Aspergillus fumigatus. FTGM contains a linear α-mannan structure called core-mannan composed of 9 or 10 α-(1 → 2)-mannotetraose units jointed by α-(1 → 6)-linkages. However, the enzymes involved in core-mannan biosynthesis remain unknown. We speculated that two putative α-1,2-mannosyltransferase genes in A. fumigatus, Afu5g02740/AFUB_051270 (here termed core-mannan synthase A [CmsA]) and Afu5g12160/AFUB_059750 (CmsB) are involved in FTGM core-mannan biosynthesis. We constructed recombinant proteins for CmsA and detected robust mannosyltransferase activity using the chemically synthesized substrate p-nitrophenyl α-d-mannopyranoside as an acceptor. Analyses of CmsA enzymatic product revealed that CmsA possesses the capacity to transfer a mannopyranoside to the C-2 position of α-mannose. CmsA could also transfer a mannose residue to α-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose and showed a 31-fold higher specific activity toward α-(1 → 6)-mannobiose than toward α-(1 → 2)-mannobiose. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel filtration chromatography of isolated FTGM revealed that core-mannan structures were drastically altered and shortened in disruptant A. fumigatus strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB. Disruption of cmsA or cmsB resulted in severely repressed hyphal extension, abnormal branching hyphae, formation of a balloon structure in hyphae, and decreased conidia formation. The normal wild type core-mannan structure and developmental phenotype were restored by the complementation of cmsA and cmsB in the corresponding disruptant strains. These findings indicate that both CmsA, an α-1,2-mannosyltransferase, and CmsB, a putative mannosyltransferase, are involved in FTGM biosynthesis.Fungal-type galactomannan (FTGM) is a polysaccharide composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues located at the outer cell wall of the human pathogenic fungus  In Aspergillus fumigatus, a major pathogenic fungus causing invasive pulmonary aspergillosis, the structure of GM has been elucidated in detail7. The GM polysaccharide is composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues and is located at the outermost layer of the A. fumigatus cell wall8. There are two types of GMs in A. fumigatus, fungal-type galactomannan (FTGM) and O-mannose-type galactomannan (OMGM)9. FTGM contains a linear α-mannan structure called core-mannan in which 9 or 10 α-(1 → 2)-mannotetraose units are concatenated by α-(1 → 6)-linkages7. Moreover, FTGM also contains galactofuran side chains of β-(1 → 5)-galactofuranotetraose units concatenated by β-(1 → 6)-linkages and bound to the core-mannan by β-(1 → 2), β-(1 → 3), and β-(1 → 6) linkages7. The FTGM is found in three forms: soluble and released into the extracellular medium, cross-linked to other cell wall polysaccharides, and in membranes anchored to a glycosylphosphatidylinositol10. Part of the FTGM is bound covalently to a branched β-(1 → 3)-glucan11. Kudoh et al. reported that FTGM is attached to the nonreducing terminus of N-glycans in A. fumigatus6. Alternatively, the OMGM consists of β-(1 → 5)-galactofuranosyl chains bound to the nonreducing terminal side of an O-mannose-type glycan, where mannosyl chains are attached to a hydroxyl group of serine or threonine in the proteins12. Like FTGM, the galactofuranosyl residues of OMGM are elongated by β-(1 → 6)-Galf 6. Recently, we reported that GfsA is a β-1,5-galactofuranosyltransferase involved in the biosynthesis of β-(1 → 5)-galactofuranosyl chains, major structures in both FTGM and OMGM13. However, the enzymes involved in the biosynthesis of core-mannan remain unidentified.Galactomannan (GM), consisting of Saccharomyces cerevisiae14, Candida albicans15, and Schizosaccharomyces pombe16. The KTR/MNT mannosyltransferase gene family in the budding yeast S. cerevisiae includes KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6, and KTR714. The Mnt1/Kre2, Ktr1, and Ktr3 enzymes required for the addition of the second and third mannose residues to O-mannose-type glycans in S. cerevisiae17, and Mnt1/Kre2, Ktr1, Ktr2, Ktr3, and Yur1 may be involved in the elaboration of N-glycan outer chains19. The other members Ktr4, Ktr5, and Ktr7 were found to rescue defects in the C. albicans N-glycosylation pathway20.Alpha-1,2-mannosyltransferases have been well studied in the yeast species A. fumigatus homologs of Mnt1/Kre2 include α-1,2-mannosyltransferases involved in core-mannan chain biosynthesis. Based on the Blast algorithm, three putative α-1,2-mannosyltransferase genes were found in the A. fumigatus strain Af293/A1163 genome21: Afu5g10760/AFUB_058360, Afu5g02740/AFUB_051270, and Afu5g12160/AFUB_059750. The Afu5g10760/AFUB_058360 gene is reported as afmnt1, a putative α-1,2-mannosyltransferase involved in cell wall stability and virulence in A. fumigatus21. However, the functions of Afu5g02740/AFUB_051270 and Afu5g12160/AFUB_059750 have not been characterized.We speculated that the Escherichia coli and measured the mannosyltransferase activity. Specific mannosidase analyses of the enzymatic products revealed the CmsA can catalyze the transfer of a mannopyranoside to the C-2 position of α-mannose. Moreover, we demonstrated by proton nuclear magnetic resonance (1H-NMR) spectroscopy that CmsA is involved in the biosynthesis of the FTGM core-mannan structure. Finally, we show that disruptant strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB (∆cmsAB) show abnormal growth phenotypes and altered sensitivity to antifungal agents.In this study, we hypothesized that the latter two putative α-1,2-mannosyltransferase genes Afu5g02740/AFUB_051270 and Afu5g12160/AFUB_059750 are involved in biosynthesis of the FTGM core-mannan and are tentatively termed CmsA and CmsB, respectively. To verify this hypothesis, we constructed recombinant CmsA protein for expression in N-linked protein glycosylation22. A phylogenetic tree of the three putative α-1,2-mannosyltransferase genes of A. fumigatus and the homologous sequences from S. cerevisiae and C. albicans classified Afu5g02740/AFUB_051270 and Afu5g12160/AFUB_059750 into two gene clusters separate from that containing ScMnt121, suggesting functions distinct from Mnt1. The Afu5g02740/AFUB_051270 and Afu5g12160/AFUB_059750 genes encode predicted 397- and 506-amino acid proteins with molecular masses of 46.5 and 58.9 kDa, respectively , which is necessary to coordinate the divalent cation24. The Mnt1/Kre2 and Ktr4 proteins of S. cerevisiae do not contain the archetypal DXD motif26, but the EPD (amino acids 247–249) and EPN (amino acids 262–264) sequences have been claimed to serve the same purpose26. The equivalent sequences in CmsA and CmsB were found in EPK (amino acids 205–207) and EPE (amino acids 245–247), as shown in Fig. N-glycosylation27, while Afu5g12160/AFUB_059750 is closely related to the genes encoding the putative mannosyltransferases ScKtr5 and ScKtr720. We hypothesized that these two putative α-1,2-mannosyltransferase genes are involved in biosynthesis of FTGM core-mannan. Therefore, we tentatively named Afu5g02740/AFUB_051270 core-mannan synthase A (CmsA) and Afu5g12160/AFUB_059750 core-mannan synthase B (CmsB) and attempted to elucidate their functions.The genes Afu5g02740/AFUB_051270 and Afu5g12160/AFUB_059750 are annotated in the Aspergillus genome database (AspGD) as putative transmembrane α-1,2-mannosyltransferases with predicted functions in ely Fig. . TMHMM pins Fig. 23. SeverE. coli expression system. Transcripts encoding the putative catalytic domains of CmsA (amino acids 27–397) and CmsB (amino acids 71–442) were introduced into an E. coli expression vector fused in-frame with a 6 × histidine (6 × His) tag at the N-terminus. CmsA was successfully expressed as a soluble protein in an E. coli, but CmsB was unfortunately expressed as an insoluble protein in an E. coli. Recombinant 6 × His-tagged CmsA protein was purified by Ni+ affinity chromatography and gel filtration chromatography and analyzed using SDS-PAGE  as a sugar acceptor, guanosine diphosphate-α-d-mannose  as a sugar donor, and 1.5 mM Mn2+ as a cofactor -specific mannosidase but not by α-(1 → 6)-specific mannosidase -α-Man-pNP. Thus, CmsA has GDP-Man: α-mannoside α-1,2-mannosyltransferase activity in vitro. In addition, we attempted to evaluate the elongation activity of CmsA at 30 °C for 60 min using α-(1 → 2)-mannobiose (α-Man-(1 → 2)-α-Man) or α-(1 → 6)-mannobiose (α-Man-(1 → 6)-α-Man) as sugar acceptor substrates -mannobiose and α-(1 → 2)-mannobiose were 473 and 15.1 (nmol/min/mg protein), respectively. CmsA showed a 31-fold higher specific activity in the presence of α-(1 → 6)-mannobiose than in that of α-(1 → 2)-mannobiose under the same reaction conditions, indicating that CmsA recognizes α-(1 → 6)-mannobiose as a preferred acceptor substrate rather than α-(1 → 2)-mannobiose   strain A1151 after cultivation on minimal medium (MM) at 37 °C for 36 h   Fig. ; Table 1msB Fig. ; Table 1msB Fig.  and 5A,B6 h Fig. , and colcmsA, ∆cmsB, and ∆cmsAB were sparse and scattered compared with WT conidiophores    Fig. , which w04) Fig. .A. fumigatus grew linearly, whereas ∆cmsA, ∆cmsB, and ∆cmsAB strains exhibited abnormal swelling  and branching , caspofungin (CPFG), amphotericin B (AMPH-B), flucytosine (5-FC), fluconazole (FLCZ), itraconazole (ITCZ), voriconazole (VRCZ), and miconazole (MCZ)  Table . The ∆cmin Table . Howeverty Table .Table 2S6, we investigated the influence of cmsA and cmsB disruption on OMGM structure. Since the presence of β-(1 → 5)-/β-(1 → 6)-galactofuranosyl residues in OMGM prevents the determination of mannosyl chain length, we constructed ∆glfA∆cmsA and ∆glfA∆cmsB strains to reveal alterations in the length of mannosyl chain by cmsA or cmsB disruption  -mannosyl residues are present in both FTGM and OMGMion Fig. . We extrad) Fig.  with detins Fig. , indicatcmsA and cmsB gene disruption on FTGM structure, we extracted and purified total GMs from WT A1151, ∆cmsA, ∆cmsB, ∆cmsAB, ∆cmsA::cmsA, and ∆cmsB::cmsB strains. The extracted total GMs containing both FTGM and OMGM (designated FTGM + OMGM) were then subjected to β-elimination to remove OMGM to designate FTGMs. First, FTGMs were analyzed to determine their carbohydrate compositions -β-Galf-(1 → 5)-β-Galf-(1 → 5)- according to a previous report6. This result indicates that the Galf sugar chain in FTGM remains even if cmsA, cmsB, or both genes are disrupted. Thus, to remove galactofuranosyl residues from FTGM, FTGM fractions were subjected to acid hydrolysis and the products (designated FTGM-HCls) analyzed by 1H-NMR spectroscopy , 5.233 ppm , 5.216 ppm , and 5.054 ppm  in the WT spectrum - , -(1 → 6)-α-Man-(1 → 2)-α-Man-(1 → 2)-α-Man-(1 → 2)-α-Man-(1 → 6)- , -(1 → 6)-α-Man-(1 → 2)-α-Man-(1 → 2)-α-Man-(1 → 2)-α-Man-(1 → 6)- , and -(1 → 6)-α-Man-(1 → 2)-α-Man-(1 → 2)-α-Man-(1 → 2)-α-Man-(1 → 6)- , respectively, according to a previous report6. In contrast, these core-mannan signals were absent or substantially truncated in the 1H-NMR spectra for ∆cmsA-FTGM-HCl, ∆cmsB-FTGM-HCl, and ∆cmsAB-FTGM-HCl, indicating that core-mannan structures are altered and/or lost in the absence of cmsA or cmsB -α-Man- and t-Man-(1 → 2)-α-Man-30. Thus, A′ and D′ indicate the presence of α-(1 → 6)-linked and α-(1 → 2)-linked terminal mannosyl residues, respectively, suggesting that shorter oligomannan structures are present in the deletion mutants. The signals A, B, C, and D found in the spectrum from WT-FTGM-HCl were restored in the 1H-NMR spectra for the complementary strains ∆cmsA::cmsA and ∆cmsB::cmsB  and detection by the phenol-sulfuric acid method. Peak FTGM-HCl content in the WT A1151 strain was detected from fractions 35 to 50 Fig. . In ∆cmsard Fig. , while t WT Fig. . SimilarmsB Fig. . These rin vitro mannosyltransferase assay and substrate-specific-mannosidase analysis -mannobiose than toward α-(1 → 6)-mannobiose in vitro -galactofuranosyl chains were retained in FTGM (β-eliminated GM) extracted from ∆cmsA and ∆cmsB, indicating that not