To simultaneously optimize carrier concentration, lower thermal conductivity and improve the stability, BiCuTeO, an oxygen containing compound with lower carrier concentration, is in situ formed in Cu2Te by a method of combining self-propagating high-temperature synthesis (SHS) with spark plasma sintering (SPS). With the incorporation of BiCuTeO, the carrier concentration decreased from 8.1 × 1020 to 3.8 × 1020 cm−3, bringing the increase of power factor from ~1.91 to ~2.97 μW cm−1 K−2 at normal temperature. At the same time, thermal conductivity reduced from 2.61 to 1.48 W m−1 K−1 at 623 K. Consequently, (Cu2Te)0.95-(BiCuTeO)0.05 composite sample reached a relatively high ZT value of 0.13 at 723 K, which is 41% higher than that of Cu2Te.Because of the high carrier concentration, copper telluride (Cu With the capability of direct conversion from thermal energy to electrical energy through the movement of internal phonons and charge carriers, thermoelectric (TE) materials are widely researched and used in cogeneration, electronic refrigeration and thermal sensors ,4,5,6,7.ectively ,9. Thereiltering ,14,15,16ineering ,20,21,222Te3 [2 [2Te) [2Se) [2S) [2Se and Cu2S, Cu2Te should have more appealing thermoelectric performance owing to the lower ionicity of chemical bonds between Cu and Te due to the lower electronegativity of Te in comparison to Se or S, which is beneficial for obtaining large carrier mobility (μ) and improving its carrier conductivity. Besides, the lattice thermal conductivity (κL) of Cu2Te is expected to be lower than that of Cu2S and Cu2Se because tellurium is much heavier than sulfur and selenium. These two points make Cu2Te a theoretical potential TE material with high ZT [2Te is too high because of its severe copper deficiency, which easily forms nonstoichiometric Cu2-δTe compound [2Te is too complex, especially, it has at least five phase transitions from room temperature to its melting point [Among the TE materials, transition-metal chalcogenides, especially tellurides, such as PbTe ,23, Bi2T2Te3 ,25 and A2Te3 [2 ,27, have[2 [2Te) ,29, alone) [2Se)  and coppSe) [2S) , have em high ZT . Howevercompound . The othng point .2Te in recent years. Dabin Park, et al. [2Te nanorod composites with 1D nanostructure through a solution phase mixing process by using Cu2Te with different properties and polyvinylpyrrolidone (PVP) in 2018. Sayan Sarkar, et al. [2Te, Cu1.97Ga0.03Te, to improve the electrical conductivity and Seebeck coefficients of Cu2Te in 2019. However, the way of incorporating with oxide to optimize the thermoelectric properties of Cu2Te is rarely reported.Several researches have been made to improve the thermoelectric property of Cu, et al.  synthesi, et al.  synthesi2O2)2+ insulating layers alternately stacked with (Cu2X2)2− conductive layers as a charge reservoir along the c-axis [2Te with BiCuTeO to lower the thermal conductivity and improve the Seebeck coefficient of Cu2Te by optimizing the carrier concentration.BiCuXO  oxychalcogenides have a layered structure that is made up of 1−x-(BiCuTeO)x composites by self-propagating high-temperature synthesis (SHS) and spark plasma sintering (SPS) process to optimize carrier concentration (n) and decrease the total thermal conductivity. As a result, the PF was raised from ~1.91 to ~2.97 μW cm−1 K−2 at normal temperature, and the (Cu2Te)0.95-(BiCuTeO)0.05 composite sample reached a relatively high ZT value of 0.13 at 723 K, which is 41% higher than that of pristine Cu2Te.Herein, we synthesized (Cu2Te)1−x-(BiCuTeO)x  samples were prepared by self-propagating high-temperature synthesis (SHS) and spark plasma sintering (SPS) process. Commercial high-purity powders of Bi , Cu , Te  and Bi2O3  were weighed with a stoichiometry of (Cu2Te)1−x-(BiCuTeO)x , and then mixed uniformly in an agate mortar in air. The mixed powders were cold-pressed into a pellet using a steel die under 6 MPa in air and the bulk was put in an alumina crucible. The SHS process was started by heating the bottom of the crucible to the ignition temperature with an alcohol lamp in air. Once the reaction began, we put the lid on the cauldron and moved the alcohol lamp away. The combustion wave was persisted by the energy released by the initial reaction and it spread to the whole bulk in a few seconds. The product after SHS was crushed into powders and the obtained powders were densified by SPS furnace  for 10 min at 773 K under axial pressure of 40 MPa.(Cuσ) and Seebeck coefficient (S) were simultaneously measured by using a ZEM-3 instrument . Thermal conductivity (κ) was measured indirectly according to the equation D is thermal diffusivity as measured by the LFA 457 MicroFlash , The micro-structure of all prepared bulks was characterized by the X-ray diffraction . The fractured surface morphology of the samples was studied by field emission scanning electron microscopy , equipped with energy dispersive spectrometer (EDS). The morphology and chemical composition of the nano-powders  were characterized by high-resolution transmission electron microscopy  with an integrated energy dispersive spectrometer (EDS), and the corresponding fast Fourier transform (FFT) patterns are obtained by RADIUS software. The electrical conductivity 1−x-(BiCuTeO)x bulk samples, as presented in x = 0, the Cu2Te is composed of Cu2Te (PDF#06-0649), Cu2−δTe (PDF#10-0421) and Cu0.664Te0.336 (PDF#37-1027), similar to the previous study [x = 0.1, the peaks of BiCuTeO are detected at 33°, and become more and more pronounced with the content of BiCuTeO growing. Since x = 0.2, the peaks of Cu2Te (PDF#06-0649) start to decrease, because the Cu and Te reacted with Bi and O to form BiCuTeO. The standard Powder Diffraction File (PDF) card is calculated according to the literature data [Cuogenides . The cryure data .2Te)1−x-(BiCuTeO)x and the in situ formation of BiCuTeO, detailed microscopic investigations were made on the prepared Cu2Te samples. Two phases with obviously different contrast were presented in the cross-sectional microstructures of (Cu2Te)1−x-(BiCuTeO)x shown in 2Te, and the gathering of Bi which was revealed in 2Te.To prove structure evolution of the (Cu2Te)0.9-(BiCuTeO)0.1 sample to research microstructure information, as revealed in 2Te)0.9-(BiCuTeO)0.1. The lattice fringe space measured as 4.7 Å may correspond to the (002) plane of BiCuTeO, while differently oriented lattice spacing of about 7.2 Å, 3.3 Å and 2.9 Å may correspond to (003), (106) and (204) planes of Cu2−δTe, respectively. Actually, the lattice spacing of about 7.2 Å and 3.3 Å could also be assigned to the (031) and (162) planes of Cu2Te, because of their similar crystal structure.TEM investigation was performed on 1−x-(BiCuTeO)x as a function of temperature, where (a)–(c) are the in plane electrical conductivity, Seebeck coefficient and power factor, respectively. As presented in x is less than 0.1 and more than 0.2, the electrical conductivity is decreased as the content of BiCuTeO increases, while the electrical conductivity increases from x = 0.1 to x = 0.2. The reason should be ascribed to the variable carrier concentration and mobility, as demonstrated in x = 0.2, Cu2Te (PDF#06-0649), one of the main phases, begins to decrease until it almost disappears, while the phase of BiCuTeO increases. That leads to the decrease of the carrier concentration and increase of the carrier mobility, as Cu2Te has a high carrier concentration and BiCuTeO has a low carrier concentration but high mobility.Since Cung point , the ele2Te, as shown in −1 at 723 K. Such a low Seebeck coefficient is related to the high carrier concentration. Among all the samples, (Cu2Te)0.95-(BiCuTeO)0.05 and (Cu2Te)0.9-(BiCuTeO)0.1 composites show the highest Seebeck coefficient values of 26.94 μV K−1 and 27.33 μV K−1 at room temperature, respectively. Besides the appropriate carrier concentration, the DOS effective mass (m*) should be another reason. Thus, it is necessary to calculate the m*. The calculations are based on assumption of acoustic phonon scattering and the single parabolic band (SPB) mode [2Te)0.95-(BiCuTeO)0.05 and (Cu2Te)0.9-(BiCuTeO)0.1 composites have the highest m* of 0.785 m0 and 0.716 m0. The PF of (Cu2Te)1−x-(BiCuTeO)x as a function of temperature is displayed in 2Te)1−x-(BiCuTeO)x. The PF of pristine Cu2Te is 4.05 μW cm−1 K−2 at 723 K, and the PF of (Cu2Te)0.95-(BiCuTeO)0.05 and (Cu2Te)0.9-(BiCuTeO)0.1 composites increase to 4.49 μW cm−1 K−2 and 4.43 μW cm−1 K−2 at 723 K, respectively. The difference of PF at high temperature is not so obvious, but the improvement of PF is large at room temperature. The PF increased from 1.91 (for pristine Cu2Te) to 2.97 μW cm−1 K−2 (for the (Cu2Te)0.95-(BiCuTeO)0.05) at normal temperature. 2Te)1−x-(BiCuTeO)x composites have nearly isotropic transports.All the samples display p-type conducting within the measured temperature range on account of the intrinsic copper deficiencies in CuPB) mode . Althougκtot) of (Cu2Te)1−x-(BiCuTeO)x composites shown in 2Te and the high hole concentration caused by unavoidable deviation from stoichiometry. They show complicated temperature dependencies in the temperature ranged from 300 to 723 K, first decreasing and then increasing as a whole over the entire temperature range, which is the effect of the phase transition of Cu2Te [2Te)0.95-(BiCuTeO)0.05 and (Cu2Te)0.9-(BiCuTeO)0.1 composites have the lowest thermal conductivity due to the lower carrier thermal conductivity (κe). As displayed in κe is dominant in the total thermal conductivity (κtot). The κe of (Cu2Te)1−x-(BiCuTeO)x changes, affected by its electrical conductivity according to Wiedemann law, κe = LσT, where L, σ and T are Lorentz coefficient, electrical conductivity and temperature, respectively. Besides, electrical conductivity could be calculated according to σ = neμ, where n, e and μ are carrier concentration, elementary charge and carrier mobility, respectively. As exhibited in x = 0 to x = 0.1, the carrier concentration of (Cu2Te)1−x-(BiCuTeO)x is decreased, which causes the reduction of the σ and further results in the depression of the κe. When the content of BiCuTeO changes from 0.1 to 0.2, the carrier mobility is improved a lot, bringing about a high σ and further causing the enhancement of κe. The reduction of the range from x = 0.2 to x = 0.4 is owing to the dropping of the μ. The κtot decreases to 1.48 W m−1 K−1 at 623 K with increasing BiCuTeO content, while the thermal conductivity of Cu2Te is 2.61 W m−1 K−1 at 623 K.The total thermal conductivity (of Cu2Te . (Cu2Te)κtot, the ZT values of (Cu2Te)0.95-(BiCuTeO)0.05 and (Cu2Te)0.9-(BiCuTeO)0.1 are higher than that of Cu2Te, as illustrated in ZT values of (Cu2Te)0.95-(BiCuTeO)0.05 and (Cu2Te)0.9-(BiCuTeO)0.1 are 0.13 and 0.12 at 723 K, respectively, while the ZT value of pristine Cu2Te is 0.09 at 723 K.Consequently, combining the results of the higher PF and lower 2Te)1−x-(BiCuTeO)x composites by SHS combined with SPS. The carrier concentration has been optimized with the incorporation of BiCuTeO, which increased the power factor of (Cu2Te)1−x-(BiCuTeO)x and largely decreased carrier thermal conductivity resulting in a lower total thermal conductivity.In this study, we successfully synthesized (Cu2Te)0.95-(BiCuTeO)0.05 composite sample reached a relatively high ZT value of 0.13 at 723 K, which is 41% higher than that of pure Cu2Te.Consequently, the (CuZT of (Cu2Te)1−x-(BiCuTeO)x is not high enough, this study lays a foundation for better performance system research.Although the


