Furthermore, THOC5 is preferentially recruited to its target genes in slow polymerase II cells compared with fast polymerase II cells. Importantly chromatin-associated THOC5 interacts with CDK12 (a modulator of transcription elongation) and RNA helicases DDX5, DDX17, and THOC6 only in slow polymerase II cells. The CDK12/THOC5 interaction promotes CDK12 recruitment to R-loops in a THOC6-dependent manner. These data demonstrate a novel function of THOC5 in transcription elongation.THOC5, a member of the THO complex, is essential for the 3′processing of some inducible genes, the export of a subset of mRNAs and stem cell survival. Here we show that THOC5 depletion results in altered 3′cleavage of >50% of mRNAs and changes in RNA polymerase II binding across genes. THOC5 is recruited close to high-density polymerase II sites, suggesting that THOC5 is involved in transcriptional elongation. Indeed, measurement of elongation rates   •THOC5 depletion decreased transcription elongation rates globally•3′ end cleavage of more than 50% of genes was altered in THOC5-depleted cells•The rate of transcription elongation determines polyA site usage•THOC5/THOC6 recruit CDK12 and DDX5/17 to regulate R loop structures and elongation rate Molecular biology; Cell biology Saccharomyces cerevisiae as a five-protein complex ,,Drosophila melanogasterThe THO complex, which is a sub-member of TREX (transcription/export) complex, was originally identified in ,,,,The THO complex controls the 3′processing of RNA, inhibiting R-loop formation and export of a subset of mRNAs; however, the molecular functions of individual members of this complex are still unclear. Recent data suggest that each member of the TREX complex plays different roles during transcription/export of mRNA.,,,,,,THOC5 plays a key role in stem cell and cancer cell biology and has been shown to be post-translationally regulated by stem cell ligands (CXCL12), oxidative stress, and the downstream actions of oncogenes.chem-seq), we show that THOC5 influences 3′end processing of 50%–60% of alternatively cleaved mRNAs and also participates in transcription elongation by recruiting CDK12 to RNA polymerase II (Pol II) where R-loops are formed. R loops can be formed during cellular stress and as such targeting molecular pathways that regulate their formation or removal, such as THOC5-/THOC6-mediated actions, could provide new approaches for developing therapies.In this study, using RNA sequencing by nanopore technology and 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB)/transient transcriptome sequencing . Four hundred ninety-three TSS and 176 gene body peaks were detected. Upon THOC5 depletion the average peak intensity significantly increased more than 1.4-fold at both TSS (p = 8.4E-93) and gene body (9.9E-15) of THOC5-dependent genes. The depletion of THOC5 also induced a Pol II accumulation of 1.4-fold at the TSS of THOC5 independent genes  in THOC5-depleted and control cells. In parallel, we performed tandem affinity purification (TAP)-THOC5 CHIP sequencing. Upon depletion of THOC5, Pol II density clearly increases at the TSS site, whereas only a slight increase was observed in gene body and at the 3′end of 626 THOC5-dependent genes A. We thent genes C, suggesent gene H. In addent gene I but notent gene J. These ent gene A, suggesent gene . We thenTo examine whether changes of elongation rates influence 3′end cleavage, we utilized inducible cell lines expressing α-amanitin-resistant mutants of the human Pol II large subunit that accelerate or decelerate elongation.To examine whether all members of TREX participate in transcription elongation and 3′processing, we examined which members of TREX are associated with chromatin. Notably, only THOC5, THOC6, UAP56, and CHTOP were clearly detected in the chromatin-associated fraction at levels comparable to the nucleoplasmatic fraction E. We theImportantly, one of the proteins unique to the slow Pol II fraction is CDK12, a protein that controls Pol II elongation rate by phosphorylation of the Pol II carboxy-terminal domain (CTD).NDUFAF4) was examined. Similar to THOC5, THOC6 was found to be recruited in the promoter region and the gene body of THOC5-dependent genes  is involved in control of the elongation rate, we depleted THOC2, a key member of THO complex D. THOC2 It has been shown that transcription pausing/slowing may cause R-loop formationin vitro. Next, we examined which of the two directly interacts with R-loop. For that we synthesized a blunt-ended DNA-RNA heteroduplex (C4D and OSD) and a DNA-RNA hybrids with a 5′ ssDNA flap at one end as an R-loop mimetic structure (C4R) derived from the THOC5 target C4ORF46 and OSBPL3 , oxidative stress, and the downstream actions of oncogenes. This set of facts indicate the later development of this protein is critical in higher organisms, although the precise role for the protein has not been elucidated. Here mechanistic details on THOC5 and THOC6 function are elucidated. Complementing the finding by Yan et al. who state that R-loops have a role in cell fate determination, memory, and plasticity,c-MYC.Previously, we have shown that THOC5 plays a role in the 3′processing of inducible genes, such as c-FOS, IER2 or ZFP36 with fast elongation rates are processed and exported in the absence of THOC5.We have previously shown that the most rapidly induced IEGs such as ,,It has been reported that THOC5 is also involved in digestion of left-over RNA strands after cleavage to avoid R-loop formation.,,,,,Is the THO complex one functional unit? Recent data from our and other groups suggest that each member of the THO complex plays different roles during transcription/export of mRNA.C. elegans,,,,in vitro data suggested that THOC5/THOC6 complex binds to R-loops in general .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Doan Duy Hai Tran . DMEM were supplemented with 10% bovine growth serum (Thermo Fisher) and penicillin-streptomycin-glutamine (Gibco). Cells were grown at 37°C in standard cell culture incubators. All cells were routinely tested and confirmed to be free of mycoplasma contamination. A full list of cell lines used in the paper is included in the Coding sequences of THOC5, THOC6, DDX5 and DDX17 were cloned into pcDNA3.1+/C-(K)-DYK and purchased by Genscript . THOC5, THOC6, DDX5 and DDX7 were transiently overexpressed in HEK293 and purified after two days using anti-FLAG M2 affinity gel .For GST pulldown assay, the coding sequences of FLAG tagged THOC6, DDX5 and DDX17 were subcloned into pGEX-4T1 vector. Protein expression was induced in BL21 (DE3) strain by adding IPTG and proteins were purified using Glutathione Sepharose . GST fusion proteins and GST tag were separated using TEV protease. TEV protease was then removed using NEBExpress® Ni-NTA Magnetic Beads (NEB).HEK293 cells were grown and transduced with control shRNA (shCr) and shRNA targeting THOC5 (shTHOC5) as previously described.Cytoplasmic RNA was isolated as previously described.PolyA + RNA (500 ng) were used for the nanopore direct RNA sequencing. A library was prepared using a direct RNA sequencing kit  according to the manufacturer’s protocol. Libraries were sequenced on R9.4 flow cells for 48 h. Base calling was performed using Guppy (Oxford Nanopore). Basecalled data were aligned to human reference genome GRCh38 using minimap2.For the identification of 3′cleavage sites the tool NanoFiltChIP experiments were performed as previously described.www.usegalaxy.org) was used for subsequent data analysis. Reads were mapped to the human reference genome (GRCh38) using Bowtie2 . PCR duplicates were detected and removed using MarkDuplicates (Picard). Metagene analysis was performed using deepTools 2.0.GSE173374.5 ng of ChIP derived DNA were subjected to library preparation using TruSeq ChIP Library Preparation Kit . Libraries were prepared and indexed according to manufacturer’s protocol. Indexed libraries were pooled and sequenced on an Illumina HiSeq 2500 (Illumina). FASTQ files were generated by CASAVA (v1.8.2). Galaxy workflow  of non-overlapping protein coding genes 60–300 kb in width from standard chromosomes (4591 genes). The read coverage was then calculated and normalized to the sequencing depth.To calculate the elongation rates the module “wave peak calling for single gene” was applied. This module filtered out poorly expressed genes  and genes with a wave peak <2 kb in the first  sample to reduce noise from the TSS region.7) were lysed by cytoplasmic lysis buffer . After centrifugation (800xg for 2 min) supernatant (cytoplasmic fraction) was removed and the pellet was resuspended in 60 μL NUN1 buffer (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA and 50% (v/v) glycerol). Six-hundred μl of ice-cold NUN2 buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% (v/v) NP-40 and 1 M urea) was then added to the suspensions, vortexed and further incubated on ice for 15 min. After centrifugation, supernatant was collected as nucleoplasmic fraction. The pellet was incubated with Turbo DNAse I  in high salt buffer ) for 20 min at 37°C (chromatin associated fraction). For co-immunoprecipitation study the chromatin associated fraction was diluted 1:3 with 50 mM Tris-HCL, pH 8 buffer containing 0.5% NP-40.HEK293 cells (10Chromatin associated extracts were diluted in 50 mM Tris-HCl pH 8 to a concentration of 150 mM NaCL and then NP-40 was added to a concentration of 0.5% (v/v). The diluted extracts were immunoprecipitated by Protein G Agarose-PLUS with mouse monoclonal anti-THOC5, FLAG M2 antibodies or control IgG in the presence of RNAse A. Following 8 h rotation at 4°C, the beads were washed five times. Input and binding samples were supplied for immunoblot or interactome analysis.m/z 300–1700 was acquired in