Candida auris infections are difficult to treat due to acquired drug resistance against one or multiple antifungal drug classes. The most prominent resistance mechanisms in C. auris are overexpression and point mutations in Erg11, and the overexpression of efflux pump genes CDR1 and MDR1. We report the establishment of a novel platform for molecular analysis and drug screening based on acquired azole-resistance mechanisms found in C. auris. Constitutive functional overexpression of wild-type C. auris Erg11, Erg11 with amino acid substitutions Y132F or K143R and the recombinant efflux pumps Cdr1 and Mdr1 has been achieved in Saccharomyces cerevisiae. Phenotypes were evaluated for standard azoles and the tetrazole VT-1161. Overexpression of CauErg11 Y132F, CauErg11 K143R, and CauMdr1 conferred resistance exclusively to the short-tailed azoles Fluconazole and Voriconazole. Strains overexpressing the Cdr1 protein were pan-azole resistant. While CauErg11 Y132F increased VT-1161 resistance, K143R had no impact. Type II binding spectra showed tight azole binding to the affinity-purified recombinant CauErg11 protein. The Nile Red assay confirmed the efflux functions of CauMdr1 and CauCdr1, which were specifically inhibited by MCC1189 and Beauvericin, respectively. CauCdr1 exhibited ATPase activity that was inhibited by Oligomycin. The S. cerevisiae overexpression platform enables evaluation of the interaction of existing and novel azole drugs with their primary target CauErg11 and their susceptibility to drug efflux. Candida species is an emerging problem. While nosocomial invasive candidiasis is most often caused by Candida albicans, an increased incidence of serious infections is due to non-albicans spp., such as C. glabrata, C. tropicalis, C. parapsilosis, and C. auris [Aspergillus fumigatus, were recently highlighted by the World Health Organization as priorities for new research into diagnosis and treatment [The global incidence of invasive infections caused by non-albicans C. auris ,2,3,4. Treatment .C. auris is a readily transmitted pathogen with a high mortality rate and resistance profiles that can compromise antifungal therapy and the operation of healthcare environments. First reported in 2009 as a clinical isolate from the external auditory canal of a Japanese patient [C. auris were then obtained on three continents almost simultaneously, while a retrospective study identified a C. auris strain isolated in Korea in 1996 [T  for FLC. This MIC difference may be due to a gene duplication encoding a putative phospholipid-translocating P-type ATPase and a putative cytochrome P450 [ patient , clinica in 1996 . The firMYA-5001 ) and it n B11220 ,10 but, C. auris strains shows greater genetic diversity between continents than within such geographic boundaries [C. auris clades as: clade IV > clade I > clade II = clade III [C. auris as an urgent health threat due to: (1) misidentification and inappropriate patient management, (2) the number of outbreaks and their practical consequences in healthcare settings, and (3) multidrug-resistance phenotypes that make C. auris infections difficult to treat. The CDC suggested that novel antifungals and the use of combination therapy should be investigated for pan-resistant strains [Consistent with separate emergence inside continents, the current collection of undaries . Genome undaries ,11,12. Alade III . In 2019 strains .C. auris (C. auris Erg11 (CauErg11) and it has been found in clades I, IV, and V. While K143R is found in clades I and IV only, most of these mutations belong to clade I [C. auris clinical isolates, CDR1 and MDR1 are highly expressed, with CDR1 the main contributor to triazole resistance [TACK1b, the gene encoding the transcription factor driving CauCDR1 expression, were shown to be a principal cause of FLC resistance, with A640V the most prominent Tac1b substitution [C. auris isolates [Clade-associated mutations conferring resistance to azole, echinocandin, and polyene antifungals have been found in C. auris . Y132F i clade I ,15,16,17 clade I . Among tsistance . Gain-oftitution ,23. The isolates . The N64isolates . C. auris are lethal [C. auris that selectively inhibit Erg11 or efflux pumps without being compromised by mutations that either directly or indirectly affect the azole target enzyme or drug efflux.Up to 50% of systemic infections caused by e lethal ,33,34. DSaccharomyces cerevisiae, C. albicans, C. glabrata and C. parapsilosis, Cryptococcus neoformans, as well as the S. cerevisiae Pdr5 and C. albicans Mdr1 and Cdr1 drug efflux pumps, all expressed constitutively from the PDR5 locus of a hypersensitive S. cerevisiae strain with seven drug efflux pumps removed [This laboratory has implemented tools for biochemical and phenotypic analysis and structure-directed screens by expressing functional, recombinant full-length Erg11 from  removed ,37,38,39S. cerevisiae host strain ADΔΔ  unless otherwise stated. S. cerevisiae was grown and maintained in Yeast Extract Peptone Dextrose medium . Synthetic defined (SD) histidine dropout medium  was used to select transformants. Drug susceptibility testing assays used SD medium complemented with 0.79 g/L complete supplement mixture (CSM) and containing 10 mM MES  and 20 mM HEPES , buffered with UltraPureTM Tris  to pH 6.8.All reagents used in culture media were purchased from FormediumS. cerevisiae strains was performed using the Alkali-CationTM Yeast Transformation Kit . DNA sequence analysis of transformation cassettes and genes of interest in genomic DNA were performed at the Genetic Analysis Services facility . The presence of recombinant enzyme in Coomassie blue R250-stained SDS-PAGE separated protein bands was verified by mass spectrometry of tryptic fragments using an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) at the Centre for Protein Research .Nile Red, 2-deoxy-D-glucose, Oligomycin (OM), and the antifungals FLC, Voriconazole (VRC), Posaconazole (POS), Itraconazole (ITC), and AMB were purchased from Sigma-Aldrich. ISA was purchased from BOC Sciences , and micafungin (MFG) was a gift from Astellas Pharma Inc. . The novel tetrazole antifungal VT-1161 and the Mdr1 inhibitor MCC1189 were synthesized by MicroCombiChem . Beauvericin was purchased from Cayman Chemicals . Desalted oligonucleotides were purchased from Integrated DNA Technologies . Phusion Green Hot Start II High-Fidelity PCR master mix (2x)  was used for all PCR amplifications, except the use of TaKaRa Ex Taq DNA polymerase  for colony PCR. NucleoSpin Gel and PCR clean-up kits  were used for DNA gel extraction and PCR purification. Genomic DNA was isolated using the Yeast DNA Extraction Kit (Thermo Fisher Scientific). Transformation of C. auris ERG11, MDR1, or CDR1 from the S. cerevisiae PDR5 locus. The host strain ADΔΔ (Y1857) has the PDR3 transcriptional regulator gene and seven ABC transporters removed while the mutant gain-of-function transcriptional regulator pdr1-3 gene enables constitutive expression from the PDR5 locus [Recombinant strains were constructed to overexpress R5 locus . StrainsERG11, CauMDR1, and CauCDR1 were amplified from genomic DNA of the clade II wild-type clinical isolate B11220 using the primers listed in CauERG11-6×His (strain Y2767), CauMDR1-6×His (Y2765), or CauCDR1-6×His (Y2766), with each ORF containing a C-terminal GGR linker and hexahistine tag sequence, followed by the PGK transcription terminator and a downstream HIS1 marker flanked by two LoxP sites. Each transformation cassette was bordered by PDR5-specific arms to enable homologous recombination at the PDR5 locus of the host strain S. cerevisiae Y1857. The endogenous ERG11 gene was also deleted in strain Y2767 using a disruption cassette with the recyclable selection marker URA3 flanked by modified LoxP sites [ERG11 mutants Y2768 (CauERG11 Y132F-6×His) and Y2769 (CauERG11 K143R-6×His) were constructed by incorporating the mutations Y132F and K143R, respectively, into CauERG11-6×His using the primers listed in Table S2. For this, the HIS1 marker of CauERG11-6×His was deleted from the PDR5 locus using the Cre-expressing plasmid pSH69 . The galactose-inducible Cre recombinase mediated recombination between the two LoxP sites, allowing HIS1 to be removed [HIS1 marker was incorporated into the PDR5 locus of the CauERG11-6×His-expressing strain via homologous recombination. The nucleotide sequences in the PDR5 locus of transformants  and crude membranes isolated by differential centrifugation, as described previously [TM Hybond® polyvinylidene difluoride (PVDF) membrane , were decorated with a mouse monoclonal anti-6×His tag antibody conjugated with horse radish peroxidase (Sigma-Aldrich), as previously described [TM Western ECL, Bio-Rad Laboratories Inc., Hercules, CA, USA). Gel imaging was performed using a ChemiDoc MP gel imager (Bio-Rad) and the relative expression of the recombinant His-tagged proteins on the Western blots was evaluated using Image Lab Software Version 6.1 (Bio-Rad).Strains of interest were cultured in YPD at 30 °C with shaking at 200 rpm and harvested at ODeviously . The peleviously  with boveviously  on 8% Trescribed , and visw/v) glycerol, 250 mM NaCl, 20 mM Tris, 0.5 mM PMSF, 1 tablet cOmpleteTM Mini, EDTA-free Protease Inhibitor cocktail  per 50 mL plus 16 mM n-decyl-β-D-maltopyranoside . The detergent-solubilized protein affinity purified by binding to 2 mL of Ni-NTA Agarose matrix  per g of protein in a disposable Econo-Pac® Chromatography column (Bio-Rad) was washed with 2 mM L-histidine and eluted with 100 mM L-histidine. The eluted protein was concentrated to 500 µL using Amicon® Ultra–4 Centrifugal Filter Units with a 50 kDa cutoff . Samples (1 µL) were separated by SDS-PAGE to obtain