Analysis of the TCRα repertoire indicated it was generally reduced in diversity in the absence of Themis, whereas the diversity of sequences using the TRAV9N-3 V-region element was increased. In wild type mice, Vα3.2+ cells showed higher CD5, CD6 and CD44 expression than non-Vα3-expressing cells, and this was more marked in cells from Themis-deficient mice. This suggested a virtual memory phenotype, as well as a stronger response to self-pMHC. The Vα3.2+ cells responded more strongly to IL-15, as well as showing bystander effector capability in a Listeria infection. Thus, the unusually large population of Vα3.2+ CD8+ T cells found in the periphery of Themis-deficient mice reflects not only altered thymic selection, but also allowed identification of a subset of bystander-competent cells that are also present in wild-type mice.Deletion of the gene for Themis affects T cell selection in the thymus, which would be expected to affect the TCR repertoire. We found an increased proportion of cells expressing Vα3.2 (TRAV9N-3) in the peripheral CD8 Thepression , 26. Howelf pMHC , 28, sugnfection , such asnfection . Bystandnfection , 32, andnfection –35.Ptpn6) and Shp2 (Ptpn11), although the precise mechanism is controversial , f/f.CD4-CreThemis– and f/f.CD4-Cre+, Themisf/f.dLck-CreThemis– and f/f.dLck-Cre+Themis (Rag1–/– and CD45.1 mice on C57BL/6 background were bred in our restricted flora (RF) facilities at Comparative Medicine, NUS. Mice were treated under Institutional Animal Care and Use Committee-approved guidelines in accordance with approved protocols.stm1Gasc  crossed tm2Tch/J , Themis+Lck-Cre+ , Rag1–/Mice were euthanized and dissected. Peripheral (p)LN  were excised and mashed upon a 70 μm cell strainer into 5 ml cRPMI. For cell surface staining, cells were spun at 1200 rpm at 4°C for 5 minutes and the resulting cell pellets were resuspended in 100 μl PBS with 0.5% BSA (FWB: FACS wash buffer), containing the fluorophore-conjugated antibodies’ dilutions (1:300) for the cell surface antigens and incubated on ice for 30 minutes in the dark. Cells were then centrifuged at 1200 rpm at 4°C for 5 minutes and resuspended in 300 μl of FWB for flow cytometry analysis. For biotinylated antibodies, staining with the antibody was followed by staining with 100 μl FWB containing BUV 395 labeled-streptavidin  at 1:500 dilution for 30 minutes on ice. Cells were then centrifuged at 1200 rpm at 4°C for 5 minutes and resuspended in 300 μl of FWB for flow cytometry analysis.For intracellular staining, the cells from the previous step were resuspended in 0.2 ml IC fixation buffer  while being vortexed, followed by an incubation at room temperature for 20 minutes. The cells were then washed twice with 2 ml 1X permeabilization buffer , resuspended in 100 μl FWB containing the fluorophore-conjugated antibodies’ dilutions (1:250) for the intracellular antigens and incubated at room temperature for 30 minutes. The cells were then washed once with 2 ml 1X permeabilization buffer and then with 2 ml FWB. The cells were then resuspended in 300 μl FWB for analysis on a flow cytometer. 25 μl Count Bright beads  were added to each sample for cell count analysis. Cells were analyzed on BD LSR Fortessa X-20 flow cytometer . Flow cytometry data were analyzed using FlowJo software . All antibodies used for flow cytometry purposes are described in °C for an hour. The cells were then washed thrice with FWB. The resulting cells were then stained with Ki67  antibody at 1:1000 dilution and other cell surface antigens for 30 minutes at room temperature. The cells were then washed with 2 ml FWB and analyzed on a flow cytometer.Cell pellets were washed twice with FWB for 5 minutes at 350g. The resulting cell pellet was vortexed after discarding the supernatant. 3 ml ice-cold ethanol was added to the cell pellet drop by drop while vortexing. Cells were votexed for another 30 seconds and then incubated at -20Mice were euthanized and dissected. Lymph nodes were excised and pooled together from several mice of the same genotype to prepare the samples for sorting. These were then mashed upon a 70 μm cell strainer into 5 ml cRPMI. The resulting cell suspensions were then spun down at 1200 rpm for 5 minutes at 4°C. The resulting cell pellet was surface stained by resuspending in 0.5 ml cRPMI per mouse containing fluorescently-conjugated antibodies at 1:500 dilution, followed by incubation at 4°C for 30 minutes on a shaker. They were then washed with cRPMI and then resuspended in 0.5 ml cRPMI per mouse for sorting. The cells were sorted on either Sy2000  or Facsfusion .® High-Fidelity DNA Polymerase  according to the manufacture instruction, with primers: tcgtcggcagcgtcagatgtgtataagagacagcaactactACTGC, GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGggtacacagcaggttctgg (first round), and CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC where i7 and i5 represent Illumina Nextera V2 indexes . Libraries were purified using AMPure XP , and molar concentration of amplicons was quantified using Qubit DNA Assay  and KAPA Library Quantification Kit . Sequencing of the TCR’s amplicons was performed on MiSeq platform using MiSeq Reagent Kits v2 .For the synthesis of the NGS libraries covering entire TCR repertoires, the total RNA was isolated from similar number of sorted SP CD8 thymocytes and peripheral CD8 T cells. The Vα3.2 TCR repertoires were retrieved from sorted Vα3.2+ CD8 SP thymocytes and Vα3.2+ CD8 peripheral T cells. The reverse transcriptase reaction was performed according to the previously published protocol  with temExtraction of the sequences corresponding to the TCRs was performed using MiXCR platform . Further+CD44lo cells were labeled with Cell Trace Violet . Cells were spun down and resuspended in PBS at a concentration of 2x106/ml. Cell Trace Violet was then added to the cell suspension at the concentration of 5 μM. Cells were vortexed immediately and incubated at 37°C for 10 minutes while vortexing every 5 minutes. After the incubation, media was added to quench the reaction at 5 times the original staining volume and further incubated for 5 minutes at 37°C. The cells were then spun down at 500 g for 5 minutes at 4°C. The cells were then used for the subsequent experiments.Sorted CD86/ml. 100 μl of the cell suspension was seeded into a 96 well plate. For IL-15 stimulation, 100 μL of media with recombinant mouse IL-15  at the concentration of 2 μg/ml was added to the cells. The cells were incubated at 37°C for 5 days and then analyzed for proliferation by the CTV dilution on a flow cytometer. For IL-7/12/18  stimulation, 100 μl media with either IL-7 or a combination of either IL-7 and IL-12 or IL-7 and IL-18  was added to the cells. The cells were incubated at 37°C for 7 days and then analyzed for proliferation by the CTV dilution on a flow cytometer.After CTV labeling, live cells were counted and adjusted to the concentration of 1*106/ml. CD45.1 recipient mice were sublethally irradiated at 6 Gy. 0.2 ml cell suspension was injected retro-orbitally (intravenous i.v.) into either Rag1–/– or the sublethally irradiated CD45.1 mice. 5 recipient mice were injected per donor genotype and un-injected mice were used as controls. After one week, lymph nodes and spleens were excised from the CD45.1 mice or Rag1–/– mice after euthanizing them. The single cell suspensions from lymph nodes and spleens were stained for the congenic markers CD45.1, CD45.2 to distinguish donor and recipient cells. The proliferation of the donor cells was analyzed based upon CTV dilution as determined using a flow cytometer.After CTV labeling, live cells were counted and adjusted to the concentration of 2.5*10Themis–/– and Themis+/+ mice were infected with 104 colony-forming units of LM-OVA  (100 ng/ml) for 6 hours in the presence of Brefeldin A  at 1:500 dilution and stained for IFNγ via intracellular staining protocol mentioned above.f LM-OVA  via retrb-OVA monomers in the dark. The tetramer was then used at 1:50 dilution for cell surface staining.3.18 μl of PE labeled Streptavidin (1 mg/ml)  was added every 10 minutes for a total of 10 times to 10 μl of 2 mg/ml biotinylated H-2 KPrism  and Excel  were used for all statistical analysis and graphical representations. Normality of data was tested using Shapiro-Wilk test. All data sets were found to pass the normality test. Data are presented as means ± s.d., and we determined significance by two-sided Student’s t test. We considered a p-value of equal to or less than 0.05 as statistically significant.Themis–/– and Themis+/+ mice. The biggest difference was in the proportion of Vα3.2+ CD8+ T cells  and post-selection (dLck-Cre) Themis conditional knockout mice  repertoires from SP CD8αβ+ thymocytes and CD8αβ+ lymph node T cells that developed in the Themis–/– and Themis+/+ mice. After the reconstruction of TCR sequences from raw NextGen sequence datasets, we were able to identify 437 and 599 different clonotypes originating from Themis+/+ and Themis–/– SP thymocytes, respectively . In the periphery (Themis+/+ and Themis–/– LN), these differences dwindled. A plausible explanation of this observed phenomenon is that analysis of the thymic repertoire is restricted to the single wave of the SP thymocytes passing through at that time. In contrast, the datasets from the lymphocytes depict a more prolonged process, which includes clonal or homeostatic proliferation together with accumulation of clones over time.To more precisely define the development of the TCR Vα3.2Themis+/+ and Themis–/– SP thymocytes acquire a public character in the peripheral lymphatic organs , but not if it occurred after selection (dLck-Cre) . These cells also express more CD44 in the periphery  repertoire and the phenotype of the Vα3.2-expressing cells raised the possibility of TCR-driven mechanisms orchestrating the function and peripheral molding of this CD8he MHC-I , 7, 9. AN-3 reper T cells , 62.