the Orbitrap at a resolution of 60,000 at m/z 400 with an automatic gain control target of 106. Each full scan was followed by the selection of the 20 most intense ions; CID (collision-induced dissociation) and MS/MS analysis was performed in the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific). Selected ions were excluded from further analysis for 60s. Ions with an unassigned charge or a charge of +1 were rejected.Precipitated THOC5 protein complexes and control IgGs were subjected to electrophoresis on 10% (w/v) polyacrylamide gels and stained with simply Blue Coomassie safe stain . Precipitated THOC5 protein complexes and control IgGs were subjected to electrophoresis on 10% (w/v) polyacrylamide gels and stained with simply Blue Coomassie safe stain (Thermo Fisher Scientific). Protein bands were excised and destained with repeated incubation in 200 mM ammonium bicarbonate, 40% (v/v) acetonitrile. Gel pieces were dried with 3 washes in acetonitrile and then trypsinized  acetonitrile) overnight at 37°C. Peptides were extracted from the gel pieces by incubation in 50% (v/v) acetonitrile, 0.1% (v/v) formic acid, and peptides were desiccated and resuspended in 3% (v/v) acetonitrile, 0.1% (v/v) formic acid, 20 mM citric acid, pH 2.7. For each analysis, 10% of the peptide sample was loaded onto a nanoACQUITY UPLC Symmetry C18 Trap  , and flow was set to 15 μL/min of 3% (v/v) acetonitrile, 0.1% (v/v) formic acid, and 20 mM citric acid for 5 min. Analytical separation of the peptides was performed using a nanoACQUITY UPLC BEH C18 column  (Waters Corporation). Briefly, peptides were separated over a 91-min solvent gradient from 3% (v/v) acetonitrile, 0.1% (v/v) formic acid to 40% (v/v) acetonitrile, 0.1% (v/v) formic acid on-line to an LTQ Orbitrap Velos (Thermo Fisher Scientific). Data were acquired using an information-dependent acquisition method where, for each cycle one full MS scan of Data were analyzed using Mascot  software; the parameters were: Uniprot database, trypsin with up to 1 missed cleavage allowed, variable modifications oxidized methionine, phosphorylated serine, threonine, and tyrosine modifications permitted and peptide tolerance of 0.025 and 0.03 Da for MS/MS tolerance.Proteins which were detected in both control IgG and precipitated THOC5 complexes were discarded. Then, THOC5 interaction partners which had Mascot >30 were further selected for Ingenuity® Pathway Analysis (IPA).Details of immunoblotting have been described previously.Semi-quantitative RT-PCR and qRT-PCR were performed as previously described.Genomic DNA was isolated by using the QIAmp DNA Mini kit . Genomic DNA (500 ng) was treated with 10U of RNase H  for 8 h at 37°C before loading on the dot blot and transferred onto an N+ membrane . Following UV crosslinking at 1200 μJ, membranes were blocked in 5% milk/PBS-Tween (0.05% Tween 20) and incubated overnight with S9.6 (1:1000) or ssDNA  antibody. After the last wash, the membrane was incubated for 1 h at RT with secondary antibody (goat anti mouse horseradish peroxidase). After washing three times for 10 min with PBS-Tween (0.05% Tween 20), the membrane was briefly dried and incubated with Super-Signal West Femto chemiluminescent substrate (Thermo Fisher Scientific) for 2 min. The chemiluminescent signal was detected by exposure of the membrane to ImageQuant™ LAS 4000 .Recombinant GST, GST-THOC6 WT and GST-THOC6 mutant were incubated with purified THOC5, 50 pmol of R-loop or ssDNA for 2 h rotation at 4°C. After washing, input and bound fractions were analyzed by THOC5, R-loop (S9.6 antibody) or Biotin specific immunoblot.Biotinylated R-loop or ssDNA were incubated with purified FLAG-THOC5, THOC6 WT or THOC6 RK-mutant for 16 h rotation at 4°C or for 2 h at 37°C. Input and bound fractions were analyzed by FLAG M2, R-loop (S9.6 antibody) or Biotin specific immunoblot.DRIP was performed as previously described.2). DDX5 or DDX17 and labeled DNA-RNA duplexes (100 nM) substrates were incubated in MOPS buffer for 60 min at 37°C, followed by deproteinization in one-fifth volume of stop buffer  for 30 min at 37°C. The resolution of R-loop was confirmed by immunoblotting using Biotin antibody.Unwinding assays were performed in MOPS buffer (25 mM MOPS (morpholinepropanesulfonic acid), pH 7.0, 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 5 mM ATP, 5 mM MgClCells were fixed in ice-cold methanol for 10 min and then incubated in staining buffer (TBST with 0.1% BSA) for 10 min at RT. Enzymatic treatments were done in RNAse H buffer with 1:100 dilutions of RNAse H (NEB) and ShortCut RNase III for 4h at 37°C. Samples were subsequently washed by incubating with staining buffer for 10 min with rocking. For primary immunolabeling, samples were incubated in staining buffer with 1:200 dilutions of mouse S9.6 antibody (Kerafast) for 2h at RT with rocking. Samples were then washed once with staining buffer and incubated with 1:200 dilutions of secondary anti-mouse TRITC conjugate (Sigma) and counterstained with DAPI. Nikon NIS elements D 3.0 Software was used to quantify the fluorescence. Only the foci and nuclear S9.6 signal intensity were quantified.Nuclear extracts in RIPA buffer containing 1x Protease inhibitor cocktail (Sigma) were sonicated using Covaris AFA™  technology according to the manufacturer’s instructions. After centrifugation , extracts were immunoprecipitated using Protein G Agarose-PLUS  pre-coated with S9.6 antibody or control IgG. Following 4 h rotation at 4°C, the beads were washed three times in RIPA buffer. Precipitated R-loops or bound proteins were visualized using immunoblot. For S9.6 dot blot, aliquots of input, IgG and S9.6 IP were treated with proteinase K before subsequent R-loop purification.HeLa cells were transfected with FLAG tagged THOC5, THOC6 and empty vector for two days. Cells were fixed in 4% PFA for 15 min at RT and permeabilized with 0.2% Triton X-100 for 5 min. Samples were blocked in blocking buffer of Proximity ligation assay for 1h at 37°C. After blocking, samples were incubated with S9.6 and FLAG antibody  (1:200 dilution in antibody dilution buffer) for 2h at 37°C. After 2x washing with wash buffer A, the samples were incubated with pre-mixed Duolink PLA plus and minus probes for 1 h at 37°C. The subsequent steps in proximal ligation assay were carried out using the Duolink® PLA Fluorescence kit (Sigma) according to the manufacturer’s instructions.200 nM of biotin labeled R-loop mimic substrate (C4R) were incubated with 10 μM THOC6 in the binding buffer containing 20 mM Tris pH 7.5, 2 mM DTT, 12 mM MgCl2, 100 mM NaCl, and 10% glycerol for 30 min at RT. The complexes were separated by native PAGE and visualized by Biotin immunoblot.Cell experiments were performed in a minimum of three independent experiments (n ≥ 3). Data are displayed as the mean with error bars reported as +/− SD. The statistical significance was determined by the Student´s test (two-sided) (Excel) for


The CdO/HfS2 vdW heterostructure has a 1.19 eV indirect bandgap with type-II band alignment. Importantly, the CdO/HfS2 vdW heterostructure possesses an intrinsic Z-scheme photocatalytic characteristic for water splitting by obtaining decent band edge positions. CdO donates 0.017 electrons to the HfS2 layer in the heterostructure, inducing a potential drop to further separate the photogenerated electrons and holes across the interface. The CdO/HfS2 vdW heterostructure also has excellent optical absorption capacity, showing a promising role as a photocatalyst to decompose the water.Using two-dimensional (2D) heterostructure as photocatalyst for water splitting is a popular strategy for the generation of hydrogen. In this investigation, the first-principles calculations are explored to address the electronic performances of the 2D CdO/HfS TMDs matructure , thermaltructure , thermoetructure , and opttructure  performatructure , field-etructure , and photructure . It is wtructure , physicatructure . Besidestructure , suggesttructure .3N4/Ca2Nb2TaO10 nanocomposite with a mass ratio of 80:20 has the highest hydrogen precipitation efficiency, which is more than 2.8 times that of single-layer g-C3N4  based on density functional theory (DFT)  . The genry (DFT) . The DFTry (DFT) . Furtherry (DFT) . In the 2 monolayers are optimized by the lattice parameters of 3.68 Å and 3.64 Å, respectively, demonstrated by Γ with the valence band maximum (VBM) in 2 monolayer has an indirect bandgap with the CBM between the Γ and M, the VBM is found near the Γ point, as shown in 2 monolayers are 2.07 and 2.05 eV, respectively. The results are in good agreement with the previous studies , which is obtained by E = (Eh—ECdO—EHfS2)/S, where Eh, ECdO, EHfS2, and S represent the energy of the CdO/HfS2 heterostructure, original CdO, HfS2 monolayers and the area of the CdO/HfS2 heterostructure, respectively. Importantly, the obtained lowest binding energy is about −43.93 meV/Å−2 for CH-5 configuration, which is smaller than that in graphites of about −18 meV Å−2, revealing van der Waals (vdW) interactions between the interface of the heterostructure  to the CBM, resulting in holes at VBM. Some photogenerated electrons (or holes) will be promoted from CBM (or VBM) of the CdO (or HfS2) to the CBM of the HfS2 (or CdO) by the conduction-band offset, CBO . Moreover, the remaining photogenerated electrons at the conduction band of the HfS2 and the photogenerated holes at the valence band of the CdO can make recombination at the interface of the CdO/HfS2 vdW heterostructure because of that specific band energy between the −4.44 and −5.67 eV at pH 0  occurs between the interface of the heterostructure, which is decided by Δρ = ρh—ρCdO—ρHfS2, where ρh, ρCdO, and sHfS2 represent the charge density of the CdO/HfS2 