Coomassie-blue-stained protein bands for mass spectrometry of tryptic fragments. Tryptic fragments from the SDS-PAGE separated CauCdr1-6×His-containing band in crude membranes were used for mass spectrometry. For protein identification, the obtained fragments were searched against the Swiss-Prot database, the sequence of interest, and S. cerevisiae database to exclude possible background contamination.Cells were harvested from 2 L YPD cultures, crude membranes prepared, and CauErg11-6×His, CauErg11 Y132F-6×His, CauErg11 K143R-6×His, or CauMdr1-6×His partially purified by nickel–nitrilotriacetic acid (Ni-NTA) affinity chromatography, essentially as described by Monk et al. . The cruS. cerevisiae. The assays were carried out in flat-bottom 96-well plates  containing 100 µL of antifungal in serial dilution in SD medium or 100 µL of SD medium with 0.5% DMSO for the no-drug control. The plates were seeded with 100 µL of SD pH 6.8 grown cells at an OD600nm = 0.02 and incubated with shaking at 100 rpm under 75% humidity  for 48 h at 30 °C. The OD600nm was read using a Synergy 2 plate reader . For all antifungals, the MIC was read as ≥80% growth inhibition (MIC80) of the non-drug control after subtraction of the medium background. Three separate clones of each construct were tested in at least three biological replicates.Antifungal susceptibility profiles of recombinant strains were determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) of Yeasts version 7.3.2 with modifications. SD medium buffered to pH 6.8 was used instead of RPMI-1640 to provide the inoculum and growth conditions optimized for 600nm = 0.5 and incubated for ~7 h until an OD600nm ~2 was achieved. The pellet was washed 3 times with 10 mL of ice-cold phosphate-buffered saline (PBS) via centrifugation at 3900× g for 5 min and incubated on ice overnight. Cells were warmed to room temperature for 30 min then resuspended in 10 mL 50 mM HEPES buffered with 0.1M NaOH to pH 7.0. The cells were starved by incubation with 5 mM 2-deoxy-D-glucose (2-DOG) for 30 min at 30 °C with gentle agitation (50 rpm). Nile Red  was added, and the cells were incubated for 30 min at 30 °C. The cells were washed twice with ice-cold HEPES buffer, adjusted in the HEPES buffer to OD600nm = 10, and kept on ice for 2 h. Flat-bottom 96-well plates were prepared containing 50 µL of the HEPES buffer. A 100 µL sample of Nile-Red-loaded fungal cells was added to each well. Efflux pump activity was initiated by adding 50 µL 80 mM glucose while the addition of 50 µL of HEPES buffer served as a control. Fluorescence intensity was measured using a Synergy 2 plate reader at excitation wavelength 485/20 nm and emission wavelength 528/20 nm at 35 s intervals for 10 min, with 5 s shaking between readings. Nile Red fluorescence intensity was expressed as a percentage relative to the intensity at time T0.Nile Red efflux was measured as previously described ,46. Cell2SO4). The amount of free phosphate was measured using a Synergy 2 plate reader at 750 nm with KH2PO4 as standard. The ATPase activity of crude membranes from the negative control strain Y2411, which lacks Cdr1, was subtracted from the activity of Cdr1-overexpressing strains.The ATPase activity of recombinant strains overexpressing Cdr1 was measured in the presence and absence of OM, using crude membranes isolated from 40 mL YPD cultures, as previously described ,48,49,50−1cm−1 [The in vitro function of recombinant Erg11 in strains Y2767, Y2768, and Y2769 was analyzed in type II drug-binding studies. The concentration of functional wild-type enzyme was determined using a carbon monoxide (CO) binding assay . Protein−1cm−1 .d was calculated using GraphPad Prism 9 software by applying a non-linear regression analysis (Levenberg-Marquardt method) and obtaining the best fit of the data for the Hill and Michaelis–Menten binding equations.Type II binding of azoles was measured using difference spectra between 350 and 500 nm, with 1 µM enzyme titrated with POS, VRC, or ISA. The antifungals were dissolved in DMSO and added to the sample cuvette. The same amounts of DMSO were added to the reference cuvette, with the total amount of DMSO in the cuvette not exceeding 2%. The trough–peak difference in the spectra was used to plot the binding curves. The dissociation constant KStatistical analyses were performed with GraphPad Prism 9 .S. cerevisiae strains generated in this study are listed in C. auris ERG11, MDR1, or CDR1 from the S. cerevisiae PDR5 locus. The recombinant strains showed similar generation times and morphologies compared to the parental host strain ADΔΔ during culture in SD medium pH 6.8 , CauErg11 (Y2767) and its mutants Y132F (Y2768), and K143R (Y2769). As expected, the overexpression of CauERG11-6×His significantly reduced susceptibility to VRC and VT-1161, but not to AMB or MFG, consistent with the two azole drugs targeting CauErg11. The two CauErg11 mutants exhibited significantly higher MIC80 values for FLC and VRC in comparison to the host strain Y1857. The Y132F mutant also gave MIC80 values significantly higher than the host strain when tested against the tetrazole VT-1161, while the K143R mutant appeared susceptible to VT-1161 and showed MIC values comparable to the host strain. The MIC80 values for the strains expressing recombinant wild-type CauErg11 and its Y132F and K143R mutations were identical for the remaining antifungals tested.Susceptibility profiles of the host strain ADΔΔ and the recombinant strains were evaluated by measuring MIC80 values obtained for the strains overexpressing drug efflux pumps. Compared to the host strain Y1857, the expression of CauMdr1 (Y2765) or CauCdr1 (Y2766) increased FLC resistance >155-fold. VRC resistance increased >100-fold on expression of recombinant CauMdr1 and >200-fold on expression of recombinant CauCdr1. The expression of either efflux pump gave significantly higher MIC values with the midlength-tailed azole ISA, a more modest increase of 7-fold for CauMdr1 and a very strong increase of >800-fold for CauCdr1. Expression of CauCdr1 but not CauMdr1 elevated MIC80 levels against POS 4-fold, ITC > 47-fold, and VT-1161 > 2500-fold compared to the host strain. The control antifungals MFG and AMB gave essentially unchanged MIC80 values for the host and recombinant strains.S. cerevisiae strains that overexpress C. albicans Mdr1A (Y525) or Cdr1B (Y570) provided positive controls [Nile Red was used as a substrate to confirm the efflux activity of strains overexpressing CauMdr1 (Y2765) and CauCdr1 (Y2766). The host strain ADΔΔ (Y1857) was used as negative control , and thecontrols . Pump-spFor the MFS-pump-expressing strains Y2765 and Y525 preloaded with Nile Red, proton motive force-dependent efflux activated with glucose decreased the relative fluorescence intensity of membrane-associated Nile Red by about 35% within 3 min. After 10 min, a steady-state reduction of 40% was seen a. A prevC. albicans Cdr1 and Cdr2 [Strains Y2766 and Y570, expressing ATP-dependent ABC pumps, showed weaker glucose activation of Nile Red efflux than the MFS strains. After 3 min of glucose exposure, the membrane-associated relative fluorescence intensity was reduced by 25% for Y2766 and 20% for Y570, with similar or slightly lower steady-state levels of fluorescence intensity maintained for 10 min b. The adand Cdr2 , almost 50 values of Y2766 and Y570 Cdr1 ATPase activity for OM were 0.128 and 0.068, respectively.To demonstrate the ATP-dependent efflux activity of the CauCdr1-overexpressing strain, the ATPase activities of crude membranes containing recombinant CauCdr1 (Y2766) and CaCdr1B (Y570) were measured. Crude membranes from strain Y2411 were used as a negative control to correct for background ATPase activity in the Cdr1-expressing strains. Strains Y570 and Y2768 had 4-fold (Y570) or 7-fold (Y2766) higher ATPase activity, respectively, than the negative control Y2411 (57 ± 13 nmol/min/mg protein), confirming the functionality of CauCdr1. −1 cm−1 [The function of the recombinant CauErg11 was investigated by measuring its ability to bind the azoles POS, VRC, and ISA. CauErg11-6×His was partially purified from DM extracts of crude membranes using Ni-NTA affinity chromatography eluted with L-Histidine . The amo−1 cm−1 . The affKd values < 1 µM in the presence of 1 µM enzyme.Type II binding of the azoles  with Kd −1 cm−1 [Kd values < 1 µM obtained in the presence of 1 µM enzyme indicated tight binding of both POS and VRC, comparison of the maximal trough–peak absorbance difference  <1 indicative of tight 1-1 binding. Previous studies of overexpressed C. albicans and C. parapsilosis CYP51 gave Kd values in a similar range for the antifungal POS [d = 0.74) than for CaErg11-6×His (Kd = 0.29) [Kd = 0.36) than for VRC (Kd = 0.7), in both the wild-type and Y132F mutant enzyme. While the binding of POS to CauErg11 declined by 2-fold, VRC binding decreased 9-fold between the wild-type and mutant enzymes, suggesting that VRC is less able than POS to occupy the CauErg11 Y132F-6×His active site  and VRC (9-fold), and weakly for VT-1161 (2.6-fold), but not for ITC or POS. The binding of ISA, which contains a tertiary alcohol group, was not affected by the CauErg11 Y132F mutation. This is presumably because of compensatory interactions with the active site of the enzyme, possibly due to the 2,5-difluorphenyl group of ISA, its stereochemistry around the tertiary alcohol, and/or due to interactions of its butan-2-yl-1,3-thiazol-4-ylbenzonitrile tail with the entry channel of Erg11. Interestingly, the homologous S. cerevisiae Erg11 Y140F mutation does not confer resistance to ISA.In sistance . The CaEsistance . Both muS. cerevisiae conferred resistance to FLC (16-fold MIC80 increase