+ TCRα repertoire , we selected TCRs that use TRAV14 (Vα2), TRAV6 (Vα4), and TRAV12 (Vα8) as a representative fraction of the lymph node- and thymus-derived CD8+ TCRs, the fifth position was more frequently occupied by hydrophobic amino acids  T cells from Themis–/– and Themis+/+ mice (CD45.2) into sublethally irradiated CD45.1 recipients. After a week, the recipients were euthanized and lymph nodes and spleens were analyzed. Overall, Themis+/+ CD8+ T cells showed enhanced LIP compared to Themis–/– CD8+ T cells  Vα3.2+ cells also suggested better responses to IL-15 stimulation, as IL-15 is required for maintenance of the memory pool    and sacr(LM-OVA) . We confKG2D+  . We anal 6 hours , 69. OnlThemis–/– mice might be different than that of Themis+/+ mice. Our results show that there are indeed changes in TCR repertoire in Themis–/– mice. We observed that the proportion of Vα3.2+ CD8+ T cells and Vα3.2+ Vβ5.1/5.2+ cells was triple or more in the periphery of Themis–/– mice than Themis+/+ mice. We have previously found in rearranging TCR minigene mice that co-expression of Vα3.2 and Vβ5.2 is a favored combination  subset. Like the CD8+ Vα3.2+ compartment, Tregs retain a highly diverse TCR repertoire in the periphery , were even more frequent than usual in the CD8fections , 35 and fections , so a behttps://www.ncbi.nlm.nih.gov/geo/, GSE162963.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by National University of Singapore Institutional Animal Care and Use Committee (NUS IACUC).Listeria infection experiments and performed the CD6 validation experiments. YC and DT assisted in the mouse breeding and irradiation of mice. JY generated the biotinylated H-2 Kb-OVA monomers. VR and JH were involved in optimization of the experimental protocols. NG supervised the study and did data interpretation. All the authors contributed to writing and editing the manuscript. All authors contributed to the article and approved the submitted version.MP, LW, and JB performed the literature review, planned the experiments, and did data interpretation. MP performed most of the experiments. LW performed the TCR sequencing and CDR3 analysis. JB assisted in the This research was supported by the Singapore Ministry of Health’s National Medical Research Council under its CBRG/0097/2015 and by the Singapore Ministry of Education’s grant 2014-T2-1-136, to NG. Work performed at Scripps Research was supported by NIH grants AI073870 and DK094173 to NG.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Increased expression of KDM1A and decreased expression of DACT1 in cervical cancer cells were noticed in a previous study. This study is aimed at exploring the mechanism behind the KDM1A regulation on DACT1 in cervical cancer cells.  The expression profile of KDM1A and DACT1 in cervical cancer tissues was searched in TCGA database. In vitro experiments verified the effect of KDM1A and DACT1 on proliferation and migration ability of cervical cancer cell lines after cell transfection. The interaction of KDM1A with HDAC1 was identified by coimmunoprecipitation (Co-IP). The expression levels of KDM1A and DACT1 in cervical cancer cell lines were determined by qRT-PCR and western blot.  TCGA database showed that cervical cancer tissues had elevated expression of KDM1A and decreased expression of DACT1, which was consistent with the observation in cervical cancer cell lines. KDM1A was found to negatively regulate DACT1 through histone deacetylation. Meanwhile, the downregulation of KDM1A or overexpression of DACT1 could suppress the cell proliferation and migration ability in HeLa and SiHa cells. Cotransfection of KDM1A and DACT1 overexpression could reverse the increased cell proliferation and migration ability induced by KDM1A overexpression.  KDM1A can downregulate DACT1 expression through histone deacetylation and therefore suppress the proliferation and migration of cervical cancer cells. As the second most common malignant cancer in female, cervical cancer is characterized by poor prognosis in the advanced stage due to metastasis or recurrence . FortunaAcetylation refers to posttranscriptional modification, mainly including lysine acetylation, protein acetylation, and histone acetylation, among which histone acetylation is of critical importance for gene regulation . FurtherTypical searching in TCGA database identified that KDM1A was highly expressed in cervical cancer tissues. Further investigation in cervical cancer cell lines confirmed the highly expressed profile of KDM1A in cervical cell lines and also identified DACT1 as a target gene of KDM1A. In light of recent studies, overexpression of DACT1 was reported to suppress the proliferation, invasion, and migration ability of cervical cancer cells . Therefo2 and 95% humidity. Cells in logarithmic phase were harvested for following experiments.Human cervical cancer cell lines HeLa, Ca Ski, SiHa, C-33A, and C4-1 and immortalized human cervical epithelial cell line H8 were purchased from China Center for Type Culture Collection . All cells were maintained at DMEM  with high glucose, in which 10% of fetal calf serum (FCS) and 1% mycillin were supplemented. The cells were cultured in a 37°C incubator with 5% COhttp://ualcan.path.uab.edu/) and GEPIA (http://gepia.cancer-pku.cn/detail.php?).The expression levels of TGFRB2 and DACT1 in human cervical cancer tissues were obtained from UALCAN (5/well) for cell culture of 24 h. After that, sh-KDM1A (2 μg), pcDNA3.1-DACT1 (2 μg), and corresponding negative controls  were transfected into cells and named as the sh-KDM1A group, sh-NC group, pcDNA3.1-DACT1 group, and pcDNA3.1 group. Cells cotransfected with pcDNA3.1-KDM1A (2 μg), pcDNA3.1-DACT1, (2 μg) or negative controls were named as the pcDNA3.1 group, pcDNA3.1-KDM1A group, and pcDNA3.1-KDM1A+pcDNA3.1-DACT1 group. Lipfectamine 2000 transfection kit  was used for cell transfection based on instructions on the kit.HeLa and SiHa cells in logarithmic phase were seeded in 6-well plates (2 × 10ΔΔCt- [(target gene) − Ct]experimental group − [Ct(target gene) − Ct]control group. The primer sequences are listed in TRIzol Reagent  was used to extract the total RNA, whose purity and concentration were measured using a microplate reader (Biotek Synergy 2). The RNA was reversed into cDNA template on a PCR amplifier for real-time quantitative RT-PCR with the application of PCR analyzer . The expression of mRNA was relative to that of GAPDH. The PCR reaction was conducted at the condition of predenature of 95°C for 10 min, followed by 40 cycles of denature at 95°C for 10 s, annealing of 60°C for 20 s, and extension at 72°C for 34 s. Data were calculated using 2-ΔΔCt : ΔΔCt = μL/50 mL cell lysate. Then, the cell lysis was centrifuged at 4°C and 12000 rpm for 10 min with supernatant collected. BCA kit  was used to detect the concentration of proteins. The proteins  were separated on a 10% SDS-polyacrylamide gel and transferred into PVDF membrane  to terminate the unspecific reaction with 5% skim milk powder for 1 h. After that, the membranes were incubated with rabbit anti human KDM1A , rabbit anti human DACT1 , and rabbit anti human Anti-Histone H3  for overnight at 4°C. The membranes were then washed with TBST for 3 × 10 min before incubation with horseradish peroxidase labeled goat anti rabbit IgG  at room temperature for 1 h. After TBST washing for 3 × 10 min, the membranes were analyzed using a chemiluminescence imaging system . GAPDH was used as an internal control.Cells were washed in precold PBS for 3 times before cell lysis for 30 min at ice with 100 3/well) were seeded into 96-well plates for cell culture for 24 h, 48 h, 72 h, and 96 h, respectively, before incubation with CCK-8 solution for 2 h. The optical density was measured at the wavelength at 450 nm using a microplate reader .CCK-8 kit  was used to assess the cell growth of HeLa and SiHa cells. After cell transfection, cells (5 × 10Cells in logarithmic phrase were digested with 0.25% trypsin and made into single cells. Cells floated in culture medium containing 10% FCS were seeded into a 37°C prewarmed culture dish (10 mL) at the density of 50, 100, and 200 cell per dish. The dishes were gently shaken to ensure cells were scattered evenly. Cells were cultured at 37°C and 5% humidity for 2~3 weeks. Cell culture was terminated when cell clones were visible by naked eyes. The supernatants in the culture dishes were abandoned, and cells were washed with PBS 2 times before fixation with 5 mL acetic acid/methanol (1 : 3) for 15 min. Abandon the fixation solution. Giemsa staining was performed for 10~30 min before cells were washed in running water and dried. The culture dishes were inverted to calculate the cell clones by the naked eyes or by a microscope with low power lens (10 or more cloned cells were counted). Cell clone formation rate was calculated after cell clone number was counted.5/well) for cell culture. Once cell confluence reaches 90%, cells were treated by mitomycin (1 μg/mL) for 1 h, and a new 200 μL pipette tip was used to make a straight scratch to the cells. Cells were washed with PBS twice and further cultured in high-glucose DMEM without serum. The cell migration ability was assessed and photographed under an inverted microscope at 0 h and 24 h, respectively: cell migration rate = ((0 h migration distance − 24 h migration distance)/0 h migration distance) × 100%.Cells in logarithmic phrase were seeded into 6-well plates  in the protein complex were measured by western blot.HeLa and SiHa cells in logarithmic phrase were added with precold cell lysis at 4°C for 15 min before centrifugation at 14000 g for 15 min. The supernatant was transferred into a new centrifuge tube for incubation with negative IgG for anti-KDM1A antibody  or anti-H3 antibody  for overnight at 4°C. The pretreated 10 t-test, and data among multiple groups were compared using one-way analysis of variance (ANOVA) and post hoc Tukey's tests. p < 0.05 was considered having statistically significance.GraphPad Prism 6.0  was used for data analysis. All data were calculated from averaged value of three repeat experiments and expressed as the mean ± standard deviation (SD). Comparison between two groups was analyzed using a http://ualcan.path.uab.edu/). The results showed that KDM1A was highly expressed and DACT1 (305/3) was lowly expressed in cervical cancer tissues . Similar expression pattern was also found in GEPIA database (http://gepia.cancer-pku.cn/detail.php?), in which increased KDM1A expression and deceased DACT1 expression (306/13) were found .To fully understand the implication and effect of KDM1A and DACT1 in human cervical cancer tissues, we first searched the expression profile of those two factors in the UALCAN database (p < 0.01) and protein (p < 0.01) expressions