all β-(1 → 5)-galactofuranosyl chains are bound to the core-mannans of FTGM and OMGM  is a putative α-1,2-mannosyltransferase and is required for cell wall stability21. A ∆afmnt1 strain exhibited increased sensitivity to cell wall stress, high temperature, and calcofluor white and Congo red staining21. However, the ∆afmnt1 mutant normally grows under normal growth conditions21. In contrast, both ∆cmsA and ∆cmsB abnormally grow, and their growth defects are considerable. This observation suggests that the function of AfMnt1 is different than that of both CmsA and CmsB. However, the enzymatic function(s) of AfMnt1 have yet to be elucidated, necessitating further detailed analysis. Recently, a multiple deletion mutant of 11 putative mannosyltransferases other than cmsA and cmsB that is responsible for establishing α-(1 → 6)- and α-(1 → 2)-mannose linkages in yeast was constructed in A. fumigatus31. The mutant did not reduce the mannan content of the mycelium cell wall but reduced the mannan content of the conidial cell wall31. The mutant did not exhibit drastic mycelial growth defects31. Therefore, core-mannan constructed by CmsA and CmsB seemingly plays an important role in mycelial cell wall maintenance rather than these 11 mannosyltransferases.Wagener cmsA and cmsB are also present in many pathogenic fungi that cause plant and animal diseases, such as the animal pathogens Trichophyton rubrum, Paracoccidioides brasiliensis, and Ajellomyces capsulatus and the plant pathogens Magnaporthe grisea, Fusarium graminearum, and Botrytis cinerea. There are a few previous reports on phenotypic changes conferred by disruption of cmsA and/or cmsB orthologs. In Neurospora crassa, strong growth inhibition was observed by disruption of the cmsA ortholog32, suggesting common functions for CmsA in filamentous fungi. However, in the insect pathogenic fungus Beauveria bassiana, disruption of the cmsA ortholog ktr4 or cmsB ortholog ktr1 did not cause drastic growth inhibition33, suggesting that cmsA and cmsB have different functions in B. bassiana or that core-mannan elaborated by CmsA and CmsB does not play an important role in B. bassiana development.Orthologs of 36. A similar balloon structure was also reported in other cell wall-deficient strains, for example, strains missing chitin-synthase with myosin domain (csmA)37, protein O-mannosyltransferase A and C (pmtA and pmtC)40. Given that core-mannan deficiency decreases the physical strength of the cell wall, strains ∆cmsA and ∆cmsB may not be able to withstand the osmotic pressure of the external environment, resulting in formation of the observed balloon structure. Disruption of cmsA and cmsB also repressed hyphae extension, suggesting that CmsA and/or CmsB inhibitors may be effective antifungal drugs. FTGM is a sugar chain structure specific to fungi and does not exist in humans or plants (that do not possess homologous enzymes). Thus, we assume that the α-1,2-mannosyltransferases involved in FTGM biosynthesis are potential targets for use in novel antifungal therapies. Moreover, cmsA and/or cmsB disruptant strains demonstrated increased susceptibility to several clinical antifungal drugs , suggesting that CmsA and/or CmsB inhibitors may enhance the therapeutic efficacy of these compounds  and grown on MM composed of 1% glucose, 0.6% NaNO3, 0.052% KCl, 0.052% MgSO4·7H2O, and 0.152% KH2PO4 plus Hunter’s trace elements . Aspergillus strains were transformed using standard procedures. Plasmids were amplified in E. coli DH5α and proteins expressed in E. coli strain Rosetta-gami B (DE3) .The cmsA (AFUB_051270) and cmsB (AFUB_059750) genes from A. fumigatus A1163 were synthesized by Bioneer (Korea). The cmsA gene was delivered by Bioneer cloned in a pBT7-N-His vector with an N-terminal 6 × His tag, termed pBT7-CmsA. The pBT7-CmsA was used for recombinant protein production. The cmsB gene was amplified by PCR and cloned into pET15b-KAI9. The cmsB gene was amplified by PCR using the cloned synthesized cmsB gene as a template for the primers pET15b-CmsB(CO)-IF-F and pET15b-CmsB(CO)-IF-R  to yield pET15b-CmsB. All PCR reactions were performed using Phusion High-Fidelity DNA Polymerase . The pBT7-CmsA and pET15b-CmsB plasmids were transformed into Rosetta-gami B (DE3) cells.Bacterial codon-optimized -R Table . The amp9. Protein concentrations were determined using the Qubit Protein Assay Kit , and CmsA and CmsB were analyzed by SDS-PAGE to assess purity and molecular weight.Protein purification was performed as previously describedp-nitrophenyl α-d-mannopyranoside (α-Man-pNP) was purchased from Sigma-Aldrich (USA). Alpha-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose were purchased from Dextra Laboratories Ltd (United Kingdom). Standard assays were performed with α-Man-pNP, α-(1 → 2)-mannobiose or α-(1 → 6)-mannobiose as acceptor, GDP-Man as donor, and purified CmsA protein in a total reaction volume of 20 µl. The mixture was incubated at 37 °C and stopped by heating (99 °C) for 5 min. Para-nitrophenol derivatives were detected by UV300 absorbance and analyzed by HPLC using a Shodex Asahipak NH2P-50 4E amino column  as previously described9. Alpha-(1 → 2)-mannobiose or α-(1 → 6)-mannobiose derivatives were labeled using 2-aminopyridine (PA) after reaction as previously described, with slight modifications41. Reaction mixtures were dried using a centrifugal concentrator, and the resulting samples were dissolved in 20 µL of PA reagent  and incubated at 90 °C for 1 h. The samples were then mixed with 70 µL of PA reducing reagent  and incubated at 80 °C for 35 min. Next, 500 µL of 0.2 M ammonia solution and 300 µL of chloroform were added to the resulting sample. Following centrifugation, the supernatant was designated as the PA-sugars. The PA-sugars were purified with a Monospin PBA spin column  as previously described42. The purified PA-sugars were detected via fluorescence at excitation and emission wavelengths of 310 nm and 380 nm, respectively, and analyzed using HPLC with a Shodex Asahipak NH2P-50 4E amino column (250 × 4.6 mm) as follows. Elution was performed using two solvents: eluent A, 93% acetonitrile in 0.3% acetate (pH adjusted to 7.0 with ammonia), and eluent B, 20% acetonitrile in 0.3% acetate (pH adjusted to 7.0 with ammonia). The gradient program was set at a flow rate of 0.8 mL/min (expressed as the percentage of solvent B): 0–5 min, isocratic 3%; 5–30 min, 3–40%; 30–35 min, 40–3%; 35–60 min, isocratic 3%. Enzymatic activity was evaluated by determining the area of products via HPLC. Oligosaccharide levels were compared to α-Man-pNP, α-(1 → 2)-mannobiose or α-(1 → 6)-mannobiose as a standard. Alpha-1,2-mannosidase and α-1,6-mannosidase were purchased from ProZyme (USA) and New England Biolabs (USA), respectively, and used according to the manufacturers’ instructions.The artificial acceptor substrate A. fumigatus A1160 was used as the parental strain  of cmsA were PCR amplified from genomic DNA with primer pairs AFUB_051270-1/AFUB_051270-2 and AFUB_051270-3/AFUB_051270-4. The 5′- and 3′-flanking regions (approximately 1.0 kb each) of cmsB were PCR amplified from genomic DNA with primer pairs AFUB_059750-1/AFUB_059750-2 and AFUB_059750-3/AFUB_059750-4. The AnpyrG gene used as a selective marker was amplified using plasmids pSH1 as template13 and the primer pairs pyrG-5/pyrG-6. The three amplified fragments were purified, mixed, and subjected to a second PCR without specific primers to assemble each fragment as the overhanging chimeric extensions act as primers. A third PCR was performed with the nested primer pairs AFUB_051270-7/AFUB_051270-8 for cmsA or AFUB_059750-7/AFUB_059750-8 for cmsB and the products of the second PCR used as templates to generate the final deletion constructs. The amplified final deletion constructs were purified using the Fast Gene Gel/PCR Extraction Kit  and used directly for transformation of A. fumigatus. Transformants were grown on MM plates containing 0.6 M KCl as an osmotic stabilizer under appropriate selection conditions, and single colonies were isolated twice before further analysis. Correct replacement of the DNA fragments for gene complementation was confirmed by PCR using the primer pairs AFUB_051270-1/pyrG-R and pyrG-F/AFUB_051270-4 for cmsA and the primer pairs AFUB_059750-1/pyrG-R and pyrG-F/AFUB_059750-4 for cmsB . The cmsB gene was disrupted in A. fumigatus ∆cmsA strain by ptrA insertion  as template and the primer pair ptrA-5/ptrA-6. Disruption of cmsB was confirmed by PCR using the primer pairs AFUB_059750-1/ptrA-R and ptrA-F/AFUB_059750-4  to construct a complementation cassette for cmsB. The regions of the cmsA and cmsB gene were PCR amplified from genomic DNA using the primer pairs AFUB_051270-1/AFUB_051270-complement-2 and AFUB_059750-1/AFUB_059750-complement-2, respectively. The region of the pyrG gene from A. nidulans (AnpyrG) was PCR amplified from pSH1 using the primer pair complement-3/complement-4. The ptrA genes used as selective markers were amplified using the pPTR-I plasmid as a template and the primer pair ptrA-5/ptrA-6. The three amplified fragments were purified, mixed, and subjected to a second PCR assay without specific primers to assemble each fragment as the overhanging chimeric extensions acted as primers. A third PCR assay was performed using the products of the second PCR as a template and the nested primer pair AFUB_051270-7/complement-8 for cmsA and AFUB_059750-7/complement-8 for cmsB to generate the final DNA constructs. The amplified final deletion constructs were purified using the Fast Gene Gel/PCR Extraction Kit and used directly for transformation. Transformants were grown on MM plates containing 0.6 M KCl as an osmotic stabilizer under appropriate selection conditions, and single colonies were isolated twice before further analysis. Correct replacement of the DNA fragments for gene complementation was confirmed by PCR using the primer pairs AFUB_051270-1/ptrA-R and ptrA-F/AFUB_051270-4 for cmsA and AFUB_059750-1/ptrA-R and ptrA-F/AFUB_059750-4 for cmsB  using a previously described method44. A CTAB fraction precipitated at pH 9.0 with NaOH in the presence of borate was recovered as the galactomannoprotein fraction. A β-elimination reaction was performed by exposing the fractionated FTGM + OMGM to reducing alkali conditions 30. After neutralized with 50% acetic acid solution, resultant samples were dialyzed overnight against distilled water. The purified samples were then lyophilized, resuspended in distilled water, and clarified using 0.45-µm pore filters. These β-eliminated FTGM + OMGMs were designated the purified FTGMs9.Preparation of total GM (FTGM + OMGM) from f sugar chains, FTGMs were treated with 0.1 M hydrochloric acid at 100 °C for 60 min. Samples were then neutralized with 10 M NaOH and dialyzed overnight against dH2O6. These acid-treated FTGMs, designated FTGM-HCls, contained only core-mannan without Galf sugar chains.To remove the Gal2O, and clarified using 0.45-µm pore filters. These samples were designated as OMGMs.To prepare OMGM, a β-elimination reaction was performed against the fractionated FTGM + OMGM under nonreducing alkali conditions . Samples were neutralized with 50% acetic acid solution, lyophilized, resuspended in dH6. Briefly, 5 mg FTGM-HCls were applied to the column at 0.5 ml/min. Gel filtration chromatography was performed using a Sephacryl S-200 HR (1 × 75 cm) column  with 0.8 M NaCl as eluent. One hundred 1-ml fractions were collected. The eluted sugars were monitored using phenol-sulfuric acid method45.Separation of FTGM-HCls by gel filtration chromatography was conducted using a method described previously with minor modifications6. Briefly, 200 mg OMGMs were applied to a Bio-Gel P-2 (2 × 90 cm) column  at 0.5 ml/min and eluted using dH2O. One hundred 5-ml fractions were collected. The eluted sugar chains were monitored using the phenol-sulfuric acid reaction45.OMGMs were separated by gel chromatography using a previously described method with minor modifications6. Briefly, 1 