versus wild-type CauErg11) and VRC (2.3-fold), the level of resistance was lower than for the CauErg11 Y132F mutant, presumably due to the indirect effect of the K143 mutation on Y132. As expected from the analysis of the Y132F mutant, the CauErg11 K143R mutant did not confer resistance to ISA, ITC, and POS. It also failed to confer resistance to VT-1161. The reason for this difference is not clear but the indirect action of the K143R mutation may have been insufficient to affect the key hydrogen bond network involving CauErg11 Y132. The K143R mutation is also predicted to affect the catalytic efficiency of the enzyme [In the CaErg11 crystal structure (PDB #: 5V5Z) and in a homology model of CauErg11, K143 lies in a position proximal to the heme iron, forming an ionic bond with the heme propionate that is hydrogen bonded with Y132. The K143R mutation is, therefore, predicted to indirectly affect the binding of substrates interacting with Y132 and affected by the Y132F mutation. While the CauErg11 K143R mutant expressed in e enzyme . As the C. albicans and C. auris contribute in similar fashions to intermediate and high-level azole resistance, respectively. Our data suggest that CauMdr1-overexpressing strains will mainly contribute to FLC and VRC resistance, whereas the overexpression of CauCdr1 is the major driver of pan-azole resistance. Constitutive high-level expression of these efflux pumps conferred by GOF mutations in the Mrr1 and Tac1b transcription factors, mimicked in our S. cerevisiae model by the Pdr1-3 transcription factor acting on the PDR5 locus, can be expected to dominate the azole resistance in C. auris Clades I and IV conferred by overexpression of CauErg11 and by the mutations Y132F and K143R. Our successful use of the pdr1-3 GOF transcriptional regulator gene as a surrogate is much simpler than, for example, engineering S. cerevisiae strains in which the ScMRR1 or ScPDR1 loci are replaced with their wild-type and mutant CauMRR1 or CauTAC1 counterparts, as well as requiring modification of the PDR5 promoter in anticipation that functional heterologous transcription complexes will be formed. In contrast, reversion of the pdr1-3 gene to PDR1 in the S. cerevisiae CauMDR1 and CauCDR1 recombinant strains might enable mimicry of wild-type CauMdr1 and CauCdr1 expression and function.The overexpression of CauMdr1 confers strong resistance to FLC and VRC, and weaker resistance to VT-1161, but not to ITC or POS , Table 2S. cerevisiae-based expression system with background drug efflux pumps deleted was used to functionally overexpress the recombinant azole target CauErg11, its Y132F and K143R mutants, the MFS drug efflux pump CauMdr1, and the ABC transporter CauCdr1. This approach, in a host system hypersensitive to azole drugs, enabled characterization of the azole drug target CauErg11, an essential contributor to sterol biosynthesis, the main target-based mutations (CauErg11 Y132F and K143R), plus the CauMdr1- and CauCdr1-dependent drug efflux pumps that contribute to azole resistance in C. auris. This platform extends our previous studies with key molecules from other fungal pathogens expressed in S. cerevisiae [C. auris clinical isolates, it creates an opportunity for the structure-based discovery needed to identify novel azole antifungals not susceptible to target-based resistance as well as screens for inhibitors of the MFS and ABC transporter-based drug efflux mechanisms that contribute high-level azole resistance.A revisiae ,65,66,67


The choice of storage method to ensure the quality of mulberry leaves is a challenge in the supply process. (2) Methods: The differences in primary metabolites of freeze-dried mulberry leaf powder after 30 days of storage under different storage conditions  were investigated. (3) Results: A low temperature and vacuum had better preservation effects on the types and activity of the primary metabolites of mulberry leaves, with vacuum preservation being the best. However, the types of primary metabolites in mulberry leaves were significantly reduced after non-vacuum storage at room temperature compared to those with other storage methods. Among the metabolites detected, including dehydroascorbic acid, various phenolic acids, amino acids, lipids, and carbohydrates showed a significant decrease in their contents of more than 40%, and there was a significant increase in the contents of various compounds of the muconic acid biosynthetic pathway compared to those in other storage methods. Moreover, the antioxidant activity of mulberry leaves stored at room temperature under non-vacuum conditions was also significantly reduced. (4) Conclusions: Vacuum storage is the most ideal storage method for preserving mulberry leaves. Morus alba L.) is a woody plant of the genus Morus in the family Moraceae [3H7NO2), proline (C₅H₉NO2), asparagine (C4H8N2O3), and γ-aminobutyric acid (C4H9NO2)), three organic acids (acetic acid (CH3COOH), succinic acid (C4H6O4), and fumaric acid (C4H4O4)), two carbohydrates (glucose (C6H12O6) and sucrose (C12H22O11)) [9H8O4), gallic acid (C7H6O5), protocatechuic acid (C7H6O4), vanillic acid (C8H8O4), chlorogenic acid (C16H18O9), and ferulic acid (C10H10O4)) [Mulberry  mode, UPLC-MS/MS was used to evaluate compounds in each of the four samples in G1 and G2 (eight subgroups). Qualitative analysis indicated that 611 primary metabolites  were present in G1, while 609 primary metabolites  were present in G2 A. Among To confirm the preservation of mulberry leaf quality under various storage conditions, cluster analysis was used to compare the primary metabolites of mulberry leaf according to the storage conditions. Hierarchical clustering analysis revealed that T2-A was clearly distinct from the other groups . The figTo determine the differences in the primary metabolites of mulberry leaf according to the storage conditions, the DAMs of G1 and G2 were analyzed with variable importance in projection (VIP) ≥ 1 and a fold change of ≥2 or ≤0.5, and volcano plots were generated .In G1 A–F, for In G2 G–L, for Three measurement methods were used to evaluate the effects of the storage method on the overall antioxidant activities of mulberry leaves. FRAP assays were performed to measure the trivalent iron reduction ability. The DPPH radical scavenging capacity (DRSC) and ABTS radical scavenging capacity (ARSC) analyses were based on the single-electron transfer capacity of antioxidants to ABTS/DPPH radicals, which thus formed electron pairs.The FRAP data for the mulberry leaf extracts subjected to the four storage conditions are shown in The ARSC values of the mulberry leaf extracts subjected to the four storage conditions are shown in The DRSCs of the leaf extracts subjected to the four storage conditions are shown in The experimental results showed that the antioxidant activity of the mulberry leaves was mainly influenced by the vacuum level and, secondarily, by the temperature. Vacuum storage of mulberry leaves at room temperature was the best condition for preserving antioxidant activity (T2-V > T1-V > T1-A > T2-A) in G1, and vacuum conditions also showed better preservation in G2 (T2-V ≈ T1-V > T2-A ≈ T1-A) .From the above results, we can see that both vacuum storage and refrigeration can better preserve the primary metabolites in mulberry leaves. However, the storage methods had different trends in their effects on the antioxidant activity of mulberry leaves at different growth stages. The reason for these results may be due to the changes in the types and contents of primary metabolites in mulberry leaves during the growth process.UPLC-MS/MS was used to evaluate the compounds between G1 and G2. PCA, which was used to confirm the overall metabolic differences, revealed scores for the first two main components of 73.5% and 19.5%, respectively. The results showed that the G1 and G2 samples each formed a cluster A, indicaIn total, 153 DAMs were identified between G1 and G2. Volcano plots were generated to depict the DAMs between G1 and G2 A. OveralThe KEGG database was used to annotate the functions of the DAMs in G1 and G2. The results of the metabolic pathway analysis are shown in p-value of the enrichment analysis. The darker the color is, the smaller the p-value is and the more significant the enrichment degree is. The lighter the color is, the greater the p-value is and the lower the significance of the channel is.Note: Each bubble in the bubble diagram represents a metabolic pathway. The abscissa represents the size of the impact in the topology analysis; the larger the value, the higher the enrichment degree. The vertical coordinate and bubble color represent the negative natural log of the 2), and oxygen (O2) during storage affect the rate of food spoilage [At present, mulberry leaves are mainly consumed as vegetables, tea, noodles, mulberry powder additives, and mulberry juice additives . Howeverspoilage . Approprspoilage . Vacuum spoilage  and presspoilage . Therefo6H8O6). In the absence of fresh vegetables, dehydrated vegetables can provide the necessary vitamins. During vegetable storage, temperature, oxygen, light, moisture, pH, storage time, enzymatic modification, and trace metal elements can have various effects on vitamins, including degradation reactions [20H30O) and E (C29H50O2) may also be destroyed [6H6O6) was detected. Ascorbic acid and dehydroascorbic acid can be interconverted through redox reactions, and both have biological activities [Vitamins are essential components for the metabolism of protein, carbohydrates, and fat . In daileactions . Vitaminestroyed . During estroyed . Althougtivities . As antitivities  found th3H4O2)) [Lipid oxidation is a primary mechanism underlying the deterioration of vegetable