE was obtained from Guangdong Microbial Culture Collection Center. Murashige and Skoog (MS) medium  medium  was bougC. paliurus calluses with excellent appearance and loose texture were inoculated into a 100 mL culture flask containing 40 mL MS medium supplemented with 1.0 mg/L kT, 0.3 mg/L NAA, 0.3 mg/L 2,4 -D, 30 g/L sucrose and 0.7% agar. The cultures were cultivated in a rotary orbital shaker at 115 rpm and (25 ± 2)°C under constant light, and sub-cultured with an inoculation amount of 6.0 g per culture bottle every 7 days.th day using suction filtration, and then dried in an oven at 50°C until a constant weight was achieved. The weight of the harvested cells  was measured after being cooled down to the room temperature, which was recorded as a biomass increment indicator (DW).The cultured cells were collected on the 6O-galactoside) were dissolved in 1% (v/v) hydrochloric acid water to prepare 1.0 mg/mL of standard solution, which were used to prepare series of standard solutions with concentrations from 10 to 400 μg/mL, 20–400 μg/mL, 5–200 μg/mL, respectively.Polyphenol extraction was performed according to O-galactoside) was calculated and used as total polyphenol content (TPC) and total polyphenol yield (TPY) of the cultured C. paliurus cells. The standard curves for the detection of polyphenols were shown in Polyphenol extraction and standard solution were determined with HPLC  on a C18 column . The assay procedure was performed in accordance with that described by . In thisReferring to In the evaluation of the promotion effects of each elicitor on the polyphenol biosynthesis in the cultured cells, the effects of concentration and addition time of the elicitors were all taken into consideration, which were designed and described in the The co-induction concentrations of 5-ALA and SA were optimized by comprehensive test (completely random design), using TPC as the evaluation indicator. The test concentration of 5-ALA and SA were designed and described in th day, and these samples were frozen with liquid nitrogen quickly and then stored at −80°C for the subsequent detection. Each group was sampled in six replicates, and the treated and untreated cells were named CPTG and CPCG, respectively. Sample cells were fully ground in liquid nitrogen with 400 µL cooled methanol/acetonitrile/aqueous solution . After vortex oscillation mixing, the extracted samples were stored at −20°C for 60 min, and then centrifuged at 14,000 g for 20 min under 4°C . Afterward the supernatant was recovered, and then evaporated in a vacuum rotary evaporator at 55°C until a constant weight was observed. The recovered extracts were re-dissolved in the acetonitrile aqueous solution (the ratio of acetonitrile to water was 1 to 1 (v/v)), followed by another centrifugation at 14,000 g for 15 min under 4°C. Thereafter, the collected supernatant was filtered through PTFE filters (0.22 μm) and then kept in an auto-sampler vial for the LC-MS/MS analysis .The cultured fresh cells of the control group and treated group co-induced by 5-ALA and SA were sampled on the 6All the detection was performed in a 1,290 series UHPLC . Chromatographic separation was carried out in a ACQUITY UPLC BEH C18 column , and the column temperature was maintained at 40°C. The flow rate was typically set at 0.3 mL/min. The acidified water (0.1% formic acid (v/v), solvent A) and acetonitrile (solvent B) were used as mobile phases. The elution gradient was programmed as following: 0–0.5 min, 5% B; 0.5–1.0 min, 5% B; 1.0–9.0 min, 5–100% B; 9.0–12.0 min, 100% B; 12.0–15.0 min, 5% B. During the whole analysis process, the samples were placed in a 4°C automatic injector, and the volume of each injection was 5 µL .The samples were separated by UHPLC and analyzed by q-exact-four-stage rod electrostatic field orbital trap high-resolution mass spectrometer . Electrospray ionization (ESI) mode of