mg of each FTGM was hydrolyzed with 4 M trifluoroacetic acid (TFA) at 110 °C for 3 h. After removal of the TFA via evaporation, resulting monosaccharides were reduced overnight with 1% NaBH4 at room temperature. The resulting alditols were acetylated with pyridine/acetic anhydride  at 40 °C for 12 h. The reagents were then removed via evaporation with toluene, dissolved in chloroform, and washed thrice with water. The resulting alditol acetate derivatives were analyzed via gas chromatography-mass spectrometry (GC-MS) using a capillary column  with helium as the carrier gas and a temperature program of 160 °C–200 °C at 3 °C/min. The GC-MS analyses were performed using the JEOL JMS-K9 mass spectrometer (Japan).Carbohydrate composition analysis was performed as described previously, with slight modifications2O with intervening lyophilization and were then dissolved in D2O (99.97% atom 2H). The NMR spectra were recorded using a JNM-LA600 spectrometer (JEOL) at 45 °C. The proton and carbon chemical shifts were referenced relative to internal acetone at δ 2.225 and 31.07 ppm, respectively.Samples for NMR were exchanged twice in D13. Briefly, conidia from each strain were point inoculated into the center of agar plates and colony diameters measured after 24, 48, 72, 96, and 120 h of incubation at 37 °C. The growth rates were determined for each colony in millimeters per hour during each of the incubation intervals  and then averaged across the entire time interval. Growth rate measurements were repeated 20 times for each individual strain.Colony growth rates were measured as described previously13. Briefly, approximately 105 conidia were spread onto MM plates (90 mm diameter). After 3 days of incubation at 37 °C, the conidia were suspended in 15 ml of 0.01% (w/v) Tween 20 and counted using a hemocytometer. Efficiency was quantified as the number of conidia formed per unit area.The efficiency of conidiation was analyzed as described previously46, with minor modifications. Briefly, dried plates were used for antifungal susceptibility testing 47.Drug susceptibility was tested in triplicate according to the Clinical and Laboratory Standards Institute reference broth microdilution method (document M38-A2)http://www.cbs.dtu.dk/services/TMHMM/)23. BLAST searches were performed by a tool in Aspergillus genome database (AspGD) (http://www.aspergillusgenome.org)22.The transmembrane regions of CmsA and CmsB were predicted by the TMHMM tool (Supplementary-materials


The higentclass1pt{minimand Kenig , 34, 35 vergence  follows f being a Schwartz function in For In 2014, Sjolin and Soria  obtainedAssumeThen, for everya, inequality –(H3). Ifandthen the global estimate (holds.estimate  holds.Letand setAssume thatandsatisfies (H1)–(H3) withThen, for everym, inequality –(H3), for instance, the fractional Schrödinger equation –(H3) withandThenforandHere the constantCmay depend onsandandμbut not onx, t, orN.The proof of Lemma Assume thatϕsatisfies (H1)–(H3) withandThenforandwhereHere the constantCmay depend onαandandμbut not onx, d, andN.Applying the result of Lemma g continuous and rapidly decreasing at infinity. We take a real-valued function σ, which is defined byAssume that tor (see ) to provAssume that equality  holds ifestimate  it suffig continuous and rapidly decreasing at infinity. We take a real-valued function Let ξ)dξ. By  it is suestimate  in the pTo prove Lemma Assume thatforand thatis monotonic onI. Thenwhere C does not depend onF, ψ, orI.(ii)Assume thatforThenwhere C does not depend onF, ψ, orI.C such that for C depends only on α, μ.By conditions (H1) and (H2), there exist positive constants Without loss of generality, we may assume M such that δ is a small positive constant such that To estimate , we choo we show  by dividCase (I): λ such that ξ.Case (I-a): Case (I-b): herefore  follows m2−1. By , we havestimates , (4.17),stimates .Case (II): α for ation of  into thrCase (II-a):  follows .Case (II-b): Case (II-c): Summing up all the above estimates, we complete the proof of estimate  and finiIn this paper, by linearizing the maximal operator and duality methods, and applying the results of Lemma ualities  and (1.9ualities  and (1.7


Pseudotaxus chienii is an endangered coniferous plant that is endemic to China. Because P. chienii is sessile and has a long life cycle, its options for responding to drastic or rapid changes in climate are limited. To survive locally, P. chienii must be able to adapt, and the species shows variations in leaf size along an environmental gradient from east to west. It is important to determine whether this phenotypic variation is driven by DNA methylation. Therefore, we performed a preliminarily survey using methylation-dependent restriction-site associated DNA sequencing (MethylRAD) to investigate the methylation status of three P. chienii individuals from heterogeneous ecological niches. In total, 372,611 CCGG tags and 726,332 CCHGG tags were obtained. The rate of high quality methylation tags for a specific site in the genome varied from 42.31% (Gxdms3-4) to 50.01% (Jxbj3-4) and 50.18% (Zjdxg3-6). The level of CCHGG methylation (16.63%) was higher than that of CCGG (13.60%), which may be why P. chienii has low levels of phenotypic variation. The methylation data can be accessed using the Sequence Read Archive (SRA) database (SRP128155). Specifications Table MethylationValue of the data•This dataset provides valuable information that could help predict epigenetic adaptations to future changes in climate.•These data enhance our understanding of the mechanisms of natural phenotypic variation and may be used to provide guidance for the management of genetic resources and species conservation.1Pseudotaxus chienii individuals from heterogeneous ecological niches. The clean data were deposited in the National Center for Biotechnology Information SRA database (SRP128155).This article provides methylation data for three 22.1P. chienii individual from Daxiagu in Zhejiang Province (Zjdxg3-6), one from Bijiashan in Jiangxi Province (Jxbj3-4), and one from Damingshan in Guangxi Zhuang Autonomous Region (Gxdms3-4). P. chienii individuals across these regions possess high genetic diversity and show strong local adaptations to rapid environmental changes, which are suitable for methylation analysis In this study, we selected one 2.2A 20 µL ligation mix that included ligation master mix, 5 µM adaptor A, 5 µM adaptor B, and digested DNA was incubated at 16 °C for 1 h. The sequences of the two adaptors were as follows:slx-ada1-BsaXI: 5′–CAAGCAGAAGACGGCATACGACCGCGCGAGTNNN–3′.3′–GGCGCGCTCA–5′.slx-ada2-BsaXI: 5′–CGACAGGTTCAGAGTTCTACAGTCCGACGATCNNN–3′.3′–TGTCAGGCTGCTAG–5′.Ligation products were amplified in 30.4 µL of reaction mix containing 8 µM of each primer , 1.6× High-Fidelity DNA Polymerase Buffer, 0.39 mM dNTPs, 0.4 U of Phusion High-Fidelity DNA polymerase , and 18 µL of ligated DNA. PCR amplification was performed for 1–16 cycles using the following conditions: 98 °C for 5 s, 60 °C for 20 s, 72 °C for 10 s, and a final extension step of 5 min at 72 °C. The PCR products were separated using 8% polyacrylamide gel electrophoresis along with a 100-bp DNA ladder . The target bands were excised, the DNA was allowed to diffuse out of the gel in nuclease-free water for 30 min at 37 °C and then amplified using the PCR conditions described above. PCR products derived from the three individuals were mixed, further purified, eluted, and quantified.2.3Sample barcodes were introduced using PCR. Each 85-µL PCR reaction mix contained 5× High-Fidelity DNA Polymerase Buffer, 10 mM dNTPs, 10 µM Primer3, 10 µM Index Primer, 1.6 U Phusion High-Fidelity DNA polymerase , and 50 ng of gel-extracted PCR product. A total of 1–16 cycles of PCR were performed as described above, and following purification, the PCR products were subjected to single-end sequencing (100–150 bp) using an Illumina Hiseq X Ten sequencer  , Table 2


BAM15 is a novel mitochondrial protonophore uncoupler capable of protecting mammals from acute renal ischemic-reperfusion injury and cold-induced microtubule damage. The purpose of our study was to investigate the effect of BAM15 on apoptosis during 5-day transportation of human-induced pluripotent stem (hiPS)-differentiated retinal tissue.Retinal tissues of 30 days and 60 days were transported with or without BAM15 for 5 days in the laboratory or by real express. Immunofluorescence staining of apoptosis marker cleaved caspase3, proliferation marker Ki67, and neural axon marker NEFL was performed. And expression of apoptotic-related factors p53, NFkappaB, and TNF-a was detected by real-time PCR. Also, location of ganglion cells, photoreceptor cells, amacrine cells, and precursors of neuronal cell types in retinal tissue was stained by immunofluorescence after transportation. Furthermore, cell viability was assessed by CCK8 assay.Results showed transportation remarkably intensified expression of apoptotic factor cleaved caspase3, p53, NFkappaB, and TNF-a, which could be reduced by supplement of BAM15. In addition, neurons were severely injured after transportation, with axons manifesting disrupted and tortuous by staining NEFL. And the addition of BAM15 in transportation was able to protect neuronal structure and increase cell viability without affecting subtypes cells location of retinal tissue.BAM15 might be used as a protective reagent on apoptosis during transporting retinal tissues, holding great potential in research and clinical applications.The online version of this article (10.1186/s13287-019-1151-y) contains supplementary material, which is available to authorized users. Successful establishment of using human-induced pluripotent stem cell (hiPSC) to produce functional retinal tissues holds great potential for future therapies –3. It haBAM15 is a novel mitochondrial protonophore uncoupler, which is able to transport protons into the mitochondrial matrix through a pathway without activating ATP synthase, thus uncoupling oxidative reaction from ATP production . In addiPrecise coordination of proliferation and apoptosis is essential for retinal development and maintenance, whereas its disruption leads to abnormal structure and early onset of retinal degeneration . As a ceAlso, the preservation of integral microtubule cytoskeleton is indispensible for neuronal elaborate dendritic and axonal processes as well as proper synaptic connections. Hence, failure to maintain neuronal structural integrity leads to defections in neural functions –34. NeurHere, we report for the first time the influence of transportation on iPS-induced retinal tissue, inspiring novel pharmacological strategies to attenuate apoptosis in the process. We analyzed the appropriate conditions for transporting retinal tissue and revealed that supplement of BAM15 attenuated 5-day transportation-induced apoptosis. In addition, BAM15 was able to increase cell survival rate and protect neural structural integrity in transportation without affecting subtypes of cells’ location of retina. Prospectively, protective reagent BAM15 holds great potential for decreasing apoptosis of transportation for medical and research applications.The hiPSC line was obtained from Cellapy®  and maintained on Matrigel (BD Biosciences)-coated plates with StemFlex medium  according to WiCell protocols. The hiPS cells were passaged every 5–6 days by enzymatic digestion with 1 mM EDTA at 80% confluence. The hiPSCs were differentiated to retinal tissue based on previously established protocol with minor modifications , 38. BriMitochondrial uncoupler N5, N6-bis (2-Fluorophenyl)- oxadiazolo  pyrazine-5, 6-diamine  was utilized in the assay at the concentration of 100 μM . BAM15 wAt the beginning of transportation, drugs were added at appropriate concentrations and transported for 5 days at room temperature on the bench without vibration, stress forces, and concessive forces to mimic transporting environment. Five days later, the cups were transferred back to culture dish in incubator at 37 °C