quality. This mechanism is affected by many factors, including storage temperature and oxygen concentration. The oxidation level and type of fat/oil in vegetables may greatly affect the stability and sensory properties of primary metabolites. Differences in fat/oil types may lead to differences in vegetable quality . Both lo3H4O2)) . Phenolic acid is positively associated with free radical scavenging activity. It can inhibit lipid oxidation by scavenging free radicals and converting phenol free radicals into non-oxidative, low-energy forms . Gallic 4H6O6), oxalic acid (C2H2O4), citric acid (C₆H₈O₇), and malic acid (C21H28O3), are common organic acids with good anti-browning effects [30H48O3) were significantly downregulated, and the contents of their downstream amino acid products (tyrosine (C9H11NO3), phenylalanine (C₉HNO2), and tryptophan (C11H12N2O2)) were also significantly down-regulated. However, the products of the muconic acid biosynthetic pathway , methyl protocatechuate (C8H8O4), p-hydroxybenzoic acid (C₇H₆O3), muconic acid (C6H10O8), and adipic acid (C6H10O4)) were significantly upregulated. This may have been due to the conversion of phosphoenolpyruvic acid (C3H4KO6P) and 3-dehydromuconic acid into 3,4-dihydroxybenzoic acid (C7H6O4) in the presence of air, water, sunlight, and oxygen in a non-vacuum storage environment and the upregulation of a range of downstream compounds. Nevertheless, the principles that produce the conversion of the above compounds are not yet known.The concentration and variation of organic acids are considered to play an important role in maintaining the nutritional value and quality of foods, allowing for changes in flavor, color, and aroma, and they are important indicators of food maturity, spoilage, and fermentation . Aliphatacid C21HO3, are cacid C21HO3, are c6H9N3O2), lysine (C6H14N2O2), arginine (C6H14N4O2), phenylalanine, and their derivatives) were significantly reduced. However, the contents of N-acetyl-L-phenylalanine (C11H13NO3), methyldopa (C10H13NO4), and lysine butyrate (C10H22N2O4) were markedly increased. These results indicated that the proteins and amino acids, among other substances, in mulberry leaves may undergo decomposition and transformation reactions during storage. Proteins are hydrophilic, which enables them to extensively interact with other compounds. The decomposition of proteins and amino acids during storage may affect mulberry leaves’ flavor and stability [The amino acid compositions and contents also changed substantially during storage. These changes might have been caused by altered protein structures or metabolic reactions. Storage temperature, time, and light conditions can also influence amino acid composition . After mtability . 5H12O5), ribitol (C5H12O5), arabitol (C5H12O5), glucurono-6,3-lactone (C6H8O6), sorbitol (C6H14O6), and mannitol (C6H14O6) significantly increased, indicating that the long-chain carbohydrates  in mulberry leaves were degraded into monosaccharides. Moreover, free sugars were partially degraded into sugar derivatives during storage. These findings were similar to the results of a brown rice storage experiment [Carbohydrates are closely associated with protein, lipid, nucleic acid, and secondary biomass metabolism . After mperiment . In addiperiment . The malperiment . Moreoveperiment . The orgThe experimental results showed that the type, content, and antioxidant activity of the primary metabolites of mulberry leaves were influenced by the storage methods. Vacuum packaging was the most effective storage method. A study by Rinaldi et al.  showed t15H20O4)) [7H6O2), gallic acid, ferulic acid, coumaric acid (C6H4O4), p-hydroxybenzoic acid, caffeic acid, and coumarin (C₉H₆O2)) may be related to the adaptation and defense mechanisms of plants [After defining the storage conditions, it is also essential to determine the harvesting period to ensure the intake of nutrients from mulberry leaves. The results of the MMN analysis indicated no obvious differences in the types and numbers of primary metabolites throughout the mulberry tree growth process, although the differences in their expression levels were statistically significant B. By ana5H20O4)) . Fourth,f plants ,36. In summary, storage methods and harvesting periods can affect the types, contents, and activities of primary metabolites in mulberry leaves. UPLC-MS/MS revealed that compared with the other three groups, the primary metabolite contents changed significantly in the mulberry leaves subjected to storage at room temperature under non-vacuum storage conditions. In contrast, the analysis of antioxidant activity showed that the activity of the primary metabolites of mulberry leaves was significantly reduced under paper bag storage conditions. These changes presumably occurred because the paper bag could not completely prevent the entry of room air, such that the mulberry leaves came into contact with oxygen and microorganisms, which resulted in oxidation or decomposition reactions that led to the deterioration of the mulberry leaves. Therefore, vacuum storage conditions are ideal for preserving mulberry leaves’ primary metabolites. Although there were no significant changes in the types of primary metabolites in mulberry leaves at different picking stages, the antioxidant activity of young mulberry leaves was significantly higher than that of old leaves. When the consumption of fresh mulberry leaves is not guaranteed, people can ensure the intake of nutrients by consuming young mulberry leaves that have been vacuum-preserved after freeze-drying.Morus alba L.) leaves were harvested from an experimental field of South China Agricultural University . The first harvest was performed after 3 months of growth  after the mowing of the mulberry trees, and the plants were allowed to regrow. The second harvest was performed 6 months after the first harvest  as a parallel group. Mulberry leaves were harvested from all mulberry plants in the experimental field, and 20 leaves were randomly harvested from each mulberry tree, pooled into a total sample, and then randomly grouped for the experiment.White mulberry (n = 3 per condition) for 30 days.Harvested mulberry leaves were completely freeze-dried in a freezer  at −60 °C to a constant weight. After they were dried, the samples were evenly divided into 12 parts. Each part weighed 150 g and was stored under four conditions (T1-V: Samples were packed in polyamide bags and stored in a vacuum at 4 °C.T1-A: Samples were packed in non-vacuum paper bags and stored at 4 °C.T2-V: Samples were packed in polyamide bags and stored in a vacuum at room temperature (25 ± 2 °C).T2-A: Samples were packed in non-vacuum paper bags and stored at room temperature (25 ± 2 °C).http://www.metware.cn/) and analyzed using a primary-metabolite-targeted metabolomic method. Samples were pulverized by a mixing mill with zirconia beads  at 30 Hz for 1.5 min to obtain a powder. The resulting powder (100 mg each) was extracted with 1.0 mL of 70% methanol aqueous solution at 4 °C overnight and then centrifuged at 10,000× g for 10 min. The extracts were absorbed  and filtered  before ultra-performance liquid chromatography tandem mass spectrometry ) analysis [Samples were sent to Wuhan Metware Biotechnology Co., Ltd.  . Principal component analysis (PCA) and other multivariate statistical analysis methods were used for sample comparisons. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.kegg.jp/kegg/pathway.html) was used to annotate differential metabolites. The least significant difference (LSD) in a one-way analysis of variance (ANOVA) was employed to analyze the differences among the results by applying the SPSS software  [p < 0.05 was considered statistically significant.Each sample was analyzed by using three biological replicates, and the results were expressed as means ± standard deviations  . p < 0.0Mulberry leaves’ primary metabolite contents and antioxidant activities were considerably affected by storage methods. Good preservation of mulberry leaves can provide better flavor and nutritional value. Among the methods investigated, vacuum storage at room temperature was optimal for the preservation of mulberry leaves’ primary metabolite contents and antioxidant activities.


RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model.RBM20 variant without a DCM phenotype were identified.• Two human patients with the I536T-TTN splicing.• A splicing reporter assay demonstrated that the variant affected the Rbm20I538T mice showed neither DCM nor cardiac dysfunction.• Rbm20I538T mice showed different splicing patterns and the gene expressions.