heterostructure, original CdO, and HfS2 monolayers, respectively. The charge density difference of the CdO/HfS2 vdW heterostructure is addressed in 2 layer. The charge density amount is investigated by Bader-charge analysis (V) of the CdO/HfS2 vdW heterostructure is also obtained in 2CO2 (0.66 eV) (2CO2/GaN (3.75 eV)   and Hf2C3.75 eV) . It is w3.75 eV) .2 vdW heterostructure are calculated by α is the absorption coefficient. The angular frequency and the speed of light are expressed by ω and c, respectively. The real and imaginary parts of the dielectric constant are represented by 2, and CdO/HfS2 vdW heterostructure are demonstrated by the absorption peaks of 3.56 × 105 cm−1, 4.19 × 105 cm−1, and 3.51 × 105 cm−1 at the wavelength of 342, 323, and 351 nm, respectively, in the ultraviolet region. Importantly, the CdO/HfS2 vdW heterostructure possesses excellent visible light absorption capacity by the absorption peak of 7.21 × 104 cm−1 locating at the wavelength of 465 nm, which is higher than other reported 2D heterostructures as photocatalyst, such as g-GaN/Mg(OH)2 (5.33 × 104 cm−1) (4 cm−1)   and ZnO/(4 cm−1) . Besides2 is constructed by vdW interactions proved by first-principles calculations. The electronic properties of the CdO and HfS2 monolayers are calculated. In contrast, the CdO/HfS2 vdW heterostructure possesses a type-II band structure to prevent the recombination of the photogenerated charges. Furthermore, the decent band alignment of the CdO/HfS2 vdW heterostructure demonstrates a Z-scheme photocatalytic mechanism near the interface. Besides, the CdO/HfS2 vdW heterostructure shows pronounced visible light absorption performance. These results explain that the CdO/HfS2 vdW heterostructure can be used as a candidate for an excellent photocatalyst for water splitting.In this work, the CdO/HfS


MgH2 + 10 wt% MgFe2O4 is prepared by using the ball milling method. The MgFe2O4-doped MgH2 sample started to release H2 at approximately 250 °C, 90 °C and 170 °C lower than the milled and pure MgH2 respectively. At 320 °C, the isothermal desorption kinetic study has shown that the doped sample has desorbed approximately 4.8 wt% H2 in 10 min while the milled MgH2 desorbed less than 1.0 wt% H2. For isothermal absorption kinetics, the doped sample can absorb approximately 5.5 wt% H2 in 10 min at 200 °C. Meanwhile, the undoped sample absorbs only 4.0 wt% H2 in the same condition. The activation energy of 10 wt% MgFe2O4-doped MgH2 composite is 99.9 kJ mol−1, which shows a reduction of 33.1 kJ mol−1 compared to the milled MgH2 (133.0 kJ mol−1). X-ray diffraction spectra display the formation of new species which are Fe and MgO after dehydrogenation, and these new species are believed to act as the real catalyst that plays a crucial role in improving the sorption performance of the MgFe2O4-doped MgH2 system by providing a synergetic catalytic effect.In this study, the effect of nanolayer-like-shaped MgFe 2O4-added MgH2 composite showed an improvement in de/absorption properties compared to that of un-doped MgH2.The nanolayer-like-shaped MgFe Humanity is expected to use 40 TW of power (40 billion of kW) in the future. To satisfy this demand, different sources of renewable energy, such as hydrogen, are needed. Sustainable hydrogen is an ideal clean energy carrier because there is no carbon dioxide or other greenhouse gas emission at the end-user level. Commonly, there are 3 forms of storing hydrogen which is high-pressure gas, cryogenic liquid hydrogen in tanks (stored at 21.2 K) and as solid state hydrogen storage by either reacting with chemical compounds or absorbing. Among these approaches, solid-state hydrogen storage has higher potential for higher hydrogen density and may yield greater utility towards the practical implementation of hydrogen storage. Among the various materials for solid-state hydrogen storage, MgH2.15,25–35 Zhang et al.25 demonstrate that ferrite nanoparticles  can greatly lower the decomposition temperature of MgH2. CoFe2O4 provides the best catalytic effect compared with other ferrites. New by-products are found after the dehydrogenation process, and its phase shows a great catalytic effect on the properties of hydrogen storage of MgH2. Furthermore, Li et al.26 shows a significant improvement in the desorption performance of MgH2 when catalysed with MnFe2O4. X-ray photoelectron spectroscopy and X-ray diffraction (XRD) tests show that Fe0.872O and Mg2MnO4 phases take part as significant role in enhancing the dehydriding performance of MgH2. Meanwhile, we showed in our previous study that MnFe2O4 synthesised via a simple hydrothermal method provides a remarkable effect in improving the hydrogen storage performance of MgH2.27 Interestingly, our result showed that Fe metal formed after dehydrogenation instead of Fe0.872O species, as claimed by Li et al.26 This variation paved the way for the debate on how ternary metal oxides, particularly ferrites, work as catalysts in improving the hydrogen sorption performance of MgH2. Moreover, the difference in the synthesis method of the catalysts may also provide a different effect in the catalytic role.Previous research has proved that catalyst based on ternary metal oxides greatly improved the hydrogen storage performance of MgH2-ternary metal oxides catalyst system, it is quite interesting to investigate the use of other ferrites (e.g. MgFe2O4) as catalysts to improve the hydrogen sorption performance of MgH2. Therefore, in this work, MgFe2O4 was synthesised by using a simple hydrothermal method, and its catalytic effects on the hydrogen sorption performance of MgH2 were systematically studied. To the best of authors' knowledge, this paper is the first to study the hydrogen sorption performance of MgH2 catalysed with MgFe2O4. The possible catalysis mechanisms of MgFe2O4 in the sorption performances of MgH2 are also discussed in this paper.Inspired by the role of active species that formed during the heating process in the MgH2.2O4 was synthesised via a hydrothermal method. In a typical synthesis, a stoichiometric amount of Mg(NO3)2·6H2O (Sigma-Aldrich) and Fe(NO3)3·9H2O (Sigma-Aldrich) were dissolved in 50 ml distilled water. A total of 10 ml of H4N2·H2O (Sigma-Aldrich) was added dropwise to the above solution to attain the resultant pH of >9. The mixture was then transferred into a sealed Teflon lined stainless-steel autoclave (125 ml capacity) and heated for 12 h at 180 °C. The final product was washed several times with deionised water and dried overnight at 60 °C under vacuum. A total of 10 wt% of as-prepared MgFe2O4 was mixed with 300 mg of MgH2  and undergo intensive ball milling for 1 h in a planetary ball mill at the rate of 400 rpm. For comparison, pure MgH2, and MgH2 added with 10 wt% Fe (Alfa Aesar) and 10 wt% MgO , respectively were also prepared under the same conditions. All preparations, including loading and weighing, were conducted in an argon atmosphere glove box (MBraun Unilab).The nanolayer-like-shaped MgFe−1 in vacuum chamber. Meanwhile, the sorption kinetics was conducted under 1.0 atm at 320 °C for desorption kinetic measurement and under 33.0 atm at 200 °C for absorption kinetic measurement. The thermal properties of the doped and undoped samples were performed using differential scanning calorimeter (DSC)/thermogravimetric analysis from Metler Toledo. With a flow of 50 ml min−1 argon, the samples were heated with 15, 20, 25 and 30 °C min−1 heating rate from 25 °C to 500 °C.The onset decomposition temperature and sorption kinetic measurement for doped and undoped samples were characterised by using Sievert-type pressure–composition–temperature apparatus . For onset decomposition temperature measurement, the samples were heated from room temperature to 450 °C at a heating rate of 5 °C minvia a Rigaku MiniFlex X-ray diffraction apparatus equipped with Cu Kα radiation. Data were collected in the 2θ range 20° to 80° at 2° min−1. The morphologies of the samples were observed by scanning electron microscopy (SEM) (JEOL JSM-6350LA). Fourier transform infrared (FTIR) spectrometry was recorded on an IR Shimadzu Tracer-100 between 400 and 2000 cm−1. Raman spectra were recorded on Renishaw Raman spectroscopy (532 nm radiation) extended with 0.1% power laser measurement at room temperature.The phase composition of the samples was analysed by XRD 3.2, the phase structure of MgFe2O4 was confirmed by XRD, as shown in θ of 30.1°, 35.4°, 43.1°, 53.5°, 57.0°, 62.6° and 74.1°, respectively. This result is in good agreement with the standard cubic spinel structure (JCPDS 71-1232). No other peaks were detected in the sample. The average crystallite size of MgFe2O4 was approximately 19 nm, as determined by using Scherrer's formula:L is the average crystallite size (nm), θ is the angle of diffraction, k is Scherrer's constant (k = 0.94), λ is the X-ray wavelength (0.15405 nm) and B is the full width at half maximum of the diffraction peak in radian (FWHM). The SEM image . The result indicates that addition of MgFe2O4 reduces the decomposition activation energy and boost the desorption performances of MgH2.The improvement in desorption behaviour is correlated with the kinetic barrier of the hydrogen desorbed from the MgHalysis41  was condDSC data , it can 2, and MgFe2O4-doped MgH2. From the images, it can be seen clearly that the particle size of the pure MgH2 is around 50–100 μm . The XRD exploration displays the formation of new species of Fe and MgO after the dehydrogenation process, and these species remained unchanged after rehydrogenation. It is believed that the new species of Fe and MgO play a synergistic role in enhancing the hydrogen storage performances of MgH2.In this study, nanolayer-like-shaped MgFeThere are no conflicts to declare.