and recovered for 5 days before subjected to experiments. To analyze actual transportation course, we used express company to deliver the retinal cups for 5 days and monitored changes of temperature by recorder  during the process.On day 5 after recovery, the retinal cups were fixed with 4% paraformaldehyde for 30 min, sucrose gradient dehydration and embedded in optimal cutting temperature (OCT) compound overnight at − 80 °C. The cups were sliced at 10 μm using a Leica microtome. After being permeabilized with 0.5% Triton X-100  and blocked with normal goat serum , sections were stained with primary antibodies against Ki67 , NEFL , Laminin , and cleaved caspase-3  Brn3  HuC/D  Recoverin  OTX2  AP2α , Iowa, USA) PAX6 , Iowa, USA) at 4 °C overnight. The secondary antibodies used included the corresponding species-specific Alexa Fluor-555-, and Alexa Fluor-647-conjugated antibodies  and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Photos were captured with a fluorescence system .p value less than 0.05 was considered significant.Expression of p53, NFκB, and TNF-α in retinal cups was measured after transportation by real-time PCR analyses using the SYBR Green system . Total RNA from cups was isolated with TRIzol reagent . One microgram of total RNA was subjected to reverse transcription with the SYBR Prime-Script RT-PCR kit in accordance with the manufacturer’s protocol. The following primer pairs were used: for β-actin, 5′-caccacaccttctacaatgag-3′ (sense) and 5′-tagcacagcctggatagcaac-3′ (antisense); for p53, 5′-gaggttggctctgactgtacc-3′ (sense) and 5′-tccgtcccagtagattaccac-3′ (antisense); for NFκB, 5′-cgagacagtgacagtgtctgc-3′ (sense) and 5′-gctctctgagcacctttggatg-3′ (antisense); and for TNF-α, 5′-gaggccaagccctggtatg-3′ (sense) and 5′-cgggccgattgatctcagc-3′ (antisense). The experiments were repeated at least three times, and The viability of retinal cups after transportation was assessed using the Cell Counting Kit-8 (CCK8) assay . Five days after recovery from transportation, the retinal tissues were plated into 48-well plates (3 cups/well). Each condition was performed in three replicate wells. Subsequently, the cups were incubated at 37 °C for 3 h after CCK8 reagent was added to each well. The absorbance was measured at 450 nm. The viability was calculated using the optical density ratio of a treated group relative to the control. Each set of experiments was carried out in triplicate.P value < 0.05 was considered significant.All experiments were repeated three times or more. Data are expressed as means ± SE. All calculations and statistical tests were analyzed using GraphPad Prism 6 for Mac version 4.02  or Microsoft Excel 2003 . Multiple-group comparisons were carried out using one-way ANOVA. The experimental procedures are as follows Fig. a: hiPSC As shown in Fig. + cell population, which was similar to the blank group. Quantification of Ki-67+ was presented in Additional file +cell/1000 μm2 for D30: blank, 10.13 ± 1.19, con, 26.26 ± 2.80, DMSO, 23.17 ± 1.15, BAM15, 15.79 ± 1.58; Ki67+cell/1000 μm2 for D60: blank, 6.10 ± 0.53, con, 13.50 ± 0.59, DMSO, 14.16 ± 0.50, BAM15, 8.22 ± 0.25; p < 0.05). As a stimulating factor, transportation increased expression of Ki-67+ cell, which could be reduced by addition of BAM15 supplement  Thus, DMSO and BAM15 protected retinal cups from apoptosis in transportation. Laminin was mainly stained in the center of the NR-like structures and varied slightly with different treatments, suggesting BAM15 imposed little effect on expression of Laminin.These immunofluorescence results indicated cleaved caspase-3 expressed occasionally in normal retinal tissues in the developing stage , 40–42. p = 0.005, 0.001, 0.025 respectively; for TNF-α: blank, 1-fold, con, 3.26 ± 0.60-fold, DMSO, 2.28 ± 0.06-fold; BAM15, 1.46 ± 0.28-fold, p = 0.016, 0.001, 0.040 respectively; for P53: blank, 1-fold, con, 3.63 ± 0.49-fold, DMSO, 2.26 ± 0.23-fold, BAM15, 1.29 ± 0.01-fold, p = 0.017, 0.016, 0.001 respectively). Similarly, retinal tissue of 60 days exhibited the same trend . According to results of CCK8 assay . Similarly, the presence of 100 μmol/L BAM15 improved cell survival in the real transportation condition . Thus, BAM15 is clinically safe.After 5 days of transportation, retinal tissue of 30 days and 60 days was extracted for real-time PCR analysis. Expression of apoptotic-related genes p53, NFkappaB, and TNF-a was elevated by transportation, and supplement of DMSO and BAM15 was able to attenuate their expression. As showed in Fig. +cell/1000 μm2: blank, 7.70 ± 0.23, con, 7.32 ± 0.38, DMSO, 8.50 ± 0.42, BAM15, 7.06 ± 0.50; Hu C/D+ cell/1000 μm2: blank, 6.21 ± 0.78, con, 6.04 ± 0.17, DMSO, 5.58 ± 0.26, BAM15, 6.87 ± 0.19; recoverin+ cell/1000 μm2: blank, 3.26 ± 0.29, con, 3.35 ± 0.29, DMSO, 2.49 ± 0.25, BAM15, 3.25 ± 0.37; OTX+ cell/1000 μm2: blank, 5.24 ± 0.18, con, 5.50 ± 0.25, DMSO, 5.28 ± 0.10, BAM15, 5.32 ± 0.26; AP2α+cell/1000 μm2: blank, 0.63 ± 0.06, con, 0.49 ± 0.04, DMSO, 0.52 ± 0.01, BAM15, 0.59 ± 0.02; PAX+ cell/1000 μm2: blank, 8.03 ± 0.15, con, 8.59 ± 0.46, DMSO, 7.79 ± 0.31, BAM15, 8.46 ± 0.13). Thus, 5 days of transportation imposed little influence on location of subtypes of retinal cells. To find out whether transportation has delayed effects on differentiation, retinal tissues after 15 days of recovery from transportation were detected and similar results were observed  transportation increased apoptosis in retinal tissues; (2) transportation damaged neural structural integrity; and (3) mitochondrial uncoupler BAM15 was able to attenuate apoptosis, increase cell viability and protect neural structural integrity during transportation.Although protocols adopted for shipment of viable cells varied among laboratories, the main consideration is to attenuate apoptosis, protect morphological intactness, and preserve cell viability , 10. FilBAM15 has a broad range of efficacy without toxicity , 45, 46.BAM15 might attenuate transportation-induced apoptosis by targeting relevant signaling pathways. The mitochondrial membrane regulates expression of caspase pathway, and previous evidence indicated caspase-3 and caspase-9 participated in iPSC apoptosis . Also, oFive days of transportation imposed little effect on expression of laminin. Laminin presents in the developing human eyes and plays an important role in ocular differentiation . In addiSurprisingly, in our investigation, elevation expression of proliferative marker Ki67 was found to co-occur with increased apoptotic-related markers. The increased expression of proliferation marker was in response to stimulation of transportation, manifesting proliferation potential of stem cells was maintained during transportation. In BAM15-treated group, we surmised supplement of BAM15 reduced the influence of transportation, and thus staining of Ki67+ cells was less than the control group.Previous investigation revealed that addition of BAM15 greatly enhanced tubulin preservation in the cold possibly by reducing mitochondrial stress . SimilarIn the subsequent work, the full mechanism of how BAM15 functions remains to be further explored. Moreover, we dedicated to investigate influence of BAM15 on retinal development and differentiation in the future.Addition of mitochondrial uncoupler BAM15 is capable of attenuating apoptosis, protecting neural structural integrity and increasing cell viability in 5-day transportation of three-dimensional retinal tissue.Additional file 1:Figure S1. Relationship between area and cell number in three-dimensional retinal tissue. X-axis stands for area of organoid (μm2) and y-axis stands for cell number. (TIF 11557 kb)Additional file 2:Figure S2. (A) Quantification of Ki67 in Fig. Additional file 3:Figure S4. Retinal cells were stained by immunofluorescence after 15 days of recovery in three-dimensional retinal tissues. Staining of ganglion cells , photoreceptor precursors , amacrine cells  was presented in different groups. (TIF 11747 kb)Additional file 4:Figure S6. In real transportation, trend of changes was similar to mimic transportation. (A) In the Con group and BAM15 group, morphology of retinal tissue varied slightly before and after transportation. (B-D) Immunofluorescence results presented the staining of Caspase-3 (B), NEFL(C) and Ki67(D) after real transportation. (E-I) Staining of ganglion cells , photoreceptor precursors , amacrine cells  was presented in the Con and BAM15 group. Scale bar = 50 μm (TIF 54539 kb)Additional file 5:Figure S7. The box used in real express transportation (A) and temperature at different time point during real transportation (B). (TIF 2677 kb)Additional file 6:Figure S3. Effects of BAM15 on cell proliferation and differentiation. (A) Outward appearance of retinal tissue with concentration at 20 μmol/L, 50 μmol/L and 100 μmol/L BAM15 in incubator at different time point. (B) BAM15 imposes little effects on cell proliferation in retinal organoid . (C) Quantification of retinal cell marker after transportation in Fig. 2: blank, 7.70 ± 0.23, con, 7.32 ± 0.38, DMSO, 8.50 ± 0.42, BAM15, 7.06 ± 0.50; Hu C/D+ cell/1000 μm2: blank, 6.21 ± 0.78, con, 6.04 ± 0.17, DMSO, 5.58 ± 0.26, BAM15, 6.87 ± 0.19; recoverin+ cell/1000 μm2: blank, 3.26 ± 0.29, con, 3.35 ± 0.29, DMSO, 2.49 ± 0.25, BAM15, 3.25 ± 0.37; OTX+ cell/1000 μm2: blank, 5.24 ± 0.18, con, 5.50 ± 0.25, DMSO, 5.28 ± 0.10, BAM15, 5.32 ± 0.26; AP2α + cell/1000 μm2: blank, 0.63 ± 0.06, con, 0.49 ± 0.04, DMSO, 0.52 ± 0.01, BAM15, 0.59 ± 0.02; PAX+ cell/1000 μm2: blank, 8.03 ± 0.15, con, 8.59 ± 0.46, DMSO, 7.79 ± 0.31, BAM15, 8.46 ± 0.13). (TIF 7512 kb)Additional file 7:Figure S8. Compared with the blank group, BAM15 at 20 μmol/L, 50 μmol/L and 100 μmol/L concentration imposes little effect on NEFL (A) and cleaved caspase-3 (B) expression in the incubator as negative control. (TIF 6721 kb)Additional file 8:Figure S5. (A) Outward appearance of retinal tissue after 5 days of transportation and 15 days of recovery. (B) Cell viability of retinal tissues after 5 days of transportation and 15 days of recovery . (TIF 7675 kb)


The Wnt/β-catenin pathway is involved in the osteogenic differentiation of human adipose-derived stem cells (hASCs) under cyclic strain. Very little is known about the role of microRNAs in these events.Cells were obtained using enzyme digestion methods, and proliferation was detected using Cell Counting Kit 8. Cell cycles and immunophenotypes were detected by flow cytometry. The multilineage potential of hASCs was induced by induction media. Cyclic strain was applied to hASCs  to induce osteogenic differentiation and miRNA changes. Bioinformatic and dual-luciferase analyses confirmed lymphoid enhancer factor 1 (LEF1) as a potential target of let-7i-3p. The effect of let-7i-3p on LEF1 in hASCs transfected with a let-7i-3p mimic and inhibitor was analyzed by immunofluorescence. hASCs were transfected with a let-7i-3p mimic, inhibitor, or small interfering RNA (siRNA) against LEF1 and β-catenin. Quantitative real-time PCR (qPCR) and western blotting were performed to examine the osteogenic markers and Wnt/β-catenin pathway at the mRNA and protein levels, respectively. Immunofluorescence and western blotting were performed to confirm the activation of the Wnt/β-catenin pathway.Flow cytometry showed that 82.12% ± 5.83% of the cells were in G1 phase and 17.88% ± 2.59% of the cells were in S/G2 phase; hASCs were positive for CD29, CD90, and CD105. hASCs could have the potential for osteogenic, chondrogenic, and adipogenic differentiation. MicroRNA screening via microarray showed that let-7i-3p expression was decreased under cyclic strain. Bioinformatic and dual-luciferase analyses confirmed that LEF1 in the Wnt/β-catenin pathway was the target of let-7i-3p. Under cyclic strain, the osteogenic differentiation of hASCs was promoted by overexpression of LEF1and β-catenin and inhibited by overexpression of let-7i-3p. hASCs were transfected with let-7i-3p mimics and inhibitor. Gain- or loss-of-function analyses of let-7i-3p showed that the osteogenic