positive and negative ions were both adopted for the MS detection. MS detection conditions were as follows: ion source gas1 (gas1): 60, ion source gas2 (gas2): 60, curtain gas (cur): 30, source temperature: 320°C, ionsapary voltage floating (isvf) ± 3,500 V. Declustering potential (DP): ±60 V, collision energy: 35 ± 15 eV, exclude isotopes within 4 Da, candidate ions to monitor per cycle 6 .http://personalbio.bioon.com.cn/). Multi-dimensional statistical analyses such as PCA , orthogonal partial least squares discriminant analysis (OPLA -DA) were conducted. And single-dimensional statistical analysis like volcanic map was drawn by R software (https://www.r-project.org/).Qualitative and quantitative analyses were all carried out in Shanghai Personalbio Technology Co., Ltd.  and fold change obtained by OPLA -DA model were combined to analyze and screen differential metabolites. VIP >1, abolites .Cell samples were prepared according to the method described in http://www.personalmedicine.cn/TechnicalPlatform) in Shanghai Personalbio Technology Co., Ltd.Total RNA was extracted from cells in a total RNA Extractor  following the supplier’s instructions. The quality and quantity of extracted RNA were evaluated using agarose gel electrophoresis and 2,100 Bioanalyzer . The quality-checked RNA was purified and then used as a template to synthesize cDNA first strand followed by cDNA second strand with Primscript™ First-Strand cDNA Synthesis Kit . PCR amplification was subsequently conducted to establish cDNA libraries. RNA-sequencing was performed on the IlluminaHiseq™ 2,500 platform  were spliced by software Trinity (software version number: 2.4.0) . All the2(Fold Change)| ≥ 1 and q-value <0.05. In order to analysis the metabolic and signal transduction pathways involved in DEGs, the DEGs enrichment of KEGG (https://www.genome.jp/kegg/genome) was performed by comparing DEGs with the entire background of all KEGG-associated unigenes with a hypergeometric test (p < 0.05).DESeq was adopted to analyze DEGs with filtering conditions including |log® qPCR SYBR® Green Master Mix (Vazyme). The reaction system was 20 μL, including 10 μL 2 × SYBR real-time PCR premixture, 1 μL cDNA and 0.8 μL primers. The qPCR procedures were as follows: initially denatured at 95°C for 5 min, followed by 40 cycles of 15 s at 95°C and 30 s at 60°C  assay. The RNA and cDNA required for this analysis were obtained according to the method in  at 60°C . Gene ren method .C. paliurus cells were analyzed through KEGG database (https://www.genome.jp/kegg/).To reveal the inducement mechanism of the co-induction of 5-ALA and SA on the polyphenol biosynthesis, the differential metabolites and DEGs between the treated and untreated p ≤ 0.05).All the experiments were performed in triplicate and the results were given as the mean ± standard deviation (SD). The data was analyzed by DPS statistical software. Statistical analysis was performed with ANOVA followed by Duncan’s multiple range test  - catechin, cyanidin-3-O-galactoside and procyanidin B1 reached 49.28, 4.92 and 24.21 mg/L, respectively  were aligned with six public databases, and 45.36, 21.58, 15.99, 21.35, 44.13% and 30.10% of these unigenes had been annotated in NR, GO, KEGG, Pfam, eggNOG and Swiss-prot through BlastX, respectively   . All of ectively . It sugg(21.44%) . A total(21.44%) . When blA total of 5,233 genes changed significantly under the co-induction of 5-ALA and SA . As showCpMYB44s, one CpMYB12, two CpERF105s, three CpMYB10s, four CpWD40s, one CpORG2 and two CpMYC2s changed significantly under the co-induction of 5-ALA and SA. Among these significantly changed DETFs, CpMYB10 and CpERF105 showed a remarkable upregulation expression, on the contrary CpMYB44 and CpMYC2 presented a downregulation expression. TGA family is an important regulator of the signal transduction pathway related to SA biosynthesis. Results