Saccharomyces cerevisiae Rsm22 are reported, a low-resolution SAXS structure of the protein is revealed, and SAM-dependent RNA methyl transferase activity of the protein is demonstrated.Rsm22-family proteins are conserved putative SAM-dependent methyltransferases with important functions in mitochondrial translation. Here, the results of a comparative bioinformatics analysis of Rsm22-type proteins are presented, the expression, biophysical characterization and crystallization of  Saccharomyces cerevisiae Rsm22 protein (Sc-Rsm22), encoded by the nuclear RSM22 (systematic name YKL155c) gene, is a distant homologue of Rsm22 from Trypanosoma brucei (Tb-Rsm22) and METTL17 from mouse (Mm-METTL17). All three proteins have been shown to be associated with mitochondrial gene expression, and Sc-Rsm22 has been documented to be essential for mitochondrial respiration. The Sc-Rsm22 protein comprises a polypeptide of molecular weight 72.2 kDa that is predicted to harbor an N-terminal mitochondrial targeting sequence. The precise physiological function of Rsm22-family proteins is unknown, and no structural information has been available for Sc-Rsm22 to date. In this study, Sc-Rsm22 was expressed and purified in monomeric and dimeric forms, their folding was confirmed by circular-dichroism analyses and their low-resolution structures were determined using a small-angle X-ray scattering (SAXS) approach. The solution structure of the monomeric form of Sc-Rsm22 revealed an elongated three-domain arrangement, which differs from the shape of Tb-Rsm22 in its complex with the mitochondrial small ribosomal subunit in T. brucei (PDB entry 6sg9). A bioinformatic analysis revealed that the core domain in the middle (Leu117–Asp462 in Sc-Rsm22) resembles the corresponding region in Tb-Rsm22, including a Rossmann-like methyltransferase fold followed by a zinc-finger-like structure. The latter structure is not present in this position in other methyltransferases and is therefore a unique structural motif for this family. The first half of the C-terminal domain is likely to form an OB-fold, which is typically found in RNA-binding proteins and is also seen in the Tb-Rsm22 structure. In contrast, the N-terminal domain of Sc-Rsm22 is predicted to be fully α-helical and shares no sequence similarity with other family members. Functional studies demonstrated that the monomeric variant of Sc-Rsm22 methylates mitochondrial tRNAs in vitro. These data suggest that Sc-Rsm22 is a new and unique member of the RNA methyltransferases that is important for mitochondrial protein synthesis.The  In order to carry out OXPHOS, mitochondria must maintain a functional respiratory chain (Murphy Saccharomyces cerevisiae Rsm22 (Sc-Rsm22) is a nuclear-encoded protein localized to the mitochondria. Our group has found the S. cerevisiaeRSM22 gene to genetically interact with mitochondrial fatty-acid synthesis (mtFAS), a process that has been suggested to coordinate mitochondrial biogenesis with acetyl-CoA availability , which is a distant homologue of Sc-Rsm22 -dependent methyltransferase (SAM-MTase) protein family  region, which is important for SAM binding. The substrate of Sc-Rsm22 has not been reported. According to previous bioinformatics studies of the yeast ‘methyltransferome’, Sc-Rsm22 was initially predicted to be a protein MTase  studies shows Sc-Rsm22 as an elongated monomer which can also further dimerize to form a homodimer. The possible structure and key residues for substrate recognition by a proposed OB-fold are also discussed. Our 2.2.1.S. cerevisiae strain W1536 5B was isolated using the protocol developed by Hoffman & Winston  and 0.3 g acid-washed glass beads (0.45–0.52 nm diameter) were added in the last step and vortexed thoroughly for 3–4 min. 0.3 ml Tris–EDTA (TE) was added to the mixture, which was centrifuged at 17 900g for 5 min. The aqueous layer (containing the DNA) was transferred to a new Eppendorf tube and 1.0 ml 100% ethanol was added. The tube contents were mixed by inverting and then centrifuged at 17 900g for 2 min. The supernatant was discarded and the pellet was resuspended in 0.4 ml TE plus 30 µg RNase. The solution was incubated for 5 min at 37°C and 10 µl 4 M ammonium acetate and 1 ml 100% ethanol were then added; the tube contents were mixed by inverting and centrifuged at 17 900g for 2 min. The pellet containing the DNA was resuspended in 50 µl TE.The genomic DNA of ston 1987. Yeast c2.2.RSM22 open reading frame encodes 628 amino-acid residues. Analysis of the Sc-Rsm22 protein sequence using MitoProt II and Target P 1.1  using forward and reverse primers introducing an NcoI site and an XhoI site, respectively, for cloning into the NcoI/XhoI sites of vector pET-23d (Novagen/EMD Biosciences). Primer sequences are listed in Supplementary Table S1. Use of the NcoI site introduces an ATG start codon followed by a G. To complete the codon and ensure that the RSM22 ORF was translated in frame, one C and one T residue were added. Hence, the first codon following the translation start site is GCT, encoding alanine. The vector introduces six histidines at the C-terminus of Sc-Rsm22, followed by a stop codon. Total DNA isolated from the W1536 5B yeast strain . For expression of the Sc-Rsm22 protein, the transformed cells were precultured in 10 ml LB medium supplemented with 100 mg l−1 ampicillin at 37°C overnight. The next day, 5 ml of the preculture was added to 1 l autoinduction medium  supplemented with 100 mg l−1 ampicillin. The cells were grown at 37°C until the OD600 reached 1.5; the culture was then switched to a lower temperature (18°C) and incubation was continued overnight for overexpression in autoinduction medium. After overexpression, the cells were harvested using a JS4.2 rotor , after which the supernatant was discarded and the cells were resuspended in lysis solution  with one EDTA-free protease-inhibitor tablet (Roche) per 50 ml. 5 mM MgCl2, 0.1 mg ml−1 lysozyme and 0.025 mg ml−1 DNase I  were added and incubated at room temperature for 20 min. Finally, cell lysis (50 ml) was performed by sonication with a digital sonifier (Branson) on ice (15% amplitude for 3 min using cycles of 15 s on and 30 s off to avoid overheating the sample). The total cell lysate was then subjected to centrifugation for 50 min at 15 000 rev min−1 using an SS-34 rotor at 4°C. The cell pellet and supernatant were separated. 5 mM imidazole  was added to the supernatant followed by filtration with a 0.45 µm filter. An Ni–NTA immobilized metal-affinity chromatography (IMAC) column was prepared with 2 ml 50% Ni–NTA agarose (Qiagen). The matrix was washed with 20 ml distilled water followed by equilibration with 10 ml initial wash solution . Filtrated protein solution was applied onto the equilibrated column. The column was consecutively washed with 30 ml wash solution with increasing imidazole concentrations  and each flowthrough fraction was collected. Finally, the protein was eluted three times using 3 ml elution solution .The pET-23d-M Tris pH 7.5, 500 mM NaCl, 5% glycerol, which was the optimal buffer based on our dialysis and dynamic light-scattering (DLS) experiments  was slowly added to the eluted protein and the solution was stirred at 4°C for 30 min. The precipitated protein was collected by centrifugation and dialyzed against 40 mSupplementary Table S2). Soluble Sc-Rsm22 was found in one condition , which was the initial purification condition. Further DLS experiments were performed to test additional buffer conditions (Supplementary Table S3). All of the conditions showed aggregation of the protein except for the original purification condition. We thus excluded cation-exchange chromatography as a possible additional purification step and continued the purification of Sc-Rsm22 by SEC in 40 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol after IMAC.An attempt to use cation-exchange chromatography as an additional purification step was unsuccessful. This step required the salt concentration of the solvent to be changed. We tested the solubility of the protein at different pH values and salt concentrations. We noticed precipitation and aggregation of the purified protein during the change from the initial solvent to another solvent with low salt concentration. Overnight dialysis of Sc-Rsm22 was performed for buffer exchange. We tested four buffer conditions varying in pH value and salt concentration .2.5.Bruker BioTools (Bruker) and Mascot (Matrix Science). All of the MS experiments and analysis were performed in the Biocenter Oulu Proteomics and Protein Analysis core facility.MS was used to confirm the identity of the purified protein as Sc-Rsm22. After IMAC and SEC, the solution containing the purified protein was subjected to SDS–PAGE and stained with Coomassie Blue. Protein bands were cut out of the gel, washed with water and treated with trypsin overnight. After the tryptic digestion, 2% trifluoroacetic acid was added, mixed and run on an ultrafleXtreme MALDI TOF-TOF mass spectrometer (Bruker). The data were analyzed using 2.6.M Tris pH 7.5, 500 mM NaCl, 5% glycerol. Overnight dialysis of purified Rsm22 was carried out with four buffer conditions (Supplementary Table S2) at 4°C. Soluble protein was only found in 40 mM Tris pH 7 5, 500 mM NaCl, 5% glycerol after dialysis. Dialyzed protein was concentrated and used in DLS experiments. Buffer solutions were prepared using a Freedom Evo pipetting robot (Tecan) for DLS experiments (Supplementary Table S3). 