Influenza A viruses have undergone rapid evolution with virulent; however, complete and comprehensive data on gene evolution and amino acid variation of HA and NA in immunosuppressed patients was few. In this study, we analysed molecular epidemiology and evolution of influenza A viruses in immunosuppressed population, and immunocompetent population were used as controls.Full sequences of HA and NA of A(H1N1)pdm09 and A(H3N2) were acquired through reverse transcription-polymerase chain reaction (RT-PCR). HA and NA genes were sequenced using the Sanger method and phylogenetically analysed using ClustalW 2.10 and MEGA software version 11.0.During the 2018–2020 influenza seasons, 54 immunosuppressed and 46 immunocompetent inpatients screened positive for influenza A viruses by using the quantitative real-time PCR (qRT-PCR) were enrolled. 27 immunosuppressed and 23 immunocompetent nasal swab or bronchoalveolar lavage fluid samples were randomly selected and sequenced using the Sanger method. A(H1N1)pdm09 were detected in 15 samples and the remaining 35 samples were A(H3N2) positive. By analyzing the HA and NA gene sequences of these virus strains, we found that all A(H1N1)pdm09 viruses shared high similarities to each other and the HA and NA genes of these viruses exclusively belonged to subclade 6B.1A.1. Some NA genes of A(H3N2) viruses were not in the same clade as those of A/Singapore/INFIMH-16-0019/2016 and A/Kansas/14/2017, which may have led to A(H3N2) being the dominant strain in the 2019–2020 influenza season. Both A(H1N1)pdm09 and A(H3N2) viruses showed similar evolutionary lineages patterns of HA and NA between immunosuppressed and immunocompetent patients. Compared with the vaccine strains, there were no statistically significant of HA and NA genes and amino acid sequences of influenza A viruses in immunosuppressed and immunocompetent patients. However, the oseltamivir resistance substitution of NA-H275Y and R292K have been observed in immunosuppressed patients.A(H1N1)pdm09 and A(H3N2) viruses showed similar evolutionary lineages patterns of HA and NA between immunosuppressed and immunocompetent patients. Both immunocompetent and immunosuppressed patients have some key substitutions, which should be of note monitored, especially those with potential to affect the viral antigen.The online version contains supplementary material available at 10.1186/s12985-023-02067-2. Influenza viruses include A, B, C and D, and among these four types, type A is the most infectious, which can even cause a life-threatening pandemic . A (H1N1The previous literatures and surveillances of the Centers for Disease Control and Prevention (CDC) of influenza at the molecular level were mainly from immunocompetent patients , 9. WithVariations in HA and NA may lead to the emergence of new clinical features, such as the HA-D239 site mutation is associated with severity of illness, while NA-H275, E119, R292, Q136, and I223 substitutions can lead to neuraminidase inhibitors (NAIs) resistance –16. CompClinical characteristics and antiviral therapy for immunosuppressed influenza patients were analysed in our prior study , and in Peking University People’s Hospital (PKUPH) is a national influenza surveillance sentinel unit, receiving at least 100,000 inpatients from all Beijing districts annually. During the 2018–2020 influenza seasons (November to the following March), 54 immunosuppressed and 46 immunocompetent inpatients screened positive for influenza A viruses by using the quantitative real-time PCR (qRT-PCR) method were enrolled in this study. Original nasal swab or bronchoalveolar lavage fluid (BALF) samples from these patients were collected and immediately placed in virus transport media tubes for analysis. Immunosuppressed patients were defined as the presence at least one risk factor as follows: congenital/genetic immunocompromise, HIV infection, SOT, HSCT, malignancies receiving chemotherapy, aplastic anemia, chronic haemodialysis, chronic steroid use, immunosuppressive agents use and biological drug use . Full seData on demographic factors , clinical presentation and complications , mechanical ventilation, time to fever clearance, death) and laboratory test results  were collected through medical records.We extracted RNA from samples using the QIAamp Viral RNA Mini Kit  and performed the reverse transcription with a commercial kit , following the manufacturer’s instructions. The complementary DNAs (cDNAs) generated from the reverse transcription were stored at − 20 °C until use.4 (0.6 μl), 10 mM dNTP Mix (0.4 μl), 10 µM forward primer (0.4 μl), 10 µM reverse primer (0.4 μl), and Platinum® Taq DNA Polymerase High Fidelity (0.1 μl of 5U/µL). The PCR conditions were: 94 °C for 3 min, followed by 40 cycles of 94 °C for 15 s, 60 °C for 30 s and 72 °C for 2 min, with extension at 72 °C for 10 min. PCR products were analyzed by the method of electrophoresis. 27 immunosuppressed and 23 immunocompetent samples were randomly selected and PCR products were directly sequenced using the Sanger method .For sequencing the HA and NA genes, high-fidelity thermostable DNA polymerase  was used. Specific primers for HA and NA were shown in Table The nucleotide sequences generated were aligned with publicly available sequences of influenza A viruses available in the National Center for Biotechnology Information (NCBI) and Global Initiative of Sharing All Influenza Data (GISAID) database. Full-length reference sequences, which were phylogenetically related to the H1N1 and H3N2 viruses, were included with our sequences for phylogenic analysis using MEGA v.11.0. Sequences with (a) evidence of lab errors and (b) 100% similarity were discarded. All HA and NA gene sequences were aligned using ClustalW 2.10. Phylogenetic analysis of HA and NA gene sequences and substitution analysis of HA and NA proteins were performed using MEGA software version 11.0. Sequences of HA and NA genes in our study have been deposited into NCBI with the accession number OQ455958-455972 and OQ456032-456116.2 test. P values < 0.05 were considered statistically significant.Statistical analysis was performed using SPSS statistical software version 22.0 . Two-group comparisons of normally distributed data were performed with the independent samples t-test. Frequency comparisons were made with the χAccording to the weekly influenza data released by Chinese National Influenza Center, the epidemiology of influenza virus in northern China from April 2018 to May 2020 is different in the 2018–2019 and 2019–2020 influenza seasons. As shown in Fig. During the 2018–2020 influenza seasons, 54 immunosuppressed and 46 immunocompetent inpatients were verified using RT–PCR. Our hospital was not designated hospitals of HIV infection, so patients with HIV infection were not involved in this study. The immunosuppressed factors in this study were as follows: 38 patients with malignancies receiving chemotherapy, 8 patients with chronic steroid use , 6 patients with HSCT (including 1 patient with aplastic anemia), and 2 patients with chronic haemodialysis. There was no congenital/genetic immunocompromise.Twenty-seven inpatients with immunosuppression and 23 immunocompetent inpatients of our study were randomly selected and sequenced using the Sanger method. A(H1N1)pdm09 were detected in 15 samples and the remaining 35 samples were A(H3N2) positive. HA and NA gene sequences were compared with other A(H1N1)pdm09 and A(H3N2) sequences on NCBI and GISAID.Phylogenetic analyses of the HA genes showed the tested A(H1N1)pdm09 viruses felled together with the vaccine strain A/Brisbane/02/2018 rather than A/Michigan/45/2015 (H1N1), and belonged to subclade 6B.1A.1  viruses felled together with the vaccine strain A/Singapore/INFIMH-16-0019/2016 except A/Beijing/32003/2018 which felled into A/Kansas/14/2017 pdm09 and 35 AH3N2) viruses were fully sequenced and analysed of homology. HA genes of these A(H1N1)pdm09 viruses shared 98.00% to 100.00% nucleotide similarity and 97.53% to 100.00% amino acid identity to each other. NA genes of these A(H1N1)pdm09 viruses shared 98.47% to 100.00% nucleotide similarity and 96.81% to 100.00% amino acid identity to each other. As shown in Additional files 1 and 2 Supplementary Tables  viruses HA genes of these AH3N2) viruses shared 94.59% to 100.00% nucleotide similarity and 93.83% to 100.00% amino acid identity to each other. NA genes of these A(H3N2) viruses shared 95.82% to 100.00% nucleotide similarity and 95.11% to 100.00% amino acid identity to each other. As shown in Additional files 3 and 4 Supplementary Tables N2 viruseFurther comparisons between these two groups, we found that there were no statistically significant of HA and NA genes and amino acid sequences of influenza A viruses in immunosuppressed and immunocompetent patients pdm09 virus in this study exhibited 11 key substitutions, and NA of A(H1N1)pdm09 virus exhibited 12 key substitutions, as shown in Table Compared with the vaccine strains of A/Hong Kong/4801/2014, A/Singapore/INFIMH-16–0019/2016, and A/Kansas/14/2017, HA of AH3N2) virus in this study exhibited 10 key substitutions, and NA of A(H3N2) virus also exhibited 10 key substitutions, as shown in Table N2 virus Both immunocompetent and immunosuppressed patients have some key amino acid variations. The oseltamivir resistance substitution of NA-H275Y and NA-R292K have been observed in immunosuppressed patients. One A(H1N1)pdm09 virus strain showed V190I mutation at the HA receptor binding site 190 helix. HA-A158E mutation of A(H1N1)pdm09 and I156L/K mutation of A(H3N2) were also be found. V190I and I156L mutation came from immunocompetent patients, while A158E and I156K mutation came from immunosuppressed patients. N222K and G298A near enzyme active center of NA in A(H1N1)pdm09 were also derived from immunosuppressed patients.In this study, we described clinical characteristics of influenza A infections with vital amino acid variations pdm09 include Sa, Sb, Ca, Cb, Pa and Pb, and antigenic epitopes of A(H3N2) include A, B, C, D and E. Both A(H1N1)pdm09 and A(H3N2) have same receptor-binding domain (RBD) on HA, which is composed of 190 helices, 130 rings and 220 rings . In thisActivity of NA protein is an important component of influenza virus infection. The epitopes of NA protein consist of 83–143, 156–190, 252–303, 330, 332, 340–345, 368, 370, 387–395, 400, 431–435 and 448–468. The enzyme catalytic sites of NA protein include the central site of enzyme activity  and the auxiliary site  [One NA-H275Y mutation and two NA-R292K mutations were found, which were the first time that our team found drug-resistant mutations at H275 and R292 sites. In order for oseltamivir to bind correctly, NA must be rearranged to form a pocket, and the key of rearrangements is E277 rotation so as to bind to R225. Vitro modeling and X-ray crystallography have shown that H275Y inhibits the rotation of E277 residues, thereby preventing the formation of pockets . In thisThis study provided complete and comprehensive data on gene evolution and amino acid variation of HA and NA in immunosuppressed patients with influenza A infections, and described clinical characteristics of key amino acid variations of HA and NA. A(H1N1)pdm09 and A(H3N2) viruses showed similar evolutionary lineages patterns of HA and NA between immunosuppressed and immunocompetent patients. Both immunocompetent and immunosuppressed patients have some key substitutions, which should be of note monitored, especially those with potential to affect the viral antigen. However, the number of this study may not be sufficient to draw the firm conclusions, further studies with a larger sample size will be needed to confirm and extend our findings.Additional file 1: Table 1. Nucleotide similarity of HA and NA genes of Apdm09 compared to vaccine strains.Additional file 2: Table 2. Amino acid similarity of HA and NA genes of Apdm09 compared to vaccine strains.Additional file 3: Table 3. Nucleotide similarity of HA and NA genes of Acompared to vaccine strains.Additional file 4: Table 4. Amino acid similarity of HA and NA genes of Acompared to vaccine strainsAdditional file 5: Table 5. Clinical characteristics of influenza A infections with key amino acid variations.