indicated that CpTGA1, CpTGA2, CpTGA4, and CpMYC2 were all downregulated under co-induction, among which CpTGA1 and CpTGA2 decreased significantly, while CpWRKY18, CpWRKY28, CpWRKY70, and CpTGA6 were upregulated, especially CpWRKY28  and two CpAIM1s (recombinant Absent In Melanoma 1) were annotated in SA biosynthesis pathway, meanwhile seven CpNPR1 genes (non-expresser of PR genes 1) and three CpPR1 genes (PR genes 1) were annotated in SA signal transduction pathway  were further verified by QRT-PCR. The determination results , at this moment the sample cells were in the middle and late stage of the co-induction, in which the expression of upstream genes such as CpPAL, CpC4H, and CpCHI had already retreated, and the midstream and downstream genes like CpANS, CpLAR, CpF3′H, CpFLS, Cp4CL, CpF3H, and Cp3GalT were highly expressed , while increased 3.3 times when added on the 10th day  in suspension culture of Origanum vulgare cells. Consequently, we deduce that cells in different culture phase have different physiological states, and therefore might present different responses to the external stimulus. Thirdly, elicitor concentration has a profound effect on the induction, which may act on cell growth or on the regulation of polyphenol synthesis. Our experiment data indicated that both 50 μM 5-ALA and SA showed the best stimulatory effect on polyphenol synthesis in the series of our test concentrations. Similar results had been reported by Gardenia jasminoides Ellis cells . When treated by low temperature, ERF105 expression was positively correlated with the accumulation of polyphenols in Arabidopsis thaliana , the content and yield of total polyphenols in the treated cells reached 8.0 mg/g and 147.12 mg/L, respectively. (+)-Catechin was the most abundant polyphenol, whose content and yield were as high as 5 mg/g and 91.67 mg/L, respectively. Integration detection and analysis of transcriptome and metabolome showed that the co-induction of 5-ALA and SA significantly changed the expressions of 14 pathway genes and 7 TFs related to polyphenol biosynthesis. Under the co-induction, the expressions of TFs such as CpERF105, CpMYB10, and CpWRKY28 increased significantly, which might further upregulated the expressions of CpANS, CpLAR, CpF3′H, CpFLS, Cp4CL, CpF3H, and Cp3GalT and downregulated the expressions of CpPAL, CpANR, and CpF3′5′H, and finally enhanced the polyphenol accumulation.Five elicitors were used to stimulate the polyphenol synthesis in the cultured


To the editor,Measuring glomerular filtration rate by iohexol plasma clearance has become a preferred method in clinical practice and research –9, but a90) and an at least substantial CCC of ≥ 0.95 were considered optimal. R 4.2.1 [Data from the BIS, including 546 individuals with data obtained up to 300 min post-injection, were evaluated , 8. One- R 4.2.1  and NONM R 4.2.1  were use90 goal was achieved with Eqs. BM- and Ch-like equations performed similarly well Table , with an


Ryanodine receptor (RYR) regulates intracellular calcium liberation. Calcium mobilization is critical in the regulation of MC functional programs. However, the role of RYR in MRGPRX2-mediated pseudo-allergic skin reaction has not been fully addressed. To study the role of RYR in vivo, we established a murine skin pseudo-allergic reaction model. RYR inhibitor attenuated MRGPRX2 ligand substance P (SP)-induced vascular permeability and neutrophil recruitment. Then, we confirmed the role of RYR in an MC line (LAD2 cells) and primary human skin-derived MCs. In LAD2 cells, RYR inhibitor pretreatment dampened MC degranulation (detected by β-hexosaminidase retlease), calcium mobilization, IL-13, TNF-α, CCL-1, CCL-2 mRNA, and protein expression activated by MRGPRX2 ligands, namely, compound 48/80 (c48/80) and SP. Moreover, the inhibition effect of c48/80 by RYR inhibitor was verified in skin MCs. After the confirmation