• The online version contains supplementary material available at 10.1007/s00109-022-02262-8. Sudden unexpected death in a young adult is a rare event, but elucidating its mechanisms is one of the most important issues in medical science. Recently, genetic analysis has been improved and adopted in the field of postmortem examination, and there are many reports and studies in which postmortem genetic analysis uncovered the mechanism of autopsy-negative sudden death –4. Most RBM20, encoding RNA Binding Motif Protein-20 (RBM20), is the regulator of heart-specific pre-mRNA splicing -Puro-CAG > hRBM20[NM_001134363.3] were purchased from VectorBuilder Inc . Using overlapping PCR mutagenesis, the mutations of c.T1607C were inserted into the wild-type cDNA in constructs of pRP-hRBM20I536T. For all constructs, sequencing was performed over the entire region of the amplified sequences. Plasmid DNA was purified using a HiSpeed® Plasmid Maxi Kit .The 2. The TTN reporter minigene and the expression vectors were transiently co-transfected in a 1:4 mixture into Hela cells using Lipofectamine  in accordance with the manufacturer’s protocol. Fluorescence images of fluorescent proteins were acquired 24 h after transfection using EVOS (Thermo Fisher Scientific), and then the cells were harvested for total RNA preparation and RT-PCR analysis.HeLa cells were cultured in DMEM Medium  supplemented with 10% fetal bovine serum at 37 °C with 5% COTotal RNAs from HeLa cells involved in transient transfection experiments were extracted using the RNeasy mini kit (Qiagen). Complementary DNAs (cDNAs) were prepared with the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific) using 1 µg of RNA with a random primer, in accordance with the manufacturer’s protocol. Semi-quantitative PCRs were performed using PrimeStarGXL , and the PCR products were analyzed using the Bioanalyzer 2100 Expert with DNA7500 Kit . Primers and conditions of RT-PCR were described previously .Mice were kept in a temperature-controlled facility under specific-pathogen-free conditions with ad libitum access to food and water in a 12-h light/dark cycle. C57BL/6 J mice were purchased from CLEA Japan .https://crispr.dbcls.jp) to target exon 6 of Rbm20. The gRNA sequence was as follows: Rbm20 gRNA 5′-GGAGAATGACGTCATTAACC-3′. The corresponding crRNA (5′-GGAGAAUGACGUCAUUAACCGUUUUAGAGCUAUGCU-3′), tracrRNA , and Cas9 nuclease were purchased from Integrated DNA Technologies . The crRNA and tracrRNA were annealed and complexed with Cas9 nuclease according to the manufacturer’s instructions.The gRNA was designed using CRISPRdirect (Single-strand DNA (ssDNA) with c.T1613C mutation was ordered as PAGE Ultramer from IDT. The ssDNA sequence was as follows: Rbm20 ssDNA 5′-GCACATCTGCAATCTCCCGGAGGGCAGCTGCACGGAGAATGACGTCACTAACCTGGGGCTGCCCTTTGGCAAGGTCACTAATTACATCCTCATGAAGTCA-3′.Male and female C57BL/6 mice (CLEA Japan) were used as sperm and oocyte donors. Female mice were superovulated, and then zygotes were prepared by in vitro fertilization. They were cultured in KSOM medium  before and after electroporation. Electroporation was performed using a 1-mm gap electrode (CUY501P1-1.5) and a super electroporator NEPA21 . Zygotes were washed with Opti-MEM (Thermo Fisher Scientific) and then placed in the electrode gap filled with 5 µl of Opti-MEM solution containing 200 ng/µL Cas9 protein, 6 pmol/µL gRNA (crRNA/tracrRNA complex), and 10 pmol/µL ssDNA. They were cultured in KSOM medium overnight, and surviving 2-cell-stage embryos were transferred to the oviducts of pseudopregnant Jcl:ICR female mice (CLEA Japan).Genomic DNA was extracted from F0, F1, and F2 mice. PCR was performed using Taq DNA polymerase . The primer sequences are shown in Supplementary Material, Table S1. The PCR products were purified using the Gel/PCR Extraction Kit . The purified PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) through an Applied Biosystems 3500xL Genetic Analyzer (Thermo Fisher Scientific), or restriction enzyme digestion was performed by RspRS II enzyme (Takara Bio Inc.), according to the manufacturer’s instructions. In some cases, PCR products were cloned into TA vector  before sequencing.After euthanasia, total RNAs from mouse hearts at the ages of 1, 12, and 36 weeks were extracted using the RNeasy Plus Universal Mini Kit (Qiagen). Nine representative libraries were generated for the nine groups  by RNA-seq with SMART-seq v4 Ultra Low Input RNA kit for sequencing (Takara Bio Inc.) and Nextera XT DNA Library Prep Kit  and sequenced on a NovaSeq 6000 instrument (Illumina).Splicing analysis with Percentage Spliced In (PSI) was performed as described previously . DiffereComplementary DNA was synthesized from total RNA (1 µg) using a Prime-Script RT PCR Kit (Takara Bio Inc.), according to the manufacturer’s instructions. The confirmation of the quantification of the splicing variant was performed as previously described . In detaTtn exon50 or Gapdh. Statistical significance was assessed by Prism9.Quantitative real-time PCR (qRT-PCR) was performed using TB Green® Premix Ex Taq™ II (Takara Bio Inc.) and a Thermal Cycler Dice Real Time System (Takara Bio Inc.). Sequences of the PCR primers used are presented in Supplementary Material, Table S3. Expression levels were normalized with For the histological analyses, heart tissue at the age of 12 weeks was fixed in paraformaldehyde, 4% in phosphate-buffered saline, embedded in paraffin, sectioned, and subjected to hematoxylin–eosin staining and Masson’s trichrome staining. Stained sections were photographed using Virtual Slide System VS120 .Ultrasound echocardiography (UCG) was performed in a blinded manner. In detail, mice at the age of 12 weeks were anesthetized with isoflurane and placed on a heated platform. Cardiac functions, such as left ventricular fractional shortening (LVFS), end-diastolic dimension (LVDd), and diastolic wall thickness of the LV posterior wall (LVPWth), were assessed using a high-frequency ultrasound system . A two-dimensional parasternal short-axis view was obtained at the levels of the papillary muscles, and M-mode tracings were recorded.RBM20 variant were identified. The first patient was reported previously [Two Japanese sudden death patients who possessed the I536T-eviously . The secRBM20 variant  is expressed from the product containing the fused exon E51E218, and red fluorescent protein (mCherry) is translated from the E51E218-skipped mRNA, corresponding to the splicing patterns of the N2A and N2B types, respectively , were generated . Through electroporation of the Cas9/gRNA complex and donor ssDNA into zygotes, 36 founder (F0) mice were born. Of them, 14 mice (39%) had the intended point mutation. These F0 mice were crossed with C57BL/6 J mice (CLEA Japan), and heterozygous F1 mice, which were confirmed by Sanger’s sequencing, were obtained.Using the CRISPR/Cas9 system, KI mice harboring the c.T1613C (p.I538T) point mutation in The KI mice were born according to Mendel’s law and were all healthy, fertile, and indistinguishable from their wild-type littermates.Rbm20 expression on splicing of other genes, qRT-PCR was performed, and there were no differences in Rbm20 expression among the three genotypes , and various abnormal splicing patterns in Ldb3, Camk2d, and Ryr2 were also detected  between the WT and Rbm20 KI mice (270 upregulated and 809 downregulated genes) were identified , which is different from the human sudden death cases with the same RBM20 variant. Heart weight was not different among genotypes . The clinical significance of the increase in these genes remains unknown, but Casq1, Mybpc2, and Myot were also increased in the other studies [RBM20 variants are susceptible to arrhythmias such as ventricular tachycardia, leading to cardiac transplantation, implantation of an implantable cardioverter-defibrillator, or sudden death [RBM20-associated abnormal splicing of ion channels genes such as RYR2 were also reported [Rbm20I538T/I538T mice have susceptibility to arrhythmia.In the present study, handling , 26, 27, factors . RNA-seq studies , and upr studies . Human pen death –36. Ventreported . Thus, iRbm20I538T/I538T mice did not show DCM, although RBM20 cardiomyopathy often shows DCM phenotypes. Nmrk2, which was highly expressed in DCM [Nppa and Nppb, were also not upregulated. Considering the results of biophysical and morphological examinations, heart failure did not happen in Rbm20I538T/I538T mice. In this respect, the human I536T patient and I538T mice are not typical.On the contrary, Rbm20I538T/I538T mice showed abnormal Ldb3 splicing of exons 3, 4, 5, and 6, but they did not show a significant difference in the exon 11 inclusion/exclusion ratio. Abnormal splicing of exons 3 to 6 was reported in RBM20 cardiomyopathy [RBM20 variant but was possibly caused by myotonic dystrophy.In addition, myopathy . The preRbm20I538T/I538T mice were generated, and it was demonstrated that Rbm20I538T/I538T mice mimicked human I536T-RBM20 heterozygous patients in morphological phenotype and abnormal splicing, but they differed in that they did not suffer sudden death. This mouse model will have a great advantage in further studies of elucidating mechanisms of sudden death. Patient 1 had another genetic background, myotonic dystrophy. We would like to generate a double-transgenic mouse with Rbm20 and Dmpk in the future. Investigating the genetic background, as well as uncovering the trigger of sudden death, is an important issue to diagnose the cause of death from congenital diseases. On the other hand, molecular biological analyses and morphological investigations were performed, but electrocardiograms were not analyzed. Further examination would be necessary.This study has some future implications and limitations. This variant has not been reported, but the fact that these two patients were encountered at two independent institutes, and they were not consanguineous, means that there are possibly more sudden death cases with this variant. Both patients had a heterozygous I536T variant, which was thought to have been inherited from one of the parents without a familial history of cardiomyopathy or sudden death, though familial genetic analysis was not permitted. Thus, just having this variant does not seem to cause sudden death, and an unknown factor seemed to be a trigger. RBM20 variant were presented. Splicing analysis of patient 2 was not performed directly, but the reporter assay showed an abnormal Ttn splicing. The mouse model showed abnormal splicing and different gene expressions without sudden death or cardiac failure. These splicing and expression changes in Ttn and Ca handling genes do not cause DCM morphology.In conclusion, two sudden death cases of human I536T-Supplementary file1 (PPTX 830 KB)Below is the link to the electronic supplementary material.