S)-hydroxycholesterol reduced co-immunoprecipitation of both TMEM97 and PGRMC1 in immune pellets of immunoprecipitated TSPO in MP cells. To the best of our knowledge, this is the first suggestion of a  interaction between TSPO and TMEM97 that can be affected by S2R ligands.Sigma-2 receptor (S2R) is a S2R ligand-binding site historically associated with reportedly 21.5 kDa proteins that have been linked to several diseases, such as cancer, Alzheimer’s disease, and schizophrenia. The S2R is highly expressed in various tumors, where it correlates with the proliferative status of the malignant cells. Recently, S2R was reported to be the transmembrane protein TMEM97. Prior to that, we had been investigating the translocator protein (TSPO) as a potential 21.5 kDa S2R candidate protein with reported heme and sterol associations. Here, we investigate the contributions of TMEM97 and TSPO to S2R activity in MCF7 breast adenocarcinoma and MIA PaCa-2 (MP) pancreatic carcinoma cells. Additionally, the role of the reported S2R-interacting partner PGRMC1 was also elucidated. Proximity ligation assays and co-immunoprecipitation show a functional association between S2R and TSPO. Moreover, a close physical colocalization of TMEM97 and TSPO was found in MP cells. In MCF7 cells, co-immunoprecipitation only occurred with TMEM97 but not with PGRMC1, which was further confirmed by confocal microscopy experiments. Treatment with the TMEM97 ligand 20-( The sigma-2 receptor (S2R) is an 18–21 kDa membrane protein that is a potential biomarker of the proliferative status of solid tumors . Many S23H]DTG binding, the same was not true of PGRMC1 DTG -yl)propyl]-5-methoxy-1,2,3,4-tetrahydroquinoline hydrochloric salt) PK 11195, in the absence  and in the presence of 10 µM PK11195 as reference compound (non-specific binding) were suspended. The samples were incubated for 90 min at 25 °C. The incubation was stopped by rapid filtration on Whatman GF/C glass microfiber filters (pre-soaked in 0.3% polyethylenimine for 20 min). The filters were washed with 3 × 1 mL of ice-cold buffer . The dissociation constant KD and the receptor density Bmax were measured by the nonlinear fitting of the specific binding vs. the radioligand concentration using Prism software.In 0.5 mL of incubation buffer , 100 µg of tumor cells membranes, different concentrations of the radioligand [3H]DTG, 1 µM (+)-pentazocine to mask sigma-1 subtype, in the absence  and in the presence of 10 µM DTG as reference compound (non-specific binding) were suspended. The samples were incubated for 120 min at 25 °C. The incubation was stopped by rapid filtration on Whatman GF/B glass microfiber filters (pre-soaked in 0.5% polyethylenimine for 60 min). The filters were washed with 3 × 1 mL of ice-cold buffer . The dissociation constant KD and the receptor density Bmax were measured by the nonlinear fitting of the specific binding vs. the radioligand concentration using Prism software.In 0.5 mL of incubation buffer , 400 µg of tumor cells membranes, different concentrations of the radioligand [3H]PK1195 and [3H]DTG were purchased from Perkin-Elmer Life Sciences . (+)-Pentazocine and PK1195 were purchased from Sigma Aldrich ; DTG was from Tocris. The KD values and SEM were obtained using non-linear curve fitting .[7), seeded in 10 cm dishes, were washed with PBS before lysis in 1 mL of cold low-stringency lysis buffer  and treated as previously reported [Cells (1 × 10reported . Briefly2 flask until they reached approximately 70% confluence. Cells were washed twice with ice-cold PBS and incubated with 750 µL radioimmunoprecipitation assay buffer (RIPA buffer)  supplemented with protease and phosphatase inhibitor cocktail . Lysed cells were harvested with a scraper and centrifuged at 8000× g for 20 min  at 4 °C. Protein concentration was measured using the Pierce BCA protein assay kit  following the manufacturer’s instructions. Proteins were denatured after mixing with 2× Laemmli loading buffer (1:1) at 95 °C for 5 min in a dry bath heater . Lysates (30 µg) were loaded to 10% SDS-PAGE 15-well gels  and electrophoresed at 150 V for 45 min. Wet transfer by chilled Towbin buffer  methanol (pH 8.3)) was used to transfer the protein to the PVDF membranes  at 20 V for 1.5 h on mini trans-blot cell .Cells were maintained in complete media in a T75 cmMembranes were incubated with TBS-T  containing 5% instant skimmed milk powder  for an hour. After washing twice with washing buffer TBS-T, the PVDF membranes were incubated with anti-PBR Rabbit , anti-TMEM97 , anti-PGRMC1  and anti-β-actin  and mouse anti-actin  at 1:1000 and 1:2000 respectively overnight at 4 °C. Next day, and the PVDF were washed twice with TBS-T and incubated with Donkey anti-Rabbit IgG H&L (HRP)  or Donkey F (ab’) 2 Anti-Mouse IgG H&L (HRP)  at 1:4000 for 1 h at room temperature. The bands’ detection was performed using Clarity Max Western ECLt-test was performed for pairwise comparisons. Differences between more than two groups were evaluated by one-way ANOVA followed by Dunnett’s multiple comparisons tests. For multiple cell lines and multiple treatments, 2-way ANOVA was used. For each experiment, the statistical method employed is specified in the figure legends. The asterisks in the figures denote statistical significance .Statistics tools implemented in the GraphPad Prism software were used for statistical analyses. Data are represented as the mean ± standard deviation (SD) from at least three independent experiments. Student’s Escherichia coli Top10 strain cells. Briefly, plasmids were mixed gently with Competent E. coli Top10 cells. The mixture was incubated for 30 min on ice. After incubation, the tube was placed in a digital dry path heater (Corning® LSE™) set at 42 °C for 45 sec without shaking. Next, the tube was transferred to an ice bath for 5 min. Then, 900 µL of 2% Luria Broth  was added. The solution was mixed and incubated at 200 rpm for 60 min at 37 °C. After incubation, the solution was spread on LB agar  plates containing 100 µg/mL ampicillin and incubated at 37 °C overnight. Plasmid extractions were performed using the QIAprep Spin Miniprep Kit  according to the manufacturer’s protocol. The plasmids concentration was measured using the NanoDrop 2000 .Lentiviral-delivered shRNAs were constructed using Mission TRC1.5-pLKO.1-Puro series Lentiplasmids  targeting TSPO/PBR . A total of 0.4 µg Lentiviral plasmids encoding the TSPO  was transformed into Competent We generated lentivirus particles as described . BrieflySense and antisense strands for the siRNA  that include:254_GAGCUCUACCCAGUCGAGUUUAGAA254_CUAAACUCGACUGGGUAGAGCUCUUControl_254 GAGUCCACCGACUAGUGUUAUCGAAControl_254 CGAUAACACUAGUCGGUGGACUCUU5 MIA-PaCa-2 cells were seeded overnight in 2 mL complete media containing 4 µL Lipofectamine RNAiMAX Transfection Reagent  and 10 nM of control or siRNA in a 6-well plate. The next day, cells were treated with the same combination of media and transfection reagents. On the third day, 10% of the cells were harvested to measure the expression of the TMEM97 gene by RT-PCR [For transfection, 5 × 10y RT-PCR .6 cells in 10 mL of growth medium in 10 cm Petri dishes and incubated at 37 °C overnight. Cells were transfected with 17 µg of the pLKO.1 vector containing sh_RNA targeting TMEM97, as per standard protocol using FuGENE HD Transfection Reagent in Opti-MEM medium without serum. Vector-silencing cells were selected using puromycin. After transfection, cells were placed in a normal DMEM growth medium. After 1 day, cells were detached with trypsin/EDTA and replated into DMEM growth medium containing puromycin (2 µg/mL) and cultured for 25 days. Surviving cell clones were picked out and propagated separately in 6-cm Petri dishes in the same medium with 2 µg/mL puromycin. To suppress the reversion of the phenotype, all subsequent cell culture was carried out in DMEM growth medium as described above, supplemented with 2 µg/mL puromycin.The procedure to develop an MCF7 cell line with reduced TMEM97 (shMCF7TMEM97 cells) was carried out according to Abate et al.  with minRNA extraction was performed using the Total RNA mini kit  following the manufacturer’s instructions. RNA concentration was measured by NanoDrop 2000 . One µg of RNA was used to synthesize cDNA using a cDNA synthesis kit  in C1000 Touch™ Thermal Cycler. Primers for TMEM97 and Actin were synthesized at Monash University, Melbourne. TMEM97; Forward 5′-TACTTCGTCTCGCACATCCC-3′Reverse 5′-TTGCTGAACTCCTGCGGGTA-3′. Actin primers were Forward 5′-ACGACAT GGAGAAAATCTG-3′ Reverse 5′-ATGATCTGGGTCATCTTCTC-3′.® Green Supermix .RT-PCR was performed at CFX96 Touch™ Real-Time PCR Detection System using iTaq™ Universal SYBR5 cells) were seeded in 6-well plates overnight. The next day, cells were incubated with either DMSO (control), 10 nM DTG  to block the S1R, or 4 nM RHM-1 (supplied by R.H.M.) to block the S2R. Then, the cells were incubated with 30 nM SW120 for 30 min. The cells were washed briefly with PBS three times to remove non-specific binding. The cells were trypsinized immediately and transferred to a falcon tube. Next, the cells were centrifuged at 1300× g for 3 min. The supernatant was discarded, and the pellets were washed twice with PBS and centrifuged. The supernatant was discarded, and the cells were resuspended in 500 µL PBS. The flow cytometry was performed by implementing a 488 nm laser for the excitation of FITC.Cell lines  of the labeled cells was measured by flow beck flow cytometry. The control cells were incubated with 30 nM of SW120 for 30 min and measured by flow cytometry.2 in a 150i CO2 incubator . After attachment to the coverslips, cells were washed twice with ice-cold PBS. Next, the cells were fixed using 3.6% paraformaldehyde in PBS pH for 10 min at room temperature. Subsequently, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. PLA was performed using a Duolink PLA in situ starter kit . The cells were blocked with 1 drop (~40 µL) of Duolink blocking solution for 1 h in a heated humidity chamber for 60 min at 37 °C. After blocking, the cells were incubated with diluted primary antibodies 1:100 in