differentiation of hASCs was promoted by decreased let-7i-3p expression and inhibited by increased let-7i-3p expression. Furthermore, high LEF1 expression inactivated the Wnt/β-catenin pathway in let-7i-3p-enhanced hASCs. In contrast, let-7i-3p inhibition activated the Wnt/β-catenin pathway.Let-7i-3p, acting as a negative regulator of the Wnt/β-catenin pathway by targeting LEF1, inhibits the osteogenic differentiation of hASCs under cyclic strain in vitro. Bone tissue engineering can be used to repair bone defects induced by various factors by construction of functional bone tissue in vitro . Seed ceBone marrow mesenchymal stem cells (BMSCs) are currently the most widely used seed cells in the field of bone tissue engineering . HoweverHow to promote the osteogenic differentiation of ASCs in vitro has become an important research area for bone tissue engineering. Mechanical loading plays an essential role in cellular biological responses, such as the regulation of protein synthesis, cell proliferation, and differentiation . Some stThe Wnt signaling pathway is involved in the regulation of many critical biological processes, including embryogenesis and development, stem cell maintenance and differentiation, energy metabolism balance, and tissue stability maintenance. The Wnt/β-catenin pathway plays a complicated role in bone biology. Some syndromes show low bone mass and, consequently, frequent bone fractures caused by loss of functional mutations of the coreceptor LRP5 , while gMicroRNAs (miRNAs) are highly conserved single-stranded noncoding RNA molecules consisting of 18–22 nucleotides. MiRNAs bind to the 3′UTRs of their target mRNAs, inhibiting their translation or promoting their degradation . miR-138In this study, a gene microarray was used to screen the altered expression levels of miRNAs in hASCs during osteogenic differentiation under cyclic strain. We analyzed miRNAs associated with Wnt/β-catenin by bioinformatics methods and found that the target gene regulated by hsa-let-7i-3p is most likely lymphoid enhancer factor 1 (LEF1) in the Wnt/β-catenin pathway. We hypothesize that cyclic strain activates the Wnt/β-catenin pathway by downregulating hsa-let-7i-3p, thereby promoting the osteogenic differentiation of hASCs. This study aimed to validate this hypothesis using cell and molecular biology methods.g for 5 min, after which the supernatant was discarded, and the pellet was resuspended twice. Resuspend cells were cultured in alpha-modified Eagle’s medium  containing 10% fetal bovine serum  at 37 °C in a 5% CO2 incubator. In this study, fourth-passage cells were used in the following experiments.Fat tissue was obtained from 6 donors who underwent flap reconstruction in the Oral and Maxillofacial Surgery Department at the Affiliated Stomatological Hospital of Nanjing Medical University, and all patients signed informed consent. The procedures were approved by the ethics committee of Nanjing Medical University. Adipose tissue was isolated from flaps and then washed three times with phosphate-buffered saline (PBS) to remove red blood cells. The adipose tissue was digested with 0.1% collagenase type I  at 37 °C for 1 h. After tissue digestion, the tissue was centrifuged at 3002 incubator. Cells in 6 wells were measured by adding 10 μl of Cell Counting Kit-8 solution to each well, followed by incubation at 37 °C for 2 h. Absorbance was measured by spectrophotometry  at 450 nm. Cell counting was performed for 8 consecutive days. The growth curve of the cells was drawn according to the above results.Cell proliferation was detected following the manufacturer’s manual by Cell Counting Kit-8 assays  according to the manufacturer’s instructions. Cells were seeded into 96-well plates at 3000 cells per well and then cultured in α-MEM supplemented with 10% FBS at 37 °C in a 5% COCells were collected by centrifugation and fixed with precooled 70% ethanol overnight. Cells were added to 50 μL of RNase stock solution (100 μg/mL), stained with 500 μL of PBS containing 50 μg/mL ethidium bromide (PI), and incubated at 4 °C for 30 min in the dark. Finally, the fluorescence intensity of DNA-PI was detected by a FACSVerse flow cytometer  for analysis of cell cycle changes.Immunophenotyping of the hASCs was performed by staining for 30 min on ice in the dark with the fluorochrome-labeled monoclonal antibodies CD29-APC , CD34-PE , CD45-PE , CD90-FITC , and CD105-PerCP cy5.5 . Untreated cells served as the negative control to adjust for the compensation of fluorochrome overlap. The fluorescence intensity was examined by a FACSVerse flow cytometer , and flow cytometry analysis was performed with FlowJo software 7.6.1 .hASCs were induced toward adipogenic, osteogenic, and chondrogenic differentiation to confirm their multilineage potential. Adipogenic medium  was used to induce adipogenic differentiation of hASCs; after 14 days, hASCs were stained with Oil Red O to determine the formation of lipid droplets. Osteogenic medium  was used to induce osteogenic differentiation of hASCs; 21 days later, hASCs were stained with Alizarin Red to confirm the existence of mineralized nodules. Chondrogenic medium  was used to induce chondrogenic differentiation of hASCs; 14 days later, hASCs were stained with Alcian Blue to verify the existence of proteoglycans.5 cells/ml and incubated in α-MEM containing 10% FBS at 37 °C in a 5% CO2 incubator. Twenty-four hours later, stable adherent cells in the BioFlex® Culture Plates were exposed to uniaxial cyclic strain  by a Flexcell® FX-5000™ Tension System . The control groups were cultured in α-MEM supplemented with 10% FBS at 37 °C in a 5% CO2 incubator for 6 days without cyclic strain loading. On the sixth day, hASCs were collected for real-time quantitative PCR (qPCR) and western blot analyses.hASCs were seeded on silicone rubber BioFlex® Culture Plates  at a density of 1.0 × 10mirVana™ miRNA Isolation Kit  according to the manufacturer’s instructions. Fluorescent labeling using the T4 RNA ligase labeling method was followed by precipitation with absolute ethanol, and the samples were then blown dry. The isolated RNA was dissolved in hybridization solution and incubated overnight at 42 °C. Then, the samples were washed with 0.2% sodium dodecyl sulfate (SDS) and 2 × SSC. After drying, the slides were scanned by the LuxScan 10K-A Dual Laser Scanner .Total RNA was isolated from hASCs by TRIzol Reagent  after cyclic strain for 6 days. miRNAs were isolated from total RNA using an Ambion® http://www.targetscan.org), miRWalk , and miRBase (http://www.mirbase.org).The conservative characteristics of let-7i-3p, the 3′UTR binding site for LEF1, and the targets of let-7i-3p were analyzed by the following three databases: TargetScan (The following genetic research tools were used in this study: LEF1 expression plasmid (EX-LEF1), LEF1 siRNA (siLEF1), negative control expression plasmid (EX-Ctrl), negative control siRNA (siR-Ctrl), let-7i-3p mimic, let-7i-3p inhibitor, negative control of the let-7i-3p mimic (miR-Ctrl mimic) and negative control of the let-7i-3p inhibitor (miR-Ctrl inhibitor), LEF1 3′UTR-wild type (wt-LEF1), and LEF1 3′UTR-mutant (mu-LEF1). These vectors were transfected into fourth-passage hASCs using Lipofectamine™ 2000 Transfection Reagent  according to the manufacturer’s instructions. The let-7i-3p mimic and inhibitor, siLEF1, miR-Ctrl mimic, miR-Ctrl inhibitor, and siR-Ctrl were synthesized by GenePharma Corporation . EX-LEF1 (pEZ-M02 vector), EX-Ctrl (pReceiver-M02 vector), wt-LEF1, and mu-LEF1 were supplied by GeneCopoeia Corporation . ORF sequence information for EX-LEF1 and EX-Ctrl is listed in the supplemental materials. Other sequences transfected in this process are listed in Table To change the expression of β-catenin, we transduced lentiviral particles containing an shRNA targeting β-catenin or an shRNA control  into SKOV3 cells. Lentiviral-transduced SKOV3 cells were selected with puromycin (1.5 μM/mL). Transient transfection was performed using a pool of 4 short interfering RNAs (siRNAs) targeting β-catenin  or individual siRNA sequences  and DreamFECT transfection reagent . The scrambled siRNA pool (Dharmacon) was used as a control.Renilla luciferase reporter gene. The sequences mentioned above are listed in Additional file Renilla luciferase activities 48 h after transfection. The firefly values were normalized to Renilla luciferase.Sequence fragments, such as LEF1 3′UTR-WT and LEF1 3′UTR-Mu, were inserted between the NotI and XhoI cleavage sites of the psiCHECK-2 vector  downstream of the The fourth-passage hASCs transfected with the let-7i-3p mimic and inhibitor were used to detect the effect of let-7i-3p on LEF1 by immunofluorescence. Two days after transfection, hASCs were seeded on coverslips in 9-well plates. After 48 h, hASCs were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. Triton X-100  was added to each well for 20 min at room temperature. After rinsing three times with PBS, hASCs were blocked with goat serum for 2 h. Primary antibodies specific for LEF1  were added to the cells and then incubated overnight at 4 °C. Fluorescent Cy3 secondary antibodies  were added and incubated for 1 h at 37 °C in the dark after rinsing three times with PBS Tween-20. The nucleus was then restained with 4′,6-diamidino-2-phenylindole . Cells were subsequently viewed by fluorescence microscopy .Total RNA was isolated from hASCs by TRIzol Reagent and detected by a NanoVue™ Plus spectrophotometer  to determine purity at 260/280 nm. cDNA was synthesized from RNA using PrimeScript RT Master Mix . PCR was performed on the ABI 7300 Real-Time PCR System . The following markers associated with osteogenesis and the Wnt/β-catenin pathway alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), secreted protein acidic and cysteine-rich (SPARC), LEF1, β-catenin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All primer sequences used in this study are shown in Table g, 20 min). The protein concentrations were determined using a BCA Protein Assay Kit . The protein was boiled at 100 °C for 5 min after the addition of 5 × SDS loading buffer . Protein samples were loaded onto SDS polyacrylamide gel electrophoresis gels and then electrophoresed at 100 V for 1.5 h. Then, the protein blots were transferred onto membranes , which were blocked with TBS containing 5% nonfat milk overnight at 4 °C. Then, the membranes were incubated with primary antibodies specific for ALP , RUNX2 , SPARC , LEF1 , β-catenin , and GAPDH  overnight at 4 °C. Afterward, the membranes were washed three times with TBS-0.05% Tween 20 and incubated with the corresponding secondary antibodies for 1 h at room temperature. Next, ECL solution  was added to the membranes and incubated for 1 min. The protein blots were visualized by exposure to enhanced chemiluminescence reagents . Grayscales on the blots were analyzed by Quantity One software .Adherent hASCs were lysed with cell lysis buffer for western blot and immunoprecipitation (IP) analyses  and Nuclear and Cytoplasmic Protein Extraction Kit  respectively. Then, samples centrifuged at 4 °C  One-way analysis of variance (ANOVA) was used for multiple group comparisons. Two-way ANOVA was used for two-group comparisons. All of the results are presented as the mean ± sem, and statistical significance was set at As shown by a light microscope , the fourth-passage hASCs had a typical fibroblast-like morphology , CD90 (98.43% ± 1.77%), and CD105 (92.21% ± 6.48%) and negative for CD34 (2.39% ± 0.72%) and CD45 (0.97% ± 0.41%) , 2.57 ± 0.09-fold (p = 0.004), and 2.13 ± 0.60-fold (p = 0.018), respectively; LEF1 and β-catenin, which are associated with the Wnt/β-catenin pathway, were increased 1.89 ± 0.45-fold (p = 0.012) and 2.22 ± 0.51-fold (p = 0.017), respectively , 1.39 ± 0.09-fold (p = 0.010), and 1.79 ± 0.21-fold (p = 0.005), respectively; LEF1 and β-catenin levels were increased 1.55 ± 0.39-fold (p = 0.030) and 1.63 ± 0.20-fold (p = 0.004), respectively . Among them, 150 miRNAs exhibited decreased in