ST3GAL5 gene in humans, is a sialyltransferase responsible for synthesis of GM3 from its precursor, lactosylceramide. ST3GAL5 mutations cause an autosomal recessive form of severe infantile-onset neurological disease characterized by progressive microcephaly, intellectual disability, dyskinetic movements, blindness, deafness, intractable seizures, and pigment changes. Some of these clinical features are consistently present in patients with ST3GAL5 mutations, whereas others have variable expression. GM3S knockout (KO) mice have deafness and enhanced insulin sensitivity, but otherwise do not display the above-described neurological defects reported in ST3GAL5 patients. The authors present an overview of physiological functions and pathological aspects of gangliosides based on findings from studies of GM3S KO mice and discuss differential phenotypes of GM3S KO mice versus human GM3S-deficiency patients.Gangliosides  are highly expressed in neural tissues in vertebrates, and four species  are predominant in mammalian brains. GM3 is the precursor of each of these four species and is the major ganglioside in many nonneural tissues. GM3 synthase (GM3S), encoded by  Ugcg gene in mice) (Ugt8 gene). Synthesis of GlcCer occurs on the cytosolic surface of Golgi, whereas GalCer synthesis occurs on the luminal surface of endoplasmic reticulum  cholesterol. In small intestines of both shlApoE and shlApoE GM3S KO, in the absence of cholesterol feeding, NPC1L1 was localized mainly on the apical side of enterocytes. When cholesterol feeding occurred, NPC1L1 was internalized in shlApoE but not in shlApoE GM3S KO. Therefore, GM3s (or related gangliosides) are clearly essential for NPC1L1-dependent intestinal cholesterol absorption.Intestinal cholesterol absorption, which is necessary for maintenance of cholesterol homeostasis, is mediated by the transmembrane protein Niemann-Pick C1-like 1 (NPC1L1) , which aST3GAL5 variants led to disrupted synthesis of a- and b-series gangliosides, and consequently to severe infantile-onset neurological disorders characterized by progressive microcephaly, intellectual disability, choreoathetosis, blindness, deafness, intractable seizures, and/or pigment changes [ST3GAL5 mutations in patients with GM3S deficiency are summarized in GM3S deficiency was first reported by A.H. Crosby’s group as an autosomal recessive infantile-onset epilepsy syndrome associated with developmental stagnation and blindness in Old Order Amish . Biallel changes ,57,58,59ST3GAL5 constructs revealed an absence of enzymatic activity in these variants [The human GM3S protein consists of 418 amino acids and includes sequences for L- (large), S- , and VS-  sialyl motifs , which avariants . A recenvariants . Plasma variants . Plasma In contrast to phenotypes of human GM3S-deficiency patients, GM3S KO mice do not display neuronal abnormalities, most likely because total brain ganglioside amount is roughly maintained through alternative expression of o-series gangliosides GM1b and GD1α, indicating that other gangliosides can compensate for lack of GM3 and its downstream biosynthetic derivatives see . WhetherPhysiological functions and pathological aspects of gangliosides revealed by studies on GM3S KO mice and human GM3S-deficiency patients are summarized in this review article. The cause of differential GSL expression patterns under GM3S KO conditions in humans versus mice is unknown but is presumably responsible for the striking differences in neurological phenotypes. Possible human-versus-mouse differences in expression levels or substrate specificity of enzymes involved in GSL synthesis are described in ST3GAL5 variants have been detected in GM3S-deficiency patients. Four of them  are listed in the Genome Aggregation Database  and have allele frequencies < 0.0001. In view of these very low frequencies, it is likely that many other deleterious ST3GAL5 variants exist. Only four additional ST3GAL5 variants in GM3S-deficiency patients have been reported since the first one. The 2022 study by C. Mignot’s group mentioned in Sec. 4 revealed four novel variants from 16 non-Amish patients  [Nine distinct zygotes) . The autzygotes) , indicat


Drosophila melanogaster midgut is commonly studied as a model epithelial tissue for many reasons, one of which is the presence of a diverse population of secretory cells called enteroendocrine cells. Subpopulations of these cells secrete various combinations of peptide hormones which have systemic effects on the organism. Many of these hormones are also produced in the Drosophila brain. The split-GAL4 system has been useful for identifying and manipulating discrete groups of cells, but previously characterized split-GAL4 drivers have not driven expression in high proportions of enteroendocrine cells. In this study, we screened candidate split-GAL4 drivers for enteroendocrine cell expression using known reference drivers for this cell type and discovered a new split-GAL4 driver pair that confers expression in a greater number of enteroendocrine cells than previously characterized driver pairs. The new pair demonstrates less brain expression, thereby providing better tools for disentangling the physiological roles of gut- and brain-secreted peptides. We also identified additional split-GAL4 drivers that promote expression in discrete subpopulations of enteroendocrine cells. Overall, the tools reported here will help researchers better target enteroendocrine cell subpopulations.The  Drosophila melanogaster intestine is a widely studied model tissue due to its many parallels to mammalian digestive systems, the extensive genetic manipulations available in Drosophila, and the simplicity of the tissue relative to the digestive tracts of other organisms  in place of GAL4AD because p65AD induces stronger transcriptional activation  is controlled by different regulatory sequences than expression of the GAL4 DNA-binding domain (DBD). When both the AD and the DBD are expressed in the same cell, these domains can bind via attached leucine zipper tags to reconstitute functional GAL4, which can then activate expression of To improve the utility of the split-GAL4 system, recent work characterized the expression patterns of 7,304 experimental split-GAL4 drivers in the Drosophila midgut by both cell type and region via 2 distinct screens . The genotypes of all starting stocks used in this study are listed in PBac{UAS-DSCP-6XEGFP}VK00018 strain used for reporter comparisons was a gift from Steve Stowers (Montana State University).Fly strains were cultured at 25° on standard Bloomington media ], blocked in 0.5% bovine serum albumin and 5% normal goat serum in PBT for 1 h, incubated with DAPI and anti-HRP overnight, washed 5 times in PBT and mounted with VectaShield Plus mounting medium .To analyze the fluorescence from Adult brains were prepared following the same protocol of fixation, washing, and DAPI staining used for gut dissection. No antibody staining was performed on adult brains.Images of whole dissected intestines and brains were taken on a Zeiss Axio Zoom microscope. Images of immunostained intestines for quantification were taken on a Zeiss Axio Observer microscope. Image files were prepared using Adobe Photoshop and figures were assembled using Adobe Illustrator.UAS-Stinger in each of the 11 intestinal subregions  42 R20C06 enhancer fragment to direct p65AD expression. To evaluate the utility of R20C06 for detection of EEs, we examined UAS-Stinger expression in detail when R20C06-p65AD was combined with R20C06-GAL4DBD. We observed expression directed by this driver pair in substantially more EEs than induced by the previously characterized R57F07.A-p65AD and -GAL4DBD driver pair  R57F07.A split-GAL4 drivers together drove no expression in non-EE cells in R2, R3, and R4, and drove expression in non-EEs in 1% of Stinger-positive cells in R1 and 24% in R5, with many of the R5 Stinger-positive, Pros-negative cells having large nuclei (likely ECs). This indicates that both driver pairs conferred quite low levels of UAS-Stinger expression in non-EEs, while the R20C06 pair drove expression in a greater proportion of EEs (approximately 45% more on average). Although some GAL4 drivers with equivalent or superior EE detection capabilities have been identified, such as V1P{GawB}pros  when combined with the reference EC-p65AD driver. This expression was observed primarily in R1, but it was also seen sporadically in other regions (UAS reporter expression in cells with large nuclei (likely ECs) when several split-GAL4 drivers were combined with the reference R57F07.A drivers, including when the R57F07.A-p65AD and -GAL4DBD reference drivers were combined , indicating that expression of the R20C06 drivers was more specific to EEs than expression of the R57F07.A drivers.To further confirm the EE specificity of the  regions . AriyapaR20C06-p65AD and R20C06-GAL4DBD drivers directing EE expression when combined with the EC reference drivers. In these combinations, we saw UAS-Stinger expressed in populations of small cells in R3, particularly near the boundaries with R2 and R4 . escargot gene called KITI{sfGFP}esg  is different than that in Progenitor-p65AD (VT004241). We similarly saw no UAS-Stinger expression in these cells when R57F07.A-GAL4DBD was combined with Progenitor-p65AD, even though we saw expression when R57F07.A-p65AD was combined with Progenitor-GAL4DBD that was very similar to the expression we saw when we combined R20C06-p65AD with Progenitor-GAL4DBD (R10F08-p65AD) that detected this EE subpopulation in combination with the Progenitor-GAL4DBD driver as well. All together, these results suggest that the Progenitor-GAL4DBD reference driver is a useful tool for marking this subpopulation.In contrast, pulation  inset, a-GAL4DBD . AriyapaPBac{UAS-DSCP-6XEGFP} reporters combined with R57F07.A drivers. The extremely bright fluorescence of these UAS-6xGFP reporters, arising from 20 copies of UAS and 6 copies of the GFP coding sequence, was appropriate for that screen but was not necessary for the current study. Instead, we used the UAS-Stinger reporters because they are clear and easy to detect without the need for antibody staining yet are not so bright that it is difficult to distinguish individual cells. To evaluate whether these different reporters provided consistent results when combined with the EE reference driver pairs, we examined EE fluorescence when the R57F07.A and R20C06 driver pairs were combined with the UAS-6xGFP and UAS-Stinger reporters. When both driver pairs were characterized, we saw similar numbers of fluorescent EE cells with UAS-Stinger and UAS-6xGFP in all midgut regions, but with slightly more fluorescent cells observed with UAS-Stinger  gene and there are no other genes within 16 kb of this fragment . Based on these observations, the R20C06 split-GAL4 drivers behave differently than GAL4 drivers containing the same enhancer fragment and the protein and gene traps for the nearby nAChRα5 gene. Therefore, these other genetic elements do not present promising avenues for future pan-EE tool development. More broadly speaking, these results reemphasize that isolated enhancers or enhancer fragments are often not sufficient to recapitulate the native expression of genes and that the expression patterns driven by enhancers also depend on adjacent sequences and genomic contexts.The r midgut . When weing lexA , we obseR20C06 split-GAL4 driver pair, we collected expression pattern data for all the split-GAL4 drivers screened with R57F07.A. We later combined all drivers from that initial screen with the R20C06 drivers so that we could evaluate differences and similarities in expression patterns and explore the utility of the R20C06 driver pair for EE detection in the split-GAL4 system. Overall, 88 split-GAL4 drivers were tested. There were 42 drivers (48%) positive for adult midgut expression when crossed to the R57F07.A drivers and 39 drivers (44%) positive when crossed to the R20C06 drivers  which activated UAS-Stinger expression when crossed to an R57F07.A driver but not when crossed to a R20C06 driver, while only one driver (R65D06-GAL4DBD) activated UAS-Stinger expression when crossed to a R20C06 driver but not when crossed to a R57F07.A driver. Drivers expressing with both R57F07.A and R20C06 fell into 3 classes: (1) 64 drivers showed similar patterns when crossed to either reference driver (R20C06 than the R57F07.A reference driver (R57F07.A than the R20C06 reference driver (R20C06 did not show any expression in big nuclei (likely ECs), while this was somewhat common in crosses with R57F07.A  12 e driver ; and (3)e driver . It is iUAS-Stinger expression was present, the total number of genotypes with brains scored varied slightly between R20C06 and R57F07.A drivers. We scored brains from 39 driver pairs using R20C06 and 42 driver pairs using R57F07.A on a semiquantitative scale from 0–3, with 0 indicating no expression and 3 indicating a large number of cells with expression , while 77% of genotypes from crosses involving R20C06 drivers had a score of 1 or 0. Conversely, only 5% of genotypes with R20C06 drivers received a score of 3 (large number of cells with expression) whereas 31% genotypes with R57F07.A drivers had a score of 3  or upon request.The accompanying tables contain complete characterization data. G3 online.jkac102_Supplemental_Figure_CaptionClick here for additional data file.jkac102_Supplemental_Figure_S1Click here for additional data file.jkac102_Supplemental_Figure_S2Click here for additional data file.jkac102_Supplemental_Figure_S3Click here for additional data file.jkac102_Supplemental_Table_S1Click here for additional data file.jkac102_Supplemental_Table_S2Click here for additional data file.jkac102_Supplemental_Table_S3Click here for additional data file.jkac102_Supplemental_Table_S4Click here for additional data file.