the Duolink antibody diluent overnight at 4 °C. The next day, the cells were washed twice with wash buffer A at room temperature for 5 min. After washing, slides were incubated with corresponding PLUS and MINUS PLA probes at 1:5 dilution in Duolink antibody diluent for 1 h at 37 °C. Slides were washed twice for 5 min with wash buffer A at room temperature and incubated with 1:40 ligase in 1x Duolink ligation buffer in high-purity water for 30 min at 37 °C. This was followed by washing twice with wash buffer A at room temperature for 5 min. For amplification, slides were incubated with 1:80 polymerase in an amplification buffer in high-purity water for 100 min at 37 °C. Slides were washed twice with wash buffer B at room temperature for 10 min, followed by a wash with 0.01x wash buffer B for 1 min at room temperature. Slides were mounted with a coverslip using a minimal volume of Duolink in Situ Mounting Medium with DAPI.Coverslips were sterilized by draining in 70% EtOH, washed with PBS, and left under the UV for 30 min. Wet coverslips with PBS were placed on a 6-well plate. MP cells were seeded at <50% confluence in DMEM  supplemented with 10% FBS  and 1% penicillin-streptomycin  at 37 °C and 5% CO3/spot in 50 µL) were seeded on spot slides and allowed to recover for 48 h at 37 °C and 5% CO2. Afterward, the cells were incubated with 10 µM of (+)-pentazocine as a selective S1R ligand for 2 h at 37 °C and 5% CO2. Subsequently, the cells were treated with 5 µM of CY5-labeled S2R ligand for 1 h at 37 °C 5% CO2, followed by a washing step with PBS. Then, the cells were fixated with 4% paraformaldehyde at room temperature for 15 min. For staining of TSPO or PGRMC1 expression, cells were permeabilized and blocked with 0.5% Triton-X 100 and 1% bovine serum albumin (BSA), respectively, in PBS for 15 min at room temperature, followed by incubation of the primary TSPO  or PGRMC1  antibodies diluted in 1% BSA 0.3% Triton-PBS overnight at 4 °C in a wet chamber. On the next day, the cells were washed three times with PBS and incubated with a 1:500 dilution of a secondary anti-rabbit AlexaFluor488-labeled antibody , cat.-nr.: A-11034) for 1 h at room temperature in a wet chamber. After two washing steps with PBS, the cells were incubated with a solution containing DAPI (2.5 µg/mL #D9542) for 15 min at room temperature, followed by three washing steps and embedding with Vectashield . Afterward, confocal microscopy was performed on a Zeiss LSM 700  using a Plan-Apochromat 63x/NA 1.4/Oil lens. The pinhole size was set to 1AU. The samples were illuminated with 405 nm, 555 nm, and 639 nm lasers and 1024 × 1024 pixel images were acquired using a PMTs detector. In total, 3 pictures per spot were obtained.MCF7 cells (3 × 10In conclusion, our reported co-immunoprecipitation and colocalization of TMEM97/S2R with TSPO is a novel finding whose characterization requires further investigation. In addition, PGRMC1, which has already been reported as a TMEM97/S2R interactor, co-immunoprecipitates with TSPO in MP cells but not in MCF7. The different interacting partners in the two cell lines which we employed suggest that the TMEM97 interactome is context-dependent. Importantly, the PGRMC1-TSPO-TMEM complex in MP cells is sensitive to the presence of S2R/TMEM97 endogenous ligand 20S-OHC, as well as exogenous ligands, suggesting that the complex is involved in S2R cell biology. Overall, the previously unreported protein-protein interactions of these three proteins shed new light on their biology and functions and deserve further investigation that may disclose unexplored mechanisms of action and therapeutic potentials.


The Hypoxia inducible gene domain family member 2A (HIGD2A) protein is indispensable for the assembly of the mitochondrial respiratory supercomplex, which has been implicated in cell proliferation and cell survival under hypoxic conditions. Because the liver has a naturally low oxygen microenvironment, the role of HIGD2A in the development of hepatocellular carcinoma (HCC) remains largely unknown.. In vivo and in vitro assays were performed to investigate the biological roles of HIGD2A.Gene expression data and clinical information were obtained from multiple public databases. A lentivirus-mediated gene knockdown approach was conducted to explore the function and mechanism of HIGD2A activity in HCC cellsHIGD2A was overexpressed in HCC tissues and cell lines and was associated with a worse prognosis. Silencing HIGD2A expression significantly attenuated cell proliferation and migration, caused S-phase cell cycle arrest, and decreased tumor formation in nude mice. Mechanistically, HIGD2A depletion greatly decreased cellular ATP levels by disrupting mitochondrial ATP production. Moreover, HIGD2A knockdown cells displayed impaired mitochondrial function, such as mitochondrial fusion, increased expression of the mitochondrial stress response protein, and decreased oxygen consumption. Furthermore, knockdown of HIGD2A markedly attenuated the activation of the MAPK/ERK pathway.HIGD2A promoted liver cancer cell growth by fueling mitochondrial ATP synthesis and activating the MAPK/ERK pathway, suggested that targeting HIGD2A may represent a new strategy for HCC therapy.The online version contains supplementary material available at 10.1186/s12967-023-04105-7. Hepatocellular carcinoma (HCC) is one of the most common types of primary liver cancer and is the third leading cause of cancer-related deaths worldwide . The 5-yCellular mitochondria are critical for multiple cellular functions, including cell respiration, biosynthetic metabolism, energy production, calcium homoeostasis, immune defense, and apoptosis –6. InterHypoxia inducible gene domain family member 2A (HIGD2A) is a small protein of 106 amino acids embedded in the inner membrane of mitochondria and can be induced by exposure to hypoxia and low glucose , 20. HIGThe aim of this study was to investigate the role of HIGD2A in HCC development and to explore potential mechanisms. We conducted a comprehensive evaluation of the expression of HIGD2A in HCC and examined the association between the expression of HIGD2A and the prognosis of patients with HCC. Furthermore, to explore the role of HIGD2A in liver cancer biology, the effects of knocking down HIGD2A expression on cell proliferation, apoptosis, migration, and cell cycle were evaluated in vitro using HepG2, Huh7, and MHCC97H cell lines. Additionally, the function of HIGD2A in HCC progression was further validated in murine subcutaneous tumor xenograft models. Mechanistically, we explored the effects of HIGD2A knockdown on mitochondrial structure and function, as well as on the MAPK/ERK signaling pathway. Our findings demonstrated that elevated HIGD2A expression promotes the development of HCC by enhancing mitochondrial function and activating the MAPK/ERK pathway, which can be considered as a potential therapeutic target for HCC.https://portal.gdc.cancer.gov/ and https://dcc.icgc.org/projects/LIRI-JP, respectively). The datasets of the GSE14520 and GSE64041 cohorts were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). Immunohistochemical (IHC) images of the HIGD2A protein in normal and tumor liver tissues were obtained from the Human Protein Atlas database (http://www.proteinatlas.org/) [http://www.gsea-msigdb.org/gsea/index.jsp).Datasets from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts were downloaded (as.org/) . Survivaas.org/) , 24. A gTwenty-four patients with HCC who underwent tumor resection were randomly selected. Paired samples of HCC tissue and adjacent normal liver tissue were collected at the Nanfang Hospital of Southern Medical University  from 1 September 2019 to 1 February 2020. This study was approved by the Medical Ethics Committee of Nanfang Hospital of Southern Medical University, and written informed consent was obtained from participating patients.2. The lentiviruses for shRNA-HIGD2A (shHIGD2A) and shRNA-scramble (shCtrl) were purchased from Shanghai Obio Technology Co. Ltd. (China). Lentiviral transduction was performed according to the manufacturer’s instructions. Cell transfection was performed using lipo3000  as recommended by the manufacturer. PcDNATM3.1-flag-BCL2L1 plasmid expressing human BCL2L1(GenBank No. NM_138578) was constructed by PCR from the respective cDNA clones using flag-tag encoding oligonucleotides followed by insertion into the pcDNATM3.1 vector .The human normal hepatic cell line L02 and the human HCC cell lines HepG2, Huh7, and MHCC97H were obtained from the Shanghai Cell Bank of the Academy of Chinese Sciences and Liver Cancer Institute, Zhongshan Hospital, Fudan University (China). All cell lines were cultured in complete Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin and 0.1 mg/mL streptomycin . All cells were kept in a humidified incubator at 37 °C with 5% COCell protein samples were lysed with loading buffer  and were then heated at 100 °C for 10 min. Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane  for immunoblotting with the following primary antibodies: anti-HIGD2A antibody , anti-Mfn1 antibody , anti-OPA1 antibody , anti-CD133 antibody , anti-EpCAM antibody , anti-JNK , anti-pJNK , anti-Erk1/2 , anti-pErk1/2 , anti-P38 , anti-pP38 , anti-caspase3 , anti-cleaved caspase3 , anti-BCL2L1  and anti-β-actin antibody  at 1:1000 dilution. The membranes were then incubated with secondary antibody for 1 h at room temperature. Finally, protein bands were visualized using ECL Prime Western Blot Detection Reagent  and detected with ImageQuant LAS 4000mini .Total RNA was extracted using the EZ-press RNA Purification Kit  according to the manufacturer’s instructions. Real-time PCR was performed using the EZ-press One Step qRT-PCR Kit (EZBioscience) on the Roche 480 real-time PCR machine (Roche). β-Actin served as an internal control for real-time PCR. All primers used for real-time PCR are listed in Additional file A cell proliferation assay was conducted using the Cell Counting Kit-8 . After 48 h of lentivirus infection, HCC cells were seeded in 96-well plates (1000 cells/well) and incubated at 37 °C for 24, 36, 48, 72, and 96 h. Subsequently, the medium was added with 10 µL CCK-8 per well. The absorbance at 450 nm was measured after incubation for 2 h using Gen5 software .For the colony formation assay, after 48 h of cell infection, cells were seeded in a 6-well plate at a density of 2000 cells per well and incubated for 10 days. The cells were then fixed in 4% paraformaldehyde for 15 min, washed three times with PBS, and then stained with 1% crystal violet for 15 min. The number of colonies was counted using ImageJ software after staining the plates.For the evaluation of cell apoptosis, lentivirus-infected cells were collected by trypsinization, centrifuged, and washed with PBS. The cells were then suspended in 200 µL binding buffer with 5 µL Annexin V and 5 µL 7-AAD  for 15 min at room temperature. After staining, the percentage of apoptotic cells was analyzed using the FACS Canto™ II flow cytometer .To analyze the cell cycle, cells were harvested and fixed with 70% precooled ethanol at 4 °C overnight. After two washes, the fixed cells were incubated with 0.5 mL of PI/RNase staining buffer  at room temperature for 15 min and then analyzed by flow cytometry.4 infected cells were suspended in 100 µL of serum-free medium and then seeded in the upper chamber of an 8 µm-microporous filter . The lower chamber of the Transwell was filled with 700 µL of medium supplemented with 20% FBS. After 48 h, cells that had not migrated were gently removed from the upper chambers and cells at the bottom of the chamber were fixed with polyformaldehyde for 15 min. The fixed cells were then stained with 1% crystal violet for 15 min. After two washes with PBS, the number of migrated cells was evaluated by microscope  and captured images were evaluated with ImageJ software.To explore the effects of HIGD2A on cell migration, 5 × 106 cells were used to detect intracellular ATP levels using the ATP Assay Kit  according to the manufacturer’s instructions.HepG2, Huh7 and MHCC97H cells were trypsinized and washed with PBS. A total of 1 × 104 cells were seeded in a confocal microscopy dish and incubated for 24 h. The medium was removed from the plate and Opti-MEM containing 200 nM MitoTracker® Red CMXRos  was added. After incubation for 20 min, cells were washed twice with PBS and fixed in 4% paraformaldehyde for 20 min. Following three washes with PBS, the samples were counterstained with DAPI  for 5 min. Fluorescence was detected using a ZEISS LSM 880 confocal microscope with Airyscan . 3D rendering was performed with Imaris software .A total of 5 × 10Oxygen consumption was determined using a Seahorse XF cell Mito Stress Test kit  according to the manufacturer’s instructions. Briefly, HepG2, Huh7, and MHCC97H cells were infected with the lentivirus shHIGD2A or shCtrl and seeded onto Seahorse cell culture microplates at a density of 10,000, 7200, and 9000 cells/well, respectively. After more than 24 h, the oxygen consumption rate (OCR) was determined by the Seahorse XFe96 extracellular flux analyzer. Plates were analyzed in the presence of 1.5 µM oligomycin, 1.0 µM FCCP, or 0.5 µM rotenone/antimycin. Basal respiration and respiration capacity were measured.d-glucose (2-DG) at 50 mM were used. The glycolytic proton efflux rate (GlycoPER) was calculated.The Seahorse XF Glycolytic Rate Assay kit was used to assay the glycolytic rate. HepG2 and Huh7 cells infected with lentivirus shHIGD2A or shCtrl were seeded as described for the Mito Stress Test. Rotenone/Antimycin at a concentration of 0.5 µM and 2-deoxy-2Fold change| > 1 and a false discovery rate (FDR) < 0.05. Reactome enrichment analysis on DEGs was executed by ClusterProfiler software, and P-values < 0.05 were considered significantly enriched.Total RNA was extracted from shCtrl- and shHIGD2A-infected HepG2 cells using a Trizol reagent kit  and libraries for RNA-sequencing were constructed. Differentially expressed genes (DEGs) was analyzed using DESeq2 software using the selection criteria of |log6 cells for each mouse) was injected into the right flank of each mouse. The growth of the xenografts was monitored at the indicated time points by measuring the tumor length (L) and width (W) to calculate tumor volume (V), using the following formula: V (mm3) = 0.5 × L × W2. On day 24 after inoculation, the mice were euthanized, and the excised tumors were weighed, photographed, and fixed in 4% paraformaldehyde overnight.All animal study protocols were approved by the Animal Ethics Committee of the Nanfang Hospital of Southern Medical University (China). All male BALB/c-nu nude mice (3–5 weeks) were obtained from Hunan SJA Laboratory Animal Co., Ltd (China); animals were fed in a specific pathogen-free (SPF) vivarium under standard conditions. To construct the subcutaneous xenograft tumor model, HepG2, Huh7, and MHCC97H cells were infected with the lentivirus shHIGD2A or by a shCtrl. A 50-µL volume of cell suspension  for 15 min. The sections were blocked with a PBS solution containing 5% BSA at room temperature for 1 h and incubated with Ki67 antibody  at 4 °C overnight. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide at 37 °C for 15 min. Then, an anti-rabbit/mouse IgG-HRP-linked secondary antibody  was added for 1 h at room temperature. The sections were developed using 3-diaminobenzidine tetrahydrochloride at room temperature for about 2 min. Mayer’s hematoxylin was applied to stain the nucleus. Representative field photographs were captured using the BX63 microscope (Olympus) and were analyzed using ImageJ software.The Wilcoxon signed-rank test and Kruskal–Wallis test were performed to analyze the association between HIGD2A and clinical features in HCC. Kaplan–Meier analysis was used to compare the OS rate between the high and low HIGD2A gene expression groups using the p-value determined by the log-rank test. All statistical analyses were performed using R statistical software version 3.6.3 and 4.1.2. All data relative to the in vitro and in vivo experiments were analyzed using GraphPad 9.0. The Mann–Whitney U test or Student’s t-test were used to compare differences between the HIGD2A knockdown groups and the control group. Data normality was tested using a Shapiro–Wilk test. A p-value < 0.05 was considered statistically significant.To investigate the potential significance of HIGD2A in the development and progression of HCC, we first analyzed multiple public databases, TCGA, ICGC, GSE14520, and GSE64041, and found that the expression of HIGD2A was significantly up-regulated in liver cancer tissues compared to the normal tissues  staining to determine the effects of HIGD2A depletion on cell cycle distribution. Interestingly, silencing of HIGD2A resulted in an increase in the percentage of cells in the S phase compared to controls, suggesting that depletion of HIGD2A induced the arrest of the S phase in HepG2 and Huh7 cells Fig. A, B. To Generally, the process of cell proliferation requires high consumption of ATP. Cancer cells often exhibit increased aerobic glycolysis to meet the metabolic demands of rapid cell proliferation and aggressiveness . TherefoMitochondria are considered the 'powerhouse' of the cell, as they supply most of the cellular energy demand for ATP. Mitochondrial dynamics with repetitive fission and fusion cycles is important to ensure the maintenance of normal cell functions. For example, mitochondria are fused in the G1/S phase , and becConsistently, GSEA analysis of liver cancer samples from TCGA datasets indicated that genes related to oxidative phosphorylation were enriched in patients with HCC exhibiting greater expression of HIGD2A Fig. A. To chaTo uncover the molecular mechanisms underlying HIGD2A regulation of HCC proliferation, we performed RNA sequencing on shCtrl- and shHIGD2A.1-infected HepG2 cells. A total of 124 genes (66 up- and 58 down-regulated genes) exhibited significantly altered mRNA expression in shHIGD2A.1-infected cells, and the top 10 most significantly up-regulated and 10 down-regulated genes were selected  undergo enhanced glycolysis and oxidative phosphorylation (OXPHOS) compared to nonstem cells, and reducing OXPHOS levels weakens the stemness properties of LCSCs . Given tGiven the growth retardation phenotype detected in HIGD2A knockdown cells, we subsequently sought to determine whether silencing of HIGD2A expression influenced the proliferation of HepG2, Huh7 and MHCC97H xenografted tumors in nude mice. Consistent with the in vitro results, knock-down expression of HIGD2A in HepG2, Huh7 and MHCC977H xenografted tumors resulted in growth retardation and in significantly reduced in tumor volumes and weights compared to control tumors Fig. A–F. MeanIn this study, we detected an increased expression of HIGD2A in patients with liver cancer, and these higher levels of HIGD2A were associated with a poor prognosis. Knockdown of HIGD2A expression delayed in vitro cellular proliferation and caused cell cycle arrest in the S phase of HCC cells, which was associated with compromised mitochondrial function and reduced ATP production. Furthermore, HIGD2A knock-down inhibited the activation of the MAPK/ERK pathway and the expression of BCL2L1, which could promote liver cancer cell apoptosis and suppress cell proliferation Fig. . Thus, wThe respiratory supercomplex factor Rcf1 has two mammalian homologues, HIGD1A and HIGD2A . HIGD1A Mitochondrial dynamics is tightly regulated by fusion and fission proteins . Recent Given the improved understanding of the deregulation of cancer metabolism, targeting the mitochondria may act as a potentially powerful cancer therapy , 37. TheCSCs have the capacity for self-renewal and differentiation and contribute to tumorigenesis, tumor recurrence, metastasis, and therapeutic resistance . In HCC,There were some limitations in this study that should be considered. The first is that HIGD2A is not only highly expressed in tumor tissues but is also strongly expressed in highly proliferative tissues , which pOur study determined that knockdown of HIGD2A suppressed the proliferation and growth of HCC cells by interfering with the MAPK/ERK pathway and suggested that HIGD2A may