100 µl 0.1 mg ml−1 protein samples were used in 96-well plates. DLS experiments were perfomed using a DynaPro Plate Reader II and DYNAMICS version 7.1.7.16 (Wyatt Technology). Experiments were performed at 25°C. 20 acquisitions and images were collected.Sc-Rsm22 was purified in 40 m2.7.M Tris pH 7.5, 500 mM NaCl, 5% glycerol (filtered with 0.1 µm filters and degassed). A Superdex 200 10/300 GL column connected to an ÄKTApurifier system was pre-equilibrated and used in the MALS procedure with a miniDAWN TREOS MALS device (Wyatt Technologies). The protein samples were passed through a refractive-index detector  before entering the MALS detector. Data analysis was carried out using the ASTRA software (Wyatt Technologies).MALS experiments, online with SEC (SEC-MALS), were carried out to determine the molecular masses of the purified Sc-Rsm22 oligomers. Both Sc-Rsm22 peaks obtained from the SEC run were pooled and concentrated and were subjected to SEC-MALS analysis in 40 m2.8.−1. Sc-Rsm22 was purified in 40 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol. The solvent composition in the purified protein sample was not optimal for CD measurements owing to the presence of a very high salt concentration (500 mM NaCl). The protein samples and solvent (blank) were diluted just before the CD measurements to avoid high salt concentrations in the CD measurements. The final solvent composition used in the experiment was 2.28 mM Tris pH 7.5, 28.5 mM NaCl, 0.28% glycerol. The pH of the solvent was checked after dilution. CD spectroscopy was performed using a Chirascan CD spectrometer . CD data were collected between 280 and 190 nm at 22°C using a 0.1 cm path-length quartz cuvette. CD measurements were acquired every 1 nm with a 0.5 s integration time and were repeated three times with baseline correction. Dimeric Sc-Rsm22 was stable during data collection. We used the average of three repetitions in the analysis. However, monomeric Sc-Rsm22 showed instability after dilution. Here, we did not consider the average of the three data sets, as they deviated from each other. We used the first set of data for analysis. We then waited for 30 min and found that the diluted monomeric Sc-Rsm22 became stable. We collected ten data sets for monomeric Sc-Rsm22 and used the final data for analysis. The data were processed, including baseline subtraction, using Chirascan Pro-Data Viewer (Applied Photophysics) and CDNN . The direct CD measurements  were converted to mean residue molar ellipticities ([θ]MR) by Chirascan Pro-Data Viewer. Thermal unfolding was recorded between 190 and 280 nm with a 2°C step size (from 22 to 92°C) at a 1°C min−1 ramp rate with ±0.2°C tolerance. The melting temperature was analyzed with Global 3 (Applied Photophysics) using all data recorded. The experiment was performed in the Biocenter Oulu Proteomics and Protein Analysis core facility.CD experiments were performed to elucidate the secondary structure and determine the thermal stability of both the monomeric and dimeric forms of Sc-Rsm22. The protein concentration of the sample was 0.1 mg ml2.9.M Tris pH 7.5, 500 mM NaCl, 5% glycerol, 2.5 mM DTT for SAXS studies. Monomeric (2.9 mg ml−1) and dimeric (5 mg ml−1) Sc-Rsm22 were used separately in batch-mode experiments on the B21 beamline at Diamond Light Source (DLS), UK. Scattered X-rays at a wavelength of 1 Å (at 12.4 keV) were recorded with a PILATUS 2M detector. Buffer scattering was subtracted from the protein scattering using SCÅTTER  were estimated by SCÅTTER and PRIMUS in the GNOM package ; the two monomer ab initio shapes were superimposed on the dimer ab initio shape by SUPCOMB . Porod volumes were calculated using q-range values (7.5/Rg) with PRIMUS. The molecular mass of the protein in solution (from both batch and online SAXS experiments) was estimated using the protein volume from the Porod invariant (MMqp), the SAXMoW tool (MoW), the empirical volume of correlation (Vc) and the size and shape methods implemented in DATTOOLS from the ATSAS package (PRIMUS) as described previously . Online SAXS was carried out with a Superdex 200 5/150 column  and 40 mM Tris pH 7.5, 500 mM NaCl, 5% glycerol, 2.4 mM DTT solution on the BM29 beamline at ESRF. The collected data were processed similarly as in the batch-mode SAXS analysis.Additionally, SEC-SAXS (online SAXS) experiments were performed using purified (mixture of dimeric and monomeric) Sc-Rsm22  was used as the atomic model for Sc-Rsm22. This initial model was further improved using results from analysis using the Phyre2 server .The initial amino-acid sequence alignment of four Rsm22-family proteins  and the monomeric atomic model of Sc-Rsm22 was generated using 2.11.in vitro methyltransferase assay was carried out using a modification of the protocol of Lee et al. (2007in vitro transcribed and purified for this experiment (see Sections S1–S6). Purified synthetic tRNAs were heated for 5 min at 70°C and cooled slowly to room temperature. 100 µl 0.5 mMS-adenosyl-l-(methyl-3H)-methionine stock solution was prepared by adding 2.5 µl 20 mM SAM, 10 µl 50 mM H2SO4 and 30 µl S-adenosyl-l-(methyl-3H)-methionine (320 Ci mol−1). Reactions were assembled on ice in 1.5 ml microcentrifuge tubes in a total volume of 30 µl. The reaction consisted of 20 µg monomeric Sc-Rsm22, varying amounts of tRNA depending on the experiment, 0.1 mMS-adenosyl-l-(methyl-3H)-methionine [specific activity 5.5 × 108  disintegrations per minute (dpm) per micromole] and reaction buffer . Three negative controls were used . The reaction was carried out at 37°C for different time lengths depending on the experiment . The time taken to generate data for each reaction mixture was 10 min.An  al. 2007. Yeast mAdditionally, the activity of dimeric Sc-Rsm22 was also tested using a similar method as for monomeric Sc-Rsm22.3.3.1.a and 1b). We undertook detailed bioinformatic studies using various in silico tools. Carrying out comparative analyses employing several SAM-MTase family member homologues, we have generated an Sc-Rsm22 model and identified the key secondary-structure elements of the SAM-MT fold . The SAM-MTase fold is likely to start at position Met126 (the proposed starting position of α-helix αZ) and end at residue Asp462 (the end of β-strand β7) . The conserved glycine-rich region at the end of β-strand β1 has the sequence Val-Gly168-Tyr-Gly170-Pro-Ala172 in Sc-Rsm22, so the last glycine of the Gly-X-Gly-X-Gly motif is replaced by an alanine in this protein. Between β-strands β3 and β4 and between α-helix αE and β-strand β6, the Sc-Rsm22 protein also has several amino-acid sequence extensions exceeding 30 amino acids, which are very likely to be loop regions and are not present in other Rsm22-family members . In contrast, Tb-Rsm22 has a long extension after α-helix αA which is not seen in the sequences of the Sc-Rsm22 and METTL17 proteins .Sc-Rsm22 is a 628-amino-acid protein with a molecular mass of 72.2 kDa. The protein is highly alkaline, exhibiting a calculated pI of 9.9. Our bioinformatic analysis reveals that Sc-Rsm22 shows 18% sequence identity to Tb-Rsm22 and 16.5% to Mm-METTL17, and it can be concluded that these three enzyme homologues form an Rsm22 protein subfamily in the class I SAM-MTase family of proteins with a central SAM-binding core fold  Fig. 1b. The crs Fig. 1b. In cons Fig. 1b.X-Ala-Pro-Cys-His-X-X-Cys-Pro-X coordinate the zinc ion together with a conserved cysteine at the end of the zinc-finger loop structure . In Tb-Rsm22 a fourth cysteine (Cys837) from the antiparallel β-sheet structure of the C-terminal domain also coordinates the bound zinc . It is strikingly shorter than the C-terminal domain of Tb-Rsm22 (which is 164 residues longer), but is 77 and 98 residues longer than those of Mm-METTL17 and Hs-METTL17, respectively. Because of these large differences in the lengths of the C-terminal regions, the standard sequence-alignment algorithm was unable to detect any significant sequence conservation. However, more careful interpretation of the sequences and comparison of the predicted secondary-structure elements of Sc-Rsm22 with the known structure of Tb-Rsm22 revealed a notable sequence similarity in the region Thr490–Lys530 corresponding to the region Ala815–Asp855 in Tb-Rsm22 . This region interacts closely with the rRNA in the T. brucei mitoribosome complex, and the topology of this β-sheet structure found in Tb-Rsm22 strikingly resembles the so-called OB-fold, which is a structural motif that is frequently used for nucleic acid recognition . The secondary-structure prediction suggests that the OB-fold is followed by three C-terminal α-helices, and the first two helices form a coiled-coil motif according to COILS .The C-terminal domain of Sc-Rsm22 (Asp462–Lys628) is predicted to contain both α-helices and β-strands Fig. 1b. It is22 Fig. 1b. This b). It is much shorter compared with that of Tb-Rsm22, with a length of 250 residues, but is around the same size in METTL17 proteins . Interestingly, the mitochondrial isoform of Hs-METTL17 is completely devoid of the N-terminal domain . Also, the sequence similarity is very limited within these three distant homologues. The N-terminus of Sc-Rsm22 shows the highest sequence similarity (33% with only 17% confidence) to the N-terminal region of the TrmB protein, which