S). A recent study suggested that ORF2S is not necessary for the HEV life cycle. A previously reported efficient HEV cell culture system can be used to understand the origin and life cycle of ORF2S but is not sufficient for functional research. A more rapid and productive method for yielding ORF2S could help to study its antigenicity and immunogenicity. In this study, the ORF2S (tPA) expression construct was designed as a candidate tool. A set of representative anti-HEV monoclonal antibodies was further used to map the functional antigenic sites in the candidates. ORF2S (tPA) was used to study antigenicity and immunogenicity. Indirect ELISA revealed that ORF2S (tPA) was not antigenically identical to HEV 239 antigen (p239). The ORF2S-specific antibodies were successfully induced in one-dose-vaccinated BALB/c mice. The ORF2S-specific antibody response was detected in plasma from HEV-infected patients. Recombinant ORF2S (tPA) can act as a decoy to against B cells. Altogether, our study presents a design strategy for ORF2S expression and indicates that ORF2S (tPA) can be used for functional and structural studies of the HEV life cycle.Hepatitis E virus (HEV) is an important public health burden worldwide, causing approximately 20 million infections and 70,000 deaths annually. The viral capsid protein is encoded by open reading frame 2 (ORF2) of the HEV genome. Most ORF2 protein present in body fluids is the glycosylated secreted form of the protein (ORF2 Hepeviridae family contains two HEV subfamilies, Orthohepevirinae  and Parahepevirinae (infecting fish) [Orthohepevirinae includes four genera: Paslahepevirus, Rocahepevirus, Chirohepevirus, and Avihepevirus. At least six genotypes (gt) are pathogenic in humans and fall under the species Paslahepevirus balayani and Rocahepevirus ratti ) [Paslahepevirus balayani) occur mostly in resource-poor countries and regions and infect humans exclusively [Paslahepevirus balayani and gt1 of the species Rocahepevirus ratti are zoonotic and can cause persistent infections in immunocompromised patients [Hepatitis E virus (HEV) is the leading cause of enterically transmitted acute hepatitis worldwide, causing approximately 20 million infections and 70,000 deaths annually ,2. HEV ing fish) . The sube 2022)) ,6,7,8. Hlusively ,10. Howepatients ,12.S and the capsid-associated ORF2C [S dimer was first reported by Montpellier et al. in 2018 [S protein with a signal peptide that could rapidly direct the protein into the secretory pathway without intracellular accumulation, whereas translation from the internal AUG would disrupt the signal sequence, producing a cytoplasmic ORF2C protein that is responsible for virion assembly, which is not translocated into the endoplasmic reticulum or the Golgi apparatus and lacks the glycosylation modification [S is the major antigen in hepatitis E patient serum. ORF2S does not prevent HEV entry but impairs antibody-mediated neutralization, so it has been speculated that ORF2S is likely to act as an immunological decoy [HEV is a single-stranded, positive-sense, and quasi-enveloped RNA virus, and the ~7.2 kb full-length genome expresses three open reading frames (ORFs) . ORF1 ised ORF2C ,14. The fication . In addial decoy ,14. ORF3al decoy .C and ORF2S corresponds to Leu14 and Ser34, respectively, and the last a.a. corresponds to Ser660. Although the system has enabled increased expression of viral proteins and infectious particles, allowing direct biochemical analysis of the ORF2 proteins, the study of the immunological function, biological significance, and structure of ORF2S is limited by the protein yield. A rapid and productive system for obtaining ORF2S remains to be developed.HEV is present as a naked virion in feces and bile and an enveloped virion in the serum of HEV-infected patients and HEV-infected cell culture systems ,16. A reS protein that permits rapid recombinant expression and purification of ORF2S protein at approximately 5 mg/L, permitting biological and structural research. We demonstrate a substantial antigenic overlap between ORF2S and other HEV capsid proteins and that ORF2S is immunogenic in mice. An excellent ORF2S-specific antibody response was induced in plasma from patients infected with HEV, and ORF2S could serve as a decoy to allow the study of the B-cell response against HEV.In our study, we describe a method for the expression of the ORF21-Ser660) of genotype (gt) 4 (strain subtype 4a) was obtained from a male with chronic hepatitis E with aplastic anemia (GenBank no. OP185389). Then, the original secretion signal peptide of the HEV ORF2 (a.a. Met1-Gly23) was replaced by the tPA signal peptide. The tPA signal peptide and ORF2S genes (a.a. Ser34-Ser660) were cloned into the pTT5 expression vector and named ORF2S (tPA). Then, 10× His-tags were added to the C-termini of constructed genes. The HEV p239 (p239) genes of gt4 from a swine HEV gene (GenBank no. GQ166778) were cloned into the pTO-T7 plasmid [HEV genome open reading frame 2 (ORF2) full-length sequence (a.a. Met plasmid .S (tPA) construct was transiently transfected into Expi-293F cells, in accordance with the instructions of the manufacturer . The target proteins (ORF2S) were mainly secreted into the supernatants. Cell pellets were also collected for analysis. Cell supernatants and pellets were tested for HEV antigen (Ag) by a commercial ELISA kit . The HEV Ag commercial ELISA kit was used for detecting the 10× His-tag of ORF2S with 5000-fold diluted secondary horseradish peroxidase (HRP)-conjugated anti-His mAb . Target proteins were separated by SDS/PAGE and subsequently blotted onto a nitrocellulose membrane . Mouse monoclonal anti-HEV antibody 1B7 was applied as the primary antibody (diluted 5000-fold), which recognized the linear epitope HEV ORF2 a.a. 443–457 [The ORF2 443–457 . HRP-conS proteins mixed with nonreducing and reducing 6× loading buffer were subjected to SurePAGE, Bis Tris, 8–16% gradient SDS-polyacrylamide gel electrophoresis . ORF2S proteins were stained with Coomassie blue, in accordance with standard laboratory protocols.ORF2S was subjected to high performance liquid chromatography (HPLC)  through a TSK Gel G3000 pwxl 7.8 × 300-mm column  equilibrated in 20 mM PBS. The column flow rate was maintained at 0.5 mL/min, and the run time was 30 min. Purified HEV ORF2S in PBS was detected at OD280nm.Purified HEV ORF2S of HEV gt4 and p495 of gt1 overnight at 4 °C. The wells were blocked with 0.5% (w/v) casein in PBS at 37 °C for 2 h. As described in a previous report [2SO4 after incubation at 37 °C for 15 min. The absorbance was measured at OD450nm with a reference wavelength of OD630nm.Ninety-six-well microplates were coated with 100 ng/well of E2, p239, ORF2s report ,19, 15 mS (tPA) vaccine, which had been mixed with Freund’s Complete Adjuvant . The vaccine was manufactured in 20 μg formulation mixed with Freund’s complete adjuvant. Mice immunized with PBS suspended in Freund’s complete adjuvant were used as controls. Serum samples were collected weekly and tested for anti-HEV IgG at −1, 1, and 2 weeks of vaccination by indirect ELISA according to standard laboratory protocols. Briefly, 96-well microplates were coated with 100 ng/well of p239 and ORF2S of HEV gt4. Serum diluted 10-fold was used as the first sample, and the anti-HEV IgG level was determined from 5-fold serially diluted serum samples. The median effective dose (ED50) was calculated based on ELISA data from postimmunization BALB/c mice.Nine special pathogen-free (SPF) female BALB/c mice of 6 weeks of age were randomly assigned into 3 groups of three BALB/c mice each. SPF BALB/c mice were vaccinated subcutaneously with a single dose of HEV gt4 of p239 and ORF2S of HEV gt4, as described in a previous method [2SO4.Fifty-five HEV-infected patients gave informed consent to participation in the study. This study was designed in accordance with the Declaration of Helsinki and subsequently approved by the medical ethics committee of the School of Public Health, Xiamen University. Peripheral blood was collected from patients naturally infected with HEV, and plasma was separated by centrifugation. WHO standard serum was used as a reference to quantify the titer of anti-HEV IgG in naturally infected patients . The Ants method . BrieflyS (tPA) was separately labeled with DyLight 488 NHS Ester  and EZ-Lin Sulfo-NHS-LC-Biotin , in accordance with the instructions of the manufacturer. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque gradient  centrifugation. PBMCs were obtained from HEV 239 Hecolin  vaccinated donors 1 month after completing three vaccine doses according to a standard schedule  and were incubated with fluorescent antibodies and ORF2S (tPA) for the identification of ORF2S-specific B cells. PBMCs were stained with a cocktail consisting of LIVE/DEAD Fixable Aqua Dead Cell Stain Kit , CD3-PE-Cy7 , CD19-BV786 , CD27-PE , IgM-PerCP-Cy5.5 , IgG-BV421 , and the recombinant ORF2S (tPA)-DyLight 488 (1 μg/μL) and ORF2S (tPA)-Biotin (1 μg/μL) described above. Two consecutive staining steps were conducted. The first was Aqua/CD3/CD19/CD27/IgM/IgG/ORF2S (tPA)-DyLight 488/ORF2S (tPA)-Biotin in 100 μL PBS applied at 4 °C for 30 min. The second was streptavidin-allophycocyanin  for an additional 30 min at 4 °C to target the biotin tag of ORF2S (tPA). The stained cells were washed, resuspended in PBS, and strained through 70-μM Fisherbrand Sterile Cell strainers . ORF2S (tPA)-specific IgG+ memory B cells were gated as Live/CD3−/CD19+/CD27+/IgM-/IgG+/ORF2S+.ORF2S protein expression construct is shown in S gene sequence was obtained from a chronic gt4a HEV-infected male patient with aplastic anemia (GenBank no. OP185389). The construct was named according to the expression vector and secretion signal peptide. The ORF2S (tPA) construct was transiently transfected into Expi-293F cells. To estimate the yields and features of ORF2S, the supernatants and