play a role in abnormal mitochondrial metabolism in HCC. The present study provides novel evidence for the role of HIGD2A in hepatocarcinogenesis and may also have potential significance as a therapeutic target. Further studies should elucidate the precise mechanism of HIGD2A activity in the modulation of the MAPK/ERK pathway, and explore the role of HIGD2A in HCC metastasis, and as a feasible to target for liver cancer therapy.Additional file 1: Table S1. The shRNA sequences of HIGD2A and the primer sequences used in real-time PCR analysis. Figure S1. Diagnostic and prognostic value of HIGD2A expression and its correlation with clinical features. A Representative immunohistochemistry images of HIGD2A expression in normal liver tissues and liver cancer tissues from the HPA. B The protein level of HIGD2A in normal hepatocyte L02 cell line and different liver cancer cell lines. C–F HIGD2A expression in different status of T stage (C), pathologic stage (D), vascular invasion (E) and OS event (F). G–J Kaplan–Meier plots of OS for HIGD2A expression levels in subgroups including T stage: T1 (G), N stage: N0 (H), M stage: M0 (I) and pathologic stage: stage I (J). K Nomogram for OS prediction, with T stage, N stage, M stage, histologic grade and expression of HIGD2A applied as parameters. L Calibration curves of the nomogram for 1-, 3-, 5-year survival prediction. *P < 0.05, **P < 0.01. Figure S2. Knockdown of HIGD2A impedes MHCC97H cells proliferation and migration. A Western blot assay for total HIGD2A protein expression in HIGD2A-knockdown MHCC97H cells. B Left, colony formation of MHCC97H cells transfected shCtrl or shHIGD2A.1. Right, quantification of colony formation based on three independent assays. C The proliferation ability of MHCC97H cells infected with shCtrl lentivirus or shHIGD2A.1 lentivirus was measured by CCK8 assay at the indicated time points. D, E Flow cytometry analysis of Annexin V/7-AAD double stained HepG2, Huh7 and MHCC97H cells transfected shCtrl or shHIGD2A.1. Representative flow cytometric plot (D) and the proportion of apoptosis cells (E). F Annexin V/7-AAD apoptosis assay of L02 cells after HIGD2A gene silencing. Left, representative flow cytometric plot. Right, the proportion of apoptosis cells. G Left, cell migration ability of MHCC97H cells transfected shCtrl or shHIGD2A.1 was analyzed by Transwell assay. Right, quantitative data of migrated cells. Results shown are mean ± SEM. an unpaired t-test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant. Scale bar, 100 µm. Figure S3. HIGD2A knockdown inhibited the proliferation and migration of HCC cells in vitro. A, B Colony formation experiments for the effect of HIGD2A knockdown with shRNA on the proliferation of HepG2 and Huh7 cells. C, D The effect of HIGD2A knockdown on the growth of HepG2 and Huh7 cells was detected by CCK8 assays. E The effect of HIGD2A knockdown on cell apoptosis was detected by western blot. F, G Transwell chamber was used to evaluated the effect of HIGD2A knockdown on the migration of HepG2 and Huh7 cells. Results shown are mean ± SEM. an unpaired t-test was used. *P < 0.05, **P < 0.01 and ***P < 0.001. Scale bar, 100 µm. Figure S4. Depletion of HIGD2A induces mitochondrial stress in MHCC97H. A, B Intercellular ATP level in MHCC97H (A) and L02 (B) cells transfected with shCtrl or shHIGD2A.1. C Immunofluorescent (IF) images showing mitochondria morphology in MHCC97H transfected with shCtrl or shHIGD2A.1. Left, representative IF images  and 3D mitochondria reconstruction of shCtrl- and shHIGD2A-infected MHCC97H cell. Scale bar, 5 μm; Sphericity heat map, 0.326–0.915. Right, sphericity analysis of 3D reconstructed mitochondria (results are presented as mean ± SD). D Left, oxygen consumption rate (OCR) in MHCC97H transfected with shCtrl or shHIGD2A.1 was measured by seahorse analyzer. Right, basal respiration rate and maximal respiration capacity are shown. E Comparison of Basal Respiration among L02 and HCC cells. Results shown are mean ± SEM. A Mann–Whitney test was used. *P < 0.05, **P < 0.01 and ***P < 0.001; ns, not significant.


Dai J, Liakath-Ali K, Golf SR, Südhof TC. 2022. Distinct neurexin-cerebellin complexes control AMPA- and NMDA-receptor responses in a circuit-dependent manner. Published 7 October 2022We were alerted to isolated errors in the source data files of Figure 1G and 5C by helpful comments on PubPeer and upon reanalysis of the data noticed additional isolated errors in the source data files of Figure 1B and 1F that were not noted on PubPeer. These errors were caused by copy-paste errors during the assembly of the final Excel files for publication. Additionally, we identified one error in the Figure 1—figure supplement 1 that arose during calculations of the coefficient of variation using Igor software. Since the apparent coefficient of variation is dependent on the stimulus intensities, we have systematically re-analysed all conditions to determine the average coefficient of variance as presented in the corrected dataset. None of the corrections change the conclusions of the paper. We sincerely apologize for these errors.The specific errors and needed corrections are described below:Figure 1B—Source data 1: In the source data file of Figure 1B, the EPSC amplitude values for the 100 μA stimulation intensity of cell 3 and cell 10 in the “Cbln2+/+” condition were mistakenly copied from a previously analysed dataset that used different EPSC trace selection criteria and methods. The original and corrected values are shown below. This change does not change the figure or the conclusion. The source data file has been updated accordingly.Corrected values for the data in Figure 1B in Figure 1—Source data 1:+/+” condition“Cbln2Stimulus Intensity: 100 μA.Cell 3 and cell 10 (pA): 85.332 and 69.236.Original values for the data in Figure 1B in Figure 1—Source data 1:+/+” condition“Cbln2Stimulus Intensity: 100 μA.Cell 3 and cell 10 (pA): 103.867 and 85.262.Figure 1F—Source data 1: In the source data file of Figure 1F, the PPRs values of cell 6 in the “Cbln2+/+” condition were erroneously copy-pasted from cell 5 of the same condition during assembly of the final Excel files for publication. The original and corrected values are shown below. This change does not alter the figure or the conclusion. In addition, the labels (Cbln2+/+ and Cbln2-/-) of Figure 1F in the source data were mislabelled and are now corrected. The source data file has been updated accordingly.Corrected values for the data in Figure 1F in Figure 1—Source data 1:+/+” condition“Cbln2PPR before and PPR afterCell 6 (PPR): 1.498 and 1.213.Original values for the data in Figure 1F in Figure 1—Source data 1:+/+” condition“Cbln2PPR before and PPR afterCell 6 (PPR): 1.646 and 1.390.Figure 1G—Source Data 1: In the source data file of Figure 1G, the EPSC amplitude values for the 100 μA stimulation intensity of cells 1, 2, 3, 7, 8, 13 in the “Cbln2+/+” condition were mistakenly copy-pasted from a previously analysed dataset that used different EPSC trace selection criteria and methods. The original and corrected values are shown below. This change does not change the figure or the conclusion. The source data file has been updated accordingly.Corrected values for the data in Figure 1G in Figure 1—Source data 1:+/+” condition“Cbln2Stimulus Intensity: 100 μA.cells 1, 2, 3, 7, 8, 13 (pA): 98.573, 143.800, 134.823, 133.315, 85.603, and 218.671.Original values for the data in Figure 1G in Figure 1—Source data 1:+/+” condition“Cbln2Stimulus Intensity: 100 μA.cells 1, 2, 3, 7, 8, 13 (pA): 127.587, 137.628, 130.657, 160.723, 91.116, and 212.394.Figure 5C—Source data 1: In the source data file of Figure 5C, the data for cell18 of the “Cbln12dcko” condition were erroneously copy-pasted from cell 17 of the Cbln12f/f condition during assembly of the final Excel files for publication. The original and corrected values are shown below. This change does not alter the figure or the conclusion. The source data file has been updated accordingly.Corrected values for the data in Figure 5C in Figure 5—Source data 1:dcko” condition“Cbln12Interstimulus interval (ms) 20, 80, 200, 800, 2000.cell 18 (PPR): 2.621, 1.646, 1.197, 1.036, 1.042.Original values for the data in Figure 5C in Figure 5—Source data 1:dcko” condition“Cbln12Interstimulus interval (ms) 20, 80, 200, 800, 2000.cell 18 (PPR): 2.419, 1.513, 1.329, 1.202, 1.055.Figure 1—figure supplement 1: Due to calculation errors when we used Igor software, we have conducted a comprehensive analysis of the coefficients of variation for each cell at all 7 stimulation intensities. We then calculated the average coefficient of variation for each cell and plotted them in the corrected figure and dataset. This correction does not alter the conclusion. The source data file has been updated accordingly.We show below the original and corrected versions of Figure 1—figure supplement 1.Corrected Figure 1—figure supplement 1:Original Figure 1—figure supplement 1:The corresponding figure legend has been corrected accordingly.Corrected Figure 1—figure supplement 1 legendCbln2 deletion does not alter the coefficient of variation of AMPAR-EPSCs in burst- and regular-spiking subiculum neurons .A & B) Left, sample traces of evoked AMPAR-EPSCs with 100 μA stimulus intensity ; right, summary graph of the average coefficient of variation for AMPAR-EPSCs across all 7 conditions of 10–100 μA stimulus intensity. Data are means ± SEM. Number of neurons/mice are indicated in bars. Statistical significance was assessed by unpaired two-tailed t-test revealed no significant differences..A & B) Left, sample traces of evoked AMPAR-EPSCs with 100 μA stimulus intensity ; right, summary graph of the coefficient of variation of AMPAR-EPSCs. Data are means ± SEM. Number of neurons/mice are indicated in bars. Statistical significance was assessed by unpaired two-tailed t-test revealed no significant differences.(We show below the original and corrected source data.Corrected Figure 1—figure supplement 1—source data 1Original Figure 1—figure supplement 1—source data 1The article has been corrected accordingly.