Autosomal dominant lateral temporal epilepsy (ADLTE) is an inherited syndrome caused by mutations in the leucine-rich glioma inactivated 1 (LGI1) gene. It is known that functional LGI1 is secreted by excitatory neurons, GABAergic interneurons, and astrocytes, and regulates AMPA-type glutamate receptor-mediated synaptic transmission by binding ADAM22 and ADAM23. However, > 40 LGI1 mutations have been reported in familial ADLTE patients, more than half of which are secretion-defective. How these secretion-defective LGI1 mutations lead to epilepsy is unknown.W183R in excitatory neurons lacking natural LGI1, and found that this mutation downregulated Kv1.1 activity, led to neuronal hyperexcitability and irregular spiking, and increased epilepsy susceptibility in mice. Further analysis revealed that restoring Kv1.1 in excitatory neurons rescued the defect of spiking capacity, improved epilepsy susceptibility, and prolonged the life-span of mice.We identified a novel secretion-defective LGI1 mutation from a Chinese ADLTE family, LGI1-W183R. We specifically expressed mutant LGI1These results describe a role of secretion-defective LGI1 in maintaining neuronal excitability and reveal a new mechanism in the pathology of LGI1 mutation-related epilepsy.The online version contains supplementary material available at 10.1186/s13578-023-00983-y. Epilepsy, characterized by recurrent seizures, is a common brain disorder that affects 1–2% of the population –12. Haplv1 [v1 activity using celecoxib, a drug approved by the Food and Drug Administration, ameliorates seizure susceptibility in global LGI1 knockout mice [LGI1 mutations have been found in patients with familial ADLTE [LGI1 yield either secretion-defective or -competent proteins [v1 [v1 coexist in axonal initial segment [v1 to regulate neuronal excitability.It has been shown that functional LGI1 exists in excitatory neurons, interneurons, astrocytes, and oligodendrocytes , 20, andv1 , 21–23 av1 , 25. Furv1 –29, and out mice . Althougal ADLTE , highligproteins , 26, 28, segment . AccordiLGI1 mutation, p.Trp183Arg, from a Chinese ADLTE pedigree. To investigate the function of this mutant in the central nervous system (CNS), we used Cre-Loxp-based viral infection to specifically express mutant LGI1W183R in mouse excitatory neurons that lacked native LGI1. Utilizing in vivo and in situ electrophysiological recordings and computer simulation, we found that the LGI1W183R mutation produced secretion-defective LGI1 protein, which downregulated Kv1.1 activity, caused neuronal hyperexcitability and firing irregularity, and increased seizure susceptibility. Moreover, restoring Kv1.1 in excitatory neurons was able to correct the deficits in firing and ameliorate seizure susceptibility in mice.Here, we identified a novel missense Proband clinical information and Proband family sequencing result). In brief, the form of his attacks was short-lived buzzing and tinnitus, followed by loss of consciousness, stiffness of the limbs, and foaming at the mouth, which lasted for ~ 2 min. He also suffered from isolated attacks of tinnitus, which occurred on average once a month. He was delivered naturally at full term, and the birth process was smooth, and his developmental milestones were normal. Physical examination of his nervous system was normal as well: MRI of his head showed no evident abnormality. However, video EEG monitoring showed occasional epileptiform discharges in the right temporal region. Initial treatment was valproic acid (0.5 g) twice a day, but he still had seizures. Then oxcarbazepine (0.3 g) was added twice a day, and the proband remained free of attacks for 4 years. The proband’s father had similar seizures at the age of 18, took carbamazepine (0.2 g) twice a day, and had no attacks for ~ 20 years. The proband’s grandfather had a grand mal seizure at the age of 19 and often auditory aura since that time, but did not take antiepileptic drugs, thereby living with seizures every 3–5 years.The proband, a 16-year-old man, came to the hospital due to repeated unconsciousness and generalized convulsions. The detailed clinical information of the proband and his family is shown in Additional files 11 and 12 (A 20-h EEG dataset was collected using a Nicolet V32 EEG monitor (Natus Neurology Inc.) with 21 electrodes according to the international 10–20 system. During recording, the Fz electrode served as the common reference. EEG signals were sampled at 256 Hz and bandpass-filtered at 0.01–100 Hz. To guarantee data quality, the impedance of all electrodes was calibrated at < 5 KΩ before acquisition. During EEG recording, one camera was used to monitor the behavior of the patient. The data were read and analyzed by two epileptologists independently.LGI1flox/flox mice (Loxp-flanked encompassing exons 5 and 7) were created at the Model Animal Research Center of Nanjing University. All mice were maintained at the Experimental Animal Center of Zhejiang University and kept in temperature-controlled conditions under a 12:12 h light/dark cycle with food and water ad libitum. Male mice were used in all experiments.Following antibodies and reagents were used: anti-NeuN (#MAB377) and anti-GAPDH (#MAB374) from Millipore, DTx-K (#ab141795) and anti-LGI1 (#ab30866) from Abcam, anti-CaMKIIα (#611292) from Bio-rad, anti-Iba1 (#SAG4318) from Wako, anti-GFAP (#Z0334) from DAKO, anti-PV (#235) from SWAT, anti-Flag (#T20008) from Abmart, and Goat anti-mouse/rabbit IgG horseradish peroxidase-conjugated antibodies (#31446 and #31460) from Thermo Fisher Scientific. Other reagents were from Sigma or Tocris unless stated otherwise.2/5% CO2; 37 °C). Cells were transfected in OPTI-MEM with plasmids using lipofectamine 2000. Cells were treated with CHX (50 μM) to inhibit protein synthesis at 24, 32, 40 and 44 h after the transfection [g at 4 °C for 20 min to collect supernatant fractions.HEK293 cells were cultured in DMEM and supplemented with 10% FBS in an incubator  and anti-mouse . Film signals were digitally scanned and protein levels were quantified by measuring the integrated optical densities of the bands after background subtraction using ImageJ 1.42q (NIH).LGI1-F: 5ʹ-GCT GCA GCT CTT GTT ATT TAC GTC G-3ʹ, and LGI1-R: 5ʹ-GAG CCA TTC CAC CAG CCA CTT CAA C-3ʹ. GAPDH-F: 5′-TGT TAC CAA CTG GGA CGA CA-3′, and GAPDH-R: 5′-AAG GAA GGC TGG AAA AGA GC-3′.mRNA levels were assessed by real-time PCR using an ABI PRISM 7500 sequence detection system (Applied Biosystems). cDNA was synthesized by reverse transcription using oligo (dT) as the primer and proceeded to real-time PCR with gene-specific primers in the presence of SYBR Premix Ex Taq. Quantification was performed by the comparative cycle threshold (Ct) method, using GAPDH as the internal control. Following forward (F) and reverse (R) primers were used to amplify: Coronal cortical sections were cut at 25 m and placed in a blocking solution  for 1 h at RT. After washing with PBS, the sections were incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibody for 1 h at RT. These sections were then mounted using ProLong Gold Antifade reagent with DAPI. The antibody dilutions were 1:250 (CaMKIIα), 1:500 (Iba1 and GFAP), 1:1000 , and 1:2000 (PV). All antibodies were diluted in PBS containing 1% BSA and 1% normal goat serum.^13 viral genomes/ml). After injections, pups were placed on a warming pad, and then returned to their mothers for care [P0 pups were cryoanesthetized at − 20 °C for 2–3 min before injection. A solution of AAV containing 0.05% trypan blue was injected bilaterally into the ventricles using a 10-μl syringe with a 32-gauge needle (Hamilton). The injection site was located 2/5 of the distance along a line defined between each eye and the lambda intersection of the skull at a depth of 3 mm. Viral solution (2 l) was injected into each lateral ventricle  injected with PTZ at 45 mg/kg (i.p.). The seizure activity was observed and scored by investigators who were blinded to the genotype throughout the experiments. Seizures were scored for 30 min after the injection as follows : 1, hypoMice were deeply anesthetized with pentobarbital (30 mg/kg) and placed in a stereotaxic apparatus (Stoelting). Recording electrodes  made of twisted stainless-steel wires (diameter: 0.125 mm) insulated with Teflon were implanted into the hippocampal CA1 region  according to the Paxinos and Franklin mouse brain atlas . Two scr2PO4, 2 MgSO4, 2 CaCl2, 25 NaHCO3, 10 glucose) on a vibrating tissue slicer . After recovery for 30 min at 37 °C, the slices were incubated at room temperature (RT) for 60 min and then transferred to the recording chamber and superfused at 2 ml/min with aCSF at RT. All solutions were saturated with 95% O2/5% CO2.Coronal slices of the hippocampus (300 μm) from P17–20 mice were cut in ice-cold aCSF . Currents were filtered at 2 kHz and digitized at 10 kHz. Recordings were excluded from analysis if series resistance, input resistance, or holding current varied by 15% over the course of an experiment. All electrophysiological experiments were performed at RT.Neurons were visualized under an upright microscope  with a 40 × water-immersion objective equipped with infrared differential interference contrast enhancement. Whole-cell recordings were made with an Axon MultiClamp 700B amplifier (Molecular Devices). Glass pipettes (3–5 MΩ) were filled with a solution containing (in mM): 120 K-gluconate, 20 KCl, 10 HEPES, 2 MgCl+ currents, TTX (1 μM) and CdCl2 (100 μM) were added to the aCSF to block Na+ and Ca2+ channels, and a series of voltage pulses (3 s) from − 70 mV to 40 mV (10-mV increment) were applied under the voltage-clamp configuration. Kv1.1 activation/inactivation curves were constructed from steady-state currents after the conversion to conductance, normalized to the maximum conductance, and fitted with a single Boltzmann equation [Passive neuronal properties were measured from single APs. The rheobase was defined as the minimum depolarizing current needed to elicit an AP. AP amplitude was measured as the voltage difference between peak and resting potentials. The half-width was determined at half of spike amplitude. The input–output relationship between injected currents and spikes was determined when neurons received a series of currents ranging from 20 to 200 pA (20-pA increments) with a duration of 800 ms. The fast AHP was defined as the slope of the 