of RYR2 and RYR3 expression, the isoforms were silenced by siRNA-mediated knockdown. MRGPRX2-induced LAD2 cell exocytosis and cytokine generation were substantially inhibited by RYR3 knockdown, while RYR2 had less contribution. Collectively, our finding suggests that RYR activation contributes to MRGPRX2-triggered pseudo-allergic dermatitis, and provides a potential approach for MRGPRX2-mediated disorders.Mast cell (MC) activation is implicated in the pathogenesis of multiple immunodysregulatory skin disorders. Activation of an IgE-independent pseudo-allergic route has been recently found to be mainly mediated  MRGCalcium signaling is crucial for mast cell activation and is implicated in both MC degranulation and the generation of cytokines. Calcium activation patterns by MRGPRX2 and FcϵRI are drastically diverse , 15. MRGRyanodine receptor (RYR) is one of the primary intracellular calcium release channels apart from IP3R. RYR is known for regulating the contraction of muscles and widely expresses in the sarco/endoplasmic reticulum , 20. Rya22.1C57BL/6 mice  were obtained from the Laboratory Animal Center of Xi’an Jiaotong University. Animal experiments were approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University . Animals were housed under standard conditions  with free access to water and standard dry feed. Mice were randomly divided into a control group and an experimental group.2.2C57BL/6 mice  were intraperitoneally injected with 50 µl of dantrolene (10 mg/kg in DMSO) or vehicle for 2 consecutive days. Then, each mouse was injected intravenously with 0.2 ml of 0.4% Evans blue dye in saline. After 1 h, mice were anesthetized with an intraperitoneal injection of 80 mg/kg 1% pelltobarbitalum natricum. The thickness of the ear was determined by a vernier caliper. Then, an intradermal injection of SP  was performed randomly into one side of the ear, and the vehicle  was intradermally injected into the other. After 30 min, the mice were euthanized, ear thickness was measured again, and pictures of the ears were taken. Ear tissues were collected, dried for 24 h at 60°C, and weighed. Tissues were immersed and minced in 300 μl of acetone-saline (7:3). Following by 30 min ultrasonication, tissues were centrifuged for 20 min at 3,000 rpm. Two-hundred-microliter aliquots of the supernatant were seeded into 96-well plates, and the optical density (OD) was read at 620 nm.2.3g for 10 min at 4°C. Pellet-containing cells were processed for flow cytometric analysis.C57BL/6 mice  were intraperitoneally injected with 50 µl of dantrolene (10 mg/kg in DMSO) or saline of the same volume for 2 consecutive days. SP  or vehicle were intradermally injected into one side of the ear pinna to activate MrgprB2 (murine orthologue of human MRGPRX2). Three hours later, all mice were euthanized and ears were cut and digested with 1.5 mg/ml collagenase type IV  with DNase I at 10 μg/ml  at 37°C in a shaking incubator for 75 min. Cells were filtrated and centrifuged at 400×2.4Cells were incubated with CD11b-PE (Clone:M1/70) and Ly6G-APC (Clone: RB6-8C5) for 45 min, all from liankebio . Live versus dead cells were stained using Zombie Yellow™ Fixable viability dye . The data were acquired with a BD FACSCelesta flow cytometer  and analyzed by FlowJo software version 10.7.2 . Neutrophils were gated as CD11b+Ly6G+ live cells.2.52. Hemidepletion was performed once a week and supplements of SCF (at 100 ng/ml)  and IL-4 (at 20 ng/ml)  were provided once a week.The human mast cell Laboratory Allergic Disease 2 (LAD2) cell line was kindly provided by A. Kirshenbaum and D. Metcalfe . LAD2 cells were cultured with Basal Iscove’s medium  supplemented with 10% FCS  at 37°C with 5% CO2.65/ml, supplemented with SCF (at 100 ng/ml) and IL-4 (at 20 ng/ml) twice a week.Foreskins were obtained from circumcisions with the written informed consent of the patients or