is a sugar-sensing transcriptional regulator from Pyrococcus furiosus . The structures and functions of these proteins have not been characterized.The N-terminal region of Sc-Rsm22 (residues 1–125) is also predicted to be α-helical in our bioinformatic studies Fig. 1b. It isns Fig. 1b. Interin Fig. 1a. Also,3.2.6-tagged Sc-Rsm22  was expressed in E. coli by autoinduction as described in Section 2a). The higher oligomeric form of Sc-Rsm22 eluted at a volume of 10 ml , whereas the lower oligomeric Sc-Rsm22 variant eluted at a volume of 15 ml, from a 24 ml SEC column. The purified protein fractions were analyzed by SDS–PAGE , and further with MS, which showed that the major band on SDS–PAGE (around 72 kDa) corresponded to Sc-Rsm22 carrying the C-terminal His tag. The additional band which was still present in the sample after the SEC step  was identified to be a degradation product of Sc-Rsm22. Attempts at further purification using ion-exchange chromatography were unsuccessful . The variant with the longer elution time was confirmed to be a monomer with a determined molecular weight (MW) of 72 kDa, whereas the MALS MW of the other variant corresponded to a dimeric protein (MW of 152 kDa) . Both peaks were homogeneous according to the MW distribution pattern . The light-scattering signal revealed that both samples also contained aggregated protein, as well as a soluble protein peak with a MW of around 247 kDa. However, these peaks were not visible in the UV curves , indicating that the percentages of these variants in the protein samples were very low.To assess the homogeneity of the protein preparations, both oligomeric forms of Sc-Rsm22 were individually subjected to SEC-MALS experiments (a). The spectra of the monomeric form showed a somewhat higher calculated helical content (34.9%) compared with the dimeric form, where the calculated helical content was 27.2%. The dimeric form was also more stable than the monomeric form; the thermal stability (Tm) values were 48 and 39°C, respectively . Raw data of thermal unfolding (data from 80 to 92°C are not shown as they are not significant) are presented in Supplementary Fig. S3. We observed a change in the structural fold of the Sc-Rsm22 monomer in solvent containing a lower salt concentration (28.5 mM NaCl) from that present in the purification solvent. This change was rapid at the beginning of the solvent change from higher salt concentration (500 mM NaCl) to lower salt concentration and stabilized within 30 min. We initially collected CD spectra using monomeric Sc-Rsm22 for three repetitions, but did not generate an average of these repetitions as the data deviated from each other. We used the first set of data for CD analysis . In addition, a second measurement of CD spectra after 30 min showed that the change was directing Sc-Rsm22 dimer formation from Sc-Rsm22 monomers (Supplementary Table S4). We collected CD spectra with ten repetitions after 30 min of dilution and observed that the protein became stable . A second CD analysis of the Sc-Rsm22 monomer was also performed using the last set of data (Supplementary Table S4). However, unlike those of the Sc-Rsm22 monomer, the CD spectra of the Sc-Rsm22 dimer were stable at low salt concentration . The CD spectra of the Sc-Rsm22 dimer shown in Fig. 3a) (Sc-Rsm22 dimer in gray) are the average of three independent data sets.Both variants of the full-length Sc-Rsm22 were well folded according to CD analysis Fig. 3a. The sis Fig. 3a. In ad3.4.ab initio shapes generated for both forms and SAXS analysis are shown in Fig. 4a). The pair distribution curves of the Sc-Rsm22 monomer and dimer suggested Dmax values of 138 and 181 Å for the monomer and dimer, respectively . The ab initio shapes of the Sc-Rsm22 dimer and monomer are shown in Figs. 4c) and 4d), respectively. We estimated the Porod volume for the monomer as 160 000 Å−3 (data used 1–367). In this representation, the monomeric protein displays an elongated shape with a large globular domain in the center and two smaller globular bulges at each end . The dimeric form, with a Porod volume of 521 000 Å−3 (data used 1–290), appears as an antiparallel association of the monomers, with a larger central bulge and smaller bulges at each end of the structure. Unlike the MALS data, the molecular mass determined for the dimer appears to be exaggerated in the SAXS analysis . The dimer is depicted by a green surface, while the two monomers are shown as red and salmon surfaces. We also carried out SEC-SAXS (online SAXS) experiments. The parameters calculated for monomeric Sc-Rsm22 from both the batch and online experiments are nearly the same (Table 1−3) in online experiments compared with batch experiments. Like the batch-mode experiments, superimposition of the ab initio models of two copies of monomeric Sc-Rsm22 on one copy of dimeric Sc-Rsm22 shows a good fit . The SAXS parameters calculated for the Sc-Rsm22 dimer and monomer from both batch and online experiments are provided in Table 1Sc-Rsm22 exhibits two distinct forms under the conditions that we investigated. We performed SAXS studies on the dimeric and monomeric variants in batch mode. The ly Fig. 4b. The and Fig. 4d. The ds Table 1. Howeverch Fig. 4e. The de Table 1. However3.5.S-adenosyl-l-(methyl-3H)-methionine]-dependent methyltransferase assay was performed in vitro to test the methyltransferase activity of Sc-Rsm22. Because yeast mitochondrial tRNAs are heavily methylated, whereas the rRNAs are not, we only concentrated on studying the methylation of various tRNA molecules in this study. We used 38.8 pmol of different mt-tRNAs as substrates for Sc-Rsm22, with a mixture of radioactively labeled and cold SAM as the methyl-group donor in the reactions. The amount of methyl groups transferred to the substrates was determined by measuring the radioactivity of the samples with a scintillation counter  after completion of the reaction followed by trichloroacetic acid precipitation. The radioactivity was measured in the form of disintegrations per minute (dpm). A total of 18 Mt-tRNAs were tested as substrates and it was found that all of them incorporated the radioactive label from the substrate in the presence of monomeric Sc-Rsm22. However, we were unable to detect any activity for the dimeric variant of Sc-Rsm22. The obtained dpm values for different mt-tRNAs, indicating the incorporation of tritiated methyl groups, are presented in Supplementary Table S7. The data show that the values are between 500 and 850 dpm on average after subtracting the negative control value from each sample, with no clear indication of tRNA substrate preference (Supplementary Table S7). Data for a minimum of three and a maximum of four experiments were obtained for each tRNA.A SAM [Met as an example for the enzymatic assays of tRNA methylation in vitro that we carried out. We included multiple negative control reactions to ensure the specificity of Rsm22-dependent incorporation of methyl group(s) into this tRNA. The control reaction was carried out without enzyme and with RNase-treated sample. It is seen that the dpm values for the different negative controls range between 47 and 145.Fig. 5Met as a substrate was studied by varying the amount of substrate and the reaction time. The substrate concentration-dependent methyltransferase activities measured for mt-tRNAMet are shown in a)Supplementary Fig. S5(. The methyl-group incorporation increased linearly with the increase in substrate (mt-tRNAMet) in the range 19.3–57.96 pmol. The R2 value is 0.93. For the reaction time-dependent experiments, the concentrations of all of the reagents, including substrates (32.2 pmol mt-tRNAMet), and enzyme were unchanged. We collected data at different time points  and the incorporation of methyl groups increased with time up to the 30 min mark, after which the reaction appeared to reach a steady state. This indicates that the reaction becomes saturated after about 30 min  and is consistent with an enzyme-catalyzed reaction.In addition, the methyltransferase activity of Sc-Rsm22 using mt-tRNA4.E. coli, determine a solution structure of the protein and demonstrate its enzymatic function. Sc-Rsm22 is a conserved nuclear-encoded mitochondria-localized protein in S. cerevisiae that has been shown to physically interact with the small subunit of the mitochondrial ribosome . The MTase domain in Sc-Rsm22 is compact according to the SAXS structure and does not contain any elongated loop regions as seen in the Tb-Rsm22 structure . In general, SAM-MTase structures bind their substrate with the loops between β4 and αD, β5 and αE, and β6 and β7 . Interestingly, this region does not correspond to the portion of Tb-Rsm22 interacting with the rRNA in the T. brucei mitoribosome complex  of this fold, including residues Thr490–Gly542. The corresponding motif is highly similar in all Rsm22-family members  and makes several protein–rRNA interactions in the Tb-Rsm22 structure. In Sc-Rsm22, this region includes ten positively charged residues, which are potential candidates for RNA binding. The most likely candidates for such an interaction are Lys503, Arg504, Lys505, Lys521, Arg527, Arg538 and Lys539. AspRS also interacts with the tRNA molecule (with the anticodon bases) via the OB-domain . It is noteworthy that Glu520, a previously reported point mutation (Glu520Gly) of which in Sc-Rsm22 in combination with