pellets of expressing cells were collected for analysis. HEV Ag and His tag were detected in the supernatants and pellets. Given the high sensitivity and specificity of commercial HEV Ag ELISA , the kit was used for detecting ORF2 protein levels [S (tPA) showed high levels of HEV Ag (50) in ORF2S (tPA) supernatants (ED50 = 858.6) was also stronger than that in pellets (ED50 = 6.2) (S (tPA) pellets. Boiled ORF2S protein was detected as a monomer of approximately 90 kD in supernatants of ORF2S (tPA) (S (tPA), which suggests that the ORF2S (tPA) in pellets lacks posttranslational modifications. Further glycosylation analysis confirmed that only the ORF2S (tPA) protein in the supernatant was glycosylated, and the small protein in the pellet did not undergo posttranslational modifications (S dimer in previously reported efficient HEV cell culture systems were glycosylated protein [S protein in supernatants suggest that ORF2S (tPA) supernatants are better candidates than pellets for purification. The supernatants of ORF2S (tPA) were harvested and purified by Ni-excel affinity chromatography. SDS/PAGE and WB showed that most of the ORF2S protein, corresponding to dimers of approximately >180 kD and monomers of approximately 90 kD, was eluted by 250 mM imidazole  analysis was performed. A single peak (11.6 min) demonstrated the presence of ORF2S (tPA)  B. We per2S (tPA) C, which ications D. The OR protein ,14. The midazole E. To exa2S (tPA) F, suggesS protein and ORF2C protein lacking posttranslational modifications, we performed a detailed epitope mapping, using a panel of mAbs that recognize nine different antigenic epitopes (C1–C6 and L1–L3) to compare the antigenicity of different forms of HEV capsid proteins. The panel of mAbs used was generated in a previous study [S and other HEV ORF2C capsid proteins, including p495 (a.a. 112–606), p239 (a.a. 368–606), and E2 (a.a. 394–606)  between HEV gt1 and gt4, which are recognized by C4 antibodies, resulted in decreased binding  B. Linear394–606) B. The moP domain B. To ver protein . In addireported ) that maitope C3 C. Since  binding C.S antigen is abundant in the serum of HEV-infected patients, the immunogenicity of purified ORF2S in BALB/c mice was investigated. ORF2S (tPA) and ORF2C (p239) were tested as candidates for investigation of the immunogenicity and immunogen-induced antibody response. Groups of three animals each were immunized with one dose of a 20 µg formulation of ORF2S (tPA), p239, or PBS, and serum samples were collected weekly. The immunization program is shown in S and p239 proteins were tested separately for anti-HEV IgG by indirect ELISA. The serum conversion rate of anti-HEV IgG could be stabilized at 100%, as detected by the increased antibody response to the two proteins after immunization (50 = 25.51) and ORF2S (tPA) (ED50 = 23.15) immunized groups, whereas distinct titers (ED50 = 0.64 versus 4.24) were shown for the ORF2S (tPA) protein (S (tPA) proteins were immunogenic, and ORF2S (tPA)-specific antibodies were successfully induced in the one-dose-vaccinated BALB/c mice.Given that the ORF2nization B. Intrig protein C. ORF2S S as an immunological decoy to modulate antibody responses, we collected blood samples from 55 patients infected with HEV, and plasma was separated by centrifugation. Anti-HEV IgG titers were quantified using serial plasma dilutions on ELISA plates coated with either p239 or ORF2S (tPA). Plasma showed significantly higher anti-HEV IgG titers binding to ORF2S (tPA) (mean = 23.43 WU per mL) than p239 (mean = 3.04 WU per mL) (S (tPA). Further analysis suggested a significant correlation between anti-HEV IgG titers binding to ORF2S and p239  (S (tPA) showed an overall increase compared with those of p239, the titers decreased in some samples (S (tPA), while the remainder (16.36%) showed decreased titers, suggesting excellent antigenicity of ORF2S (tPA) binding to plasma from HEV-infected patients. ORF2S is present in body fluids and is the major HEV ORF2 protein in HEV-infected patient serum. Induced antibodies mainly target the ORF2S protein. However, substantial antigenic overlap was observed between ORF2S and ORF2C , so a small subset of antibodies also targets the p239 protein. These data suggest that ORF2S may act as an immunological decoy to induce antibody response in patient serum. To further investigate this possibility, we examined ORF2S-induced B-cell responses at the cellular level. Flow cytometry was used to assess B cells that recognized the ORF2S (tPA) probe. The gating strategy for assessing ORF2S (tPA)-specific memory B cells is shown in S (tPA)-specific CD27+ IgG+ B-cell response was revealed by flow cytometry. The proportions of ORF2S (tPA)-responding B cells varied between vaccinated and naturally infected donors (S (tPA) aroused an antigen-specific adaptive humoral response and that the immunogen may induce antibodies that recognize ORF2S-distinct epitopes located in regions outside p239 (a.a. 368–606). In addition, based on serum antibody levels and B-cell responses, it appears that ORF2S plays an immunomodulatory role to induce an antibody response. Since ORF2S does not prevent HEV entry but impairs antibody-mediated neutralization, the ORF2S protein could serve as an additional mechanism by which virions evade host immunity.To validate ORF2 per mL) A. The in= 0.252) B. Althou samples C. Of thed donors D. The led donors E. These S (tPA) expression construct as an efficient tool to express and purify the HEV ORF2S protein. Protein identification experiments with ORF2S (tPA) showed that the ORF2 protein was mostly secreted into supernatants, as previously described, and the supernatant expression level of the ORF2 protein was S/CO 123,424 (approximately 5 mg/L after purification) [S (tPA) from supernatants has a relatively complete a.a. sequence, whereas C-terminally truncated ORF2 protein was detected in cell pellets. The secreted ORF2S (tPA) proteins was eluted by 250 mM imidazole and corresponded to a glycosylated monomeric protein of approximately 90 kD, consistent with the results of a previously reported cell culture system [S protein does not require disulfide bonds formation, as is the case for the ORF2C protein [S protein dimerization is currently unknown. The structure of the E2s domain of the ORF2C protein shows that Ala597, Val598, Ala599, Leu601, and Ala602 are directly involved in the dimerization of ORF2C protein [S dimerization. Importantly, the dimeric form of the ORF2C protein is essential for its HEV neutralizing function [S is essential for its potential function. Further research is needed to test this hypothesis.Here, we generated an ORF2ication) ,14,16,27e system ,14. In a protein . The mec protein . Therefofunction . Hence, S and three other HEV ORF2C capsid proteins: p495, p239, and E2. Although ORF2S shares a consistent nucleotide identity with ORF2C, antigenic epitope C3 was lost in ORF2S (tPA). The reason for the loss of the C3 epitope may be that it is masked at the one potential glycosylation site (Asn562); alternatively, there may be a difference in conformation between ORF2S and ORF2C [S and ORF2C, and additional studies are also needed to examine the biological function of ORF2S.Epitope mapping, using a panel of mAbs that recognize nine different antigenic epitopes ,19, revend ORF2C . Future S (tPA) is stable and homogenous in PBS, with a peak at 11.6 min. In contrast to previous low-yield HEV-infected cell cultures [S (tPA) at approximately 5 mg/L was sufficient for structural analysis, allowing the elucidation of several unrecognized biological aspects of ORF2S. In addition, the sites of glycosylation could be clearly revealed by the structural analysis. Thus, we provided a design strategy for future experiments to express the ORF2S protein and carry out structural studies.Our HPLC analyses showed that ORF2cultures ,31, the S (tPA). We did not detect the loss of antigenic epitope C4 with the same antibody used in the Yin at al. study [S, although Montpellier et al. [S carries a lower capacity for HEV virions formation. Further studies are needed to elucidate this issue.Similar but not identical epitope profiles were recognized by a panel of mAbs, including previously detected E2 domain epitopes C1–C6 and linear epitope regions L1–L3 . We founl. study . The difr et al.  revealedS protein circulates in body fluids (serum and urine) and is the predominant form of ORF2 protein in the serum. We found that serum from HEV-infected patients showed significantly increased titers of anti-HEV IgG binding to ORF2S (tPA) compared with p239 protein. Furthermore, ORF2S (tPA) aroused an antigen-specific antibody response in the BALB/c model, and our data indicate that an obvious ORF2S (tPA)-specific B-cell response was present in HEV-infected patients. ORF2S impairs antibody-mediated neutralization, as demonstrated by Yin et al. through an in vitro neutralization experiment, suggesting that ORF2S suppressed the effect of neutralizing mAbs [S serves as an immunological decoy and, thereby, plays a role in blocking antibody neutralization. However, the immunoregulatory action of the ORF2S protein is unknown. It would be interesting to determine whether ORF2S-protein-induced monoclonal antibodies recognize HEV virions.During its life cycle in HEV-infected patients, the secreted form of the ORF2ing mAbs . Our datS, which HEV-infected cell cultures secretes into supernatants. Given the low-yield of these cultures, we present an efficient design strategy for expressing the ORF2S protein. Complete epitope mapping of the protein was also performed. The ORF2S-induced antibody response was detected in the BALB/c mouse model and in plasma and B cells from HEV-infected patients. In summary, this study will contribute to further research on HEV ORF2S protein structure and biological function.Recent studies have defined the origin and identification of ORF2