3-ms Vm immediately following the spike , 35. Forequation , 36.I) and available number of channels (N), peak-scaled non-stationary noise analysis was applied to each ensemble of Kv1.1 current simulated with an activation command pulse, based on previous work [N corresponded to the average number of channels open at the peak. To assign similar weights to all phases of the ensemble mean waveform, from the peak to the end of the decay, the amplitude interval from the peak to the baseline was divided into an equal number of intervals [2(I) = iI − I2/N, where for any given potential, σ2 was the variance, i was the single channel current, I was the macroscopic mean current, and N was the number of channels. The values for N and i were then estimated from the fit of the variance vs mean data to the equation.In order to estimate single-channel current  were set for the entire neuron and RMP was set at − 65 mV. The kinetics and distribution of active somatic, dendritic and axonal channels, including INa, IDR (delayed rectifier K+), IA (transient K+), IKM (M K+), Kv1.1, and Ih (non-selective hyperpolarization-activated channels), were defined according to data available for CA1 pyramidal neurons [2+ and Ca2+-dependent K+ and Ca2+ extrusion machinery was also included in the neuronal model. The densities of INa and IDR were set to 450 and 200 pS/μm2, respectively, at the soma and apical dendrites, and their density in axons was increased threefold. The somatic density of IA and Ih were 300 and 0.1 pS/μm2, respectively, and they were increased linearly with distance from the soma. IKM was inserted into the soma at a density of 60 pS/μm2 and into the axon at a fourfold higher density. The conductance of Ca2+ and Ca2+-dependent K+ channels was set to 1 and 0.1 pS/μm2, respectively. Kv1.1 was set at a uniform density of 100 pS/μm2 in both the soma and the axon [v1.1 (V1/2 = − 30 mV) were defined according to previous work [The simulation was carried out using NEURON v7.5 software. In the three-dimensional computational model of a CA1 pyramidal neuron , 44, uni neurons . The conthe axon . The steous work .t test with Welch's correction for two-group comparisons or 2-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. Survival curves were analyzed by Kaplan–Meier survival estimate using the log-rank test. The accepted level of significance was P < 0.05. n represents the number of animals or cells. Data in the text and figures are presented as the mean ± SEM.Data were analyzed using GraphPad Prism 8.0 (GraphPad Software) and Igor Pro 6.0 (Wavemetrics). Statistical differences were determined using the unpaired We treated a 16-year-old Chinese man who developed recurrent epileptic seizures . His episodes were characterized by generalized tonic–clonic seizures preceded by auditory auras. His father and grandfather had similar manifestations from the puberty, strongly indicating ADLTE , 48. We WT) or mutant LGI1W183R, and transfected them into HEK cells. mRNA analysis revealed no difference in the level of transcripts between LGI1W183R and LGI1WT  treatment. The expressions of both LGI1WT and LGI1W183R proteins were reduced over time during CHX treatment, but the rate of decrease of LGI1WT was much faster than that of LGI1W183R  or CaMKII-Cre;LGI1flox/flox (cKO), where LGI1 was either haploinsufficient or deficient in excitatory neurons  recordings -positive interneurons, astroglia, and microglia  died before P21 and their median lifetime was 22 days, as shown in the Kaplan–Meier survival curves   were at stages 3/4 and displayed a shortened latency to generalized seizures, whereas the majority of Het::LGI1WT (10/15) mice were at stages 1/2  obtained from cKO::LGI1WT and cKO::LGI1W183R neurons , and found that the values were always larger in cKO::LGI1W183R neurons than in cKO::LGI1WT neurons  in a firing was shorter in cKO::LGI1W183R neurons  [2  [W183R neurons, but the difference gradually declined over time  . As showal (AHP) , 35, whial (AHP) , 52. In W183R mutation unlikely plays this role, since it yields a secretion-defective LGI1 protein. Indeed, we found that neither the amplitude nor the frequency of mEPSCs was altered by LGI1W183R expression in cKO neurons , while single-channel conductance was not affected (0.31 for LGI1WT and 0.33 for LGI1W183R)   were applied to the cell model as the low and high intensities of current injection, respectively. With 400-ms duration, 200-pA current induced 4 APs with normal Kv1.1, but 6 APs with half Kv1.1 , but tended to hyperpolarize with reduced Kv1.1 (slope coefficient: − 214)  Fig. G. Intereels Fig. G. Therefv1.1 by LGI1W183R expression is responsible for epileptogenesis, an interesting question was whether the seizures can be ameliorated and whether the lifespan can be prolonged by restoring Kv1.1. To do so, we injected AAV9-DIO-LGI1W183R-GFP with AAV9-DIO-Kv1.1-mCherry or AAV9-DIO-mCherry bilaterally into the ventricles of Het or cKO mice at P0  died before P21 with a median lifetime of 22 days  Het::LGI1W183R::mCherry mice were at stages 3/4, whereas most Het::LGI1W183R::Kv1.1 mice (10/15) were at stages 1/2 and had an increased latency to generalized seizures . Representative images for triple fluorescence of GFP, individual marker proteins , and DAPI, show that LGI1W183R is not expressed in PV-positive interneurons, astroglia (GFAP) and microglia (Iba1) in the hippocampus and temporal cortex of cKO mice (P17). Scale bars: 1 mm (whole brain) and 50 μm (magnified).Additional file 2. Figure S2. Unchanged excitatory transmission in LGI1W183R neurons. (A) Example mEPSCs from cKO::LGI1WT and cKO::LGI1W183R mice (P17). (B) Cumulative plots of mEPSC amplitude. (C) Mean values of mEPSC frequency: 0.23 ± 0.02 Hz  and 0.22 ± 0.03 Hz , P = 0.88.Additional file 3. Figure S3. Restoring Kv1.1 in excitatory neurons does not affect other types of nerve cells. AAV9-DIO-LGI1W183R-GFP and AAV9-DIO-Kv1.1-mCherry were bilaterally injected into the ventricles of cKO mice (P0). Representative images for quadruple fluorescence of GFP, mCherry, individual marker proteins , and DAPI, show that LGI1W183R is not expressed in PV-positive interneurons, astroglia (GFAP) and microglia (Iba1) in the hippocampus and temporal cortex of cKO mice (P17). Scale bars: 1 mm (whole brain) and 50 μm (magnified).Additional file 4. Table S1. The statistics for Fig. 1G, 1H, 1J, and 1K.Additional file 5. Table S2. The statistics of spontaneous seizures.Additional file 6. Table S3. The statistics for Fig. 3C, 3G, 3H, 3J and 3K.Additional file 7. Table S4. The statistics for Fig. 4C, 4D, 4H, 4I, 4J and 4L.Additional file 8. Table S5. The statistics for Fig. 7A, 7B, 7C, 7D, 7E, 7G and 7H. Additional file 9. Movie 1. A cKO::LGI1WT mouse  behaves normally, while another cKO::LGI1W183R mouse  displays epileptic seizures. MPEG-4 format, 4.8 MB.Additional file 10. Movie 2. A cKO::LGI1W183R::mCherry mouse  displays epileptic seizures, while another cKO::LGI1W183R::Kv1.1 mouse  behaves normally. MPEG-4 format, 6.2 MBAdditional file 11.  Proband clinical information. Additional file 12.  Proband family sequencing result.


The experimental results of the ceramic powder precursor indicate that the calcination of the ceramic precursors at above ~300 °C is an exothermic process, which contributes to the transition of the ceramic powder from the amorphous phase into the crystal spinel phase; the spinel phase of ceramic powders can be formed initially at ~450 °C and well-formed at ~750 °C. A high densification of ~98% relative densities and evenly distributed grains within an average size of 2~12 μm for the sintered Sn-doped specimen were obtained. The specific resistance and B-value were notably increased from 12.63 KΩ·cm to ~24.65 KΩ·cm, and from 3438 K to ~3779 K, respectively, with the Sn-doping amount. In contrast, the aging rates of the Sn-doped specimen have not changed markedly larger, waving around ~2.7%. The as-designed Sn-doped MnNiFeO4 can be presented as a candidate for some defined NTC requirements.Sn-doped MnNiFeO In contrast, ternary or plural transition metals systems can form more stabilized spinel structures, especially for ternary Mn-Ni-Fe-O4 or Mn-Co-Fe-O4 systems [4+, Si4+, Sn4+ and Ti4+ doped in Mn3−x−yNixFeyO4 were studied, in order to modify properties of the spinel thermistors by B-site ion substitution in the spinel structure, and the specific resistivity and sensitivity were increased [3+, Al3+ and Cr3+ [2+, Zn2+ and Mg2+ [4+ and Mn3+ on crystallographically equivalent lattice B sites in the spinel structure. As a consequence, the mobility of the charge carriers is relatively low and is described as small polarons conduction [Negative temperature coefficient (NTC) ceramic thermistors based on transition metal oxides, are widely applied in temperature measuring, controllers, time-delay devices, infrared detectors, and voltage regulators ,3,4,5,6.operties ,21,22,23ncreased ,21,22,23and Cr3+ ,16,17,18and Mg2+ ,11,12 we4 ceramic composition was optioned as the host matrix owing to its relatively stable properties and low cost, and Sn4+ was the selected modifier due to its bigger ionic radius with a larger ion polarity, aiming at improving the sintering behavior and modifying the electrical properties. Two Sn-doping strategies of Mn1−xSnxNiFeO4 and isomolar Sn-doping substitution for Mn, Ni, and Fe in MnNiFeO4, respectively, were designed. The samples were prepared by the low-temperature solid-phase reaction route. The phase formation of the ceramic powders, microstructure and electrical properties of the sintered samples were studied in detail.In this study, MnNiFeO1−xSnxNiFeO4 , MnNi0.91Sn0.09FeO4 and MnNiFe0.91Sn0.09O4 2·4H2O, Ni(CH3COO)2·4H2O, FeCl2·4H2O, and SnCl2·2H2O were used for synthesizing ceramic precursors. The compounds for introducing ceramic constituent cations and oxalic acid were weighed accurately according to the molar ratio 3:3 of the total metallic cations to H2C2O4·2H2O. The weighed batches were mixed and ground in agate mortar by hand for 50~70 min until yellow green paste ceramic powder precursors were obtained. The obtained yellow green pastes were dried at 75 °C for 30 h, and then ground again. The as-prepared precursor powders were heated to 750 °C at a heating rate of 5 °C/min and kept for 600 min, and the ceramic powders were acquired with particle sizes in the submicrometer range. A 