their legal guardians. The study was approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University  and experiments were conducted according to the Declaration of Helsinki Principles. Briefly, skin samples were cut into strips and digested with a 3.5 U/ml dispase  at 4°C overnight to remove the epidermis. Then, the dermis was chopped and digested with 1.5 mg/ml collagenase type 1  and 0.75 mg/ml hyaluronidase type 1-S  with DNase I at 10 μg/ml  at 37°C in a shaking incubator for 75 min. Cells were filtrated and labeled with anti-human c-Kit magnetic microbeads to positively select MC by cell preparation columns . Purified skin MCs were cultured in Basal Iscove’s medium with 10% FCS at the concentration of 5 × 102.74) treated with or without RYR inhibitor  were resuspended in 100 µl of PAG-CM buffer  and stimulated with vehicle (spontaneous release), compound 48/80 (c48/80)  or substance P (SP)  for 60 min. Cells were centrifuged and supernatants (SNs) were collected and the pelleted MCs were rapidly frozen with 100 μl of H2O. After thawing, 50 μl of SNs or cell lysates were incubated with the same volume of 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide  solution at 5 μM in citrate buffer (pH 4.5) for 60 min at 37°C. Sodium carbonate buffer  was added to stop the reaction. Fluorescence intensity was determined at excitation at 355 nm and emission wavelength of 460 nm. % β‐hexosaminidase release = [fluorescence intensity SN/(fluorescence intensity SN + fluorescence intensity lysate)] × 100. The net release was calculated by subtracting spontaneous release.Cells (5 × 102.85 cells/sample) were loaded with 2 μM Fluo-4 AM  with 0.02% Pluronic F-127  for 45 min at 37°C in the dark, followed by de-esterification for an additional 15 min at room temperature. Then, the cells were washed and resuspended with PAG-CM buffer. For assay with inhibitor, cells were loaded with dantrolene (100 µM) for 15 min before ligand stimulation. Calcium signals were determined using a Fluorescence Spectrophotometer  with an excitation wavelength of 494 nm and an emission wavelength of 516 nm. The signal was recorded every 2 s for 2 min at baseline, and additionally 5 min after stimulation.Cells  and reverse‐transcribed with a SweScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR  as detailed by the manufacturer. PCR was carried out with the 2×Universal Blue SYBR Green qPCR Master Mix . Primers were 5’-TTGCGGAGCAAGAGATTCCC and 5’-GGCAGTGCCTCAGCATTTTT for CCL-1, 5’-CCCCAAGCAGAAGTGGGTTC and 5’-TTGGGTTGTGGAGTGAGTGTT for CCL-2, 5’-CTGGGCAGGTCTACTTTGGG and 5’-CTGGAGGCCCCAGTTTGAAT for TNF-α, and 5’-CATCCGCTCCTCAATCCTCT and 5’-GATGCTCCATACCATGCTGC for IL-13. The values were normalized to the housekeeping genes β-actin, cyclophilin B, and GAPDH. The primers were 5’-CTGGAACGGTGAAGGTGACA and 5’-AAGGACTTCCTCTAACAATGCA for β-actin, 5’-AAGATGTCCCTGTGCCCTAC and 5’-ATGGCAAGCATGTGGTGTTT for Cyclophilin B, and 5’-CCTCTGACTTCAACAGCGAC and 5’-TTACTCCTTGGAGGCCATGTG for GAPDH. The 22.106 cells/ml and treated with or without dantrolene (100 µM) for 15 min and then the cells were stimulated with vehicle (spontaneous release), compound 48/80 (c48/80) (5 µg/ml), or substance P (SP) (30 μM) for 24 h. Then, supernatants were collected for IL-13, TNF-α, CCL-1, and CCL-2 measurements. ELISAs were performed according to the manufacturer’s instructions .MCs were seeded at 1 × 102.116/ml and treated with 80 nM of RYR2- and RYR3-targeting siRNA or non-targeting siRNA with RNAi-Mate  for 48 h, and employed for downstream analyses. The siRNA sequences were as follows: RYR2 forward: 5’-GGCUCUAAUUAGAGGAAAUTT, RYR2 reverse: 5’-AUUUCCUCUAAUUAGAGCCTT, RYR3 forward: 5’-GCAGAUCAACAUGCUGCUUTT, and RYR3 reverse: 5’-AAGCAGCAUGUUGAUCUGCTT.MCs were plated at 1 × 102.12t-test  or the Wilcoxon matched-pairs signed-rank test . For the difference between two groups with unpaired experimental