compromised mtFAS causes a respiratory deficiency phenotype in yeast , DLS and CD  techniques. Our bioinformatic analyses predicted 232 residues to be present in helical structure in Sc-Rsm22, which is 37% of the total residues. Monomeric Sc-Rsm22 showed a similar helical content in CD analyses. A question of the physiological form of Sc-Rsm22 thus arises. The data presented here, including our functional analyses, indicate that the monomeric variant is likely to be the physiologically relevant form of the protein. However, we cannot completely exclude the possibility of an additional physiological function of the dimeric variant of Sc-Rsm22, as MALS and SAXS analyses as well as thermal unfolding data suggest the formation of dimeric Sc-Rsm22.The recombinantly produced Sc-Rsm22 forms both monomers and dimers, which it was possible to separate by SEC Fig. 2 and charet al., 2011et al., 2014in vivo, but may simply reflect that we have measured enzymatic activity removed from the small ribosomal subunit context. The question of the physiological RNA substrate is therefore not completely settled. We do not favor the idea of rRNA as the substrate, because previous studies demonstrated that Sc-Rsm22 is a small mitoribosomal subunit, and small ribosomal subunit rRNAs in yeast mitochondria are not methylated  an N-terminal α-helical domain with unknown function, (ii) a central MTase core responsible for the methyltransferase activity and (iii) a C-terminal domain including an OB-fold that is responsible for binding the RNA substrate, probably tRNA, extended by an α-helical extension which may form a coiled-coil structure. However, many open questions still remain concerning the structure and function of this mitochondrial enzyme. Crystallographic studies have been initiated to solve the high-resolution crystal structure of this enzyme.Sc-Rsm22 monomer, SASDJS5SASBDB reference: Sc-Rsm22 dimer, SASDJT5SASBDB reference: 10.1107/S2059798321004149/jc5035sup1.pdfInformation on oligonucleotides and PCR conditions; Supplementary Tables and Figures. DOI:


In addition, a higher total volume of gas was generated when a higher ammonium bicarbonate content was decomposed. This led to the formation of porous epoxy micro-beads with a smaller particle size, lower specific gravity, and better thermal stability. At an epoxy to polyamide ratio of 10:6, many smaller micro-beads, with particle sizes ranging from 201 to 400 μm, were obtained at an ammonium bicarbonate content of 10 phr. Moreover, the porous epoxy micro-beads with open pores were shown to have a low specific gravity of about 0.93 and high thermal stability at a high ammonium bicarbonate content. Based on the findings, it was concluded that porous epoxy micro-beads were successfully produced using a single epoxy droplet in heated corn oil, where their shape and particle size depended on the content of ammonium bicarbonate and the ratio of epoxy to polyamide used.This paper explored the effects of ammonium bicarbonate and different ratios of epoxy to polyamide on the formation of porous epoxy micro-beads through a single epoxy droplet. A single drop of a mixture, consisting of epoxy, polyamide, and ammonium bicarbonate, was dropped into heated corn oil at a temperature of 100 °C. An epoxy droplet was formed due to the immiscibility of the epoxy mixture and corn oil. The ammonium bicarbonate within this droplet underwent a decomposition reaction, while the epoxy and polyamide underwent a curing reaction, to form porous epoxy micro-beads. The result showed that the higher ammonium bicarbonate content in the porous, epoxy micro-beads increased the decomposition rate up to 11.52 × 10 Porous micro-beads have been extensively commercialized in biomedical and structural applications due to their outstanding properties, such as low density, high surface area, high porosity, and economic value compared to solid micro-beads ,2,3. TheIn numerous studies, the emulsion technique was used to produce porous micro-beads as it is simple and economical . An emulIn a previous study, Leemsuthep et al.  fabricatIn this study, a single epoxy droplet was dropped into the heated corn oil to produce porous epoxy micro-beads. In order to understand the foaming mechanism, the fundamental study of the single droplet without mechanical agitation was investigated. In addition to the importance of a single epoxy droplet used, we highlight the effect of ammonium bicarbonate content and an epoxy polyamide ratio on the total micro-beads, microstructure, and particle size distribution of micro-beads produced. On the other hand, up to the present moment, we have not found any investigation that has studied production of micro-beads using a single epoxy droplet without mechanical agitation.3 and viscosity of 11–14 Pas at 25 °C. Polyamide A062 has an equivalent weight per H active of 110, with a density of 0.96 g/cm3 and viscosity of 35–45 Pas at 25 °C. White crystalline ammonium bicarbonate powder, with a density of 1.58 g/cm3, was obtained from HmbG Chemicals  for use as a blowing agent. Corn oil, which was free of polyunsaturated fatty acids, was purchased from Yee Lee Corporation Sdn. Bhd .Epoxy resin (grade DER 331) and polyamide (grade A062) were provided by Euro Chemo Pharma Sdn. Bhd . Epoxy resin DER 331 contains epoxide at an equivalent weight of 182–192 with a density of 1.16 g/cmTwo different ratios of epoxy to polyamide  were used in this study. The epoxy to polyamide ratio of 10:6 was calculated based on the stoichiometric ratio, whereas the ratio of 10:10 was similar to the stoichiometric ratio. The total weight of epoxy and polyamide was assigned as parts per hundred parts of resin (phr). The ammonium bicarbonate content varied between 0, 5, and 10 phr. First, epoxy and ammonium bicarbonate were mixed at a speed of 300 rpm for 3 min as shown in Ammonium bicarbonate with 5 and 10 phr were placed in the test tube and partially immersed in corn oil. The temperature of the corn oil was set at 100 °C. The amount of gas released through the hollow glass rod into the water in the measuring cylinder was recorded. The setup of the apparatus is shown in The microstructure of the PEMBs was observed using a scanning electron microscope (SEM)  to study the surface morphology, such as particle shape, size, and porous structure. A sputter-coating instrument was used to coat the samples with a layer of gold/palladium before the analysis to avoid electrostatic charging during the observation.A Dino-lite digital microscope (model AM5216ZT Edge Series), supplied by Dpro Vision Sdn. Bhd. , equipped with a low-resolution digital camera using Dino Capture 2.0 software  was used to capture the particle image from a single drop of epoxy mixture. The image was then analyzed using ImageJ software  to measure the particle size distribution of total micro-beads from a single epoxy droplet.SG is specific gravity, W1 is mass of pycnometer bottle, W2 is mass of pycnometer bottle filled with sample, W3 is mass of pycnometer bottle filled with sample and water, and W4 is mass of pycnometer bottle filled with water. The measurements were repeated five times.A pycnometer bottle was used to determine the specific gravity (SG) of the PEMBs based on ASTM D854-14 . The speThe thermal gravimetric analysis (TGA) was carried out using a Mettler Toledo TGA . The purpose of the analysis was to establish the thermal properties of the samples along with their compositional properties. The heating rate of the samples was set at 20 °C/min, with temperatures ranging from room temperature to 600 °C under the flow of argon gas.−3 cm3/s with increasing ammonium bicarbonate content from 0 to 10 phr. This could be implied that, at a higher ammonium bicarbonate content, the gas generated within one second will be higher. Therefore, a higher ammonium bicarbonate content will result in a higher decomposition rate.A single droplet of epoxy mixture, composed of epoxy, polyamide, and ammonium bicarbonate, was dropped into heated corn oil at a temperature of 100 °C. Once the droplet reached the desired temperature, it was blown and cured to form PEMBs. Ammonium bicarbonate played an essential role as a blowing agent, where the decomposition of ammonium bicarbonate generated gases, thereby, leading to the expansion of the droplet. 