of KDM1A were elevated, while those of DACT1 were decreased  in all cervical cancer cell lines. Among the listed cervical cell lines, the maximum expression gap between KDM1A and DACT1 expression levels was found in HeLa and SiHa cells; therefore, HeLa and SiHa cells were selected for the following experiments. The above evidence suggested the implication of KDM1A and DACT1 in the progression of cervical cancer.Thereafter, we measured the mRNA and protein expression levels of KDM1A and DACT1 in cervical cancer cell lines  and immortalized human cervical epithelial cell line H8. qRT-PCR and western blot demonstrated that compared with H8 cells, the mRNA (p < 0.01). CCK8 and clone formation assay showed the sh-KDM1A group had suppressed proliferation rate . Meanwhile, after pcDNA3.1-DACT1 transfection, the proliferation rate , clone formation numbers , and migration rate  were suppressed in both HeLa and SiHa cell in comparison to those in the control group. The comparison on proliferation rate, clone formation numbers, and migration rate between the pcDNA3.1 group and control group showed no significant difference. Those observations showed that overexpression of DACT1 can inhibit the proliferation and migration of HeLa and SiHa cells.HeLa and SiHa cells were transfected with DACT1 overexpression through lentiviral transfection. As shown in Figures https://www.genecards.org/) showed that KDM1A can bind with many histone deacetylases to form complex so as to achieve gene silence. The STING database (https://string-db.org/cgi/input.pl?sessionId=4Bvlcg3I4RKk&input_page_show_search=on) predicted the binding between KDM1A and HDAC1 (p < 0.01). Additionally, western blot showed increased deacetylation of histone 3 in sthe h-KDM1A group when compared with the control group (p < 0.01). The effect of KDM1A knockdown on DACT1 expression in HeLa and SiHa cells showed that compared with the sh-NC group, the sh-KDM1A group had increased mRNA and protein expressions of DACT1 in both HeLa and SiHa cells , indicating the regulatory role of KDM1A on DACT1 expressions. The collected evidence showed that KDM1A enhances histone 3 deacetylation and therefore suppresses DACT1 expressions.The Genecards database .To ascertain the effect of DACT1 in cervical cancer, pcDNA3.1-DACT1 and pcDNA3.1-KDM1A were cotransfected into HeLa and SiHa cells. The measurement on transfection efficiency showed that compared with the pcDNA3.1 group, the pcDNA3.1-KDM1A group had higher expression of KDM1A and lower expression of DACT1. Meanwhile, compared with the pcDNA3.1-KDM1A group, the expression of KDM1A showed no significant changes, while DACT1 expression was substantially elevated in the pcDNA3.1-KDM1A+pcDNA3.1-DACT1 group  and elevated cell clones . In comparison to the pcDNA3.1-KDM1A group, the pcDNA3.1-KDM1A+pcDNA3.1-DACT1 group had suppressed cell proliferation rate  and less cell clone . Cell migration ability by wound healing assay showed that increased cell migration ability in the pcDNA3.1-KDM1A group when compared with the pcDNA3.1 group, but the pcDNA3.1-KDM1A+pcDNA3.1-DACT1 group had suppressed cell migration ability in comparison with the pcDNA3.1-KDM1A group . No significant difference was found between the pcDNA3.1 group and control group in terms of cell proliferation, cell clones, and migration ability. Taken together, KDM1A enhances the proliferation and migration of cervical cancer cells through regulating the expression of DACT1.Measurement on cell proliferation, cell clones, and migration showed that compared with the pcDNA3.1 group, the pcDNA3.1-KDM1A group had increased cell proliferation rate  and cervical cell lines. More importantly, we further explored the mechanism of KDM1A regulation on DACT1 in cervical cancer. The collected evidence showed that KDM1A in cervical cancer cells can suppress the expression of DACT1 through histone 3 deacetylation and therefore enhance the progression of this disease.KDM1A is a well-known histone demethylase and an emerging option for the therapeutic treatment of various cancers . KDM1A iIt is not the first time to identify the implication of DACT1 in cervical cancer. A previous study by Shi et al. showed DACT1 by acting as one component of the H1FX-AS1/miR-324-3p/DACT1 axis was proved to be a novel potential therapeutic target for cervical cancer treatment . In lighIn summary, results in this study showed that KDM1A was highly expressed while DACT1 was lowly expressed in cervical cancer tissues and cells. KDM1A can lead to histone 3 deacetylation, which consequently represses the expression of DACT1 in cervical cancer cells. Additionally, KDM1A is able to negatively regulate the expression of DACT1. Therefore, KDM1A enhances the progression of cervical cancer by inducing histone 3 deacetylation and downregulating DACT1 expression.


PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here, we investigated how PBRM1 controls HIF-1α activity. We found that PBRM1 is required for HIF-1α transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1α mRNA translation, as absence of PBRM1 results in reduced actively translating HIF-1α mRNA. Interestingly, we found that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1α mRNA is m6A-modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1α mRNA and reduction of YTHDF2 results in reduced HIF-1α protein expression in cells. Our results identify a SWI/SNF-independent function for PBRM1, interacting with HIF-1α mRNA and the epitranscriptome machinery. Furthermore, our results suggest that the epitranscriptome-associated proteins play a role in the control of hypoxia signalling pathways. The cellular response to decreased oxygen tension (hypoxia) is characterised by changes in the gene expression program primarily mediated by the hypoxia-inducible factor (HIF) family of transcription factors. HIF transcription factors are heterodimers composed of an oxygen-labile HIF-α subunit and a constitutively expressed HIF-1β subunit. In well-oxygenated cells, HIF-α subunits are targeted for proteasomal degradation by sequential proline hydroxylation and polyubiquitination mediated by proline hydroxylases (PHDs) and the Von Hippel–Lindau (VHL) E3 ubiquitin ligase, respectively .In order to facilitate the extensive changes in gene expression necessary for the cellular response to hypoxic stress, changes to the chromatin structure must take place for transcription factors such as HIF to access the promoter regions of its target genes. Chromatin exists in a spectrum of closed and open conformations determined by the density of nucleosomes, with active transcription requiring an open conformation of chromatin so that protein complexes can bind to the underlying DNA . One mecThe levels and activity of SWI/SNF chromatin remodelling complexes are important for controlling gene expression changes in hypoxia . SWI/SNFWe have previously shown that the SWI/SNF components BAF155, BAF57 and BRG1 are required for HIF-1α mRNA expression, and their depletion impairs the HIF-dependent hypoxic response . More rePBRM1 is a large (193 kDa) multidomain protein containing six bromodomains which bind to acetylated histones, BAH domains which are protein-binding domains  and an HHere, we investigated the involvement of PBRM1 in regulating HIF activity. We found that PBRM1 is required for full HIF activity and HIF-1α protein levels. However, this regulation occurs through promoting efficient translation of HIF-1α mRNA rather than through direct effects on HIF-1α gene transcription. This activity is supported by PBRM1’s ability to bind selectively to HIF-1α mRNA as well as the RNA-binding protein YTHDF2, which is also required for normal levels of HIF-1α protein in specific cellular backgrounds. This reveals a function for PBRM1 distinct from chromatin regulation and the rest of the SWI/SNF complex and suggests that the epitranscriptome and its machinery are important for the cellular response to hypoxia.v/v) foetal bovine serum (FBS) , 50 units/mL penicillin  and 50 μg/mL streptomycin . HRE-luciferase reporter cell lines were maintained in these conditions supplemented with 0.5 µg/mL puromycin. All the cell lines were routinely tested for mycoplasma contamination using a MycoAlert kit from Lonza. Hypoxia treatments were performed in an InVivo 300 or InVivo2 hypoxia workstation  at 1% O2, 5% CO2 and 37 °C. The cells were treated with MG132  at the final concentration of 20 μM, cycloheximide  at the final concentration of 0.1 mg/mL, actinomycin D at the final concentration of 5 µg/mL and FG-4592  at the final concentration of 50 µM.Cell lines were obtained from the European Collection of Cell Cultures. The cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 2 mM L-glutamine, 10% . The cell culture medium was changed 24 h following transfection and the cells were harvested 48 h following transfection. GFP-PBRM1 was a kind gift of Prof. Jessica Downs and was described in [Transfections in HeLa, U2OS, H1299 and A549 were performed using INTERFERin  following the manufacturer’s instructions. The HEK293 cells were transfected with 0.12 M CaClribed in . For DNAv/v) NP40, 0.25% (w/v) Na deoxycholate, 0.1% (w/v) SDS, 10 mM NaF, 2 mM Na3VO4 and 1 tablet/10 mL cOmplete, Mini, EDTA-free protease inhibitors (Roche)), centrifuged for 10 mins at 13,000 rpm at 4 °C, collecting the supernatant for analysis. SDS-PAGE and immunoblotting were performed using standard protocols. The following primary antibodies were used for immunoblotting: HIF-1α , HIF-2α , HIF-1β , β-Actin , PBRM1 , YTHDF2 , GFP , BRG1 . Following incubation with a horseradish peroxidase-conjugated secondary antibody , chemiluminescence  was detected. All the figures are representative of a minimum of three independent experiments.The cells were lysed in the RIPA buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1%  and activity was measured using a Lumat LB 9507 luminometer . The results were normalised according to protein concentration and reported as a percentage of control.HIF-1α forward CATAAAGTCTGCAACATGGAAGGT, HIF-1α reverse ATTTGATGGGTGAGGAATGGGTT, HIF-1β forward CAAGCCCCTTGAGAAGTCAG, HIF-1β reverse GAGGGGCTAGGCCACTATTC, β-actin forward CCCAGAGCAAGAGG, β-actin reverse GTCCAGACGCAGGATG, HK2 forward AGCCCTTTCTCCATCTCCTT, HK2 reverse AACCATGACCAAGTGCAGAA, PHD2 forward GAAAGCCATGGTTGCTTGTT, PHD2 reverse TGTCCTTCTGGAAAAATTCG, PHD3 forward CTTGGCATCCCAATTCTTGT, PHD3 reverse ATCGACAGGCTGGTCCTCTA, GLUT1 forward TCAAAGGACTTGCCCAGTTT, GLUT1 reverse GATTGGCTCCTTCTCTGTGG, VEGF forward AGCTGCGCTGATAGACATCC, VEGF reverse CTACCTCCACCATGCCAAGT, BNIP3 forward GCCCACCTCGCTCGCAGACAC, BNIP3 reverse CAATCCGATGGCCAGCAAATGAGA.RNA from the HeLa cells was extracted using a peqGOLD total RNA kit , from the HEK293 cells—using a Direct-Zol RNA MiniPrep kit  according to the manufacturers’ protocols. RNA was reverse-transcribed using a Quantitect Reverse Transcription kit . Real-time PCR was performed using the Brilliant II SYBR Green kit  on an Mx3005P qPCR machine . Levels of mRNA were calculated based on averaged Ct values and normalized to β-actin mRNA levels. The following primers were used for qPCR: 2, 300 mM NaCl, 1% Triton X-100, 1 mM DTT, 0.1 mg/mL cycloheximide and RNAsein (Sigma)). Cellular debris was pelleted by centrifugation at 17,000× g for 1 min at 4 °C. The supernatant in the amount of 500 µL was added to 10 mL 10–50% sucrose gradients (with the extraction buffer without Triton X-100). The gradients were centrifuged using an SW41 rotor at 223,000× g for 2 h at 4 °C. The gradient was collected in 1 mL fractions. RNA was extracted from the solution with an RNAeasy extraction kit (Qiagen) according to the manufacturer’s protocol. RNA was converted to cDNA using a Quantitect Reverse Transcription kit (Qiagen) on a SureCycler 8800 (Agilent) and an Mx3005P qPCR machine. Brilliant II SYBR Green (Agilent) was used to perform qPCR on an Mx3005P (Agilent).The HeLa cells were transfected in 10-cm plates. Translation was stimulated by changing the media 1.5 h before lysis. Cycloheximide in the amount of 0.1 mg/mL was added 10 mins before lysis. The cells were washed twice with 5 mL ice-cold PBS containing 0.1 mg/mL cycloheximide. The cells were collected in 300 µL PBS with cycloheximide, and the PBS was removed by centrifugation. The cells were resuspended in 550 µL polysome extraction buffer (15 mM Tris-HCl (pH 7.5), 15 mM MgCl3VO4 and 1 EDTA-free protease inhibitor tablet per 10 mL buffer). The cell lysate in the amount of 500 μg per condition was incubated with 2 μg PBRM1 antibody  or 2 μg IgG control  and rotated at 4 °C overnight. Immune complexes were captured with 20 μL protein G Sepharose beads  by incubating with rotation for 1.5 h at 4 °C. The beads were washed three times with PBS, then the beads were boiled with 20 µL SDS loading buffer.The cells were lysed in the RIPA buffer and the lysate was diluted with an equal volume of the buffer (50 mM Tris-HCl (pH 8.0), 0.5 mM DTT, 20% glycerol, 5 mM NaF, 500 mM Nag. The supernatant was diluted 10-fold with the Magna-RIP wash buffer. The magnetic beads were coated with 5 µg of the appropriate antibody for 30 mins at room temperature. The beads were incubated with the cell extract with rotation overnight at 4 °C. The beads were then washed six times with the RIP wash buffer. After setting aside 10% of the beads for IP control, RNA was eluted by treatment with proteinase K according to the manufacturer’s instructions, then purified by phenol–chloroform extraction followed by an 80% ethanol wash. RNA was finally dissolved in water, converted to cDNA and quantified with qPCR comparing to 500 ng of the input.Either the HeLa or H1299 cells were grown in 15-cm plates, washed twice with ice-cold PBS, harvested in ice-cold PBS and pelleted. Of these cells, 10% were used for each condition. The cells were lysed with 100 µL RIP lysis buffer  and snap frozen. The cells were thawed and centrifuged at 15,000× 2, 1 mM DTT, 0.4 U/µL RNase inhibitor, Sigma, Gillinham, UK). RNA was conjugated to beads by mixing 100 µL of each RNA with 20 µL streptavidin agarose beads  and incubating with mixing for 30 min at 4 °C. The beads were washed twice with 5 mM Tris-HCl (pH 7.4), 1 M NaCl. Ten-centimetre plates of PC-3 cells per pulldown were lysed in a buffer ). The cells were homogenised on ice using 10 strokes of a Dounce homogeniser, then centrifuged at 16,100× g for 15 min at 4 °C. The supernatant in the amount of 1 mg was incubated with each of the bead-conjugated RNAs for 3 h at 4 °C, then washed three times with a wash buffer (25 mM Tris-HCl (pH 7.4), 150 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.5% IGEPAL, 1 mM PMSF, 0.4 U/µL RNAse inhibitor, supplemented with an EDTA-free protease inhibitor tablet ). The proteins present were analysed by SDS-PAGE and immunoblotting.For RNA bait preparation, DNA duplexes containing T7 promoter sequence (5′-TAATACGACTCACTATAG-3′) followed by either the HIF-1α UTR (CAGTGCTGCCTCGTCTGA) or the luciferase mRNA control sequence (GGAGCCCCTGCTAACGACATTTACAACGAG) were generated by PCR. RNA was generated with transcription by the T7 RNA polymerase according to the manufacturer’s instructions . Each 20-µL reaction was spiked with 200 ng DNA template and 0.5 µM biotin-labelled CTP. The reaction mixture was then treated with 2 units of DNase and incubated at 37 °C. The RNA was purified using an RNA purification column . The secondary structure was ensured by heating to 90 °C followed by 2 min of incubation on ice and adding 50 µL RNA structure buffer (10 mM Tris-HCl (pH 7.4), 100 mM KCl, 10 mM MgClE. coli BL21 DE3 (Invitrogen) cells were transformed with plasmids containing 6xHis-tagged YTHDF2  and PBRM1 (BAH1 and 2 967–1287 or HMG 1394–1462 domains). The secondary cultures in the amount of 250 mL were inoculated and grown to OD600nm 0.6, then induced with 0.5 mM IPTG and incubated with shaking overnight at 18 °C. The cells were pelleted, resuspended in a lysis buffer , then sonicated for 60 s in 10-s pulses at 80% amplitude on ice. The lysed cells were centrifuged at 40,000× g and the clarified lysate was mixed with 40 µL Ni-NTA affinity resin for 4 h at 4 °C. The beads were washed in the lysis buffer for 15 mins, then 1 mg of the HeLa cell lysate was added and the mixture was incubated at 4 °C overnight with rotation. The beads were then washed three times with a wash buffer . The beads were boiled with an SDS loading buffer and analysed by Western blotting.The As previously discussed, SWI/SNF chromatin remodeller activity has been associated with HIF-dependent and -independent responses in hypoxia ,8,9. FurHIF activity is primarily regulated by the availability of the oxygen-sensitive HIF-α subunits. This is dependent on the degradation of HIF-α by the proteosome. To investigate if PBRM has a role in regulating their abundance, levels of HIF-1α and HIF-2α were measured by immunoblotting in hypoxic cells in which PBRM1 levels were depleted using siRNA. Interestingly, depletion of PBRM1 specifically reduced the levels of hypoxia-induced HIF-1α  but did Our analyses of how PBRM1 controls HIF-1α suggest a mechanism that differs from that of SWI/SNF components BAF155, BAF57 and BRG1, regulation of HIF-1α transcription [It is known that PBRM1 can bind to DNA as part of its function on the PBAF complex , but we As PBRM1 depletion could decrease HIF-1α protein levels but not transcript levels, we next investigated HIF-1α translation. Global protein synthesis, as well as translation rates of individual mRNAs, can be measured by polysome profiling, a technique in which free ribosomes can be separated from mRNA-bound ribosomes (polysomes) on a sucrose gradient. Quantitative PCR analysis of polysome-associated HIF-1α mRNA revealed that PBRM1 depletion caused a decrease in HIF-1α mRNA association with actively translating ribosomes consistent with a decrease in HIF-1α translation efficiency G,H. ImpoDue to our unexpected observations that PBRM1 controlled HIF-1α levels through translational control, we interrogated the PBRM1 interactome using IP-MS (data not shown) to identify known translational regulators and RNA binding proteins as interactors of PBRM1. Among these, we found YTHDF2, which can recognise and bind to m6A-methylated RNA . We wereOur data suggest that HIF-1α RNA could be m6A methylated. To investigate this further, publicly available datasets were interrogated ,21,22,23Taken together, our data demonstrate a previously unknown function of PBRM1, outside the SWI/SNF complex and in cooperation with the epitranscriptome, in the control of HIF-1α mRNA fate.Here, we investigated the role of PBRM1, a component of the SWI/SNF complex, in the regulation of the HIF response. We used a variety of cancer and non-cancer cell lines in this study. Components of the SWI/SNF chromatin remodeller are frequently mutated in cancer , and as There is a precedent for RNA binding activity within the SWI/SNF complex with the catalytic subunits BRG1 and BRM interacting with lncRNA and mRNA ,26. SomeIn hypoxia, global translation is reduced, regulated at the stage of initiation , posing Our data suggest that PBRM1 is required for efficient HIF-1α protein translation. However, in hypoxia, we observed a reduced interaction between PBRM1 and YTHDF2, suggesting that, potentially, another m6A reader might come into play. Further analysis investigating m6A levels in hypoxia and PBRM1 interaction could provide answers to these remaining questions. However, the importance of PBRM1 is still present in hypoxia, as reduced HIF-1α protein translation in the absence of PBRM1 is still observed under this condition. This indicates that reductions in transcription ,37 and tAlthough the analysis of the epitranscriptome has not been conducted in hypoxia, there are existing links between these two aspects. The m6A eraser proteins, FTO and ALKBH5, are 2-oxoglutarate dioxygenases, meaning they use 2-oxoglutarate, iron and oxygen as cofactors. Although their oxygen affinity might be too high to participate in the hypoxia response , ALKBH5


Daqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown.Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region.In this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily  was successfully heterologous expressed by This study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.The online version contains supplementary material available at 10.1186/s12934-021-01571-w. Paenibacillus panacisoli [Geobacillus stearothermophilus, Xyn2A from Trichoderma viride [Aplysia kurodai [Aspergillus niger [Aspergillus oryzae [Normally, to efficiently degrade polysaccharides in nature, microbes produce a set of enzymes with collaborative activities, such as endoglucanase, cellobiohydrolase, and β-glycosidase on cellulose , xylanasnacisoli , the xyla viride , and the kurodai . Among c kurodai , 8. A nuus niger  and two s oryzae . HoweverBacillus in Daqu [Nong flavor (NF) Daqu is the most important fermentation starter for Chinese liquor, with a large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years . However in Daqu , with mo in Daqu . Therefo in Daqu . Subsequ in Daqu , and an  in Daqu , were miAspergillus oryzae [Aspergillus niger [In addition, amylases are the main contributor to the liquefying and saccharifying processes of Daqu, which have a significant effect on the quality of Daqu . Based os oryzae . On the us niger . HoweverA novel fungal α-amylase (NFAmy13B), recognized as a GH13_5 member, was successfully obtained from the high-temperature stage of NF Daqu and characterized in this study. The first α-amylase previously characterized, NFAmy13A , was assEscherichia coli Trans5ɑ and BL21(DE3) were purchased from TransGen Biotech  and used for gene cloning and protein expression, respectively. Sonics Vibra-Cell™ used for cell disruption was purchased from Sonics & Materials . Ni-NTA Sefinose™ Resin was obtained from Sangon Biotech  for protein purification. Amicon® Ultra-15 Centrifugal Filter Devices  were used for protein concentration. NanoDrop 2000c  was used to analyze the concentrations of DNA and protein. High-performance anion-exchange chromatography (HPAEC) (Dionex ICS-5000) with a pulsed amperometric detector (PAD) was obtained from Thermo Fisher Scientific . Kanamycin sulphate, isopropylthio-β-galactoside (IPTG), imidazole, and other chemical reagents used in this study were purchased from yuanye Bio-Technology .Mix (Green) for PCR amplification and pDE2 Directional TOPO Expression Kit for expression vector construction were purchased from TsingKe . The Universal DNA Purification Kit used for PCR product purification was purchased from TianGen . CACCATGAAGTCCCTCCTCTGCTGC) and NFABr (CTAGTGCTTGTAGATATCCGAGTC)). The gene fragments were analyzed on 1% agarose gel and purified using a Universal DNA Purification Kit. The purified DNA fragments were ligated into the pDE2 vector according to the manufacturer’s instructions. Trans5α was used as the host strain for the transformation of recombinant pDE2 with the NFAmy13B gene. Transformants were selected on Luria-Bertani (LB) medium containing kanamycin (50 µg/mL). Positive clones with the correct inserts were verified by DNA sequencing (Sangon Biotech).The gene product of ORF 22,984 was the second α-amylase characterized in the GH13 family in the NF Daqu system, and was named NFAmy13B. Similar to previous studies , the genNFAmy13B plasmid was transformed into BL21 (DE3) for gene expression. After an overnight culture on LB plate supplemented with the same antibiotic, a single colony was picked and inoculated into 3 mL LB at 37 °C with shaking (200 rpm) for incubation overnight. Subsequently, 1 mL of pre-culture was inoculated into 1 L of LB medium containing the same antibiotic as described above at 37 °C and 200 rpm. When OD600 reached 0.5–0.8, 0.1 mM IPTG was added to the culture to induce gene expression, and the temperature was decreased to 16 °C. After 24 h of cultivation, the cells were harvested by centrifugation at 8000 rpm and 4 °C for 3 min, then suspended in binding buffer . The cells were disrupted by sonication, and the ruptured cells were clarified by centrifugation at 8000 rpm for 10 min at 4 °C to remove cell debris. The supernatant was then applied to Ni-NTA agarose resin for further purification, according to the manufacturer’s instruction with some modifications. The loaded protein was washed with binding buffer supplemented with 20 mM imidazole until only small amounts of protein were eluted. The bound protein was eluted using binding buffer supplemented with 250 mM imidazole, and fractions were collected for further analysis. The purity and molecular mass of the protein were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to the results of SDS-PAGE, the fractions with the desired proteins were collected and concentrated in a protein storage buffer  using Amicon® Ultra-15 centrifugal filters. The concentration of the purified protein was analyzed using NanoDrop 2000c.The recombinant pDE2-To investigate the potential activity of NFAmy13B, a plate-based activity assay was performed. Solid plates were prepared by dissolving 0.1% AZCL-dextran, -arabinoxylan, -HE-cellulose, -pullulan, -amylose, -xylan, or -debranched arabinan (Megazyme) in phosphate buffer at pH 6.0 supplemented with 1% agarose. Approximate 20 µg of purified protein was added to the center hole of the plates. The plates were subsequently incubated at 54 °C overnight, and amylase activity was determined by the blue zone around the hole of the plates.pHBAH; Sigma-Aldrich, St. Louis, MO) method [The NFAmy13B activity was performed at 54 °C and pH 6.0 for 30 min throughout this work, and the assay without enzyme was used as a blank control. The amount of reducing ends released was detected with glucose as the standard using the para-hydroxybenzoic acid hydrazide  method .2CO3–NaOH buffer for pH 9.5–1.0, 25 mM Na2HPO4–NaOH buffer for 11.0–12.0, and 50 mM KCl–NaOH buffer for 12.0–13.0) at 20 °C for 1 h. Furthermore, to evaluate the alkali-resisting characteristic of NFAmy13B, 600 nM enzyme was pre-treated at pH 11.0, 12.0, and 13.0 for different times, respectively. Then, the residual activity was measured by incubating 40 nM enzyme and 2 mg/mL corn starch at 54 °C and pH 6.0 for 30 min. The residual activity was calculated by assuming the untreated control as 100%.To evaluate the pH stability of NFAmy13B, 600 nM purified enzyme was incubated in various pHs, ranging from 3.0 to 13.0  was obtained by plotting the natural logarithms of the residual activity values versus incubation time and calculated from linear regression.To determine its thermostability at different temperatures, 150 nM enzyme was first pre-treated in 50 mM phosphate buffer (pH 6.0) at 40 to 64 °C for 30 min on a 96-well thermal cycler . After cooling on ice, the residual activities were determined at 54 °C and pH 6.0 for 30 min. To detect the half-life of thermal inactivation at 45, 50, 55, and 60 °C, 75 nM enzyme was pre-treated at the stated temperatures for different periods of time with or without 1.25 mM CaClpHBAH assay, and the relative activities were expressed as a percentage of the activity of the unadded control, designated as 100%.The effect of various additives  on NFAmy13B activity was investigated by incubating the enzyme with corn starch in the presence of different chemical compounds at 54 °C for 30 min. The released reducing ends were determined using the pHBAH method. Secondly, its specific activities were determined by incubating a low concentration of 20 nM enzyme with various substrates (5 mg/mL) containing 1.25 mM CaCl2 for different times, where the slopes of the reducing products against time were linear. One unit of enzyme activity was defined as the amount of enzyme required to release 1 µmol glucose equivalents (reducing ends) from a specific substrate per minute under standard conditions. Lastly, to investigate the kinetic parameters, 20 nM enzyme was incubated with starch substrates  ranging from 0.1 to 16 mg/mL at 54 °C for 20 min. The velocities were kept in a constant scope under every condition by using low concentration of 20 nM enzyme. The initial velocities were plotted against the substrate concentrations, and their kinetic values were determined by applying the Michaelis-Menten equation using a nonlinear regression method (Origin 9).Firstly, the digesting capacities of NFAmy13B on various substrates were determined by incubating 40 nM enzyme with 5 mg/mL substrates  at 54 °C for 30 min. The released amount of reducing sugars was estimated using the 2 at 54 °C and pH 6.0 for different time intervals . The released reducing ends  were quantified using the pHBAH method. Meanwhile, a constant incubation time of 12 h and various concentrations (1 to 16 mg/mL) of starchy substrates were studied under the same conditions. The end-products were analyzed by HPAEC-PAD using a Dionex CarboPac PA20 analytical column (3 × 150 mm) and Dionex CarboPac PA20 guard column (3 × 30 mm). The mobile phase comprised component A (250 mM NaOH) and B . Glucose (M1), maltose (M2), maltotriose (M3), maltotetraose (M4), and amylopentaose (M5) were used as standards.Initially, the experiments were performed by incubating a constant concentration of 8 mg/mL starchy substrates  with a high concentration of 1 µM protein in the presence of 1.25 mM CaCl2 at 54 °C for 30 min. The soluble end-products were analyzed by HPACE-PAD. To confirm the hydrolysis patterns of these two enzymes, the hydrolysis of maltooligosaccharides was studied at 54 °C and pH 6.0 for various time periods  by incubating 1 µM NFAmy13B or NFAmy13A with 5 mM maltose (M2), maltotriose (M3), maltotetraose (M4), and amylopentaose (M5), respectively, and analyzed using the HPAEC-PAD method.To effectively evaluate the synergistic hydrolysis of NFAmy13B and NFAmy13A on wheat starch, different concentrations of NFAmy13B and NFAmy13A were used to release the same reducing ends from wheat starch. Thus, group of 20 nM NFAmy13B and 1000 nM NFAmy13A were set with equivalent relative activity. Different ratios of 20 nM NFAmy13B and 1000 nM NFAmy13A were combined to get the equivalent relative activity with each of 20 nM NFAmy13B and 1000 nM NFAmy13A. Therefore, their synergistic hydrolysis was carried out by incubating different ratios of 20 nM NFAmy13B and 1000 nM NFAmy13A with 5 mg/mL wheat starch in the presence of 1.25 mM CaClThe amino acid sequences used for phylogenetic tree construction in the phylogenetic analysis are listed in Additional file https://swissmodel.expasy.org/) using PDB 3KWX and 1WPC as a template, respectively. All of the predicted structures were visualized by PyMOL software version 2.3.3 .The three-dimensional (3D) structure of NFAmy13A and NFAmy13B was constructed by homology-based modeling on SWISS-MODEL (NFAmy13B obtained from the N3 cDNA library was deposited in the NCBI GenBank database under Accession Number MT849765.The nucleotide sequence of NFAmy13B was successfully obtained by specific amplification with primers NFABf/r from N3-cDNA and expressed in E. coli BL21(DE3). The recombinant NFAmy13B with a C-terminal 6×His tag was purified by affinity chromatography using Ni2+-NTA resin. Based on our previous metatranscriptomic analysis of Daqu N3 [The gene sequence of  Daqu N3 , ORF 22,After pre-treatment at various pH values at 20 °C for 1 h, NFAmy13B was found to be very stable at a pH range of 5.5–12.5, with over 90% residual activity  of Ca5 mM of C2+; slightly inhibited by K+ at both concentrations, and Na+ only at 1 mM; moderately inhibited by Ba2+ and Li+ at 1 mM; largely inhibited by the remaining metal ions. None of the detected additives showed any stimulatory effect on its activity.The effects of additives on enzymatic activity were determined with final concentrations of 1 mM and 10 mM , NFAmy13A showed a lower specific activity for wheat starch (4.85 U/mg) than NFAmy13B (74.0 U/mg), indicating that NFAmy13A had a lower degrading capacity for the long-chain starchy substrates. Therefore, different concentrations of NFAmy13B 20 nM) and NFAmy13A (1000 nM) were set to release the same reducing ends from wheat starch  with the characterized intracellular fungal