5% PVA solution was used for granulation of fine calcined powders. The green compacts were formed by pressing the granulated powder in a uniaxial press to a size of 5 mm diameter × 2 mm thick discs. The green compacts were sintered at 1250 °C for 300 min in air and cooled naturally.NTC ceramic Mn1/2 precise digital multimeter . The tested pellets were subjected for reliability studies by giving an accelerated aging at 150 °C for 1000 h, and the resistance measurements for aged specimens were repeated. There were 5 specimens per composition for electrical test.The dried precursor powders were characterized by thermogravimetric analysis and differential scanning calorimetry  and Fourier transform infrared spectroscopy . The phase purity of the calcined powders and sintered samples was analyzed using X-ray diffraction . The unit cell dimension was calculated from XRD data by least-squares refinement. Scanning electron microscopy  was used for the microstructural characterization of the sintered pellets. X-ray photoelectron spectra  analysis was performed to identify the presence of different ions in the crystal sublattice of spinel structure. Monochromatic Al Kα was chosen as the radiation source in the XPS instrument and operated at 11 kV and 10 mA. For electrical characterization, the sintered discs were silvered using a thermistor grade silver paste and annealed at 850 °C for 40 min in a small muffle furnace. The electrical resistance was measured at 25 °C and 85 °C, respectively, using self-made equipment with a temperature uniformity ± 0.1 °C using a 6The TG-DSC curves of S1 and S4 ceramic precursors (green paste dried at 75 °C for 30 h) heated to 800 °C at 10 °C/min in air are shown in 2O4)3⋅4H2O is 16.6%; that of S4 precursor Mn0.91Sn0.09NiFe (C2O4)3⋅4H2O is 16.4%. At elevated temperature stages responsible for crystal water removal, the weight loss of S1 precursor is 19.78% at 124.31~242.06 °C, whereas that of S4 precursor is 15.22% at 147.58~235.71 °C. Theoretically, the crystal water content of S1 precursor MnNiFe  stretching vibration of the oxalate, and 1443 cm−1 only occurring to S4 precursors could be assigned to the asymmetric ν(O−C=O) stretching band induced by Sn-doping. The band appearing at 818 cm−1 is assigned to the bending vibration of asymmetric δ(O-C-O) [−1 should be associated to the stretching vibration of M-O .δ(O-C-O) . The ban1−xSnxNiFeO4  in 4. Based on the theoretical densities, the calculated relative densities of all as-prepared specimen are larger than 96%  is cleanest and smoothest, which consists of uniform plain-plate-shape grains of 2~12 μm with a concave grain boundary. In contrast, S6 (MnNiFe0.91Sn0.09O4) has more large grains of 8~14 μm, and the grain boundary also exhibits a concave shape.The surface microstructures of the sintered samples S1~S6 are shown in 1−xSnxNiFeO4 , associated with the densification degree of sintered samples (see 4), it can be concluded that the effect of Sn-substituted for Ni in MnNiFeO4 is the best, which coincides with the aging electrical property. From the morphologies of S1 to S4  of these materials strongly depend on the distribution of the cations in the lattice . Variousd by XRD . In this of SnO2 , and is r S4 see .1−xSnxNiFeO4  increases monotonously with the amount of Sn substituting for Mn. This can be explained by the B-occupied Mn3+/Mn4+ pairs being decreased by the introduced Sn4+ ions entering into B-sites, which coincides with the results of XPS. In contrast with S4 (MnNi0.91Sn0.09FeO4) and S6 (MnNiFe0.91Sn0.09O4), S5 (MnNi0.91Sn0.09FeO4) has the largest resistivity, which can be explained by the Sn substitution for Ni in nominal composition decreases the content of Ni2+ locating B-sites, which can yield Mn4+ in the B-site from valence balance. In contrast, S4  and S6  exhibit no more obvious effects on specific resistivity. All the B-values of S1~S6 show the same trend with the specific resistivity, which complies with the common rules for the spinel NTC thermistors.The specific resistivity and B-values of S1~S6 tend to increase with the increase of Sn-doping amount, in which S5 has a maximum specific resistivity of 32.62 KΩ·cm and a maximum B-value of 3928 K. The specific resistivity for S1~S4 Mn2O4, the distribution of the ions over the oxygen interstitial sites: Mn3+ will occupy predominantly the B-site  while Mn2+ will be on the A-site  and almost all Ni2+ will go to the B-site [2 atmosphere showed a much smaller aging rate than the air cooled samples [The aging behavior of NTC ceramics in the system Mn-Ni-Fe-O are dependent on composition and the oxidation which occurs during the cooling after sintering . In the e B-site . At highe B-site . It was  samples .25°C/R25°C of all as-prepared samples no longer show a difference and waver around 2.7%, in which S2 and S3 have slightly less average aging rates (see 4+ under hexa-/tetra-coordination number situation is 0.83 Å/069 Å, and that of Mn4+ under hexa-coordination situation is 0.67 Å; that of Mn3+ under hexa-coordination situation is 0.72 Å and 0.758 Å at low-spin and high-spin state, respectively; that of Ni2+ under hexa-/tetra-coordination is 0.83 Å/0.69 Å; that of Fe3+ under hexa-/tetra-coordination is 0.78 Å/0.63 Å.The average aging rates ΔRates see . Accordi4+ that induces the pinning effect under oxygen hexa-coordination inhibiting the exchange of the A-site and B-site ions; on the other hand, the higher densification contributes to oxygen penetration during the aging at the elevated temperature of 150 °C, leading to the stoichiometry uniformity in the whole grain volume. The higher densification also favors stoichiometry uniformity of the grains during cooling. S4~S6 demonstrate larger aging rates of ~3.08% for more Sn introduction. The largest aging rate of S4 is ascribed to excess Sn introduction of 9% molar resulting in the larger lattice distortion, yielding lattice stress. In contrast with S4 and S6, S5 has a relatively small aging rate, which should be owed to the good sintering behavior, leading to a higher relative density with a uniform microstructure.It is simply the larger radius of Sn4-based NTC ceramics with a single spinel structure were prepared successfully by LTSPR. The TG-DSC and XRD results of as-synthesized ceramic powders indicate that, at above ~300 °C, the calcination of the ceramic precursors is an exothermic process, favoring a solid-state reaction; the spinel phase of ceramic powder is formed initially at ~450 °C and well-formed at ~750 °C. A high densification of ~98% relative densities and evenly distributed grains with an average size of 2~12 μm for the sintered doping Sn specimen can be obtained simply by the conventional process. The improved sintering behavior and distinct microstructures for the Sn-doped specimen can be attributed to the sintering promoted effect of the larger radius Sn ion inclusion. The specific resistance and B-value with Sn-doping amount are notably increased from 12.63 KΩ·cm to ~24.65 KΩ·cm, and from 3438 K to ~3779 K, respectively, which can be attributed to the decrease of Mn4+/Mn3+ pairs due to Sn4+ substitution for Mn4+ in the spinel B sites. In contrast, the aging rates of the Sn-doped specimens have not changed markedly, waving around ~2.7%, which can be ascribed to the combined effect of Sn ion inclusion: advantageous effects lie in the pinning effect from inhibiting the exchange of the A-site and B-site ions, and the oxidation inhibition effect during the aging and cooling process for the higher densification with a more uniform microstructure, and the disadvantageous effect lies in the induced lattice stress by the introduction of larger radius Sn ions. The Sn-modified MnNiFeO4 with improved sinterability presents a promising candidate for some defined preparation and property requirements.Sn-doped MnNiFeO


Genetic and environmental factors are implicated in the etiology of neuropsychiatric diseases. Inbred mouse strains, including the 129S1/SvImJ (S1), constitute important models to study the influence of genetic factors in these conditions. S1 mice displayed anxiogenic-like behavior, impaired fear extinction, and increased prepulse inhibition (PPI) of startle reflex compared to C57BL/6J (BL6) mice. Given the role played by the endocannabinoid system (ECS) in these responses, we evaluated the expression of the ECS components in different brain regions in S1 mice. Gene expression levels of the cannabinoid type-1 and type-2 receptors (CB1R and CB2R) and the endocannabinoid metabolizing enzymes varied depending on the brain region evaluated. Notably, CB2R expression markedly increased in the amygdala, prefrontal cortex and hippocampus in S1 mice. Moreover, CB2R blockade with SR144528 partially rescued the anxiogenic phenotype in S1 mice, while CB2R activation with JWH133 potentiated the deficits in fear extinction and the PPI of startle reflex in this mouse strain. These data suggest that CB2R is involved in the behavioral alterations observed in S1 mice and underline the importance of this cannabinoid receptor subtype in the regulation of certain central nervous system disorders. Genetic predisposition and environmental factors contribute to the development of psychiatric disorders . HoweverConsidering the range of possible neurobiological mechanisms involved in the S1 mice phenotype, the endocannabinoid system (ECS), composed of two main receptors, the cannabinoid type-1 and type-2 receptors , their ligands, i.e., the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and the enzymes involved in endocannabinoid metabolism  could beThe aim of this study was to analyze the expression of the main components of the ECS in several brain areas of S1 strain compared to C57BL/6J (BL6) mice. Considering the main change observed, we also evaluated the consequences of the modulation of CB2R in S1 mice in key neurobehavioral responses such as anxiety, fear conditioning and extinction, and sensorimotor gating.ad libitum. All experiments were performed during the light phase. Mice were handled daily for 3 days before the beginning of the experiment. Experimental procedures were conducted in the animal facilities of Universidad Francisco de Vitoria in Madrid, Spain, in accordance with the guidelines of the European Communities Directive 2010/63/EU and the Spanish Regulations RD 1201/2005 and 53/2013 regulating animal