4HCO3) produces ammonia (NH3), carbon dioxide (CO2), and water vapour (H2O). The decomposition of ammonium bicarbonate is shown in Equation (4) [The total volume of gas generated after the decomposition increased as the ammonium bicarbonate content increased. The decomposition of ammonium bicarbonate (NHtion (4) . When a tion (4) . Thus, tThe curing of the droplet started on the outer surface once it came into contact with the heated corn oil, and further curing was then propagated to the inner core . Scheme A single epoxy-polyamide droplet without ammonium bicarbonate was dropped into heated corn oil to produce a big particle. As no gases were produced in this droplet, it was unable to blow or expand, thus, producing a solid macro-bead after curing with a size of 8.86 mm for the sample with an epoxy to polyamide ratio of 10:6, and a size of 8.37 mm for the sample with an epoxy to polyamide ratio of 10:10. The addition of ammonium bicarbonate to the single droplet caused it to be blown into many smaller micro-beads, as can be seen in Furthermore, the ratio of epoxy to polyamide could have greatly affected the microstructure of the PEMBs. From Furthermore, there were more PEMBs with a smaller particle size when the ammonium bicarbonate content was 10 phr than when it was 5 phr for both epoxy to polyamide ratios. This indicated that the higher ammonium bicarbonate content produced micro-beads with a smaller particle size. This may have been due to the high heat transfer caused by the presence of a large amount of ammonium bicarbonate. The probability of this heat being around the droplet was high due to the high volume of ammonium bicarbonate inside the droplet as compared to a low amount of ammonium bicarbonate, which tended to be encapsulated within the epoxy droplet. Therefore, 10 phr of ammonium bicarbonate tended to decompose and burst earlier, thereby, producing smaller particles as compared to the 6 phr of ammonium bicarbonate. Another reason could be that the 10 phr of ammonium bicarbonate produced a higher volume of gases. This induced a high internal pressure in the droplet, causing it to burst into many smaller micro-beads.With regard to the effect of the epoxy to polyamide ratio, it was clear that the epoxy to polyamide ratio of 10:6 resulted in slightly smaller particles as compared to the epoxy to polyamide ratio of 10:10 with 5 phr of ammonium bicarbonate. Additionally, the epoxy to polyamide ratio of 10:10 produced a large, flexible, crosslinked, three-dimensional network because it had excessive polyamide in the epoxy-polyamide system. In other words, epoxy polyamide of a 10:10 ratio had more free volume in the structure . This chAdditionally, different epoxy to polyamide ratios tended to influence the specific gravity as well. Based on Porous epoxy micro-beads were successfully produced by a single droplet in heated corn oil. The time taken to complete the decomposition reaction was faster with 10 phr of ammonium bicarbonate and a higher total volume of gases was generated. As a result, porous epoxy micro-beads with a smaller particle size, and lower specific gravity and thermal stability were produced. The epoxy to polyamide ratio of 10:6 produced many smaller-sized particles between 201 and 400 μm for both samples of 10:6_5AB and 10:6_10AB. This smaller particle size with a small, open pore decreased the specific gravity and increased the thermal stability of the micro-beads.


Arteriovenous fistula (AVF) is the most important vascular access for hemodialysis; however, preventive treatment to maintain the patency of AVFs has not been developed. In endothelium, β-catenin functions in both the intercellular adherens complex and signaling pathways that induce the transition of endothelial cells to myofibroblasts in response to mechanical stimuli. We hypothesize that mechanical disturbances in the AVF activate β-catenin signaling leading to the transition of endothelial cells to myofibroblasts, which cause AVF thickening. The present study aimed to test this hypothesis.Chronic kidney disease in mice was induced by a 0.2% adenine diet. AVFs were created by aortocaval puncture. Human umbilical vein endothelial cells (HUVECs) were used in the cell experiments. A pressure-culture system was used to simulate mechanical disturbances of the AVF.Co-expression of CD31 and smooth muscle alpha-actin (αSMA), loss of cell–cell adhesions, and the expression of the myofibroblast marker, integrin subunit β6 (ITGB6), indicated transition to myofibroblasts in mouse AVF. Nuclear translocation of β-catenin, decreased axin2, and increased c-myc expression were also observed in the AVF, indicating activated β-catenin signaling. To confirm that β-catenin signaling contributes to AVF lesions, β-catenin signaling was inhibited with pyrvinium pamoate; β-catenin inhibition significantly attenuated AVF thickening and decreased myofibroblasts. In HUVECs, barometric pressure-induced nuclear localization of β-catenin and increased expression of the myofibroblast markers, αSMA and ITGB6. These changes were attenuated via pretreatment with β-catenin inhibition.The results of this study indicate that mechanical disturbance in AVF activates β-catenin signaling to induce the transition of endothelial cells to myofibroblasts. This signaling cascade can be targeted to maintain AVF patency.The online version contains supplementary material available at 10.1186/s10020-022-00436-1. Arteriovenous fistula (AVF) is the recommended vascular access for patients on maintenance hemodialysis because of superior outcomes  and vimentin, known as myofibroblast, is one of the main causes of AVF patency loss . Male C57BL/6 mice aged 7 weeks  were used for all animal experiments. After a week of adaptation, baseline plasma creatinine levels were measured to define baseline renal function. From 8 weeks old until the time of euthanasia, the mice were fed a 0.2% adenine diet  to induce chronic kidney disease (CKD)  in saline and administered at 200 μg/kg/day by oral gavage. Oral gavage of 4% DMSO was used as a negative control for pyrvinium pamoate. Mice were treated with pyrvinium pamoate or DMSO from 1 week after operation to euthanasia.After euthanasia of mice, en bloc resection from the bifurcation of the common iliac veins to the lower end of the hepatic segment of the IVC was performed to include the IVC, abdominal aorta, underlying psoas muscles, and overlying peritoneum. The specimens were immediately fixed in paraformaldehyde and embedded in paraffin for tissue sectioning. Tissue was transversely sectioned at 3 μm at the puncture site, which had been premarked as stated previously. Additionally, the kidneys were resected for histological analyses to confirm the successful induction of CKD . IF images were obtained and merged using a TissueFAXS system  or Stellaris 8 confocal microscope . For confocal microscopic imaging, the optical slice thickness was 0.5 μm. DAB expression in IHC images was automatically analyzed using HistoQuest software. The expression of fluorescence in IF images was automatically analyzed using TissueQuest software. AVF thickening was quantitated by the maximal thickness of Masson Trichrome staining. Nuclear localization was defined on serial confocal microscope images, in which the target object surrounded by the signal of dapi suggests its nuclear localization. Analyses of the images were performed by a specialized technician in blinded manner.Human umbilical vein endothelial cells (HUVECs)  from passages 2 to 5 were used. The cell experiments were conducted in an incubator with supplementary carbon dioxide . A pressure–culture system was used to simulate the effects of mechanical disturbance in AVF. In this system, HUVECs cultivated in tissue culture flasks were continuously subjected to barometric pressures of 50–70 mmHg for 4 h during the experiment, whereas the nonpressurized groups were cultured in flasks under normal pressures. For the inhibition of β-catenin signaling, HUVECs were pretreated with pyrvinium pamoate for 30 min before pressurization. Cells were treated with 100 nM final concentration of pyrvinium pamoate dissolved in 4% DMSO. Controls were treated with 4% DMSO only.Cells were collected immediately after pressurization and stored at − 80 °C. Cells were lysed in RIPA Lysis Buffer and fractionated with a Membrane, Nuclear & Cytoplasmic Protein Extraction Kit (Bio Basic #BSP002). For western blotting, 20 μg of protein was loaded per well. Chemiluminescence  was used to detect the signals. The following primary antibodies were used: αSMA , ITGB6 , glyceraldehyde 3-phosphate dehydrogenase , β-catenin , and lamin B1 . The following secondary antibodies were used: antirabbit IgG  and antimouse IgG .Continuous variables were expressed as medians, first and third quartiles, and minimum and maximum values as shown in boxplots. Statistical comparisons were made using the Wilcoxon signed-rank test. Bonferroni correction applied in cases of multiple comparisons, such that statistical significance was defined as a P value < 0.05/number of hypotheses tested. All data analyses were conducted using SAS 9.4 software . All data used in this study are available from the corresponding author for research purposes.To examine the process of AVF thickening, AVF specimens were obtained at 2, 4, and 6 weeks post-operation. Sham operated IVC was used as the negative control (n = 6 per group). The tunica media of sham operated IVC contained a single layer of smooth muscle cells Fig. A. In conTo investigate the origin of myofibroblasts in AVF thickening, sham operated IVC and AVF were double stained for CD31 and αSMA to determine if the myofibroblasts carried the endothelial marker CD31. In sham operated IVCs, both CD31 and αSMA distributed linearly with rare overlap, demonstrating the discriminating ability of these two markers for endothelial and mesenchymal cells Fig. A. In AVFVE-cadherin was evaluated as a marker of endothelial cell–cell adhesion. The endothelium of sham operated IVCs expressed VE-cadherin linearly and strongly Fig. E. The exTo confirm the origin of myofibroblasts, double staining for Tie2, an endothelial marker, and αSMA was performed. IF images were then obtained using a confocal microscope to confirm their co-localization. In the sham operated mice, cells double-positive for Tie2 and αSMA were rarely observed Fig. A. In conWhen β-catenin signaling is activated, the β-catenin destruction complex decomposes