α-amylase (AmyD) from Aspergillus niger [Bacillus sp.707 [http://www.cbs.dtu.dk/services/SignalP/)  to the putative α-amylase XP_028489551.1) from 551.1 frous niger , and 47.s sp.707 . Based o7.6% to t8-barrel domain and C-terminal eight-stranded β-sandwich domain  of Chinese NF Daqu by metatranscriptomics . To mineNFAmy13B gene may originate from a fungal genus, such as Byssochlamys. Besides, NFAmy13B may be an intracellular fungal α-amylase based on the phylogenetic analysis. Interestingly, NFAmy13B was very stable at a pH range of 5.5–12.5 , another α-amylase with high level of expression at the same stage of NF Daqu. The degradation patterns on maltooligosaccharide except maltotriose were observed same between them. Meanwhile, NFAmy13B showed a higher catalytic efficiency on long-chain starches compared to short-chain maltooligosaccharides for NFAmy13A. Thus, we postulate that NFAmy13B plays the dominate role in hydrolyzing starch into maltooligosaccharides, similar to a liquefying α-amylase, subsequently NFAmy13A plays main contribution to the hydrolysis of maltooligosaccharides in NF Daqu. Therefore, our proposed strategy is an effective approach for directly mining novel enzymes from Daqu, and NFAmy13B together with NFAmy13A could be applied as candidate α-amylases in industries.A novel α-amylase, designated as NFAmy13B, was successfully obtained from Daqu using metatranscriptomics and heterologous expression. Its biochemical properties were comprehensively investigated, and compared with other intracellular fungal α-amylases in the GH13_5 subfamily, NFAmy13B showed different properties in degrading starchy substrates, including a high stability over a wide pH range (5.5–12.5), a higher degrading capacity on potato starch, and higher levels of activity on amylopectin than on amylose. In addition to these characteristics, its thermostability, but not enzyme activity, was stimulated by CaAdditional file 1: Table S1. Amino acid sequences used for phylogenetic tree construction. Table S2 The hydrolysis of NFAmy13B on various substrates. Table S3. Degradation capacity of NFAmy13A and NFAmy13B on wheat starch. Table S4. The hydrolysis of NFAmy13A and NFAmy13B on maltooligosaccharides (M3−M5). Table S5. Comparison of alkali-resisting α-amylase. Fig. S1. Effect of the concentrations of calcium divalent ion on NFAmy13B thermostability. Enzyme (75 nM) was incubated with different concentrations of CaCl2 at pH 6.0 and 55 °C for 30 min. The residual activities were determined by incubating 30 nM enzyme with 5 mg/mL potato starch at 54 °C and pH 6.0 for 30 min. Each experiment was performed in triplicate. Fig. S2. Kinetic studies of the NFAmy13B on the different substrates. 20 nM enzyme with 1.25 mM CaCl2 was incubated with various concentrations substrate between 0.1 and 16 mg/mL at 54 °C for 20 min. Each experiment was performed in quadruplicate . Fig. S3. Hydrolysis of maltooligosaccharides by NFAmy13A and NFAmy13B. 5 mM M2 and M3 were hydrolyzed by 1 μM NFAmy13A or NFAmy13B in the presence of 1.25 mM CaCl2 at pH 6.0 and 54 °C for 2 h. The reaction products were analyzed by HPAEC-PAD. M1, glucose; M2, maltose; M3, maltotriose. Fig. S4. Phylogenetic analysisof NFAmy13A and NFAmy13B. The amino acid sequences of enzymes from GH13 used for the construction of the phylogenetic treewere selected according to the similarities with NFAmy13A or NFAmy13B. Each enzyme was indicateda numbercorresponding to theGH13 subfamily, its abbreviation, and source. Detailsareprovided in Table S1. Fig. S5. Amino acid sequences alignment in NFAmy13A/B and high-similarity α-amylases.


Rhodobacter sphaeroides. We used cryo-electron microscopy(cryo-EM) to determine the 2.1 Å resolution structure of thisLH2 antenna, which is a cylindrical assembly of nine αβheterodimer subunits, each of which binds three bacteriochlorophyll a (BChl) molecules and one carotenoid. The high resolutionof this structure reveals all of the interpigment and pigment–proteininteractions that promote the assembly and energy-transfer propertiesof this complex. Near the cytoplasmic face of the complex there isa ring of nine BChls, which absorb maximally at 800 nm and are designatedas B800; each B800 is coordinated by the N-terminal carboxymethionineof LH2-α, part of a network of interactions with nearby residueson both LH2-α and LH2-β and with the carotenoid. Ninecarotenoids, which are spheroidene in the strain we analyzed, snakethrough the complex, traversing the membrane and interacting witha ring of 18 BChls situated toward the periplasmic side of the complex.Hydrogen bonds with C-terminal aromatic residues modify the absorptionof these pigments, which are red-shifted to 850 nm. Overlaps betweenthe macrocycles of the B850 BChls ensure rapid transfer of excitationenergy around this ring of pigments, which act as the donors of energyto neighboring LH2 and reaction center light-harvesting 1 (RC–LH1)complexes.Light-harvesting 2 (LH2) antennacomplexes augment the collectionof solar energy in many phototrophic bacteria. Despite its frequentrole as a model for such complexes, there has been no three-dimensional(3D) structure available for the LH2 from the purple phototroph  The intracytoplasmic membranes that housethese arrays have been imaged by atomic force microscopy (AFM),Rba. sphaeroides have been usedto model the interlinkedbioenergetic processes of excitation and electron transfers, formationof a protonmotive force, and its consumption by the ATP synthase.7 A structure of the Rba. sphaeroides LH2 complex would improve our understanding of LH2-enriched membranedomains in chromatophores,9 provide a molecularinterpretation of previous site-directed modifications of the LH2complex, and also underpin future manipulations of the pigment andprotein components. Earlier structural studies of LH2 complexes usedX-ray crystallography, and the first, iconic, structure was determinedfor the LH2 from Rhodoblastus (Rbl.) acidophilus (formerly Rhodopseudomonas (Rps.) acidophila) strain 10050 by Richard Cogdell and co-workers over 25 years ago.10 There was a subsequent improvement in resolution,11 and this LH2 structure was joined by others,from Rhodospirillum molischianum,of schianum,Rbl. acidaliphila. A cryo-E16 driven by a series of developments in sample and grid preparation,microscopes, detectors, and software.18 These advancesoffered new possibilities, circumventing the obstacles to crystallographicwork presented by poorly ordered crystals. A series of papers reportedthe cryo-EM structures of RC–LH1 complexes from several purplephototrophic bacteria,21 and recently, the first cryo-EM structure of an LH2complex, from Marichromatium (Mch.) purpuratum, was determined at a resolution of 2.4 Å.22 This LH2 comprises a ring of seven αβ heterodimersubunits, unlike the 8-fold and 9-fold rings from other bacteria reportedearlier.15 Here, we presentthe cryo-EM structure of the Rba. sphaeroides LH2 complex at a 2.1 Å resolution, which reveals the intricatenetwork of pigment and protein interactions that form the basis ofits stability and its function as an antenna. The results are placedin the context of other LH2 structures23 and previousprotein engineering studies of the Rba. sphaeroides LH2 complex.Recently, there has been a remarkable upsurgein determinationsof protein structures by single-particle cryo-EM,Rba. sphaeroides 2.4.1 mutant B7 (Δpuc2BA ΔcrtA) was grown photosyntheticallyat 30 °C with stirring in 20L M22+ medium under a light intensity of 150 μmol photons s–1 m–2 provided by Osram 116 W halogenbulbs. When the culture reached an absorption at 680 nm of 1.6, thecells were harvested by centrifugation at 3 000 g for 30 min and then washed once using working buffer . The pellet was stored at −80 °C until required.g, photosyntheticmembranes were harvested from the interface, pelleted, then resuspendedin working buffer, and adjusted to an absorbance of 100 at 875 nm.For solubilization, the 875 nm absorbance of membranes was 60, andthe final concentration of N,N-dimethyldodecylamine N-oxide (LDAO) detergent was 3% (w/w). The mixture was stirredat 4 °C for 30 min, and then unsolubilized material was removedby centrifugation for 1 h at 211 000 g (Beckman70.1 Ti rotor). The supernatant was applied to a five-layer (20/21.25/22.5/23.75/25%w/w) sucrose gradient in running buffer (working buffer containing0.1% LDAO). After 16 h of centrifugation at 125 000 g in a Beckman SW41 rotor, the LH2 band, which was separatedfrom the core RC–LH1–PufX complex, was collected andapplied to an ion-exchange column equilibrated with running buffer. Fractions containing the LH2 complex,which eluted at ∼150 mM NaCl, were collected, and those withan A850/280 absorption ratio of >2.7 were pooled andconcentrated.LH2 was further purified on a gel filtration column (Superdex 200)pre-equilibrated in running buffer. Fractions with A850/280 > 3.3 were kept and concentrated to an A850 of 150forcryo-EM data collection.Thawed cells were resuspendedin working buffer with a few crystals of DNase and magnesium chloride,and then the suspension was passed through a French pressure cellthree times at 18 000 psi. The broken cells were layered ontoa 15/40% (w/w) sucrose density gradient. After 4 h of centrifugationin a Beckman SW32 rotor at 100 000 Rps. acidophila (∼2.5 mg/mL)was prepared in the same way. Energy-dispersive X-ray spectrometry(EDXS) was performed on an FEI Tecnai Osiris S/TEM operated in STEMmode at 200 kV at room temperature. Data were acquired on a BrukerSuper-X system comprising four EDX detectors. Spectra were acquiredby integrating for 64 s on an area of several square microns to ensuregood sampling with a current of ∼1 nA.Ten microlitersof purified LH2complex (∼4 mg/mL) were cast onto the surface of a lacey carbontransmission electron microscopy (TEM) grid. The protein sample wasdried in air under a 100 W lamp. As a control, a grid with the LH2from Protein solution  was applied on a QuantiFoil R1.2/1.3 300 mesh Cu grid, whichwas glow-discharged for 60 s before use. An FEI Vitrobot MK IV wasused for freezing the grid, using the following settings: chamberhumidity 95%; chamber temperature 4 °C; blotting time 2.5 s;blotting force 3; and wait time 30 s. The grid was plunge-frozen intoliquid ethane cooled by liquid nitrogen. It was then stored in liquidnitrogen until use.24 Themicroscope was operated at 300 kV with a normal magnification of 130 000x,corresponding to a pixel size of 0.66 Å at the specimen level.All movies were collected in super-resolution mode with an energyselecting slit of 20 eV. A total dose of 41.6 electrons per Å2 within a 1.37 s exposure time was fractionated into 40 frames,resulting in an electron fluence of 1.04 e–/Å2/frame. Automatic data