research and approved by the local ethical committee (CEEA-UFV).Experiments were performed using male 129S1/SvImJ (S1) mice (Jackson Laboratories) and C57BL/6J (BL6) mice (Charles River) at 8–10 weeks old. BL6 mice were chosen as the comparison strain in this study because they represent one of the most commonly used mouse lines in neuroscience research. Moreover, previous work evaluating fear extinction in the S1 strain typically used BL6 mice as a reference, since they exhibit proper fear extinction, acquisition and recall . Mice weThe CB2R agonist JWH133 (5 mg/kg) (Tocris) was dissolved in a solution of 10% DMSO, 10% Tween 80 and 80% saline. The CB2R antagonist SR144528 (3 mg/kg) (Sigma) was dissolved in a solution of 5% ethanol, 5% cremophor and 90% saline. Both drugs were administered by intraperitoneal (ip) route (10 ml/kg body weight). Doses were based on previous studies in mice  in mice.Anxiety-like behavior was assessed by using a black maze elevated 30 cm above the ground with four arms (25 cm × 5 cm) set in a cross from a neutral central square (5 cm × 5 cm). Two opposite arms were delimited by walls (closed arms) and illuminated with 4–6 lux, whereas the two other opposite arms had unprotected edges (open arms) and were illuminated with 40–50 lux. Pharmacological treatments were administered 30 min before the test. The total number of visits to the closed and open arms, and the cumulative time spent in each arm were observed through a videocamera system during 5 min.Mice were cued fear-conditioned as performed in preceding experiments with slight modifications . The tesPrepulse inhibition (PPI) of startle reflex, a measure of sensorimotor gating, was assessed by using the StartFear Combined System . Mice were daily habituated to a Plexiglas cylinder located inside the sound-attenuating chamber for 5 min with background white noise (65 dB) 4 days prior to the test. The test started with 5 min habituation in the cylinder and, immediately after, mice were exposed to 5 pulse trials . These trials were performed for startle accommodation and were excluded in the final analysis. The experimental protocol consisted of 10 blocks with 6 or 12 trials each, randomly presented to mice with an inter-trial interval of 7–23 s. Blocks consisted of: no stimulus (6×) (background white noise), pulse alone (12×) , pulse preceded by 4 prepulse intensities (12× each)  and the prepulses alone (6× each). A background white noise was generated throughout the whole experiment. Pharmacological treatments were administered 30 min before the test. Startle amplitude was detected by PACKWIN V2.0 software. Percent PPI was calculated as follows: 100 × (startle response – prepulse inhibited startle response) / startle response.−ΔΔCt method.Amygdala, prefrontal cortex and hippocampus tissues were extracted in basal conditions and immediately frozen at −80°C. These brain areas were chosen based on their implication in the behavioral responses evaluated in this study. Total RNA was purified with the RiboPure™ Kit (Invitrogen) for amygdala and prefrontal cortex, and the RNeasy Mini Kit (QIAGEN) for hippocampus, according to the manufacturer’s instructions. Reverse transcription was performed with 0.9 μg of total RNA and the SuperScript™ II Reverse Transcriptase (Invitrogen). PCR reactions were conducted using PrimePCR™ Probe Assay (Bio-Rad) to quantify mRNA levels for: CB1R (Unique Assay ID: qMmuCEP0038879), CB2R (Unique Assay ID: qMmuCEP0039299), DAGLα (Unique Assay ID: qMmuCIP0032590), MAGL (Unique Assay ID: qMmuCIP0042348), NAPE-PLD (Unique Assay ID: qMmuCIP0035707) and FAAH (Unique Assay ID: qMmuCEP0055480), using GAPDH expression (Unique Assay ID: qMmuCEP0039581) as endogenous control gene for normalization. PCR assays were carried out with the CFX Connect Real-Time PCR Detection System (Bio-Rad). The fold changes in gene expression of S1 in comparison with BL6 mice were calculated using the 2t tests. Multiple-group comparisons were performed by one-way analysis of variance (ANOVA). Repeated-measurement ANOVA was used for serial freezing responses and startle amplitude response between the different prepulse intensities. Subsequent Fisher’s LSD post-hoc test was only used when ANOVA interaction effects were significant. All data were expressed as mean ± SEM. The statistical analysis was performed using Statistica (StatSoft) software. The level of significance was p < 0.05 in all experiments.Comparisons between two groups were assessed by Student’s p < 0.01)  and area under the curve (AUC) (p < 0.001)  in comparison with BL6 mice (p < 0.01) and 16 dB (p < 0.001) above background of 65 dB (p < 0.01) . S1 mice showed an anxiogenic-like effect ( < 0.01)  as revea < 0.01) . Cued fe < 0.01) . However < 0.01) , fear ex< 0.001)  when comBL6 mice . This efof 65 dB . The mea < 0.01) . This ef < 0.01) , discardp < 0.01) (p < 0.001) (p < 0.001) (p < 0.001) (p < 0.05) (p < 0.001) (p < 0.001) (p < 0.01) in S1 strain  (p < 0.001)  , without < 0.01)  or the h < 0.01)  compared< 0.001) , prefron< 0.001) , and hip< 0.001)  in S1 mi < 0.05) , prefron< 0.001) , and hip< 0.001) , while M1 strain , with no1 strain  and the 1 strain . Finally < 0.01)  and the < 0.001) , while n< 0.001)  in S1 mi3,30 = 11.81, p < 0.001) and post hoc comparison between S1 groups treated with vehicle or SR144528 (p < 0.05)   and AUC  (p < 0.05)  (p < 0.01), 8 (p < 0.01), and 12 dB (p < 0.05) above background of 65 dB  (p < 0.01)  . No modi < 0.05) . Moreove < 0.05) . In cont < 0.05)  when com < 0.05) . A signi < 0.01) . Thus, pof 65 dB . An over < 0.01) . However < 0.01) . SR14452 < 0.01)  nor star < 0.01)  in S1 miOur data show remarkable changes in the expression levels of several components of the ECS in different brain regions in S1 mice. Particularly interesting, CB2R expression was strongly increased in the amygdala, prefrontal cortex and hippocampus in this strain compared to BL6 mice. These alterations suggest that CB2R could be involved in the phenotypic characteristics observed in S1 mice. Indeed, acute pharmacological modulation of CB2R induced behavioral alterations in important neurobiological processes in these mice. Future experiments evaluating the effects of chronic CB2R agonists and antagonists in S1 mice would be interesting since acute or chronic administration of CB2R ligands could result in different responses . The useGenetic differences between strains are likely to affect several phenotypic features offering a powerful tool with which to expand our knowledge about the factors that influence psychiatric conditions. S1 inbred mice showed higher innate anxiety compared to BL6 mice as revealed the decrease of the percentage of time in open arms in the EPM test. In agreement, these mice spent significantly less time in the center of the open field  and in tOur results showed a significant reduction of CB1R and NAPE-PLD expression in the amygdala of S1 strain which could in part explain the anxiogenic phenotype and resistance of fear extinction characteristic of these mice. In agreement, CB1R knockout mice are anxiogenic  and showInterestingly, CB2R expression was strongly enhanced in the amygdala, prefrontal cortex, and hippocampus in S1 related to BL6 mice. The increased expression of CB2R could be the result of a compensation of the general reduced expression of the synthesizing and metabolizing enzymes and associated possible changes in endocannabinoid levels in S1 mice. This is the case of other neurotransmission systems such as the opioid system. Thus, marked region-specific up-regulation of the mu, delta, and kappa opioid receptors was observed in mice lacking the proenkephalin and prodynorphin genes . In any S1 mice constitute a well-established model of impaired fear extinction . RecentlPPI of the startle reflex is a sensorimotor gating process that reduces the startling response when a weaker sensory stimulus precedes a sudden startling stimulus. An impairment in the PPI response is observed in patients with schizophrenia . SeveralIn summary, our data reveal important changes in the expression levels of different components of the ECS in S1 mice. Particularly significant, the increased expression of CB2R observed in this mouse strain could contribute to its behavioral alterations. Elucidating the CB2R cell type location would shed light on the role of this cannabinoid receptor in the different functions described above. However, CB2R antibody nonspecificity strongly hinders its identification . CB2R an


Ergothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy.Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes (tregt1 and tregt2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions.Here, we showed that E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.Our results showed that the overproduction of ERG in The online version contains supplementary material available at 10.1186/s12934-022-01807-3. Ergothioneine (ERG) is a thiol-containing histidine betaine derivative, that protects cells against oxidative damage caused by excess reactive oxygen species (ROS). Uniquely, ERG exists predominately in the thione tautomer at physiological pH  and possNeurospora crassa [Schizosaccharomyces pombe [Mycobacterium smegmatis into E. coli [, engineering S. cerevisiae with the Grifola frondosa egt1/egt2 genes [N. crassa egt1 and Claviceps purpurea egt2 [E. coli after 216 h in a 3-L jar fermenter by expressing five ERG biosynthetic genes (egtABCDE) from M. smegmatis, enhancing l-cysteine (l-Cys) production, knocking out metJ and optimizing the fermentation medium [S. pombe [Aspergillus oryzae [egtB, egtD in M. smegmatis and egt1 in N. crassa) revealed that there are far more bacterial species and fungal phyla capable of producing ERG than the number of ERG-producing microorganisms discovered thus far [N. crassa that requires only two genes and may be more effective in ERG biosynthesis. Until now, only a few fungal ERG biosynthesis genes have been characterized, and in terms of the ERG productivity, their potential does not seem to be fully realized in the fungal systems reported previously [E. coli that is a model microorganism commonly used for synthetic biology and industrial applications.ERG biosynthesis occurs only in certain bacteria and fungi, typically actinobacteria , cyanobaa crassa , the fises pombe  and basies pombe , but notes pombe , which