and β-catenin localizes to the nucleus to initiate cell transition. Therefore, we assessed the nuclearization of β-catenin, expression of axin2 (a component of the β-catenin destruction complex), and c-myc  in mouse AVF. In sham operated IVC, β-catenin was expressed in the endothelium, although nuclear localization was rarely observed Fig. A. In AVFTo confirm the contributing role of β-catenin signaling in AVF thickening, mice receiving AVF creation were treated with pyrvinium pamoate, an anthelmintic drug with an inhibitory effect on β-catenin signaling (Li et al. The expression of axin2 was used to confirm the inhibition of β-catenin signaling by pyrvinium pamoate. As observed in the previous experiment, sham operated IVCs strongly expressed axin2. In AVF treated with DMSO, the expression of axin2 decreased significantly. In AVF treated with pyrvinium pamoate, the expression of axin2 increased significantly compared with AVF treated with DMSO Fig. J–M. ThesTo investigate the effect of mechanical disturbance on cell transition, HUVECs were cultivated in a standard incubator or pressure-culture system and then separated into three experimental groups as follows: a negative control group with no barometric pressurization, a group treated with barometric pressure, and a group pretreated with pyrvinium pamoate followed by barometric pressure. In the negative control group, β-catenin was distributed linearly along the cell membrane. Cytoplasmic distribution and nuclear localization of β-catenin were absent. No αSMA expression was observed in the negative control group Fig. A. In preWestern blotting was used to confirm the effects of barometric pressure on β-catenin signaling and the transition to myofibroblasts. HUVECs were divided into four treatment groups as follows: nonpressurized, no pyrvinium treatment; nonpressurized, pyrvinium-treated; pressurized, no pyrvinium treatment; and pressurized, pyrvinium-treated. The pressurization protocol and pyrvinium treatment were the same as in the previous experiment. Western blotting showed that barometric pressure significantly increased nuclear β-catenin protein expression, and treatment with pyrvinium pamoate attenuated β-catenin nuclearization Fig. A, B. AddThis study showed that activated β-catenin signaling in mouse AVF may contribute to the transition of endothelial cells to myofibroblasts, leading to the formation of AVF thickening. In mouse AVF, inhibition of β-catenin signaling significantly attenuated the transition to myofibroblasts and the thickening of AVF. Our cell experiments showed that barometric pressure activates β-catenin signaling and, consequently, induces the transition of HUVECs to myofibroblasts. Taken together, these results imply that mechanical disturbances activate β-catenin signaling to induce the transition of endothelial cells to myofibroblasts, which subsequently cause AVF thickening. Hence, β-catenin signaling is a potential target for the prevention of AVF thickening.Increased αSMA-expressing mesenchymal cells have been a universal finding in different types of vascular remodeling. Although the source of these mesenchymal cells remains controversial, a body of evidence supports the endothelial cell as an origin of pathogenic mesenchymal cells in the process of vascular remodeling (Arciniegas et al. Mechanical disturbances, including tractional or tensile forces, regulate cell adhesion, migration, and differentiation (Huang and Ingber Epithelial-to-mesenchymal transition is essentially reversible (Arciniegas et al. Aiming for the same goal as the present study, Zhao et al. used a vascular smooth muscle cell lineage tracing reporter mouse to investigate the origins of AVF lesions (Zhao et al. Barometric pressure was used to simulate the mechanical signals of AVF in this study. The pressure was set at 50–70 mmHg to represent the mean arterial pressure in humans. This method had been used in several studies to investigate the effect of blood pressure on HUVECs and aortic endothelial cells, which increases angiogenesis and fibrosis (Tokunaga and Watanabe Compared with AVF induced via surgical anastomosis, aortocaval AVF created by needle puncture has no suture-related injury and forms overtly stenotic lesions less frequently due to the relatively large diameter of IVC. Several previous studies have reported an inflammation response and adverse remodeling of aortocaval AVF (Kuwahara et al. Pyrvinium pamoate is an approved anthelmintic drug with an inhibitory effect on β-catenin signaling, rather than a specific inhibitor of β-catenin activity. Nevertheless, it has been used to target β-catenin signaling in various oncology studies (Li et al. In conclusion, this study showed that mechanical disturbance in AVF activates β-catenin signaling in endothelial cells and induces the transition to myofibroblasts to cause the formation of AVF lesions. Although we showed that inhibition of β-catenin signaling attenuated AVF lesion formation, β-catenin signaling is not the only mechanism contributing to AVF lesions. An integrative strategy targeting more than one signaling pathway may be required to prevent AVF lesions.Additional file 1. Inhibition of β-catenin signaling attenuates arteriovenous fistula thickening in mice by suppressing myofibroblasts - Supplemental Information.


We examined the mechanism by which lithium chloride (LiCl) attenuates the impaired learning capability and memory function of dual‐transgenic APP/PS1 mice. Six‐ or 12‐month‐old APP/PS1 and wild‐type (WT) mice were randomized into four groups, namely WT, WT+Li , APP/PS1 and APP/PS1+Li. Primary rat hippocampal neurons were exposed to β‐amyloid peptide oligomers (AβOs), LiCl and/or XAV939 (inhibitor of Wnt/β‐catenin) or transfected with small interfering RNA against the β‐catenin gene. In the cerebral zone of APP/PS1 mice, the level of Aβ was increased and those of α7 nicotinic acetylcholine receptors (nAChR), phosphor‐GSK3β (ser9), β‐catenin and cyclin D1 (protein and/or mRNA levels) reduced. Two‐month treatment with LiCl at ages of 4 or 10 months weakened all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AβOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of β‐catenin expression lowered the level of α7 nAChR protein in these cells. LiCl attenuates the impaired learning capability and memory function of APP/PS1 mice via a mechanism that might involve elevation of the level of α7 nAChR as a result of altered Wnt/β‐catenin signalling. When Wnt receptors are not activated, the β‐catenin bound by Axin is phosphorylated in succession via glycogen synthase kinase‐3β (GSK3β) and casein kinase 1 at a range of Ser/Thr residues (regularly spaced), which are located at the N‐terminus of the β‐catenin. Consequently, ubiquitination and targeting of β‐catenin are initiated for fast disruption by proteasome, thereby depressing the expression of downstream targets such as cyclin D1.Interestingly, Wnt/β‐catenin signalling is closely linked to learning capability and memory function among mammals and this signalling is impaired both in the brains of AD patients,As is widely acknowledged, lithium is the preferred mood‐stabilizing agent for maintaining bipolar disorder.Accordingly, lithium may be of value in treating AD. However, it is currently unknown whether lithium can reverse the learning and memory impairments in murine AD model and, if so, whether this effect involves Wnt/β‐catenin signalling modulation, as well as consequent regulation of α7 nAChR. Here, we investigated these questions by treating both APP/PS1 transgenic mice and β‐amyloid peptide oligomer (AβO)‐exposed primary neurons using LiCl or XAV939, a small molecule selective inhibitor that promotes β‐catenin degradation and inhibits the transcription by β‐catenin,22.11‐42 and XAV939 (Sigma‐Aldrich Inc.); anti‐Aβ mouse monoclonal antibody (BioLegend Inc.); anti‐GSK3β, anti‐phosphor‐GSK3β (ser9), anti‐cyclin D1 and anti‐β‐catenin rabbit monoclonal antibodies, as well as horseradish peroxidase (HRP)‐conjugated anti‐rabbit IgG ; anti‐GAPDH rabbit polyclonal antibody (Genetex); anti‐nAChR α7 rabbit polyclonal antibody (Abcam); cell counting Kit‐8 (Dojindo Molecular Technologies); anti‐NeuN mouse monoclonal antibody (Merck Millipore); anti‐glial fibrillary acidic 113 protein (GFAP) rabbit monoclonal antibody (Dako); CY‐3‐labelled anti‐mouse IgG and 488‐labelled anti‐rabbit IgG (Thermo scientific Inc.); Aβ42 assaying kits (Thermo scientific Inc.); Lipofectamine RNAiMAX Reagent (Invitrogen Inc.); universal 2×PCR mastermix ; and the remaining chemicals were all procured from Sigma‐Aldrich.LiCl, Aβ2.2SWE) mice aged 4 months having a 85Dbo/Mmjax background were procured from Nanfang BioTechnology Co., Ltd. Meanwhile, wild‐type (WT) mice of identical strain were also procured from the same company. All mice were 20–30 g in body weight. Each male mouse was allowed to mate with four females. When the resulting pups were 12–20 days old, the tips of their tails were cut off for extraction of DNA and the polymerase chain reaction (PCR)‐based genotyping, where agarose gel electrophoresis (1.5%) was used for product analysis. For PCR primer design of target transcripts shown in Table B6.Cg‐Tg .Based on literature and the results of previous studies,2.3Primary neurons were obtained from the cerebral zone of newborn Sprague‐Dawley rats employing minor modifications of a published