IgG3 is unique among the IgG subclasses due to its extended hinge, allotypic diversity and enhanced effector functions, including highly efficient pathogen neutralisation and complement activation. It is also underrepresented as an immunotherapeutic candidate, partly due to a lack of structural information. Here, we use cryoEM to solve structures of antigen-bound IgG3 alone and in complex with complement components. These structures reveal a propensity for IgG3-Fab clustering, which is possible due to the IgG3-specific flexible upper hinge region and may maximise pathogen neutralisation by forming high-density antibody arrays. IgG3 forms elevated hexameric Fc platforms that extend above the protein corona to maximise binding to receptors and the complement C1 complex, which here adopts a unique protease conformation that may precede C1 activation. Mass spectrometry reveals that C1 deposits C4b directly onto specific IgG3 residues proximal to the Fab domains. Structural analysis shows this to be caused by the height of the C1-IgG3 complex. Together, these data provide structural insights into the role of the unique IgG3 extended hinge, which will aid the development and design of upcoming immunotherapeutics based on IgG3. IgG3 antibodies have potent effector functions, but are not used as therapeutics and structural data are missing. Here, the authors combine cryoEM and MS to study IgG3-mediated complement activation to provide the first structural insights into IgG3. IgG subclasses are highly similar, sharing ~95% sequence homology in the heavy chain constant domains, and all contain a fragment crystallisable (Fc) domain linked to two fragment antigen-binding (Fab) domains via a disulfide-linked polyproline hinge4. IgG1, IgG2 and IgG4 are structurally homologous, with relatively short and rigid hinge regions containing 2-4 disulfide bonds, which limit the flexibility and size of the antibody complex7. In contrast, IgG3 contains a uniquely long hinge formed from a triple repeat of 15 amino acids that introduces 11 disulfide bonds and three O-linked glycosylation sites on each of the hinge polypeptides  is the predominant human immunoglobulin class in blood and comprises four subclasses that are named after their abundance in human serum; of all IgG in plasma, ~60% are IgG1, followed by ~30% IgG2, ~7% IgG3 and ~3% IgG411. This is in stark contrast with data describing the critical role of IgG3 in protection against pathogens2, with IgG3 having more potent effector functions than other IgG subclasses regarding pathogen neutralisation13, antibody-dependent cellular phagocytosis (ADCP)15, intracellular antiviral immunity16, and complement activation19. Recent advances in understanding IgG3 functions have revealed a critical role for protection against viruses, particularly SARS-CoV-220 and HIV21, which can synergise with complement activation to both neutralise virions and inhibit their disassociation within cells21.IgG3 is relatively less studied compared to other subclasses, in part due to its structural and functional heterogeneity, which has led to IgG3 being neglected as an immunotherapeutic candidate2s2, binds to antigen-associated antibodies , which then cleaves and activates the C1s SP. C1s propagates the cascade by cleaving C4 to form C4b, which undergoes a large structural rearrangement that exposes a reactive thioester group and enables covalent conjugation of C4b onto exposed surfaces and molecules24. Complement progression results in opsonization of the target membrane and formation of the membrane attack complex (MAC) pore, which perforates the lipid bilayer and lyses the cell  complex, composed of C1q and a heterotetramer of C1rHere, we use a combination of biophysical assays, mass spectrometry and cryo-electron tomography to provide structural data of antigen-bound IgG3. We observe arrays of densely-spaced Fab domains forming on lipid membranes, which could maximise the potential for pathogen neutralisation. Above these ordered Fab domains, elevated hexameric Fc platforms are visible that are consistently far from the liposome membrane and more highly exposed than for IgG1, which may reduce steric barriers to receptor binding. Upon the addition of complement components, we observe C1 complexes exclusively binding to these elevated IgG3-Fc platforms, and C1 adopts configurations related to C1 autoactivation and C4b deposition. Subtomogram maps of IgG3-C1-C4b reveal the importance of the extended IgG3 hinge, as it allows deposition of C4b onto the IgG3 Fab-hinge interface, with significant consequences for complement activation. These structural insights provide a wealth of data for the development and design of upcoming immunotherapeutics based on IgG3, especially regarding the length of IgG hinges for a given target antigen.2) were purified and their ability to activate complement confirmed using enzyme-linked immunosorbent assays (ELISA), which consistently showed better C1q binding to antigen-bound IgG3 compared to IgG1, as well as more efficient C4 deposition and MAC pore formation  showed that IgG3 was better than IgG1 at performing complement-mediated membrane lysis  IgG1 and IgG3  above and parallel to the liposome surface were visible above the Fab domains  from the membrane, similar to without C1. Incubation of liposomes with IgG and NHS at ambient temperatures before vitrification revealed extensive opsonization of the membranes with complement proteins and formation of MAC pores, which together form a protein corona , as well as C4b, a product of C1s -1, epidermal growth factor (EGF) and CUB2 domains -1/2 domains and the SP domain, were resolved Fig. . The two24. The TED contains the reactive thioester of C4 and is responsible for covalent attachment to membranes and proteins after cleavage to C4b32. However, in this map, the C4b TED was ~8 nm away from the liposome surface, and instead appeared to be bound to the upper IgG3-Fab or hinge regions  and serine, threonine and lysine residues in tryptic peptides from IgG were identified. All figures show data from samples with oxidised methionine . Of these, only I199T is located on the surface of the Fab domain, but is not well positioned to impact Fab-Fab binding. It is therefore more likely that the different hinge regions explain the ability of IgG3-Fabs to form ordered arrays. All IgG subclasses comprise two Fab domains and are capable of divalent antigen-binding. However, IgG1 forms hexameric Fc platforms when undergoing monovalent binding to surface antigens. Furthermore, enforcing monovalent binding by forming bispecific IgG1 was found to improve complement activation of other antigen-antibody complexes30. The short upper hinge region of IgG1, between the Fab and the first interchain disulfide bridge, comprises only 5 residues 2 fragment, which includes the upper hinge of IgG3, and the difference between IgG3 and IgG1 was lost when using monomeric Fab fragments38, in agreement with our structural data.Imaging antigen-bound IgG3 on fluid liposome surfaces revealed a propensity for Fab domain clustering and formation of an ordered array Fig. . Althougers Fig. . Indeed,rms Fig. . This dirms Fig. . Indeed,n = 100) above the liposome surface, with the hinge adopting an extended conformation  complexes46. Indeed, weak density potentially from neighbouring IgG3 complexes was present in our maps  and Factor I, or extended molecules, such as complement receptor 1 (CR1)25, may have more access to IgG3-associated C4b. Furthermore, hinge length is not linearly correlated with complement activation; IgG3 hinges can be too short or too long14. This may be due to C4b being deposited either on the lipid bilayer (for short hinges found in IgG1), or the antibody Fab/hinge region (for long hinges found in IgG3). Hinge lengths shorter than IgG1, longer than IgG3, or between these two values, may have impaired C4b deposition and therefore compromised complement activation. This could also depend on the location and environment of the antigen, and there is evidence that different antigens may have different ideal hinge lengths for C4b deposition49, which can now be explained by our structural data. It will be intriguing determine whether there is a correlation between antigen size and epitope location with complement activation by antigen subclass or IgG3 allotype.C4b was deposited directly onto lipid bilayers in IgM-C1 complexes in a vertical orientationcal Fig. . This is16. Human IgG3 allotypes represent enormous variability in the length of the hinge, and more than 15 allelic variations are currently known50, with hinge lengths varying from 32-62 amino acids. Furthermore, O-linked glycosylation at three sites, each of which are partially occupied, may also contribute to IgG3 hinge rigidity and hence persistence length8. How these allotypes and glycoforms contribute to IgG3 effector functions and C4b deposition remains unclear, although our data provide structural insights into the role of the IgG3 extended hinge.The more potent effector functions of IgG3 are generally attributed to the longer hinge of IgG3 compared to other antibodies51, combined with human constant domains. Isotype variants were based on human IgG1 (G1m(f) isotype) and IgG3 (P01860), all variants were co-expressed with kappa light chain. Gene constructs for heavy and light chains were designed to include a cleavable signal sequence for secretion and were flanked by restriction sites to allow for simple DNA cloning. DNA fragments for variable and constant domains of heavy and light chains were ordered separately from GeneArt  and cloned into a pcDNA3.3 vector  behind a CMV promotor.Recombinant anti-DNP IgG antibodies are based on variable domains mouse monoclonal antibody G2a2 against the hapten dinitrophenol (DNP)TM 2000c spectrophotometer .Antibodies were produced by transfecting 12.5 µg each of heavy chain plasmid and light chain plasmid in Expi293F™  cells using the ExpiFectamine™ 293 Transfection Kit  following the manufacturer’s instructions. After 5–7 days, transfection broth was centrifuged, the supernatant harvested and sequentially filtered through 0.45 µm and 0.2 µm filters . Antibodies were then purified on protein A  or in the case of IgG3 on either a protein A/G  or protein L cartridge  followed by concentrating and re-buffering to PBS using 30 kDa Amicon centrifugal filters . Concentration of the purified antibody preparations was