les pombe , 20 and es pombe , 16, mor E. coli , engineet2 genes  or with rea egt2 . Of noten medium . Likewiss oryzae . Therefothus far . Explorieviously , 25. HenTrichoderma reesei is the workhorse for the industrial production of lignocellulolytic enzymes [T. reesei is an attractive host for the production of recombinant proteins due to its extraordinary ability to secrete proteins and its (GRAS) Generally Regarded as Safe status approved by the US Food and Drug Administration [T. reesei remain unknown, the presence of putative ERG biosynthetic genes raises the possibility that T. reesei has the potential to produce ERG.The filamentous fungus  enzymes , 27. Morstration –30. AlthT. reesei can synthesize ERG. Through heterologous expression in E. coli, we examined the role of the two putative ERG biosynthesis genes from T. reesei and investigated the possibility of synthesizing ERG in E. coli using the fungal ERG biosynthetic genes from T. reesei and N. crassa. Our research showed that high-level of ERG production in E. coli can be achieved by using only two genes from fungi. This work offers a more practical and promising approach for the industrial production of ERG.Here, we determined that T. reesei has evolved the ability to synthesize ERG. HPLC analysis showed that the extracted samples displayed a predominant peak at a retention time of 10–10.5 min, which was the same as that of the ERG standard  and Ncegt2 (NCBI Reference Sequence: XP_001728131) as query sequences revealed that two hypothetical proteins designated Tregt1 (NCBI Reference Sequence: XP_006968620) and Tregt2 (NCBI Reference Sequence: XP_006968735), respectively, were probably involved in ERG biosynthesis in T. reesei. The coding sequences of the cloned tregt1 and tregt2 genes were 2502 bp and 1413 bp  as a sulfur donor, which facilitates hercynylcysteine sulfoxide synthesis and avoids competition with the glutathione synthesis pathway [E. coli. To maximize the ERG productivity of the recombinant strain, we will conduct closer inspection of the gene expression level, intermediate product accumulation, ERG precursor supply and proportion, and further optimizations will be made.To evaluate the potential of the recombinant  pathway , 19. FroT. reesei can synthesize ERG. By bioinformatics analysis and reconstruction of the T. reesei ERG synthetic pathway in E. coli, we found that ERG biosynthesis in E. coli can be achieved by using only two genes from T. reesei, with Tregt1 being the key enzyme in this process. In addition, the recombinant E. coli strain co-expressing egt1 and egt2 genes from N. crassa also can synthesize ERG. Through fed-batch cultivation, the highest level of ERG production was achieved after 143 h of cultivation. To the best of our knowledge, this is the first report to overproduce ERG in E. coli with fungal biosynthetic genes.Here, we demonstrated that Trichoderma reesei strain QM9414  was cultivated on potato dextrose agar (PDA) or in liquid minimal medium (MM) with 5 g/L glucose and 40 g/L lactose as the carbon source. MM without peptone was prepared as described previously [E. coli strain Trans1-T1  was used for standard cloning. E. coli K12/BW25113 (rrnB3 ΔlacZ4787 hsdR514Δ(araBAD)567 Δ(rhaBAD)568 rph-1) [T. reesei and ERG production. Luria–Bertani (LB) medium  and ZYM auto-induction medium [E. coli cells and to express enzyme, respectively. A defined medium (DM) was used for whole-cell biocatalysis and fed-batch fermentation, which contained (per L) 10 g of glucose, 8 g of (NH4)2HPO4, 13.3 g of KH2PO4, 1.2 g of MgSO4·7H2O, 1.7 g of citric acid and 10 mL of a trace metal solution. The trace metal solution (per litre of 5 M HCl) consisted of 10 g of FeSO4·7H2O, 2.25 g of ZnSO4·7H2O, 1 g of CuSO4·5H2O, 0.5 g of MnSO4·5H2O, 0.23 g of Na2B4O7·10H2O, 2 g of CaCl2·2H2O and 0.1 g of (NH4)6Mo7O24. When necessary, the antibiotic ampicillin (100 mg/L)or the inducer L-arabinose (2 g/L) was added.eviously . E. coli8 rph-1)  was usedn medium  was usedTrichoderma reesei QM9414 was cultivated on PDA at 28 ℃ for 10 days, and then the conidia were suspended in 1.1 M sorbitol solution and centrifuged at 13,300 rpm for 3 min. The lower conidia pellet was frozen in liquid nitrogen and ground into a powder. After that, the conidial powder was added to an 85% methanol solution and vortexed for 1 min, and the suspension was centrifuged at 13,300 rpm for 3 min. The supernatant was collected and diluted tenfold with 70% acetonitrile solution, and then filtered through a 0.22 μm filter for ERG detection.T. reesei QM9414 were inoculated into liquid MM containing 0.5% (w/v) glucose and 4% (w/v) lactose and cultivated on a rotary shaker (200 rpm) at 28 ℃ for 6 days, and the mycelia were collected to extract ERG. The method of extracting ERG from mycelium was the same as that for the conidia.The conidia of araBAD promoter induced by arabinose and rrnB terminator. Primer pairs FpBAD/RpBAD were designed to amplify plasmid pBAD/His to obtain linearized pBAD/His for subsequent vector construction.The expression vectors were constructed with the plasmid pBAD/His  as the backbone, which includes the pBR322 origin, tregt1 and tregt2 genes were amplified using the primer pairs Ftregt1/Rtregt1 or Ftregt2/Rtregt2 with T. reesei QM9414 cDNA [tregt1 or tregt2 products were ligated with linearized pBAD/His through a Clone Express® MultiS One Step Cloning Kit  and then transformed into E. coli Trans1-T1 for cloning and sequencing. The resulting plasmids pBAD-tregt1 and pBAD-tregt2, in which tregt1 or tregt2 was under the control of the araBAD promoter and rrnB terminator, was used to transform BW25113 to obtain the recombinant strains BW-tregt1 and BW-tregt2. To construct the recombined E. coli strain co-expressing tregt1 and tregt2, the T7 RBS sequence (tgtttaactttaagaaggagatatacc) was used to link the two genes. Briefly, primer pairs Ftregt1/RpBAD-tregt1 were designed to amplify the plasmid pBAD-tregt1 to obtain linearized pBAD-tregt1 with the T7 RBS sequence attached to the 3′ end of tregt1. Additionally, tregt2 with the T7 RBS sequence at its 5′ end was amplified by PCR using the plasmid pBAD-tregt2 as template and the primer pairs FpBAD-tregt2/Rtregt2. Subsequently, plasmid pBAD-tregt1-tregt2 was constructed by ligating T7 RBS-containing tregt2 to pBAD-tregt1 harbouring T7 RBS, which was transformed into BW25113 to create the recombinant strain BW-tregt1-tregt2.The entire open reading frame of the 414 cDNA  as a temncegt1-ncegt2 harbouring egt1 (XM_951231) and egt2 (XM_001728079.2)responsible for ERG biosynthesis in N. crassa, except that the entire open reading frames of the two genes were synthesized by Tsingke Biotechnology Co., Ltd (China). The resulting plasmid . The supernatant was used for SDS-PAGE analysis.BW25113 and the engineered strains derived from it were cultivated in LB medium with ampicillin at 37 ℃ on a rotary shaker (200 rpm) until the optical density of the cultures at 600 nm reached 0.6. Expression was induced by the addition of arabinose at a final concentration of 0.2% (w/v). After 24 h of induction at 30 ℃ and 200 rpm, the cells were collected by centrifugation and resuspended in 50 mM potassium phosphate buffer (pH 7.0). The cell suspension was sonicated and centrifuged  to form a cell suspension (OD600 = 10). The whole-cell catalysis reaction was conducted in a 100-mL Erlenmeyer flask containing 30 mL of cell suspension on a rotary shaker (200 rpm) at 30 ℃ for 48 h. The extracellular and intracellular ERG content was subjected to high performance liquid chromatography (HPLC) analysis.BW25113 and the engineered strains derived from it were grown in LB medium overnight at 37 ℃ on a rotary shaker (200 rpm). Five hundred microliter of the overnight cultures were inoculated into 50 mL of ZYM auto-induction medium containing 2 g/L of arabinose. After 24 h of induction at 30 ℃ and 200 rpm, the cell cultures were harvested by centrifugation at 5,000 rpm for 5 min. The resulting cell pellets were resuspended in the reaction mixture , the inducer l-arabinose was added at a final concentration of 0.2%(w/v) to induce the expression of egt1 and egt2 at 30 ℃ with mixing for 12 h. After that, the amino acid mixture , which are the precursors of ERG biosynthesis, was constantly fed at a flow rate of 4 mL/h/L. During the whole fermentation, feeding solution  was periodically added after glucose depletion. The dissolved oxygen was kept above 20% air saturation by adjusting the agitation intensity and aeration rate. The pH was maintained at approximately 7.0 by automatic addition of 2.7 M ammonia solution or 1 M H3PO4. At the indicated time points, the cell cultures were sampled and used to detect extracellular ERG and E. coli growth as indicated by the optical density at 600 nm (OD600).Precultures of the recombinant strains co-expressing 2 column . A mobile phase of acetonitrile/deionized water  was used at a flow rate of 1.0 mL/min. The produced ERG was detected at 254 nm and identified by comparison with the retention time of the analytical ERG standard (Sigma). Quantification was conducted by dividing the slope of the standard curves by the peak area.ERG samples were diluted tenfold with a 70% acetonitrile solution. ERG standards (Std) were dissolved in a 70% acetonitrile solution. HPLC  was performed with an Agilent ZORBAX NH2 column . Analysis was performed with a mobile phase of acetonitrile/4 mmol/L ammonium acetate .HPLC-purified ERG was identified by LC–MS  with Agilent ZORBAX NHAdditional file1: Table S1 Primers used in this study. Fig. S1 LC-MS analysis of ERG. A ERG standards (10 ppm); B the ERG sample extracted from mycelia. Fig. S2 Plasmid profiles, protein expression and ERG production of BW-ncget1-ncegt2. A Schematic drawing of plasmids expressing Ncegt1 and Ncegt2 used in E. coil BW25113 transformation. B The production of ERG by 48-hour whole cell catalysis using the recombinant strains BW-ncegt1-ncegt2. Data in the figure are mean values . C Detection of Ncegt1 and Ncegt2 expression in recombinant E. coli by SDS-PAGE. M. Protein marker; 1. BW-pBAD (control); 2. BW-ncegt1-ncegt2. Data S1 Nucleotide sequences of tregt1 from T. reesei (2502 bp). Data S2 Nucleotide sequences of tregt2 from T. reesei (1413 bp). Data S3 Amino acid sequences of Tregt1 from T. reesei (833 aa). Data S4 Amino acid sequences of Tregt2 from T. reesei (470 aa).