procedure.AβOs were prepared by a procedure described earlierTransfection with small interfering RNA (siRNA) was initiated by adding a mixture (1:1) of Lipofectamine RNAiMAX Reagent diluted in Opti‐MEM Medium (15 μl:250 μl) and the siRNA , also diluted in Opti‐MEM Medium (1:50) for 5 min. Then, the cells were incubated with the siRNA‐Lipid complex for 48 h at 37°C.2.4In a 25–26°C water‐filled round pool rendered opaque using milk powder, every mouse was compelled to find an underwater escape facility.2.542 was determined utilizing a murine Aβ42 ELISA kit as per the protocol of manufacturer.The murine cerebral level of Aβ2.6Brain tissue or cultured cells in lysis buffer containing a mixture of protease inhibitors were disrupted in a glass homogenizer. The resulting homogenate was subjected to 20‐min centrifugation under 4°C and 15520 g conditions. For the resulting supernatants, the BCA protein assaying kit was utilized to measure the protein concentrations.To achieve relative protein quantization, the above PVDF membranes were then subjected to incubation using antibodies against phosphor‐GSK3β (ser9), GSK3β, β‐catenin, cyclin D1, α7 nAChR or GAPDH overnight at 4°C. Next, the membranes were rinsed and subjected to 60‐min incubation using secondary antibody conjugated to HRP. Subsequently, enhanced chemiluminescence kit (Millipore) was used to detect the protein bands, and visualization of the resulting signals was accomplished for 30 s–3 min by exposing to chemiluminescence film (hyperperformance). Signal intensity was quantified employing the Image J software.2.7−ΔΔCT  and the formula ΔΔCT = ΔCTtarget−ΔCTcontrol = (CTtarget−CTGAPDH)−(CTcontrol−CTGAPDH) with the error estimate being ΔΔCT plus and minus the standard deviation. The sequences of the PCR primers utilized are presented in Table Total RNA, which was extracted via Trizol Reagent from brain tissue or cells, was used to obtain cDNA by reverse transcription with the Prime Script™ RT Master Mix cDNA Synthesis Kit (Takara Bio.).2.8Immunohistochemical staining for senile plagues and α7 nAChR in the brains of mice was accomplished by consulting prior literature.For immunostaining of α7 nAChR in isolated cells, seeding of primary neurons was done onto six‐well plates coated with polylysine, followed by three times of washing in PBS and paraformaldehyde fixation (4%). Then, these samples were subjected to 30‐min treatment with goat serum at RT and a subsequent overnight incubation with antibody against α7 nAChR at 4°C.2.9p values of <0.05 were regarded significantly different.For mice in various groups, the statistical values were represented as means ± SDs and compared via analysis of variance, and a subsequent post hoc (least significant difference) test. Bielschowsky procedure was employed to semi‐quantitate the Aβ plaques.33.1As shown in Figure 3.2According to the Morris water maze results displayed in Figure 3.3The brains of 6‐ Figure .42 that was unaffected by treatment with LiCl. In contrast, this level was much higher in untreated APP/PS1 animals, where the level was decreased even further by treatment with LiCl  , β‐catenin and cyclin D1 levels in the cerebral hippocampus  5 nAChRs for maintaining the ability to learn and memory.d‐aspartic acid receptors (NMDARs) and glutamatergic transmission was found in the therapeutic effects of lithium.Interestingly, a possible role for the expression and activity of N‐methyl‐In the cerebral zone of APP/PS1 mice, the Aβ level was increased and those of α7 nAChR, phosphor‐GSK3β (ser9), β‐catenin and cyclin D1 (protein and/or mRNA levels) were reduced. Treatment with LiCl for 2 months at 4 or 10 months of age attenuated all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AβOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of β‐catenin expression lowered the level of α7 nAChR protein in these cells. A proposed molecular pathway involving above changes is shown in Figure The authors have no conflict of interest to declare.Jie Xiang: Formal analysis ; Writing‐original draft . Longyan Ran: Formal analysis . Xiaoxiao Zeng: Formal analysis . Wenwen He: Formal analysis . Yi Xu: Formal analysis . Kun Cao: Formal analysis . Yangting Dong: Formal analysis . Xiaolan Qi: Formal analysis . Wenfeng Yu: Formal analysis . Yan Xiao: Formal analysis . Zhizhong Guan: Conceptualization ; Writing‐review & editing .Supplementary MaterialClick here for additional data file.


To analyze the effect of these stresses, the samples were analyzed periodically through Fourier transform infrared spectroscopy (FTIR) for structure analysis; scanning electron microscopy (SEM) for surface analysis of long-term aged samples; and optical microscopy for the surface topography of short-term aged samples. The Swedish Transmission Research Institute (STRI) classification and contact angle measurement techniques were used for hydrophobicity analysis of long-term and short-term aged samples, respectively. After aging in both conditions, the nanocomposite showed better results as compared to the other samples. After 1000 h of aging, it showed HC-5 class of hydrophobicity, whereas EMC and NE degraded to the HC-6. In case of short-term aging, the contact angle decreased to the 64.15° and 75.05° from 104.15° and 114.9° for ENC and EMC, respectively. Also, in terms of structural degradation, ENC showed the highest structural stability after 1000 h of aging with the highest stable peak of aromatic ether at 1300–1500 cm−1. Microscopic observation through scanning electron and optical techniques also revealed superior performance of the nanocomposites.Epoxy resins have demonstrated remarkable properties with potential for usage as high voltage insulators. However, a loss of these properties has been observed in high temperature and humid environments. In order to enhance the hydrothermal stability of epoxy resins, micro (15% SiO In many applications, epoxy has the ability to substitute ceramic insulators that are brittle in nature . If elecoperties . Differeoperties . Inorganoperties ,22. Zhaioperties . Erosionoperties . Epoxy-soperties . Epoxy coperties  for theioperties ,28,29.Keeping in view what has been mentioned above, this study presents a fabrication of both micro and nano composites, along with the neat epoxy, and a comparison of their stability against short and long-term hydrothermal stresses. The micro and nano epoxy composites are subjected to short and long term aging conditions and their behavior in terms of structural changes and superficial properties are analyzed under different intervals of aging cycles.Nano silica having size of 12 nm was purchased from Degussa (Evonik) Chemical Co., Los Angeles, CA, USA, and micro silica 5 µm in size was procured from Wuhan Newreach Chemical Co., wuhan, China. epoxy  were purchased from General Electric Co., New York, NY, USA, Lanxess Co., Köln, Germany and Justus Kimiaraya, Jakarta, Indonesia.First of all, dispersion of nanosilica was achieved by using an ethanol solution by keeping it in ultrasonic bath for 90 min. After that, silane was added for surface functionalization of the filler. Surface functionalization is needed for better chemical interaction of fillers and epoxy material. In the next phase, the epoxy was added into the mixture with the help shear mixer at rotational speed of almost 3500 rpm for almost 17 min. The solution was again kept in an ultrasonic bath to make it more dispersed and to give it a better interactive structure. After that, the mixture was kept at temperature higher than the boiling temperature of ethanol in vacuum to evaporate it completely. Finally, a hardener was added and mixed in this solution. Then, the bubbles were removed from the solution by setting aside in vacuum at 27 mmHg. At the end, the mixture was poured into molds to attain the required dimensions. Prepared samples were circular in shape having 80 mm diameter and 3 mm thickness . An automated environmental chamber by UTSTESTER with controlled temperature and humidity was employed shown in Experimental setup for long term aging study was developed by using a glass beaker in which samples were immersed in water and subjected to accelerated heating at 65 °C to 70 °C. The experiment was designed for 1000 h, and five cycles were carried out. In this way, to check the degradation after each cycle, analysis techniques were employed after every 200 h. A schematic diagram and image of the experimental setup are given in Swedish Transmission Research Institute (STRI) classification was employed to check the hydrophobic behavior of the samples during long term aging. Water was sprayed on to the samples and high-resolution images were taken after 20 min and compared with the standard STRI classification  was used which consist of syringe filled with distilled water having droplet of 0.5 μL. As during short term aging, a very smaller change in water angle takes place. So as to measure the smaller changes, contact angle measurement was employed for the short-term aging experiment.Optical microscopy was used at a 100 µm resolution and 20× magnification. It is evident from the literature that in short-term aging, only surface roughness takes place with prolonged aging conditions, crosslinking, and chain scission . So, optTo analyze the surface morphology for long-term aging, a field emission scanning electron microscopy  was employed. Carbon coated samples were placed on stubs and images were taken. As long-term aging might affect morphology of the specimen, it is important to use sensitive technique. Thus, SEM was employed for in depth analysis.