design, the Mann–Whitney test was performed. For the difference between more than two groups, comparisons were performed by the RM one-way ANOVA with Dunnett’s multiple comparisons tests  or Friedman test with Dunn’s multiple comparison test . When the data were compared to a fixed number, differences between groups were compared using the one-sample t-test  or Wilcoxon signed-rank test . p < 0.05 was considered statistically significant.Statistical analyses were performed using PRISM 9.0 . For the difference between two groups with paired experimental design, comparisons were performed by the 33.1The mouse ortholog of MRGPRX2 is MrgprB2, which resembles the activation pattern of human MRGPRX2 . To veri3.2To study the role of RYR in MRGPRX2-mediated MC activation, we detected β‐hexosaminidase release in a human MC line, LAD2 cells, to assess MC degranulation. We found that dantrolene significantly inhibited both c48/80 and SP-triggered degranulation  was significantly inhibited by dantrolene   . To selend RYR3) . From thInterference with RYR3 suppressed the degranulation by both agonists employed, i.e., c48/80 and SP. RYR2 siRNA also decreased the secretion of LAD2 cells, although it did not reach significance  , 37, 38.Previous studies have demonstrated that MC degranulation is regulated by calcium signals. The role of ER-expressing IP3R, STIM1, Orai channels, and TRP channels has been profoundly illuminated in multiple studies , 28, 39.It is well known that calcium signaling is essential for the generation of multiple proinflammatory cytokines , 50. MCsin vivo. MC degranulation is associated with increased vascular permeability, which leads to vasodilation and vascular leakage. Decreased skin edema and vascular leakage triggered by SP with dantrolene pretreatment demonstrated the role of RYR in MRGPRX2-mediated skin allergy. In terms of immune cell recruitment, which represents the late-phase response following MC activation, the curtailed neutrophil recruitment represents the inhibited inflammation by RYR inhibition. Interestingly, in ex vivo studies, the reduction rate of β‐hexosaminidase release is less than the vascular leakage and immune cell infiltration in in vivo studies. Apart from the reason that to trigger a certain symptom needs adequate amount of stimulator, the release of the MC granule could be under the triggering threshold after the inhibition by dantrolene. Moreover, RYR expresses on multiple cell types, e.g., keratinocyte and neutrophils (ex vivo experiments.Finally, we used the mouse cutaneous anaphylaxis model to verify the role of RYR in Mrgprb2 activation trophils , 56. Preex vivo studies were verified by RYR isoform knockdown. Moreover, the pivotal role of RYR3 was identified. With the cutaneous anaphylaxis mouse model, we found that MRGPRX2-induced vascular permeability and immune cell recruitment were impaired by dantrolene. The present study suggests the potential novel therapeutic approaches targeting RYR for the treatment of allergic diseases.In conclusion, MRGRPX2 activation regulates pseudo-allergic reactions, and emerging compounds including FDA-approved drugs were identified as MRGPRX2 ligands. The role of MRGPRX2 is insinuated in the pathogenesis of urticaria, atopic dermatitis, and psoriasis. We identified RYR as a calcium channel, which is activated by MRGPRX2. The activation leads to MC degranulation and the generation of CCL-1, CCL-2, TNF-α, and IL-13. The The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of Xi’an Jiaotong University.Conceptualization, ZW. Investigation, ZW, XZ, HZ, DC, XD, and DY. Data curation, ZW and XZ. Writing—original draft preparation, ZW. Writing—review and editing, ZW, SG, and WZ. Visualization, ZW. Supervision, ZW, SG, and WZ. Project administration, ZW. Funding acquisition, ZW and SG. All authors contributed to the article and approved the submitted version.