expression, and 87 miRNAs exhibited increased expression. Some miRNAs associated with Wnt/β-catenin and their fold changes are shown in Fig. The predicted binding sequences of both the 3′UTR in LEF1 and let-7i-3p are highly conserved across species Fig. a, b. Thep = 0.007) compared with that in the control group, which was a significant difference. In the reporter gene system in which the LEF1 mRNA 3′UTR binding sites were mutated, overexpression of let-7i-3p did not affect the reporter gene luciferase activity. Inhibition of let-7i-3p expression induced the luciferase activity of the reporter gene. Compared with that in the control group, the luciferase activity in the let-7i-3p inhibition group was increased 1.58 ± 0.37-fold (p = 0.011), and the difference was significant. Then, combination with the LEF1 mRNA 3′UTR was assessed. In the reporter gene system with a site mutation, inhibition of let-7i-3p expression did not affect the reporter gene luciferase activity. The results are shown in Fig. Overexpression of let-7i-3p significantly inhibited the luciferase activity of pMIR/LEF1-WT reporter genes, and the luciferase activity was decreased by 2.19 ± 0.49-fold (p = 0.013), as shown by qPCR, compared to that in hASCs transfected with the miR-Ctrl mimic. LEF1 mRNA expression in hASCs transfected with the let-7i-3p inhibitor was increased 1.66 ± 0.07-fold (p = 0.002), as shown by qPCR, compared to that in hASCs transfected with the miR-Ctrl inhibitor , as shown by western blot, compared to that in hASCs transfected with the miR-Ctrl mimic. LEF1 protein expression in hASCs transfected with the let-7i-3p inhibitor was increased 1.43 ± 0.10-fold (p = 0.013), as shown by western blot, compared to that in hASCs transfected with the miR-Ctrl inhibitor    Fig. a. Compar01) Fig. a.Fig. 6p < 0.001), 3.38 ± 0.64-fold (p = 0.002), 2.07 ± 0.35-fold (p = 0.008), 4.67 ± 0.47-fold (p = 0.008), and 2.33 ± 0.39-fold (p = 0.023), respectively , 1.65 ± 0.68-fold (p = 0.044), 3.04 ± 1.38-fold (p = 0.039), 3.40 ± 1.18-fold (p = 0.001), and 2.72 ± 0.44-fold (p = 0.003), respectively   , 3.76 ± 2.04-fold (p = 0.021), 1.85 ± 0.49-fold (p = 0.004), 2.64 ± 0.40-fold (p = 0.006), and 4.30 ± 1.12-fold (p = 0.001), respectively , 3.46 ± 0.79-fold (p = 0.009), 2.16 ± 0.02-fold (p = 0.010), 2.52 ± 0.61-fold (p = 0.001), and 4.15 ± 1.72-fold (p = 0.004), respectively   , 1.80 ± 0.15-fold (p = 0.012), 3.07 ± 0.86-fold (p < 0.001), 2.08 ± 0.73-fold (p = 0.018), and 3.60 ± 0.57-fold (p = 0.002), respectively  and 1.74 ± 0.24-fold (p = 0.019), respectively , 2.53 ± 0.49-fold (p = 0.008), 1.83 ± 0.28-fold (p = 0.012), 2.31 ± 1.03-fold (p = 0.046), and 2.86 ± 1.68-fold (p = 0.010), respectively  and 2.97 ± 0.19-fold (p = 0.001), respectively , 2.80 ± 1.62-fold (p = 0.005), 2.06 ± 0.30-fold (p = 0.006), 3.06 ± 0.52-fold (p = 0.029), and 2.75 ± 0.68-fold (p = 0.010), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-LEF1 , 2.03 ± 0.07-fold (p = 0.002), 2.54 ± 0.60-fold (p = 0.004), 2.91 ± 0.61-fold (p = 0.004), and 1.49 ± 0.17-fold (p = 0.022), respectively  and 1.40 ± 0.15-fold (p = 0.017), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-LEF1 , 3.12 ± 1.55-fold (p = 0.002), 2.55 ± 0.25-fold (p = 0.038), 3.92 ± 0.72-fold (p = 0.018), and 2.25 ± 1.24-fold (p = 0.030), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siLEF1 , 1.54 ± 0.17-fold (p = 0.013), 1.71 ± 0.19-fold (p = 0.003), 2.24 ± 0.28-fold (p = 0.001), and 1.55 ± 0.06-fold (p = 0.012), respectively  and 2.13 ± 0.35-fold (p = 0.001), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siLEF1 , 4.27 ± 1.78-fold (p = 0.002), 3.06 ± 0.41-fold (p = 0.001), and 3.17 ± 1.09-fold (p = 0.008), the mRNA expression levels of LEF1 was decreased 1.47 ± 0.21-fold (p = 0.016), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-β-catenin , 2.10 ± 0.03-fold (p = 0.001), 3.16 ± 1.01-fold (p = 0.002), and 1.50 ± 0.04-fold (p = 0.006); the protein expression levels of LEF1 were decreased 1.40 ± 0.13-fold (p = 0.023), respectively  and 2.23 ± 0.27-fold (p = 0.008), respectively, in hASCs cotransfected with the let-7i-3p mimic and EX-β-catenin , 2.63 ± 0.69-fold (p = 0.004), 2.36 ± 0.46-fold (p = 0.006), and 3.75 ± 0.21-fold (p = 0.001); the mRNA expression levels of LEF1 was increased 1.89 ± 0.74-fold (p = 0.009), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siβ-catenin , 2.12 ± 0.14-fold (p = 0.006), 1.40 ± 0.01-fold (p = 0.020), and 1.51 ± 0.17-fold (p = 0.011); the protein expression levels of LEF1 were increased 1.53 ± 0.19-fold (p = 0.006), respectively  and 1.83 ± 0.04-fold (p = 0.004), respectively, in hASCs cotransfected with the let-7i-3p inhibitor and siβ-catenin , is involved in many different types of diseases. Falzone et al. reported that abnormal let-7i-3p expression has the potential to predict recurrence in oral cancer patients . IndersiStudies have been conducted to directly regulate the expression of LEF1 by miRNAs, mainly focusing on tumor diseases. miR-34a increased chemosensitivity in BIU87/ADR cells by targeted inhibition of the TCF1/LEF1 axis . miR-22,Currently, some studies have confirmed that some miRNAs can affect the expression of LEF1 and the Wnt/β-catenin pathway in the process of osteogenesis. Overexpression of miR-539 increased the expression of β-catenin, LEF1, c-myc, cyclin D1, RUNX2, BGP, and BMP-2 in rat osteoblasts . HoweverTo determine the role of let-7i-3p in the process of hASC osteogenic differentiation under cyclic strain, we designed serial experiments to verify its function. Inhibition of let-7i-3p and overexpression of LEF1 promoted the osteogenic differentiation of hASCs in vitro. Furthermore, gain- and loss-of-function experiments on let-7i-3p showed that let-7i-3p negatively regulates the Wnt/β-catenin pathway via LEF1, which inhibits the osteogenic differentiation of hASCs under cyclic strain in vitro Fig. .Fig. 10Additional file 1. The sequences mentioned in the studyAdditional file 2: Figure S1. Microarray for miRNA changes under cyclic strain. There were 150 miRNAs decreased and 87 miRNAs increased significantly (>2-fold).


Phellinus linteus (PL) has been used as a traditional herbal medicine owing to its immune regulatory activity. Previous studies reported that PL grown on germinated brown rice (PBR) exerted immunomodulatory, anticancer, and anti-inflammatory activities. However, role of PBR on type I hypersensitive reactions has not been studied yet. We found that PBR contained more polyphenolic compounds than PL extract. Among fractions, PBR butanol fraction (PBR-BuOH) significantly contained the most amounts of total polyphenolic contents compared with all extracts or fractions. In this study, anti-allergic activity of PBR-BuOH was examined using in vitro and in vivo models of immunoglobulin E/antigen- (IgE/Ag-) stimulated allergy. The inhibitory activity of degranulation was higher in PBR-BuOH (IC50 41.31 ± 0.14 μg/mL) than in PL-BuOH (IC50 108.07 ± 8.98 μg/mL). We observed that PBR-BuOH suppressed calcium influx and the level of TNF-α and IL-4 mRNA expression in a dose-dependent manner. The phosphorylation of Fyn, Gab2, PI3K, Syk, and IκB protein is reduced by PBR-BuOH. Oral administration of PBR-BuOH inhibited allergic reactions including the extravasation of Evans blue dye, ear swelling, and infiltration of immune cells in mice with passive cutaneous anaphylaxis (PCA). These findings suggest that PBR-BuOH might be used as a functional food, a health supplement, or a drug for preventing type I hypersensitive allergic disease. The εRI) –11. The εRI) , corticoεRI) , aspirinεRI) , progestεRI) , and antεRI) . CurrentεRI) . Many reεRI) .Phellinus linteus (PL) has been traditionally used as a natural medicine in Asian countries for immune regulatory activities [Phellinus linteus grown on germinated brown rice (PBR), which is inoculated and cultured the mycelium of PL on germinated brown rice, that is, different from regular PL. In the previous studies, PBR exhibited physiological functions such as anti-inflammatory [εRI-mediated allergic responses. In addition, previously they reported that ergosterol peroxide and atractylenolide III were found in PBR but not in wild PL [β-glucan and γ-aminobutyric acid in PBR was higher than that in regular PL and brown rice [β-glucan and γ-aminobutyric acid inhibited the allergic activity in both IgE-mediated mast cells and type I hypersensitivity animal models [tivities –22. Wildtivities . In thisammatory , 25, antammatory , 27, andammatory . Unlike ammatory . The red wild PL , 30. Theown rice . Ergosteown rice , anti-inown rice , and antown rice . β-glucal models –37. HoweMycelia of PL (voucher number Kucari 0904) produced by solid-state culture and PBR (voucher number Kucari 0905) were kindly provided by the Cell Activation Research Institute  and deposited in the Herbarium at the College of Bioscience and Biotechnology, Konkuk University (Seoul) , 26. EacμL of each extract and fractions of PBR or PL (50 mg/mL) and gallic acid (0–500 μg/mL) was incubated with 20 μL of Folin–Ciocalteu reagent for 5 min. Then, 120 μL of 7% sodium carbonate (Na2CO3) was added and incubated at room temperature (RT) for 30 min. Absorbance was measured at 720 nm using a UV-Visible spectrophotometer . TPC was expressed as mg of gallic acid equivalents (GAEs)/g of dry mass.TPC in each extract was determined using a modified Folin–Ciocalteu method . In each2 [RBL-2H3 cells  were obtained from the American Type Culture Collection (ATCC) Biological Resource Center . Cells were cultured in minimum essential medium  supplemented with 15% fetal bovine serum  and 100 U/mL penicillin-streptomycin (Gibco) and were grown in a 75T cell culture flask at 37°C under humidified air containing 5% CO5 cells/well) were sensitized with 200 ng/mL of dinitrophenyl- (DNP-) specific IgE  overnight. After washing with PIPES buffer , cells were treated with PBR fractions (25 and 50 μg/mL) or PP2 , an Src tyrosine kinase inhibitor [μL of 1 mM p-nitrophenyl-acetyl-β-D-glucosaminide  in 0.1 M citrate buffer  and incubated at 37°C for 2 h. Then, 200 μL of 0.1 M Na2CO3/NaHCO3 solution (pH 10.0) was added to stop the reaction. Degranulation was calculated with the measurement of the released β-hexosaminidase as previously described [RBL-2H3 cells  were sensitized with 200 ng/mL of DNP-specific IgE overnight and then pretreated with PBR-BuOH (25 and 50 μg/mL) for 30 min. RBL-2H3 cells were incubated with 50 μL of Fluo-4 solution  for 30 min at 37°C and then stimulated with 200 ng/mL DNP-BSA (Ag). Images were taken every 5 min on a fluorescent microscope  using filter . Then, the fluorescence intensity was quantitated by Image J software.Intracellular calcium levels were measured as previously described , 40, 41.4 cells/well) were incubated with PBR fractions or PL fraction (25 and 50 μg/mL) at 37°C for 24 h, and then 10 μL of CCK-8 solution was added and incubated at 37°C for 2 h. The absorbance was measured with a microplate reader at 450 nm .Cell viability was determined using the Cell Counting Kit-8 (CCK-8)  as described previously , 40. RBLα was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62.2°C for 10 s, and extension at 72°C for 45 s, with a final extension at 72°C for 5 min. PCR program for IL-4 was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 10 s, and extension at 72°C for 25 s, with a final extension at 72°C for 5 min. PCR program for GAPDH was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62°C for 10 s, and extension at 72°C for 25 s, with a final extension at 72°C for 5 min [ε receptor was performed as follows: initial denaturation at 95°C for 15 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 49°C for 90 s (for FcεRI α-subunit) or annealing at 51.9°C for 90 s (for FcεRI β-subunit), and extension at 72°C for 90 s, with a final extension step at 72°C for 5 min. PCR program for IFN-γ was done as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 57.3°C for 10 s, and extension at 72°C for 25 s, with a final extension at 72°C for 5 min. The