determined by NanoDropTM 200 Increase 3.2/300 column , which was equilibrated with PBS on an ÄktaTM pure system  . For reduced conditions, samples were mixed with dithiothreitol  and Laemmli buffer and heated to 99 °C for 10 min before loading. Gel electrophoresis was performed in MOPS buffer  supplemented with 0.1% w/v sodium dodecyl sulfate  on a InvitrogenTM Mini Gel Tank electrophoresis device  for 35 min and 75 min for the reducing and non-reducing conditions, respectively. Gels were stained using SimplyBlueTM SafeStain  for 20 min and de-stained in deionised water overnight before imaging using a white light conversion screen in a ChemiDoc XRS+  . Liposomes displaying 1 mol% DNP and encapsulating 20 mM sulforhodamine B , composed of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), cholesterol and dinitrophenyl-cap-dipalmitoylphosphatidylethanolamine (DNP-cap-PE) (44:5:50:1 mol%) in PBS were generated as previously described27 using the CLARIOstar software (version 5.21 R2). Briefly, purified sulforhodamine B liposomes displaying 1 mol% DNP as an antigen source were mixed with ice cold NHS  and fluorescence intensity was measured for 100 s at 21 °C before 50 nM IgG was added. Complement activation was assayed for 30 min. All experiments were performed in triplicate. Data were baseline corrected and analysed in GraphPad Prism (version 9.3.1).Antibody mediated complement activity was measured via MAC pore formation by monitoring fluorescence sulforhodamine B release on a CLARIOstar microplate reader  as previously describedg using a Sigma 2-16k centrifuge , and the supernatant removed. The liposome pellet was resuspended in ice cold PBS and kept on ice till further use. Samples were diluted to equivalent concentrations in PBS and loaded, without heating denaturation, onto 4–12% pre-cast Bis-Tris Protein Gels) in MOPS (pH 7.7) supplemented with 0.1% w/v SDS After running gels at 200 V at room temperature for 75 min, polyacrylamide gels were stained using SimplyBlueTM SafeStain for 20 min and de-stained in deionised water overnight. Gels were stored in 1% acetic acid at 4 °C before imaging using a white light conversion screen in a ChemiDoc XRS+.Liposomes containing 1 mol% DNP were mixed with anti-DNP IgG3  and incubated at RT for 15 min. Next, samples were cooled down to 4 °C for 15 min. As a source of complement proteins, NHS  was added and incubated for 30 min at 4 °C. To purify liposome-IgG-C1-C4 complexes from unbound proteins, samples were centrifuged twice at 4 °C for 15 min at 20,000 2-Amine  and N-Succinimidyl N--6-aminocaproate  were mixed in PBS and DMSO at equimolar concentrations and incubated overnight at 37 °C with agitation at 1050 rpm in a ThermoMixer F2.0 . Ion-pairing high-performance liquid chromatography (IP-HPLC) was used to purify conjugated product with 0.1 M triethylammonium acetate  as an ion-pairing agent on a C18 coated stationary phase and acetonitrile . Samples were freeze-dried  overnight and resuspended in nuclease free water. Purified DNP-Biotin concentrations were measured on a UV Vis spectrometry at 360 nm and calculated using the DNP-NHS extinction coefficient (εnm360) of 17,500 cm−1 M−1.DNP-Biotin was produced via copper free click chemistry. Briefly, Biotin-PEG2CO3, 0.1 M NaHCO3, pH 9.6) for 1 h at 37 °C. After each incubation, plates were washed three times in PBS plus Tween-20 . Wells were blocked using 100 µl 3% bovine serum albumin (BSA) in PBS for 1 h at 37 °C. As an antigen source, 100 µl DNP-Biotin  in 1% BSA in PBS was added and incubated for 1 h at 37 °C. Next, 50 µl anti-DNP IgG antibodies  and NHS  diluted in RPMI 160 Medium  or RPMI alone were added to the wells and plates were incubated for 2 h at 37 °C. Afterwards, 50 µl primary antibody was added and incubated for 1 h at 37 °C. As a positive control, anti-DNP goat antibody  1:1000 diluted in 1% BSA in PBS was added to a well with only RPMI. To detect C1q, C4 and MAC, α-human C1q rabbit , α-human C4 goat , and α-human C5b-9 mouse  antibodies were used, respectively. All antibodies were diluted in 1% BSA in PBS. Next, 50 µl secondary antibodies conjugated to HRP were added and incubated for 1 h at 37 °C. As secondary antibodies, α-goat donkey with HRP , α-rabbit goat with HRP , and α-mouse goat with HRP  were used. Finally, 50 µl 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)  in citric acid buffer  with 0.015% (v/v) H2O2 were added and absorbance at 415 nm was measured after 15 min, 30 min and 45 min using a CLARIOstar microplate reader  using the CLARIOstar software (version 5.21 R2). Different complement protein detections were analysed separately in GraphPad Prism (version 9.3.1).To detect complement protein binding to IgG subclasses, Maxisorp Nunc Immunoplates  were coated with 100 µl 10 µg/ml Streptavidin  in coating buffer  at room temperature for 20 min. Afterwards, the sample was cooled to 4 °C for 20 min, before either ice-cold PBS or NHS  was added, followed by another 20 min incubation on ice. Before vitrification, ice-cold bovine serum albumin-coated 5 nm gold colloids were added. Freshly plasma-cleaned 200 mesh copper grids, with lacey-carbon support , were loaded into a Leica EMGP  and 6 µl sample was applied. Samples were incubated for 60 sec at 4 °C at 65% humidity, followed by 1.5 s blotting from the back and vitrification in liquid ethane. Grids were clipped and stored in liquid nitrogen until usage.52. Focusing between −3 µm and −6 µm and tracking were performed before each tilt acquisition. A total dose of 60 e−/Å2 was used.For tilt series acquisition, a Talos Arctica  operating at 200 kV with a K3 direct electron detector and a Bioquantum energy filter (Gatan) was used. The energy filter was set to a slit width of 20 eV. Tilt series were acquired using Tomography 5  in counting mode at ×49,000 magnification with a pixel size of 1.74 Å using a dose-symmetric scheme from 0 to ±57°, in 3° increments in a dose-symmetric tilt scheme53. Tomograms were reconstructed after 2  × binning of tilt series using IMOD (version 4.11) with weighed back-projection. CTF estimation as well as correction and gold beat erasing was done in IMOD (version 4.11).Before reconstruction, raw frames were aligned using the “alignframes” command from IMOD (version 4.11)54 command e2spt_boxer_old.py was used to manually pick 1193 from 55 tomograms and 2561 particles from 101 tomograms for IgG3 and IgG3 plus NHS, respectively. Particles were extracted, with contrast inversion and normalisation, with a box size of 180 pixels for IgG3 alone, or 256 pixels for IgG3-C1 complexes, with a final pixel size of 3.48 Å/pixel.EMAN2 (version 2.91)55. The initial model was used as a template and particles were refined using multiple rounds of alignment and multireference classification to align all of the particles to the lipid bilayer. The resulting overall IgG3 map was dominated by the Fab-membrane density  using e2proc3d.py. Further reconstructions were performed in Dynamo (version 1.1.157)To yield the IgG3-Fab array map, e2spt_boxer_old.py was used to manually pick 211 particles from the upper surface of the liposome shown in Supplementary Fig. 1, Supplementary Fig. For the reconstruction of the IgG3-C1-C4b complex, the EMAN2 command e2spt_binarytree.py was used to generate an initial model which was then lowpass filtered to 60 Å before subtomogram averaging using e2spt_classaverage.py from EMAN2 (version 2.91). Particles were split into even and odd datasets and all gold-standard procedures were followed. Particles were aligned over 20 iterations, with no symmetry applied and using a loose spherical mask centred on the Fc hexameric domain. The resulting map of the complete IgG3-C1-C4b complex reached 44 Å map Fig. . Next, t57. We did not have sufficient resolution to identify light (L) and heavy (H) chains within each Fab, so we used the exemplary model for the interface 5TDP to model the Fab array due to the similarity between the model and map distances 58, with the sequence for anti-DNP heavy and light chain variable domains59, and placed onto the 5TDP array using UCSF Chimera (version 1.14)60. This was then briefly simulated using Isolde (version 1.5)61 within UCSF ChimeraX (version 1.5)62 to remove steric clashes to yield the model shown in Fig. Fab domains with repeating motifs were identified from the protein database (PDB). Several common Fab-Fab interfaces were identified with the approximate distances observed here, which were; for the 3.8 nm repeat, 3WE6 and 5TDP; for the 5.25 nm repeat, 5TDP, 3HR5, 4JN2 and 5TDN  to form the hexameric IgG3-Fc platform. This was also briefly simulated using Isolde to remove steric clashes. To model the extended hinge regions, two polypeptide helices were constructed in UCSF Chimera (version 1.14) before disulfides were formed. This was allowed to relax by running a simulation in Isolde, although the exact structure of the hinge could not be determined from these data. Copies of this hinge polypeptide were placed between the Fab and Fc models, which were oriented to minimise the average hinge length, before peptide bonds were formed in UCSF Chimera (version 1.14) and simulated in Isolde (version 1.5). The relative positions of the Fc and Fab array could not be determined, and therefore other hinge topologies could be equally likely. The final IgG3 model is as shown in Fig. For the IgG3-Fc region, the PDB model with accession number 6D58 was used as a monomer. Multiple copies were placed in the C6-symmetrised IgG3-Fc map Fig.  in the s24). For C4b, the PDB model with code 4XAM23 was aligned to the PDB model with the code 5JPM, which displays the interaction of C4 with the complement protein MASP-2 , and as such the same starting orientation was used here. The fit of 4XAM was then refined by fitting into the subtomogram map of IgG3-C1-C4b as a rigid body using UCSF Chimera (version 1.14). Analysis of maps, models, and creation of figures was performed using UCSF ChimeraX (version 1.5)62.The C1 complex was modelled as described in the Supplementary Methods and Supplementary Fig. Protein bands were subjected to reduction and alkylation (dithiothreitol and iodoacetamide) followed by in-gel trypsin (Worthington) digestion using a Proteineer DP digestion robot . Peptides were extracted from the gel slices, lyophilised, dissolved in 100:0.1 water:formic acid (FA) v/v and subsequently analysed by on-line C18 nanoHPLC MS/MS with a system consisting