−1. Absorbance values were obtained against wave number (cm−1) by placing the sample directly on a diamond scanner.FTIR analysis was performed after both aging methods by using Bruker platinum ATR model Alpha, Ettlingen, Germany with a spectral range of 4000–500 cmContact angle measurements were carried out by using the contact angle meter. The contact angle images of unaged NE, EMC, and ENC could be seen in The degradation in the contact angle occurred due to hydrolysis of different functional groups in epoxy matrix (further discussed in the FTIR section). It is evident from Nanosilica epoxy composite showed the least degradation in terms of contact angle. This may be due to the fact that the surface area of nanosilica is greater and shows better interaction with epoxy networkmakes it more stable against aging conditions.Hydrophobic character of all of the prepared samples were analyzed by their comparison with HC-1 to HC-7 classes of Swedish Transmission Research Institute (STRI) hydrophobicity classification standard given in Loss of hydrophobic behavior after applying hydrothermal stress can be seen clearly in all samples. Hydrophobicity class HC-2 was observed in case of unaged neat epoxy and silica micro composite, while the nanosilica composite showed hydrophobicity class HC-1 After first cycle of 200 h, neat epoxy and EMC retained the same class of hydrophobicity. After that, continuous loss of hydrophobic behavior was seen after every 200 h in the case of the neat epoxy. HC-3 and HC-4 class was seen after the third and fourth cycle and finally degraded to HC-6 after the fifth cycle. On the other hand, silica micro and nano composites showed better stability against their loss of hydrophobic behavior. Silica micro composite showed degradation after the second cycle and HC-3 class was seen. After that, a constant loss of hydrophobicity was seen and HC-4, HC-5, HC-6 classes were observed after the third, fourth, and fifth cycles, respectively. In the case of the silica nanocomposite, a continuous loss of hydrophobicity from HC-1 to HC-2 was observed, which was maintained until the second cycle and then further degraded to HC-3 and HC-5 after the third and fourth cycles, respectively, (being retained even after the last cycle).These results showed that hydrophobicity decreased due to the deposition of hydroxyl groups on the surface of the epoxy. These groups attract water molecules that lead to a decrease of hydrophobicity. Loss of hydrophobicity at early stages of aging in neat epoxy occurred due to the roughness in surface of epoxy . Loss inThe micro silica composite showed better stability of its hydrophobic behavior in comparison to the neat epoxy, but more degradation was observed in comparison with the silica nano composite.The nano silica composite showed better retention of hydrophobicity as compared to neat epoxy and silica micro composites. By the addition of fillers, better results were obtained due to the better surface area provided by the filler. An increase in surface area from micro to nano filler water repellency also occurred. Optical microscopy was employed to analyze the surface roughness during short-term aging. An unaged sample can be seen in These voids and roughness appeared due to the oxidation on surface. The degradation was minimized in case of micro composite due to higher surface area of micro and nano silica, which reduced the oxidation at the surface. The silica filler is stable under higher temperatures and forms a strong interaction with the polymer matrix, resulting in increased resistance towards oxidation. It could be seen from Analysis of optical microscopy results showed that the nano silica-based composite was more stable and retained its surface against elevated temperature and humidity. Scanning electron microscopy (SEM) was used to analyze the surface morphology of epoxy material and its degradation after applying long term hydrothermal stresses. SEM images of all of the prepared samples were taken before and after 1000 h of aging at 100 µm. SEM images of un-aged samples are given in In FTIR was employed to study the variation in absorbance of different functional groups in the epoxy, which indicated the extent of degradation in its structure, as the increase or decrease in intensities of the absorbance band corresponds to the degradation and interactions involved during hydrothermal aging. −1” corresponds to the –OH group. It is evident that the NE intensity of this band gradually increased up to the second cycle. Then, the loss of the –OH group can be seen during the third, fourth, and up to the fifth cycles. This is due to the diffusion of water into epoxy chain. After 400 h, a loss of water can be seen due to degradation in the epoxy backbone [Long-term aging results of FTIR after each cycle were recorded. The band between “2700 to 3500 cmbackbone . Trends −1 in −1) and p-Phenyl (700–800 cm−1) groups. Finally, after the 1000 h of aging the alkyl peak was 128%, 116% and 198% of the virgin peaks for NE, EMC, and ENC respectively. The degradation epoxy back bone can be divided into two steps. The first step involves a breakdown of groups, which resulted in increase in absorbance intensities of these groups and second step involves oxidation which causes decrease in absorbance intensities and leads to the complete degradation of the epoxy.Absorbance intensities of alkyl group can be seen between bands 2750–3000 cmThe increase in intensities of the alkyl, p-Phenyl, and carbonyl groups was due to the hydrolysis of the ester linkages, which resulted in increase in intensity Of –OH band. After that, the decrease might be due to the oxidation of this group, which shows the degradation in epoxy backbone . The abs−1 is the most stable bond of epoxy and it does not react with water and oxygen easily. After 1000 h of aging the intensity of aromatic ether group was 80%, 166 %, and 170% of the original peak. So, the relative absorbance intensities were calculated for this functionality by using following equation.Ir is the relative intensity.For quantitative analysis relative absorbance intensities were measured. As the aromatic ether bond at 1300–1500 cmThe comparison of relative absorbance intensities are given in p > 0.05.An increase at 600 h could be seen. It is evident that degradation in crosslinking occurred was after the second, third, and fourth cycles for NE, EMC, and ENC, respectively. A statistical tool was used to analyze the data of In the next step, samples were analyzed using one way ANOVA, which was employed to check the relative standard deviation and standard error in the absorbance values of the samples. From p-value between the groups is p > 0.05, which suggests that the difference between all three samples are not significant. However the std deviation value of absorbance is lowest in case of ENC which represents its slow aging.The FTIR analysis for short term aging was also carried out to check the degradation in structure of epoxy backbone.−1 due to hydrolysis was prominent in the neat epoxy, and there was no significant increase in the band intensity in EMC and ENC after 30 h. A small increase can be seen after 60 h. However, the increase was higher for EMC. This indicates the more stable structure of ENC. The same trend was recorded for all of the other bands.It could be seen in An epoxy neat sample (NE), epoxy 5% nano silica composite (ENC), and epoxy 15% silica micro-composite (EMC) were prepared and subjected to short term as well as long term aging using different accelerated experimental setups of hydrothermal stresses. From the FTIR results, an increase in peak of carbonyl (C=O) was recorded, showing the oxidation of the backbone. For long-term aging, detailed degradation analysis was carried out and it could be seen that maximum oxidation in terms of an increase in intensity of carbonyl group was observed. In NE, 333%, 285% in case of EMC, and 261% was recorded for ENC after t the third cycle. Also, the most stable group (which is the aromatic ether group in ENC) showed maximum stability with 170% peak intensity of virgin sample as compared to 80% and 166% for NE and EMC, respectively. Similarly for the alkyl group, after the 1000 h of aging the peaks were 128%, 116%, and 198% of the virgin peaks for NE, EMC, and ENC, respectively. Hydrophobicity classification was carried out using contact angle measurements for short-term aging. The ENC showed higher resistance towards loss of hydrophobicity. The contact angle for NE was 106.9° and was decreased to the 51° after 60 h of aging. However, EMC and ENC showed contact angle of 114.9° and 104.15 which decreased to 75.05 and 64.15, respectively after 60 h of aging. Similarly in case of long-term aging initially NE and EMC shows HC-2 and ENC shows HC-1 class of hydrophobicity and after 1000 h of aging EMC and NE shows HC-6 and ENC shows HC-5 class of hydrophobicity. In both cases, the loss of hydrophobic behavior was minimum in the case of ENC and highest in the case of NE. Results from both optical and scanning electron microscopies and SEM methods revealed that the nanocomposites exhibited least surface degradation as compared to NE and EMC.