following primers were used: TNF-α forward 5′-CAC CAC GCT CTT CTG TCT ACT GAA C-3′; TNF-α reverse 5′-CCG GAC TCC GTG ATG TCT AAG TAC T-3′; IL-4 forward 5′-ACC TTG CTG TCA CCC TGT TC-3′; IL-4 reverse 5′-TTG TGA GCG TGG ACT CAT TC-3′; FcεRI α-subunit forward 5′-AAT GGA TCC ACA ATG ATA GC- 3′; FcεRI α-subunit reverse 5′-AAT GAT GGG AAA ATG AGT TG-3′; FcεRI β-subunit forward 5′- GCA AAA GCT CTA CCA GAG AA-3′; FcεRI β-subunit reverse 5′-CTA CGC TCA AAT TCT TGT CC-3′; IFN-γ forward 5′-CA CAC TGC ATC TTG GCT TTG-3′; IFN-γ reverse 5′-TC CAC ATC TAT GCC ACT TGA G-3′; GAPDH forward 5′-CTT CAC CAC CAT GGA GAA GGC TG-3′; GAPDH reverse 5′-GAC CAC AGT CCA TGC CAT CAC TG-3′ . The PCR product was separated by electrophoresis in 1.5% agarose gels. The bands were analyzed by RT-PCR using LI-COR Odyssey .Reverse transcription PCR was performed as described previously , 40. Totor 5 min . PCR pro6 cells/well) were lysed in RIPA cell lysis buffer, according to the manufacturer's protocol . The protein concentrations were determined using a BCA Protein Assay kit . Equal amounts of proteins were loaded into each well and electrophoretically separated by 7–10% SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes and blocked in 5% nonfat milk. Samples were probed with the following primary antibodies: phosphorylated Fyn , phosphorylated-the adaptor growth-factor-receptor-bound protein 2 (GRB2)-associated binding protein 2 , Gab2 , phosphorylated-phosphoinositide 3-kinase , phosphorylated-Syk , Syk , phosphorylated-IκBα , NFκB , phosphorylated-ERK1/2 , ERK1/2 , and β-actin . The membranes were washed in TBST  buffer  and were incubated with horseradish peroxidase-labeled secondary antibody . The bands were visualized and analyzed using LI-COR Odyssey .Protein analysis was performed as described previously , 31, 40.Six-week-old female BALB/c mice, which are housed in under specific pathogen free conditions, were obtained from Orient Bio . They were maintained in cages at 22 ± 2°C and humidity 55 ± 5% and were exposed to 12 h light/12 h darkness cycles each day. Animals were randomly divided into four groups of six mice and were acclimated to laboratory conditions for 5–7 days. They were fed with standard diet and allowed free access to drinking water. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Gachon University  (GIACUC-R2017014).μg/mL) was injected into the ear of the mice. After 24 h, mice were intravenously injected with 1 mg/mL DNP-BSA (Ag) containing 1% Evans blue dye. To measure the activity of PBR-BuOH (25 mg/kg) or cetirizine , mice were orally administered the abovementioned dosages 1 h before DNP-BSA administration. The dye was extracted from each excised ear tissue with 700 μL formamide at 63°C overnight as previously described [DNP-specific IgE 1  μg/mL waescribed . The absEar samples were fixed using 10% formalin and were embedded in paraffin as previously described . Paraffit-test and one-way ANOVA followed by Dunnett's t-test or Duncan's t-test (p < 0.05). The experimental data were analyzed using the Statistical Package for the Social Sciences-12 .Results are presented as means ± standard deviation (SD). Statistical differences between the control and the treatment groups were determined by Student's ###p < 0.001) was significantly higher than that in PL  was highest among total water extract, hexane, or ethyl acetate fractions /g of dry mass, ractions . These dβ-hexosaminidase release (an index of degranulation) in IgE/Ag-stimulated RBL-2H3 cells. As shown in β-hexosaminidase release in the PBR-BuOH-treated group was inhibited at a concentration of 41.31 ± 0.14 μg/mL . IC50 values suggested that PBR-BuOH suppressed degranulation most efficiently compared to other PBR fractions. In addition, IC50 values of PBR-BuOH treatment (41.31 ± 0.14 μg/mL) are lower than those of PL-BuOH treatment (108.07 ± 8.98 μg/mL). Thus, our data showed that PBR-BuOH significantly inhibited degranulation in IgE/Ag-stimulated RBL-2H3 cells , we evaluated whether they could inhibit H3 cells  and had s  significantly suppressed the level of IgE/Ag-induced TNF-α and IL-4 mRNA expression in a dose-dependent manner  significantly suppressed the level of IgE/Ag-induced FcεRI α-subunit and FcεRI β-subunit mRNA expression in a dose-dependent manner  and downstream molecules  are involved in FcεRI signaling pathways and NF-κB signaling pathway. First, to elucidate the mechanism of its action, we investigated whether PBR-BuOH inhibited phosphorylation of Fyn, Gab2, and PI3K. PBR-BuOH decreased the expression of phosphorylated Fyn, Gab2, and PI3K in a dose-dependent manner compared with the IgE/Ag-stimulated control , DNP-BSA (Ag) containing 1% Evans blue dye was injected through the tail vein . PBR-BuOp < 0.001) or CZ , compared with that in the IgE/Ag-stimulated control group (42.4 ± 14.9)  or CZ (20 mg/kg/day)-treated mice was decreased, compared with that in the mice with IgE/Ag-mediated PCA . The num ± 14.9) . These rPhellinus linteus grown on germinated brown rice (PBR) alleviated IgE/Ag-stimulated hypersensitivity by suppressing FcεRI-mediated pathway along with improvements in antiallergic activity. Recently, several studies reported that PBR showed enhanced biological activities, including immune regulation [α and IL-4) exerted by IgE-mediated allergy-induced basophils [In the present study, we demonstrated that gulation , anti-ingulation , 25, antgulation , 27, andgulation . The levgulation . It is rgulation  and atragulation . Other ggulation , 25. In asophils –48. In tεRI, and IgE/antigen (Ag)-complex [β-hexosaminidase [β-hexosaminidase is one of the lysosomal enzymes, which may be fused with secretory lysosome and endosome in RBL-2H3 cells [β-hexosaminidase in IgE/Ag-stimulated RBL-2H3 cells  and chemokines  are secreted by IgE/Ag-stimulated basophils and mast cells within a few hours [α and IL-4 can be used as fascinating therapeutic target molecules for IgE-mediated allergic responses [α is found in secretory granules and induces endothelial activation, adhesion molecule expression, and inflammatory cell recruitment [α [α and IL-4. We found that PBR-BuOH significantly reduced TNF-α and IL-4 mRNA expression  and the signaling portion (β- and γ-subunits). Some asthmatic patients have more FcεRI-expressed immune cells, compared with non-asthmatic control patients [εRI-expressed immune cells can rapidly respond to small amounts of IgE/antigen (Ag) complex and lead to the allergic reaction by releasing cytokines, chemokines, and other mediators, leading to activation of recruited other immune cells [εRI α-subunit-deficient mice do not induce degranulation because IgE cannot bind to the cell membrane receptor of basophils and mast cells [εRI α-subunit activates the antigen-binding site of IgE molecule, induction of FcεRI α-subunit increases IgE-mediated allergy [εRI α-subunit mRNA in IgE/antigen-stimulated RBL-2H3 cells  the translocation of granules to cell membrane  and (2) εRI , 76–79. εRI , 78, 80.H3 cells  and signH3 cells . Our obstivation , 74, 75.tivation , 41. We reaction –83. Sometivation . As showH3 cells . These din vitro results. In addition, Hong et al. conducted a clinical trial in which PBR powder was orally administered for 12 weeks to an atopic dermatitis patient at an average age of 7.3 years [PCA is a representative local type I allergy that is induced in mice by injecting IgE into the ears and intravenously injecting antigen into the tails , 85, 86..3 years . The sym.3 years .α and IL-4 mRNA expression. PBR inhibited the phosphorylation of Fyn, Gab2, and PI3K in FcεRI-mediated signaling pathway and calcium mobilization. In addition, PBR-BuOH suppressed IgE-mediated type I hypersensitivity in the PCA murine model. Our findings suggest that PBR could be developed as functional food, health supplement, and drug for treating or preventing type I allergic diseases.Our study demonstrated that PBR-BuOH has more potent antiallergy activity than PL-BuOH in IgE/Ag-mediated allergic response through suppressing degranulation and decreasing the level of TNF-


Eukaryotic cells have 2 to 3 discrete nucleoli required for ribosome synthesis. Nucleoli are phase separated nuclear sub-organelles. Here we examined the role of nuclear Lamins and nucleolar factors in modulating the compartmentalization and dynamics of histone 2B (H2B-ECFP) in the nucleolus. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) of labelled H2B, showed that the depletion of Lamin B1, Fibrillarin (FBL) or Nucleostemin (GNL3), enhances H2B-ECFP mobility in the nucleolus. Furthermore, Nucleolin knockdown significantly decreases H2B-ECFP compartmentalization in the nucleolus, while H2B-ECFP residence and mobility in the nucleolus was prolonged upon Nucleolin overexpression. Co-expression of N-terminal and RNA binding domain (RBD) deletion mutants of Nucleolin or inhibiting 45S rRNA synthesis reduces the sequestration of H2B-ECFP in the nucleolus. Taken together, these studies reveal a crucial role of Nucleolin-rRNA complex in modulating the compartmentalization, stability and dynamics of H2B within the nucleolus. The nucleus houses chromatin and several non-membranous nuclear bodies involved in transcription , splicinIn vitro studies implicate Nucleolin as a histone chaperone with FACT-like activity, which regulates SWI-SNF function and ACF chromatin remodelers .,35.34,35)(ii)). This contrasts with Lamin, FBL and GNL3 depletion, which did not alter the extent of H2B-ECFP compartmentalization in the nucleolus . The inucleolus , Fig. S2ucleolus ). Of notucleolus , Fig. S2Since Nucleolin knockdown reduced H2B-ECFP compartmentalization in the nucleolus, we performed the converse experiment of overexpressing Nucleolin. Interestingly, Nucleolin co-expression showed a consistent and enhanced retention of nucleolar H2B-ECFP in ~67% cells (24 h), which declined to ~42% (48 h), and ~24% (72 h) post transfection , black bWe asked if the compartmentalization of H2B-ECFP in the nucleolus, correlates with endogenous levels of Nucleolin across cell lines . ImmunobNucleolin is a histone chaperone and evicts histones from DNA ,39. We m)). Furthermore, H2B-ECFP and NCL-GFP, co-localize in the nucleolus reiterating the association between Nucleolin and H2B-ECFP .10.1080We sought to investigate into the mechanisms of Nucleolin mediated sequestration of H2B-ECFP in the nucleolus. Towards this end, we examined the effect of co-expressing deletion mutants of Nucleolin into DLD1 cells and scored for H2B-ECFP compartments in the nucleolus. We co-expressed H2B-ECFP with (i) full length NCL FL (ii) NCLΔN  (iii) NCLΔRBD (RBD1-4 deleted) and (iv) NCLΔGAR (GAR domain deleted). We observed a comparable localization of NCLΔN in the nucleolus as that of full length NCL, while NCLΔRBD and NCLΔGAR partially mislocalized in the nucleoplasm, consistent with previous studies ) 40–42]–4240–42]Since NCLΔRBD did not enhance nucleolar H2B-ECFP localization, we determined if rRNA was necessary for NCL mediated localization of H2B-ECFP in the nucleolus. We treated DLD1 cells with Actinomycin D (0.05 µg/ml) for 4 hours, which showed a significant decrease in 45S rRNA levels ).We asked if nucleolar localization of H2B-ECFP was affected upon inhibition of rDNA transcription by Act D treatment ). H2B-ECIt is noteworthy that upon Act D treatment, Nucleolin speckles in the nucleoplasm do not colocalize with H2B-ECFP , inset).The nucleolus is a complex milieu of ribosomal DNA, RNA, proteins and non-ribosomal proteins . SequestHistones are hyperdynamic in ES cells which have a relatively open chromatin conformation . HistoneOn the other hand, Nucleostemin is highly expressed and is a marker of cancer stem cells . FurtherNucleolin exhibits a dominant role in sequestering H2B-ECFP into the nucleolus . NucleolNucleolin levels modulate H2B-ECFP retention and dynamics in the nucleolus across cell types  and 5. FWe surmise that the N-terminal domain of Nucleolin rich in acidic amino acid stretches binds to nucleoplasmic H2B-ECFP and transports