collection was carried out in ThermoFisher EPU 2.11 with two exposures per hole in aberration-free shift(AFIS) mode. In total, 3 138 movies were collected in a defocusrange from −0.8 to −2.2 μm within 10 h.Cryo-EMdata were collectedon a Thermo Fisher Titan Krios G2 cryogenic electron microscope equippedwith a Gatan BioQuantum K3 direct electron detector at the CambridgePharmaceutical Cryo-EM Consortium.25 with a binning factor of 2 on 5 × 5 patches, resulting in apixel size of 0.66 Å in the motion-corrected images. The contrasttransfer function (CTF) parameters were estimated using CTFFIND4.26 All bad images, including those with empty holes,uncorrected drift, thick ice or extensive ice contamination were rejected.Particles were picked in cisTEM27 witha box size of 180 Å. In total, 1 717 608 particleswere picked from all selected images, corresponding to an averageof 547 particles per image. All extracted particles were subjectedto reference-free 2D classification. After discarding bad 2D classes,1 476 723 (86%) were selected for 3D classification.Reference-free 2D classification showed that the LH2 from Rba. sphaeroides is a nonamer , which possesses an architecture similar to theLH2 from Rbl. acidophilus. A crystalstructure model of this complex (PDB 1NKZ), therefore, was used to build an initialmodel for 3D classification using Chimera.28 Owing to the homogeneous subunits in the current LH2 complex, aC9 symmetry was imposed starting from the 3D classification. Duringthe 3D classification, 835 641 (48.6%) particles were groupedinto the best class (3.6 Å) out of 4. After multiple rounds of3D refinement, CTF refinement  and Bayesian polishingwith the default parameter in RELION 3.1, this data set produced a2.3 Å resolution map. The selected particles were re-extractedusing 25 nm × 25 nm box for CTF refinement and further Bayesianpolishing. This resulted in a final 3D map with a resolution of 2.1Å.The initial super-resolution moviefiles were motion-corrected within RELIONa and one carotenoid taken fromLH2 of Rbl. acidophilus (PDB 1NKZ) was docked intothe C9 symmetric density map of LH2 from Rba. sphaeroides using Chimera, focusing on correct positioning of the B850 pairsand their coordinating His residues. Amino acid sequences of bothα/β polypeptides in the template were mutated in COOT29 based on the Rba. sphaeroides sequences in Rba. sphaeroides, αβBChl3Car, was constructed. After beingreal-space-refined in COOT, all symmetry copies were generated toform a preliminary model of the complete LH2 complex. A preliminaryrefinement was performed in COOT, and tightly bound detergent molecules,LDAO, were added. The model was then rebuilt and optimized in ISOLDE,17 including the addition of 230 water moleculesand a calcium ion coordinating the final turn of each β chainhelix. The rebuilt model was subjected to restrained global refinementin Phenix30 using Phenix real_space_refine.The refinement statistics are summarized in Table S1. The refined model and its map were deposited into ProteinData Bank (PDB) and the Electron Microscopy Data Bank (EMDB) withaccess codes of 7PBW and EMD-13307, respectively.An α/β subunithaving three BChl Rba. sphaeroides strain B7, which was engineeredto ensure uniformity of the complexes assembled. First, the puc2BA genes encoding the second set ofLH2 αβ polypeptides31 wereremoved by in-frame deletion, leaving only the puc1BAC operon to provide polypeptides for the LH2complex and with PucC as an assembly factor.32 Second, a crtA mutation was used to inactivatespheroidene monooxygenase (CrtA), which introduces a C2 keto groupinto the yellow spheroidene to form the red carotenoid spheroidenone.33 Restricting the carotenoid pathway in this wayremoves the possibility of LH2 complexes assembling with a mixtureof spheroidene and spheroidenone, minimizing heterogeneity. We recorded3 138 cryo-EM movies, from which 1 717 608 particleswere picked for further data processing, yielding a final resolutionof 2.1 Å (S1 and Table S1). Statistics forthe final model are in Table S1. The 2.1Å resolution allows nearly all of the α- and β-polypeptidesto be modeled into the density . Whereas bound Mg2+ was found in the Rbl. acidophilus LH2, EDX identified Mg2+ in the Rba. sphaeroides complex and new peaks appeared for Ca2+. Based on thisresult combined with the coordination bond lengths and geometry, weconclude that it is reasonable to assign the C-terminal metal as calcium.This metal is held to the C-terminus of the LH2 β-polypeptideby the three backbone oxygens of β-Ala44, Thr47, and Trp49 andby the C-terminal carboxylate of Gly51. We found some evidence inthe density map for two water molecules coordinating with the Ca , giving an approximate octahedralgeometry, but they are not assigned in the final model. The role ofthese metals is unclear; in one treatment, we incubated the Rba. sphaeroides complex with excess EDTA to removeall bound ions, in another the complex was fully reconstituted withexcess CaCl2, and a third treatment used EGTA and excessMgCl2 to strip the endogenous metal and replace it withMg2+. None of these procedures affected the absorptionspectrum of the complex .Figure S4) of the carotenoid from the adjacent αβ heterodimer and the C8–C12 region of spheroidene in darkerorange makes a series of hydrophobic contacts with ring D of the α-boundB850  in the adjacent αβ heterodimer. Theoverall effect is a circular chain of carotenoid interactions interlinkingeach αβ heterodimer with its neighbor. The extent of thecontacts between the carotenoid and surrounding pigments and proteinsaccounts for the decisive contribution made by this pigment to thestability of LH2.36Y absorptiontransition dipoles are almost parallel to the plane of the membrane,in approximate alignment with the head-to-tail QY transitionsof the paired B850 BChls , the same detergent successfully used by Rod Clayton and GeorgeFeher for isolating reaction center complexes.51 The other antenna complex in the Rba. sphaeroides photosystem, LH1, was destroyed by LDAO and its purification awaitedmore gentle solubilization by lithium dodecyl sulfate.52 This detergent had an interesting effect onLH2 by selectively removing nearly all of the B800 absorption band,which could be restored by the subsequent addition of LDAO.53 The structure of the Rba. sphaeroides LH2 complex provides a basis for these early observations and alsofor many subsequent pigment exchange studies that replaced B800 withBChl and chlorophyll (Chl) analogues.56 B800 sits within 10Å of the solvent/membrane interface, sufficiently close to makeit selectively susceptible to some detergents. Despite the networkof hydrogen bonds between B800 and LH2 α and β polypeptides,and the interaction with the carotenoid spheroidene  that coordinates the B850 BChls;counting forwards or backwards from that point allows comparison ofLH polypeptides of differing lengths (see Rba. sphaeroides LH2 mutants in which β-Arg30 (β Arg–10) was alteredto Phe, Leu, Glu, and Met41 examined thebasis for the red-shifted B800 absorption of monomeric BChls boundwithin LH2 antenna complexes, in relation to the 771 nm absorptionof BChl in ether.40 It was concluded thatthe hydrogen bond from β-Arg30 to the C3-acetyl group of theB800 BChl imparts a shift of approximately 10 nm, and another 10 nmof shift arises from the local properties of the BChl binding site.40 The structure in this region of the complex and subsequentlycalibrated against the membrane potential by Baz Jackson and TonyCrofts in 196960 and later by many workers(e.g.61). Then, it became clear that theseabsorption shifts originated in the carotenoids bound to LH2,63 and a subsequent mutagenesis study of the Rba. sphaeroides LH2 complex showed that alteration of β-Arg30 greatly attenuatedthe carotenoid absorption shift that responds to the membrane potential.57 Ultrafast carotenoid band shifts, attributedto a local electric field have been observed for the LH2 complex followingexcitation of either B800 or B850 BChls;65 these band shifts might also stem from the presence of β Arg30.While spanning the membrane, the nine carotenoids make multiple contactswith protein side chains and with B800 and B850 BChls that stabilizethe complex  fromlow-light-grown Rps. acidophila strain7050 has Phe and Leu at locations on the C-terminal region of LH2αwhere aromatic residues would normally form hydrogen bonds to B850BChls (Rba. sphaeroides, mutagenesisof C-terminal residues α-Tyr44 to Phe and α-Tyr44 andα-Tyr45 to Phe and Leu produced B800–838 and then B800–826complexes, respectively;43 Raman spectroscopyverified the disruption of this bonding arrangement.42 The progressively larger overlap between B800 and the blue-shiftedLH2 absorption bands increased the rate of excitation transfer betweenthe two rings of pigment.68 When the samemutants were examined in an intact photosystem where LH2 donates energyto the RC–LH1–PufX complex, energy transfer from B800–850,B800–839, and B800–826 LH2 complexes to LH1 slowed from4.6 ps to 6.2 and 7.0 ps, respectively, due to a decreased spectraloverlap between the LH2 donor and the 875 nm absorbing LH1 acceptor.47 Slowed rates of forward LH2 to LH1 energy transferfor blue-shifted mutants were accompanied by slower back-transferof energy from the RC–LH1–PufX core complex to the blue-shiftedLH2 antenna,69 an observation also madefor low-light Rbl. acidophilus cellscontaining B800–820 and core complexes.70 It appears that the assembly of a B800–820 antenna,achieved by swapping hydrogen-bonding residues for those that cannotparticipate in bonding, confers an advantage in low-light conditionsby minimizing back-transfer, thereby “concentrating”excitations within cores where they can be trapped at the RC.The 850nm absorption bandof the 50BChls 7C.In thRba. sphaeroides LH2 complexhas featured in numerous spectroscopic, developmental, physiological,and computational studies, aimed at understanding its absorption andenergy-transfer functions, the way in which LH2 arrays improve thecollection of solar energy under low light and how LH2 arrays influencethe architecture, organization, and shape of photosynthetic membranes.Invaluable guides to the structure of this nonameric LH2 complex wereprovided over 25 years ago by the crystallographic structure of theLH2 from Rbl. acidophilus(Rba.sphaeroides LH2 complex.15 Single-particle cryo-EM has eventually supplied the LH2 structureat a 2.1 Å resolution, high enough to reveal the details of theinternal molecular arrangement of carotenoids, BChls, and bondinginteractions that establish internal energy-transfer pathways andwhich enable LH2 to form an adaptable network for gathering excitationenergy and transferring it to RC–LH1–PufX complexes.The idophilus,11 and b