of an Easy nLC 1000 gradient HPLC system , and an Orbitrap Fusion Lumos Tribrid mass spectrometer . Samples were injected onto a homemade precolumn  and eluted via a homemade analytical nano-HPLC column . The gradient was run from 2 to 40% solvent B (20:80:0.1 water:acetonitrile:FA v/v/v) in 30 min. Solvent A was 100:0.1 water:FA v/v. The nano-HPLC column was drawn to a tip of ∼5 μm and acted as the electrospray needle of the MS source.n = 1 was set, with an exclusion duration of 10 s.The mass spectrometer was operated in data-dependent MS/MS mode with a cycle time of 3 s. In the master scan (MS1) the resolution was 120,000 and the scan range 400–1500, at an AGC target of 400,000 with maximum fill time of 50 ms. For MS2, precursors (charge stated 2–5) were selected using the quadrupole with an isolation width of 1.2 Th. Fragmentation was performed using HCD at a normalised collision energy at 32% and recording of the MS2 spectrum in the Orbitrap. Dynamic exclusion after N-glycopeptides and IgG3 O-glycopeptides were identified by manual inspection of the raw LC-MS/MS data of the corresponding tryptic peptides using XCalibur software . To compare the overall N-glycan profile on the IgG1 and IgG3 antibodies used for our studies, summed MS-spectra covering the full width of the chromatographic peak of the tryptic peptides TKPREEQYNSTYR (IgG1) and TKPREEQYNSTFR (IgG3) carrying different N-glycans were generated.IgG1 and IgG3 Fc 65 as a node in PD. The database consisted of five proteins . Trypsin was selected as enzyme and two missed cleavages were allowed. Carbamidomethylation of cysteine and oxidation of methionine were selected as fixed and variable modifications, respectively. Precursor and fragment mass tolerance were set at 2 and 20 ppm, respectively. For the identification of thioester mediated crosslinked peptides, crosslinks between Q and S/T/K were defined (loss of NH3). Only crosslinked peptides between C4B and IgG1/3 with a score above 25 were selected for further analysis by manual interpretation of the MS/MS spectra.In a post-analysis process, raw data were first converted to peak lists using Proteome Discoverer (PD) . Subsequently, cross-linked peptides were identified using XlinkX (version 2.5)Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Because glass-ceramics generally have the advantage of structural rigidity and chemical durability over sintered polycrystalline ceramics, the present study employed glass-ceramic Narpsio to perform chemically driven ion exchange of Na+ with protonated water molecules with an aim to produce a proton conductor. The ion exchange was carried out in a hydrochloric acid solution by changing immersion time, temperature, and acid concentration. The ion exchanged Na4Y0.6P0.2Si2.8O9-based glass-ceramics were analyzed by the complex impedance method, and the proton conductivity was found to exhibit 3 × 10−4 Scm−1 at 300 °C with the activation energy of 59 kJ/mol. The dependence of humidity-sensitive conductivity of the ion exchanged bulk glass-ceramics was also examined; the conductivity increased almost linearly from 0.6 × 10−4 Scm−1 in dry air to 1.5 × 10−4 Scm−1 in 75% humid ambience at 300 °C. Thus, the ion exchanged glass-ceramics can be considered to be high temperature proton conductors as well as humidity sensors.We have developed 12-membered silica-tetrahedra-ringed Na Using the first-generation ceramic sodium ion conductor of Na-β/β″-alumina -type sodium rare-earth silicophosphates in the system Na2O-R2O3-P2O5-SiO2, which will be abbreviated as Narpsio in the following [5YSi4O12-type Narpsio conductors on the basis of the composition Na3+3x-yY1-xPySi3-yO9 with appropriate parameters of x and y. Because Na+ ion glass-ceramics can be synthesized with the various combination of the composition parameters x and y, the use of Narpsio has an advantage to develop suitable materials for practical devices. Our present aim is to produce bulk ceramic protonic conductors by ion exchange. Most of the abovementioned materials were polycrystalline ceramics or their powders. As glass-ceramics generally have structural rigidity against aqueous chemicals, we employed Naollowing ,11. We o5YSi4O12 structure consists of tunneling channels of Na+ ions surrounded by 12-membered rings of SiO4 tetrahedra along the c-axis, forming conduction paths. A unit cell contains 14 mobile (15%) and 76 bonding (85%) Na+ ions. Narpsio materials were also found to be appropriate for the production of glass-ceramics without phase separation during crystallization from original glasses [+, two times larger than Na+ in volume, to some extent [4Y0.6P0.2Si2.8O9 under  =  which gives rise to the maximum conductivity of 1 × 10−1 Scm−1 at 300 °C [+ ions in grains exchange with permeating H3O+ through grain boundaries. We firstly conducted ion exchange experiments using Narpsio powders to find the optimum conditions of the processing time, temperature, and solution concentration, then performed the production of protonic conducting bulk glass-ceramics under the appropriate conditions.The Na4 mobile % and 76  glasses ,18,19,20e extent . Considet 300 °C ,12,13,144 mobile % and 76 t 300 °C ,18,19,202CO3 , Y2O3 , NH4H2PO4 , and SiO2 . Those powders were weighed according to the composition of Na4Y0.6P0.2Si2.8O9, thereafter mixed by ball-milling. The mixture was calcined at the temperatures of 400 °C and 900 °C for 0.5 h to evacuate NH3 and CO2, respectively. The calcined powders were heated up to 1350 °C and melted in a Pt crucible for 1 h in air, then rapidly poured into cylindrical carbon dice to form glass. After annealing at 500 °C for 6h for nucleation, the annealed glass specimens were crystallized at 900 °C for 5 h according to the previous reports −pH (: 10before ion exchange)−pH −pH (: 10after ion exchange)−pH  observation was also performed on the bulk samples chemically etched with 3% HF solution to evaluate the microstructure. The conductive coatings were performed using sputtering of carbon. Differential scanning calorimetry (DSC) was carried out on the prepared samples to confirm the changes in thermodynamic parameters before and after ion exchange. The sample was packed in an aluminum pan, covered with an aluminum lid, and measured using a Rigaku DSC_8230. Thermogravimetric-mass spectrometry (TG-MS) was performed to analyze the gaseous components desorbed from the sample. The sample was packed in an alumina pan and measured simultaneously using a thermogravimetric -mass spectrometer  under the conditions of the heating rate of 10 °C min−1, the He gas flow rate of 20 mL min−1, and the sampling span of 10 s.Identification of the crystalline phases was performed on the crushed powders of crystallized specimens by the X-ray diffraction  method. The XRD measurements were calculated between 10 and 40° under the scanning speed of 3°·minElectrochemical impedance measurements were undertaken by the alternate current (AC) two-probe method on cylindrical glass-ceramics with a diameter and thickness of 14 mm and 2 mm, respectively. Electrodes were formed by sputtering gold on the polished surfaces. The ionic conductivities were measured by impedance spectroscopy . The applied AC field was in the frequency range from 10 mHz to 32 MHz, with a voltage amplitude of 300 mV. The temperature dependence of the conductivity was measured similarly at several temperatures ranging from 100 °C to 300 °C.+ conductive glass-ceramics was measured under water vapor atmosphere to evaluate the effect of relative humidity on H+ conductivity. The schematic diagram of the measurement apparatus is shown in φ: relative humidity/%, e: partial pressure of water vapor/hPa, t: temperature/°C, and se (t): saturated water vapor pressure/hPa. To calculate the saturated water vapor pressure, the Tetens equation shown in Equation (5) was used.a = 7.5, b = 237.3 (for water), a = 9.5, and b = 265.5 (for ice).Furthermore, the conductivity of HIn order to calculate the amount of water vapor depending on the temperature of the mantle heater, the dew point of the generated water vapor was measured using the low-pressure cooling dew-point method. The ionic conductivities in a steam atmosphere were measured using an Agilent Technologies 4294A-Precision-Impedance Analyzer, similar to the method described above. The applied AC field had a frequency range from 40 Hz to 100 MHz, with a voltage amplitude of 300 mV. The temperature dependence of the conductivity was measured similarly at several temperatures ranging from 100 °C to 300 °C.5RSi4O12-type by XRD by comparison with the international standard diffraction pattern (ICDD No. 00-032-1204 for Na5YSi4O12).The original glass and crystallized glass-ceramic specimens are compared to demonstrate the dense bodies in 5RSi4O12-type structure was maintained in the HCl concentration range of 0.01 to 0.1 M, whereas the crystalline phase was destroyed to amorphous over 0.2 to 1.0 M  =  as the target material for conversion of sodium to hydronium ion conductors by chemical treatment in acid solutions. Taking into account the structural rigidity and chemical durability of glass-ceramics, chemically driven ion exchange of Na+ with protonated water molecules in Na4Y0.6P0.2Si2.8O9-based glass-ceramics was performed. The value of σ at 300 °C was 3.8 × 10−4 Scm−1 with the activation energy of 58.6 kJ mol−1 for the most conductive bulk specimen. The ion exchanged bulk glass-ceramics showed humidity-sensitive conductivity; σ increased from 0.6 × 10−4 Scm−1 in dry air to 1.5 × 10−4 Scm−1 in 75% humid ambience at 300 °C. Although the conductivity of glass-ceramics decreased in the order of 10−2 Scm−1 by ion exchange of Na+ with H3O+, the value of σ of ion exchanged glass-ceramics still can be anticipated to be on the level of practical applications with further efforts. The ion exchange is a promising method to convert the charge carrying ions to the different ones because the finding of a novel conductor is unimaginably beyond our effort. The use of glass-ceramics is also noticeable because the fabrication of thinner bulk specimens can be realized through the glass making process, where melts are thinned during cooling. Considering those circumstances, we are conducting the research to optimize ion exchange conditions for glass-ceramic materials to improve the proton conductivity up to ca. 10−2 Scm−1 for practical applications.The present study focused on the fundamental Na