it to the nucleolus  21,22].,22.21,22While it was previously proposed that overexpressed H2B localizes in the nucleolus, via charge based interactions between the positively charged H2B and the negatively charged nucleic acids within the nucleolar milieu, our studies for the first time unravel a novel Nucleolin guided mechanism that modulates the sequestration, retention and dynamics of H2B in the nucleolus . This imH2B-ECFP , GFP-nuc2. We authenticated cell lines by karyotyping metaphases derived from each of these cell lines. We routinely tested cells in culture and found them free of mycoplasma contamination. Transient transfections were performed using Lipofectamine RNAimax reagent and siRNA (100 nM)  in reduced serum medium OptiMEM  for 6 h, after which cells were transferred to complete medium and incubated for a total duration of 48 h at 37°C. The siRNA oligonucleotide sequences were – FBL: 5ʹ-AGGAGAACAUGAAGCCGCA-3ʹ, FBL Scramble: 5ʹ-GAAGAACGAUCAGGACAAU-3ʹ; GNL3: 5ʹ-GCUUAAAACAAGAACAGAU-3ʹ, 5ʹ-AUGUGGAACCUAUGGAAAA-3ʹ; GNL3 Scramble: 5ʹ-AUAAUCGAACGAUAAGAAC-3ʹ; LMNA/C: 5ʹ-CAGUCUGCUGAGAGGAACA-3ʹ;LMNA/C Scramble: 5ʹ-GGAGGUCGAGCCAAUAUCA-3ʹ; LMNB1: 5ʹ-AGACAAAGAGAGAGAGAUG-3ʹ; LMNB1 Scramble: 5ʹ-GAGGGAAACGUAAAGAAGA-3ʹ; LMNB2: 5ʹ-GAGCAGGAGAUGACGGAGA-3ʹ; LMNB2 Scramble: 5ʹ-GGAAGCGUAGACGGAAGAG-3ʹ. NCL: 5ʹ-UCCAAGGUAACUUUAUUUCUU-3ʹ; NCL Scramble: 5ʹ-GCUAGCUUUAUUCGUAUAUUA-3ʹ, NPM1: 5ʹ-AGATGATGATGATGATGAT-3ʹ. Transient plasmid transfections were performed using Lipofectamine LTX with Plus reagent  and cells were imaged after 24 h of transfection at 37°C. For FRAP analyses upon knockdowns, siRNA and DNA transfections were performed sequentially – siRNA transfection was performed as mentioned previously, while DNA transfections were performed after 24 h and cells were imaged by fluorescence microscopy, after 48h of siRNA transfection at 37°C.DLD1 colorectal adenocarcinoma cells  were grown in RPMI medium , HCT116 colorectal carcinoma and MCF7 breast adenocarcinoma (ATCC) cells were grown in DMEM , CRL1790 normal colon cells were grown in MEM . All media were supplemented with Penicillin (100 units/ml)/Streptomycin (100 μg/ml)  and 10% heat inactivated FBS . Cells were cultured at 37°C in the presence of 5% COhttp://imagej.nih.gov/ij/). The intensity of the band was normalized to the respective loading controls.SDS–PAGE and immunoblotting were performed according to standard protocols. Lysates were prepared in RIPA buffer, protein concentration was estimated using bicinchoninic acid (BCA) kit , resolved on SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes  for 90 min at 90 volts. Membranes were blocked with 5% non-fat dried milk in Tris-buffered saline, 0.1% Tween-20 (TBST) for 1h at Room Temperature (RT). Primary and secondary antibodies were diluted in 0.5% non-fat dried milk in 1X TBST. Primary antibodies: anti-Nucleolin , 1:1000; anti-Actin , 1:400; anti-histone 2B , 1:1000; anti-Fibrillarin , 1:1000; anti-Nucleostemin , 1:2000; anti-Lamin A/C , 1:5000; anti-Lamin B1 , 1:1000; anti-Lamin B2 , 1:400; anti-GFP , 1:1000 for 3h at RT or overnight at 4°C. Secondary antibodies: Sheep anti-Mouse-HRP , 1:10,000; Donkey anti-Rabbit-HRP , 1:10,000, for 1h at RT. Between incubations, membranes were washed three times in 1X TBST for 10 minutes each at RT. Immunoblots were developed using chemiluminescent substrate ECL Prime  and imaged with ImageQuant LAS4000. Relative levels of H2B and Nucleolin were quantified from western blots using Image J (7 cells (DLD1) were lysed in co-IP lysis buffer  vortexed and incubated on ice for 15 min, and centrifuged at 12,000 rpm and 4°C for 10 min. The lysate was precleared by incubating with Dynabeads protein A  for 1 h. Anti-Nucleolin antibody  or normal rabbit IgG was incubated with lysates overnight at 4°C. Protein A beads, pre-blocked with 0.5% BSA, were incubated with the immunocomplex for ~3 h. Beads were washed 6 times with co-IP lysis buffer to minimize non-specific binding. Bound protein was eluted from the beads by boiling in 2× Laemmli buffer for 15 min at 95°C. Samples were resolved on a 15% SDS-PAGE, followed by western blotting.For co-immunoprecipitation (Co-IP) assays, ~10ACTIN served as internal control. The primers used for qRT-PCR were:RNA was prepared by lysing cells in TRIzol (Invitrogen) followed by phenol-chloroform extraction, cDNA was synthesized using the Verso cDNA synthesis kit (Thermo Fisher Scientific). Semi-quantitative real-time PCR (qRT-PCR) was performed using SYBR green (SAF labs). 45S: Forward, 5ʹ-GAACGGTGGTGTGTCGTT-3ʹ; Reverse, 5ʹ-GCGTCTCGTCTCGTCTCACT-3ʹ and ACTIN: Forward, 5ʹ- GATTCCTATGTGGGCGAC-3ʹ, Reverse: 5ʹ-GGTAGTCAGTCAGGTCCCG-3ʹ.2) were briefly washed twice using 1X Phosphate Buffered Saline (PBS) and fixed for 10 min in 4% Paraformaldehyde  in PBS, pH 7.4 at RT, washed thrice in 1X PBS (5 min each), followed by permeabilization in 0.5% Triton-X-100 (in PBS) for 10 min at RT. Cells were blocked in 1% Bovine serum albumin (BSA)  in 1X PBS, for 30 min, washed thrice with 1X PBS and incubated in primary antibodies for 90 min at RT and secondary antibodies for 60 min at RT, with washes in between using 1X PBS. Primary antibody was diluted in 0.5% BSA (prepared in 1X PBS): anti-Nucleolin (ab13541), 1:300, anti-H2B , 1:100. Following secondary antibodies were used after diluting in 1X PBS +0.1%Triton X-100 (PBST): anti-mouse IgG-Alexa 568 (Molecular Probes), 1:1000; anti-rabbit IgG Alexa 488 (Molecular Probes) after which cells were washed thrice in 1X PBST. Cells were mounted in Slowfade Gold Antifade . Cells were imaged on a Zeiss LSM710 confocal microscope with 405 nm, 458 nm and 561 nm laser lines at 2% power using a 63X oil immersion objective, NA 1.4 at 2.5X digital zoom. X-Y resolution: 512 pixels X 512 pixels (1 pixel = 0.11 µm). Confocal z-stacks were collected at an interval of 0.32 µm.Cells grown on coverslips , CO2 independent Leibovitz L-15 medium  supplemented with 10% FBS (complete L15), was used during microscopy. Images were acquired using a 63X oil immersion objective, NA 1.4 at 2.5X digital zoom, at 2% laser power to avoid photobleaching. The acquisition parameters were adjusted to avoid bleed-through of ECFP and GFP fluorescence. A 10 pixel X 10 pixel square (1 pixel = 0.11 µm) Region of Interest (ROI) was bleached in both nucleoplasmic and nucleolar H2B-ECFP. Photobleaching was performed using the 405 nm laser line at 100% power. Laser iterations of 120 and 150 were used to photobleach labelled H2B in the nucleus and nucleolus respectively. Images were collected every 3.87 s for a total duration of 5 min. Images were analyzed using Zen 2011 FRAP Analysis module and normalized fluorescence intensity (NFI) was calculated as follows:A Zeiss LSM710 confocal microscope equipped with a heated stage at 37°C, was used for all photobleaching experiments and fluorescence image acquisitions. For live imaging, cells were grown on a 22 × 22 mmROI1 is the fluorescence intensity of the 10 px X 10 px ROI that is bleached, ROI2 is the whole nucleus fluorescence intensity and ROI3 is the fluorescence intensity of a 10 px X 10 px background region selected outside the nucleus. ROI1(t): post-bleach fluorescence intensity at time t. ROI2(t) and ROI3(t): whole nucleus and background, respectively. ROI1(t = 0): average pre-bleach fluorescence intensity. ROI2(t = 0) and ROI3(t = 0): whole nucleus and background, respectively. The NFI was plotted as a function of time to generate double normalized FRAP curves.where, Mobile fractions of H2B-ECFP were calculated as follows:Ffinal is the NFI at maximum recovery, Fbleach is the NFI at the instant of bleaching and Fpre-bleach is the NFI before bleaching.Where, 2 after which they were transferred to complete L-15 medium and imaged live. Equivalent volumes of dimethyl sulfoxide (DMSO) were used as vehicle controls.Cells transfected for 24 h were treated with 0.05 μg/ml actinomycin D (Act D) in complete medium for 4 h at 37°C with 5% COCells were fixed in 70% ethanol (in 1X PBS) and subjected to RNase treatment and propidium iodide staining for 1 hour at 37°C. Cells were scanned on FACS Calibur (BD Biosciences). Cell cycle analyses was performed by ModFit software.Two-tailed student’s t-test was used to compare the number of cells showing nucleolar H2B-ECFP compartments and mobile fractions of H2B-ECFP, p-value <0.05 was considered significant. Graphs were plotted using GraphPad Prism software.


In the title 1,4-dihydropyridine derivative, the 1,4-dihydropyridine ring makes an angle of 82.19 (13)° with the thiophene ring. In the crystal, N—H⋯O and C—H⋯O hydrogen bonds as well as C—H⋯π interactions link the molecules into a three-dimensional network. 17H21NO4S, the 1,4-dihydropyridine ring has an envelope conformation with the Csp3 atom at the flap. The thiophene ring is nearly perpendicular to the best plane through the 1,4-dihydropyridine ring, the dihedral angle being 82.19 (13)°. In the crystal, chains running along the b-axis direction are formed through N—H⋯O interactions between the 1,4-dihydropyridine N atom and one of the O atoms of the ester groups. Neighbouring chains are linked by C—H⋯O and C—H⋯π interactions. A Hirshfeld surface analysis shows that the most prominent contributuion to the surface contacts are H⋯H contacts (55.1%).In the title compound, C The best plane through the 1,4-dihydropyridine ring makes an angle of 82.19 (13)° with the plane through the thiophene ring . Both methyl C atoms are closer to the best plane through the 1,4-dihydropyridine  than the C atoms of the two ester substituents . All four of these C atoms are at the opposite sides with respect to the thiophene substituent which is in an axial position. Atoms O15, O19 and O20 are involved in intramolecular short contacts  x, y + 1, z]. The associated two-dimensional fingerprint plots . The contribution (16.4%) from the O⋯H/H⋯O contacts shows a pair of sharp spikes corresponding to the N—H⋯O interactions . In addition, C⋯H/H⋯C and S⋯H/H⋯S contacts contribute 15.7 and 9.6%, respectively, to the Hirshfeld surface. A further small contribution is from N⋯H/H⋯N contacts . The percentage contributions of the other contact types are negligible.In order to gain further insight into the packing, the Hirshfeld surface and fingerprint plots were calculated using ns Fig. 5c. In adet al.  refers to the multiple S⋯H contacts between S1 and neighbouring methyl groups (S⋯H distances ranging from 3.01 to 3.50 Å). However, the high enrichment ratio ENH must be interpreted with caution as it results from the quotient of two small numbers , the s-cis/s-cis (31%) and the s-cis/s-trans conformation (29%). The s-trans/s-trans conformation occurs only for 5% of the derivatives. In the title compound, both C=O substituents are present in an s-trans/s-cis conformation.A search of the Cambridge Structural Database -1,4-dihydropyridine-3,5-dicarboxylate:4OAc (3 mmol) in ethanol (10 mL) was exposed to microwave radiation for 3 min. at a power of 450W. The reaction mixture was cooled down and the solid product was separated by filtration and purified by recrystallization in ethanol to give the compound as yellowish transparent crystals (yield 82%), m.p. 423 K. IR : 3346, 3244 (NH), 3099, 2979 (C–H), 1699 (C=O), 1490 (C=C). 1H NMR : 19.4 , 14.3 , 34.6 (C6), 59.7 , 103.4 , 120.3 (C2), 124.6 (C4), 127.6 (C5), 144.4 (C3), 147.9 , 167.7 . Calculated for C17H21NO4S: M[+H] = 335.4 au.A mixture of thiophene-3-carbaldehyde (3 mmol), ethyl acetoacetate (6 mmol) and NHUiso(H) values of 1.2Ueq or 1.5Ueq of the parent atoms, with C—H distances of 0.93 (aromatic), 0.98 (CH), 0.97 (CH2) and 0.96 Å (CH3). In the final cycles of refinement, four outlying reflections were omitted.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019015081/sj5585sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989019015081/sj5585Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019015081/sj5585Isup3.cmlSupporting information file. DOI: 1964486, 1964486CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: