Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis. Histone proteins play pivotal roles in eukaryotic cells, compacting the DNA into chromatin and regulating gene expression by changing the DNA accessibility. As a cell divides, DNA and chromatin are duplicated and assembled in such a way that the newly replicated DNA is accompanied by the recycled parental histones and by the synthesis of new histones which reaches a peak in S-phase ,2. Upon bona fide substrates of CMA and that cytosolic histones H3 and H4 are mainly degraded by the CMA-dependent lysosomal pathway. We further investigated how histone degradation is regulated and found an interplay between HSC70 and tNASP, where they compete each other for the binding to H3, HSC70 targets histone H3 to lysosomes while tNASP protects it from degradation. This balance between two chaperones and their divergent outcomes is key for understanding how cells cope with defective newly synthesized histones and may be exploited in diseases affecting either degradation pathways or histone folding.Here, we explore the molecular mechanisms contributing to the quality control of newly synthesized histone H3 and H4. By designing a mutant histone H3 that is not correctly folded and is unable to dimerize with histone H4, we uncover the molecular mechanisms set by cells for the degradation of unfolded histone H3. Consistent with a previous report , we founThe following antibodies were used in this study: β-actin , ASF1 and NASP (from Genevieve Almouzni), Calreticulin , FLAG , HIRA , Histone H2A , Histone H3 , Histone H4 , HSC70 , HSP90 , Importin 5 , GAPDH , LAMP1 , LAMP2A , LC3B , TopoI , VDAC , Alexa 488 goat anti-mouse IgG (H + L) , anti-Mouse IgG HRP , anti-Rabbit IgG HRP , anti-Rat IgG HRP .The following plasmids were used in this study: HaloTag Histone H3 and H4 , His-H3A95P pET28a+ (made here), HaloTag-H3A95P pET28a+ (made here), His-H3 pET28a+, His-H3 Mut1 pET28a+ (made here), His-H3 Mut2 pET28a+ (made here), His-H3 DM pET28a+ (made here), HaloTag-H3 Mut1 pET28a+ (made here), HaloTag-H3 Mut2 pET28a+ (made here), HaloTag-H3 DM pET28a+ (made here), His-tNASP pET30 (Eric Campos), NASP-FLAG pCMV5 (MRC PPU Reagents and services).HeLa cells were obtained from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum . Lipofectamine 2000 (ThermoFisher Scientific) was used for cell transfections, according to the manufacturer′s instructions. For NASP knock-down, either 30 nM of negative control siRNA  or siRNA against NASP  were transfected into HeLa cells for 48 h.2 and 20 mM HEPES, pH 7.4) using 10 strokes with the type B Dounce homogenizer. Homogenates were ultracentrifuged at 100 000 g for 1 h at 4°C. Supernatants were collected and proteins quantified using the BCA Protein Assay Kit (ThermoFisher Scientific).HeLa cells were treated for 10 min with the following inhibitors of protein degradation: 10 μM Lactacystin (proteasome inhibitor), 10 mM 3-methyl-adenine  and 20 mM ammonium chloride plus 200 μM leupeptin hemisulfate (lysosome inhibitor). Then, cells were incubated with 100 μg/ml cycloheximide in the presence of the inhibitors of protein degradation as indicated. After treatment, HeLa cells were washed with ice-cold PBS, scrapped, and pelleted at 350 g for 5 min. Cytosolic extracts were prepared by resuspending the cell pellet in hypotonic lysis buffer  configured to append the HaloTag® tag to the N-terminus of histone H3. To obtain His-H3 recombinant protein from bacteria, histone H3 cDNA (NP_003520.1) was subcloned  into pET28a+ plasmid (Novagen) to add a His-Tag on the N-terminus of histone H3. Histone H3A95P mutants were generated using the GENEART site-directed mutagenesis system (Invitrogen) and confirmed by sequencing. Mutants H3Mut1 (52IRRYQ56 to 52IRAAA56) and H3Mut2 (82DLRFQ86 to 82DLAAA86), both in pFN21A HaloTag® CMV Flexi® vector or pET28a+ plasmid, were generated using the QuickChange II site-directed mutagenesis kit (Agilent) following the manufacturer instructions and confirmed by sequencing. Primers for H3Mut1: Forward: 5′cgctctccgcgagatccgcgccgccgcgaaatccaccgagctgctc3′, Reverse: 5′gagcagctcggtggatttcgcggcggcgcggatctcgcggagagcg3′; H3Mut2: Forward: 5′aggacttcaagaccgacctggccgccgcgagctcggccgttatggctc3′, Reverse: 5′gagccataacggccgagctcgcggcggccaggtcggtcttgaagtcct3′. To obtain H3DM (52IRAAA56 and 82DLAAA86 mutations), the set of primers for H3Mut2 were used over the vectors harboring H3Mut1. To express and purify recombinant His-HSC70 from bacteria, the coding sequence of human HSC70 (Addgene # 19487) was sub-cloned into the pET28a+ backbone (Novagen) by PCR amplification (Forward: 5′gatcgatcgatcgatccatatgtccaagggacctgcagttgg3′ and Reverse: 5′gatcgatcgatcgatcctcgagttaatcaacctcttcaatggtgg3′) and inserted between the NdeI and XhoI sites.The cDNA sequence of histone H3 (NP_003520.1) was synthesized  and fused to HaloTag using the pFN21A HaloTagl-lysine and transfected with plasmids containing HaloTag-H3, HaloTag-H4, HaloTag-H3A95P, HaloTag-H3Mut1, HaloTag-H3Mut2, HaloTag-H3DM. After 24 h, cells were incubated with HaloTag-TMR ligand as indicated by the manufacturer′s instructions (Promega). Briefly, cells were incubated with 1 μM of HaloTag-TMR ligand for 15 min as a pulse, washed three times with PBS, incubated at 37°C for 45 min, and fixed with 4% paraformaldehyde at RT for 20 min. Cells were then permeabilized with 100 μg/ml digitonin, blocked with 0.02% gelatin and immunostained overnight at 4°C with antibodies against LAMP1. Cells were then stained with Alexa 488-conjugated secondary antibody and DAPI at 37°C for 30 min.HeLa cells were seeded on 12 mm coverslips pretreated with 0.1 mg/ml poly-t-test.Microscopy images were acquired using an OLYMPUS FV1200 inverted confocal microscope with a 60× oil immersion objective (NA 1.4). For the analysis of lysosome location and colocalization, Z stack images of cells were captured in each channel at an interplane distance of 400 nm at optimal confocal apertures. Colocalization of H3 or H4 signal with lysosomes (LAMP1) was made in maximal projections of image stacks. First, the area of the nucleus in cells was bound by the thresholding DAPI signal and then subtracted from H3-HaloTag-TMR  and LAMP1 image channels. Then, colocalization between the cytoplasmic HT-H3 (or HT-H4) signal and LAMP1 signal in these images was estimated using the ICA  plugin of ImageJ software . Intensi2+-agarose beads (Pierce), according to the manufacturer′s instructions. Purified GAPDH from rabbit muscle was purchased from Sigma-Aldrich.Histones H3, H3A95P, H3Mut1, H3Mut2, H3DM and H4; His-tNASP, His-sNASP and His-HSC70 were overexpressed in bacteria by inducing their expression with 0.2 mM IPTG for 2 h. Histones were purified from inclusion bodies, as previously described . tNASP a2 inhalation. Livers were removed, kept at 4°C in 0.25 M sucrose pH 7.2 and homogenized using seven strokes in a plotter homogenizer. Lysosomal fractions were isolated by ultracentrifugation through a discontinuous 85.6% Nycodenz  gradient at 141 000 g for 70 min, as previously described , treated (+PI) or not (–PI) with a protease inhibitor cocktail  and incubated with 60 ng (4 pmol) of recombinant histone H3 (wild type or mutants) or 60 ng (4 pmol) of recombinant histone H4 at 37°C for 20 min, followed by centrifugation at 17 000 g for 20 min. The supernatant was removed, and the pellet was resuspended in 30 μl of MOPS buffer and analyzed by western blot. To properly compare the uptake between CMA– and CMA+ fractions, we determined LAMP2A levels in each fraction by western blot to normalize the experiment. In competition assays, 50, 100 or 200 ng tNASP (0.6–2.4 pmol) or sNASP (1–4 pmol) were pre-incubated with 60 ng of H3 or H4, followed by incubation with lysosomes, as described. Uptake of the CMA substrate into the lysosomal lumen was quantified by subtraction of the amount of protein associated with lysosomes in the absence of PI (binding) from the total amount of protein associated with lysosomal membranes and not degraded in the lysosomal lumen by the presence of PI (association), as determined in parallel assays (Six hundred pmol (10 μg) of recombinant H3WT, H3Mut1, H3Mut2 or H3DM were incubated with 600 pmol (40 μg) of recombinant HSC70 in a buffer containing 10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.4 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 0.2 mM PMSF for 30 min on ice. Then, the protein mixture was loaded onto a 5-ml 5–20% glycerol gradient in the same buffer. The gradient was ultracentrifuged at 26 000 rpm in a SW 55 Ti rotor for 16 h at 4°C. The gradient was fractionated in 250 μl aliquots and analyzed by western blot.Different molar combinations of the recombinant proteins, as indicated, were incubated in 500 μl of BC50 Buffer  supplemented with 0.05% NP-40, for 20 min on ice. Then, the protein mixture was loaded onto a Superdex 200 10/300 GL column  equilibrated with BC50 containing 0.05% NP-40. The void volume, corresponding to 7 ml, as measured by the elution of Dextran-blue, was received in one tube and then fractions of 500 μl were collected. Aliquots derived from the fractions were analyzed by western blot.in silico analysis using the GOR4 software, an algorithm which examines the proneness for each amino acid residue to form a complex structure with the surrounding residues. All alanine residues present in the α-2 Helix of H3 protein were changed to proline and analyzed one at a time. From the output, as a likelihood percentage, potential candidates were selected as the lowest percentage able to form an α-Helix structure (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_gor4.html). From this analysis we selected the mutation H3A95P.A mutation that disrupts the α-2 Helix of H3 protein was designed by Two nmol of histone H4 (∼88 μg) and 2 nmol of either wild type H3 or H3A95P (∼96 μg) were mixed in unfolding buffer  and incubated for 1 h at 4°C. Samples were then dialyzed against refolding buffer  and then centrifuged at 17 000 rpm for 20 min at 4°C. The supernatant containing H3–H4 tetramers was loaded onto a Superdex 75 10/300 GL column  equilibrated with refolding buffer, and 250 μl fractions were collected. Aliquots derived from the fractions were analyzed by Coomassie blue stained gels.P values were calculated using an unpaired Student's t-test, the error bars expressed as mean ± standard error of mean and the sample size (n) indicated in figure legends.For all western blot experiments quantified by densitometry, the statistical significance was obtained in Graphpad prism software. The To investigate whether newly synthesized histones H3 and H4 are degraded as part of their quality control mechanism in the cytosolic maturation pathway, we determined H3 and H4 protein levels in cytosolic extracts following inhibition of different degradation pathways. For this, we pre-incubated HeLa cells with Lactacystin to inhibit the proteasome, ammonium chloride plus leupeptin to inhibit whole lysosomal-dependent degradation, or 3-methyl-adenide (3-MA) to inhibit macroautophagy. We then blocked protein synthesis with cycloheximide (CHX) and followed H3 and H4 protein levels in cytosolic extracts by western blot Figure . We testWe next evaluated the direct targeting of histones H3 and H4 into the lysosomal compartment by immunofluorescence and microscopy image analysis. We expressed human H3 and H4 proteins fused to a HaloTag (HT) peptide into their N-terminal tails  and visuin vitro lysosomal uptake assays   . Togethe82DLRFQ86 motif and a putative  52IRRYQ56 motif, that are conserved in the H3.1, H3.2 and H3.3 variants , 52IRAAA56 (H3Mut2), and the double mutant 52IRAAA56 + 82DLAAA86 (H3DM)  facilitates the direct translocation of selected protein substrates to lysosomes through membrane pores formed by the LAMP2A protein . The tars Figure . No KFER) Figure . None of) Figure . We anale Figure  and D. Ce Figure . Immunofe Figure . We nexte Figure . WesternWe next explored how the CMA-mediated degradation of histones is regulated and whether an interplay between chaperones is in place. The histone chaperone NASP has two splice variants: tNASP, which is found in cancer, embryonic and germ cells, and sNASP, found in embryonic and somatic cells . For the2 by high salt dialysis  is the main degradation pathway of newly synthesized cytosolic histones H3 and H4. This degradation is regulated by a fine-tuned interplay between the tNASP-mediated stabilization of histones, and the HSC70-mediated targeting of H3 to lysosomes for degradation. Moreover, we further demonstrate that misfolded cytosolic histone H3 is targeted to lysosomes for degradation. Taking the data together, we propose a model in which CMA is a key component of the quality control of newly synthesized histones H3 and H4 and the balance between a network of chaperones and their different outcome is key for understanding how cells cope with defective newly synthesized histones H3 and H4 Figure . Recent In eukaryotic cells, the co-translational ubiquitination of nascent misfolded polypeptides is the main quality control mechanism of newly synthetized proteins ,33. In fAn important aspect to investigate is whether the CMA-mediated histone H3 and H4 degradation is restricted to a quality control step in histone biogenesis, as shown in here, or whether it can also participate in the regulation of the excess of cytosolic histones. So far, the regulation of histone excess has been linked to proteasomal degradation . In eithDNA damage-induced malignancy transformation is related to a decrease in both macroautophagy and CMA activities , which cAll data are available from the corresponding authors upon request.gkab1296_Supplemental_FileClick here for additional data file.


Vitis interspecific hybrid ‘Magarach 31-77-10′ as a female parent and Muscadinia rotundifolia × Vitis vinifera BC5 hybrid plants as male parents. The hybrid nature of the populations was assessed using RADseq high-throughput genotyping. We discovered 12,734 SNPs, which were common to all three hybrid populations. We also proved with the SSR markers that the strong powdery and downy mildew resistance of the paternal genotypes is determined by the dominant Run1/Rpv1 locus inherited from M. rotundifolia. As a result, the disease development score  for both mildew diseases in the female parent ‘Magarach 31-77-10’ was three times higher than in male parents 2000-305-143 and 2000-305-163 over two years of phytopathological assessment. The highest values of yield-contributing traits (average bunch weight ~197 g and 1.3 kg as yield per plant) were detected in the population 4-11 (♀M. No. 31-77-10 × 2000-305-163). Despite the epiphytotic development of PM, the spread of oidium to the vegetative organs of hybrids 4-11 did not exceed 20%. Some hybrid genotypes with high productivity and resistance to pathogens were selected for further assessment as promising candidates for new varieties.The Crimean autochthonous grape varieties are unique by their origin and serve as a valuable source for breeding new cultivars with increased salt and frost resistance, as well as high-quality berries. However, they suffer from fungal pathogens, as the dry and hot summer months contribute to the epiphytotic course of diseases. An increase in the resistance of Crimean grape varieties is currently achieved through interspecific hybridization. In this study, we describe the genetic and agrobiological diversity of three hybrid populations obtained using the  Vitis vinifera L. subsp. sylvestris (Gmel.) Hegi, which is considered the wild forest ancestor of cultivated grapes. Viticulture on the Crimean Peninsula has a long and ancient history; there is a hypothesis that the wild grape, preserved here from the Tertiary period, was collected for food by local tribes (Taurians) and then bred for cultivation  cross are diverse, generally less productive, but some of the hybrid genotypes are characterized by an intense accumulation of sugar.Among hybrids of population 3-11 (♀M. No. 31-77-10 × 2000-305-143), the outstanding genotype M. No. 3-11-2-41 was discovered, showing the highest score of resistance to powdery mildew, an average mass of bunches , and high average yield (349 g) per shoot .Daktulosphaira vitifoliae and pathogens Plasmopara viticola and Erysiphe necator based on the introgression of resistance genes from the highly resistant (immune) species Muscadinia rotundifolia. Among hybrids from the crossing of the hybrid genotype Magarach 31-77-10 with the donors of Muscadinia rotundifolia genes, progeny have been obtained in which genotypes with high productivity and high resistance to pathogens are distinguished. These are recommended for further agrobiological and technological evaluation as promising candidates for new varieties.The current breeding programs at the ‘Magarach’ Institute focus on creation of new varieties with complex immunity to the insect pest 2. All the 139 hybrid genotypes were represented by one plant each. The F1 progeny were maintained in an experimental vineyard that was left unsprayed with fungicides.Three hybrid populations derived from the cross of female M. No. 31-77-10 (Nimrang × Seibel 13-666) × male 2000-305-143, 2000-305-163, and a mixture of pollen from DRX-M5-734, DRX-M5-753, and DRX-M5-790 were investigated in this study. The crosses were performed in 2011. In 2012, the hybrid seeds were germinated, and in 2013, seedlings were planted as self-rooted plants in experimental fields at the Partenit village, Yalta District, Crimea (44°34′12.0″ N 34°19′44.1″ E), in a self-rooted culture with a planting scheme of 3.5 × 1 mThe area of the field is formed by valleys, ridges, and slopes with different exposure. Soils of the experimental fields are characterized as brown mountainous non-carbonate, with a heavy, loamy gravelly texture. The mild climate provides the possibility of uncovered grape culture: the average annual air temperature is 13.2 °C, the sum of active air temperatures (above 10 °C) is 3700–4055 °C, the number of days with temperature above 10 °C varies between 195 and 218, and the annual precipitation is 300–400 mm. In summer, rains are short, in the form of downpours. Plants often suffer from a lack of moisture because of the dry summer.Assessment of agrobiological traits was conducted in 2019–2020 according to the method of Lazarevsky . In shorPhytopathological field evaluation was conducted by the examination of untreated plants against a natural infection pressure. In each season, two counts were carried out: the first in 15–20 days after flowering of grapes and the second at the beginning of grape ripening.0—no signs of infestation1—single, hardly visible spots on leaves (OIV resistance—9 points)2—up to 10% of leaf surface affected (OIV resistance—7 points)3—11–25% of leaf surface affected (OIV resistance—5 points)4—26–50% of leaf surface affected (OIV resistance—3 points)5—more than 50% of leaf surface affected (OIV resistance—1 point)The nature and percentage of leaf damage were scored according to the recommended method . PreciseUsing the scale, the disease development score  in a particular genotype was calculated by the formulaFor example, of 48 leaves evaluated for a plant, 0 degree of disease development was determined for 27 leaves, 1 for 18 leaves, 2 for 3 leaves, and 3, 4, and 5 for 0 leaves. The disease development score  was calculated as follows:w/v, 20 mM EDTA (pH 8.0), 1.4 M NaCl, 100 mM Tris-HCl (pH 8.0), 5 mM ascorbic acid, 4mM diethyldithiocarbamic acid sodium salt, and 2% polyvinylpyrrolidone-40), 3.2 µL of 2-mercaptoethanol, and 0.4 µL RNase A were added. The samples were incubated at 37 °C for 30 min, followed by another 30 min at 65 °C for enzyme inactivation. Then, 800 µL of chloroform:isoamyl alcohol (24:1) was added and mixed thoroughly. The mixture was centrifuged at 10,000 rpm for 15 min at room temperature, and the upper aqueous phase was transferred into a sterile 1.5 mL Eppendorf tube. DNA was precipitated by adding 0.7 volume of ice-cold isopropanol. The solution was stored at −80 °C for 15 min and centrifuged at 12,000 rpm at 4 °C for 15 min. The DNA pellet obtained was washed in 200 µL of wash buffer (76% ethanol and 10 mM ammonium acetate) and centrifuged at 14,500 rpm for 5 min. Then, the wash buffer was removed with an FTA-1 Aspirator . The pellet was dried at room temperature and suspended in 160 µL of TE buffer (10 mM tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0)). Extracted DNA was purified by adding to each DNA sample 0.5 volume of 7.5 M ammonium acetate (pH 7.7) and incubating in ice for 20 min. After centrifugation at 12,000 rpm for 15 min at 4 °C, the supernatant was transferred into a clean 1.5 mL Eppendorf tube. The DNA in the supernatant was re-precipitated by adding 2.5 volumes of ethanol, mixed thoroughly, and centrifuged at 12,000 rpm at 4 °C for 15 min. The supernatant was removed, and the DNA pellet was washed with 200 µL of 76% ethanol, dried at room temperature for 15 min, and suspended in 40 µL of TE buffer.Fresh leaves were collected from parental genotypes and hybrid progenies and frozen in liquid nitrogen. DNA was isolated from the plant material using the protocol described by Rahimah et al.  with somThe quality of DNA samples was evaluated with a SPECTROstar Nano spectrophotometer , and the optical density was determined at A260, A280, and A350. The integrity of the DNA was further examined by electrophoresing approximately 5 µg of DNA in 1% agarose gel in 1× TAE buffer . The concentration of each DNA sample was estimated using Qubit 4.0 Fluorometric Quantitation .HindIII  in a 20 µL reaction volume. Digestion was stopped by incubating the reaction mixture at 65 °C for 20 min. Next, the barcode adapter was ligated to the end generated by HindIII, allowing pooling of the samples. The ligation reaction was carried out at 22 °C for 1 h in a volume of 50 µL with 1.6 units of T4 ligase . T4 ligase inactivation was reached by heating at 65 °C for 10 min. Next, 10 µL of each sample was pooled and simultaneously purified with AMPure XP beads. The second restriction digestion was performed with 0.7 units of NlaIII  in a 20 µL reaction volume at 37 °C for 1 h with subsequent inactivation at 65 °C for 20 min. The second common adapter was ligated to the overhanging end of NlaIII. The reaction mixture (50 µL) was dispersed into 14 aliquots, and PCR was performed on the 14 aliquots to amplify the DNA fragments. The PCR mix (50 µL) contained 1× HF buffer, 25 µL of each primer, 12.5 µM dNTPs, and 1 unit of Phusion® High-Fidelity DNA Polymerase . PCR conditions were as follows: 98 °C for 30 s, 14 cycles of 98 °C for 10 s, 65 °C for 30 s, 72 °C for 15 s, and then 72 °C for 2 min. All 14 PCR reactions were pooled and purified with AMPure XP beads. The concentration and size distribution of the prepared libraries were checked by automated electrophoresis with the 2100 Bioanalyzer . Sequencing was performed on Illumina HiSeq2500 with single end reads of 150 base pairs at the CERBALAB Company .ddRADseq libraries were prepared, as described previously . ddRAD lV. vinifera 12X genome assembly  using BWA V. 0.7.17 under default mapping parameters.Illumina reads were mapped to the The SNP calling procedure was performed using the Tassel V.5.2.40 GBS v2 plug-in  and StacFor SSR marker analysis, DNA was isolated from 100 mg of fresh young leaves using the CytoSorb kit  according to the manufacturer’s recommendations.2, and 3 units of Taq polymerase with antibodies inhibiting enzyme activity. The forward primer of each pair was 5′-end-labeled with a fluorescent dye  .Amplification reactions were performed in a total volume of 25 µL with 20 ng of template DNA, 10 pmol of each primer, and 10 µL of 2.5× PCR reaction mixture containing 0.25 mM dNTPs, 3 mM MgClPCR was carried out using a T100 thermal cycler . The cycling program consisted of the following steps: 5 min at 95 °C, followed by 35 cycles of 15 s at 95 °C, 25 s at 58–66 °C, and 25 s at 72 °C, and a final extension step of 7 min at 72 °C. Fragment analysis was performed using a 4-capillary system of the ABI 3130 Genetic Analyzer  and Nanofor 05  using SD-600 labeled with LIZ  as an internal size standard. The PCR fragments were sized with the software GeneMapper (v. 4.0) (Applied Biosystems) and the software DNA Analysis .


The catalysts were characterized by XRD, XPS, NH3-TPD, TEM, and EDS-mapping to study the influence of the introduction of W. There was an interaction between Ni and W, and strong acid sites were introduced by the addition of W. The W promoted Ni/Al2O3 showed good selectivity to HHD when used as a catalyst for the hydrogenation of HMF in water. The influences of the content of W, temperature, H2 pressure, reaction time, and acetic acid (AcOH) were studied. NiWOx/Al2O3-0.5 (mole ratio of W:Ni = 0.5) was found to be the most suitable catalyst. The high selectivity to HHD was ascribed to the acid sites introduced by W. This was proved by the fact that the selectivity to HHD was increased a lot when AcOH was added just using Ni/Al2O3 as catalysts. 59% yield of HHD was achieved on NiWOx/Al2O3-0.5 at 393 K, 4 MPa H2 reacting for 6 h, which was comparable to the noble metal catalyst, showing the potential application in the production of HHD from HMF.The conversion of 5-hydroxymethylfurfural (HMF) to 1-hydroxy-2,5-hexanedione (HHD) represented a typical route for high-value utilization of biomass. However, this reaction was often catalyzed by the noble metal catalyst. In this manuscript, W promoted Ni/Al The production of chemicals from a renewable resource is one of the essential tasks for sustainable chemistry . As the 1-hydroxy-2,5-hexanedione (HHD) was one of the high value-added compounds obtained from HMF through catalytic hydrogenation . The HHD2 pressure. The formic acid could also be used as the hydrogen source when a homogeneous catalyst was used  could be converted to HHD by Nip to now . In this2O3 with different content of W was displayed in γ-Al2O3, Ni/Al2O3 had additional two peaks at 37.3o and 43.4o. This should be ascribed to (111) and (200) diffraction peaks of NiO (PDF#47-1049). The two peaks decreased as the content of W increased for NiWOx/Al2O3 and almost disappeared for NiWOx/Al2O3-0.7 and NiWOx/Al2O3-0.9. The WOx/Al2O3 showed (001) (020) (200) (111) (021) (201), and (220) peaks of WO3 without NiO peaks . This showed that most of the NiO could be reduced to Ni by H2 reduction, which was consistent with the results of H2-Temperature programmed reduction (H2-TPR)  patterns of NiWOx/AliO peaks . W speciappeared . Two new(H2-TPR) . After t2O3 showed two peaks centered at 856.7 and 855.3 eV, which should be ascribed to NiAl2O4 and NiO, respectively  and the results were shown in ectively  , and the results were shown in 2O3 showed a sharp peak at 570 K which was ascribed to the weak to medium acid site of Al2O3. The NH3-TPD of NiWOx/Al2O3-0.5 showed three wide peaks centered at 442, 544, and 700 K corresponding to the weak acid, medium acid, and strong acid sites respectively. The NiWOx/Al2O3-0.5 posed a relatively strong acid site except for the weak to medium acid site compared to Ni/Al2O3. This desorption curve was similar to WOx/Al2O3 . The TEM images  mapping was taken for both catalysts. The results were shown in 2O3 and NiWOx/Al2O3-0.5. The content of Ni, W was close to the theoretical value. It could be seen that the distribution of Ni could be divided into two categories for Ni/Al2O3. Some of the Ni gathered together to form metal particles as shown in TEM images. While the other Ni did not agglomerate and dispersed on the support evenly. This should be ascribed to the unreduced Ni species with high interaction between Al. After the introduction of W, the NiWOx/Al2O3-0.5 had a similar Ni distribution as Ni/Al2O3 cyclopentan-1-one (HCPO) and 12% selectivity to 2,5-Bis(hydroxymethyl) furan (BHMF). Deep hydrogenation products reported in the literature  such as 2,5-bis(hydroxymethyl) tetrahydrofuran, 1,2,6-hexanetriol and 1,2,5-hexanetriol were not found, showing that the catalyst had moderate hydrogenation activity. When a small amount of W was introduced to the Ni/Al2O3, the conversion of HMF increased to 68% while the selectivity to HHD decreased a little to 25% for NiWOx/Al2O3-0.1. A small amount of W had no promoting effect for the selectivity to HHD. However, as the amount of W increased, the selectivity to HHD had a sharp increase to 66% for NiWOx/Al2O3-0.5 with a little increase in the conversion of HMF. When continued to increase the content of W, the selectivity to HHD maintained around 60% while the conversion of HMF decreased to 59%. An appropriate amount of W was needed for the high performance for hydrogenation of HMF to HHD. When no Ni was used in the catalyst (WOx/Al2O3), the HMF was almost unchanged showed that the W did not have the function for hydrogenation. The increased activity for the conversion of HMF could be caused by the interaction between Ni and W which was clued by the XRD, XPS, and H2-TPR. Firstly, the electronic state of Ni was changed by the addition of W. Secondly, the interaction between Ni and W weaken that between Ni and Al. So, the content of NiAlO4, a species that had no hydrogenation activity, decreased. Therefore, the hydrogenation activity increased after the introduction of W. The high selectivity to HHD should be ascribed to the strong acid site introduced by the addition of W. It was reported that the acids play a key role in the isomerization of furan rings in the conversion of HMF to HHD. The introduced strong acid sites by W were conducive to the rearrangement of furan rings during the reaction. Thus, the selectivity to HHD was enhanced.The hydrogenation of HMF was conducted in water under a hydrogen atmosphere. We first checked the effect of W for the reaction, and the results were shown in terature  for Ni a2O3-0.5 and Al2O3 had no obvious change after reacting at different temperature. This showed the catalysts was stable at the reaction condition. The changes in conversion could be interpreted by the different reaction pathways revealed by the change in selectivity . The results were shown in To verify the effect of acid for the conversion of HMF to HHD, the Ni/AlBased on the characterization and the hydrogenation of HMF, the reaction pathway and the role of catalyst was proposed as shown in 2O3. The Ni and W uniformly dispersed on the surface of the support. The interaction between W and Ni increased the activity of Ni/Al2O3 for the hydrogenation of HMF. The introduction of W generated strong acid sites, which were the key for the high selectivity to HHD. The role of acid was proved by the addition of AcOH to unpromoted Ni/Al2O3. A suitable temperature was needed for the transformation of HMF to HHD smoothly. After the optimization of the conditions, a 59% yield of HHD was acquired at 393 K, 4 MPa H2 reacted for 6 h on NiWOx/Al2O3-0.5. This work provided the idea for high selectivity to HHD from HMF by the introduction of suitable acid and improved the feasibility of putting this reaction into practical application by using a non-noble metal catalyst.In conclusion, the conversion of HMF to HHD was achieved by noble-metal-free W promoted Ni/Al6H6O3) was bought from Zhengzhou Alpha Chemical Co. Ltd. Aluminum oxide , n-decane  and ammonium metatungstate [99.5% metals basis (NH4)6H2W12O40·xH2O] were purchased from Aladdin Chemistry Co. Ltd. Nickel (II) nitrate hexahydrate  and AcOH  was got from Anhui Zesheng Technology Co., Ltd.HMF  was adjusted by the binding energy of C1s. The transmission electron microscopy (TEM) images and energy-dispersive spectrometer (EDS) elemental mappings were taken on a JEOL JEM-2100 F field emission transmission electron equipped with An Oxford 80T detector. The temperature-programmed desorption of ammonia (NH3-TPD) and H2-Temperature programmed reduction (H2-TPR) was conducted on the Micromeritics AutoChem II 2920 Instrument. Typically for NH3-TPD, 60.0 mg of sample were loaded into the sample tube. The sample was heated to 773 K under He flow (10 mL/min) and kept for 2 h. The tube was cooled to 373 K and 10NH3-He (30 mL/min) was introduced for 0.5 h. Then the atmosphere was switched to He (10 mL/min) and kept for 1 h to remove the physical adsorbed NH3. After that, the temperature was increased (10 K/min) from 373 to 973 K in an atmosphere of He (10 mL/min). The desorbed NH3 was detected by the thermal conductivity detector (TCD). For H2-TPR, the calcinated catalyst (85.0 mg) was degassed at 573 K under an atmosphere of Ar (10 mL/min) for 2 h. The sample was cooled to 373 K. The temperature was increased (10 K/min) from 373 K to 973 k under the atmosphere of 10H2-Ar (30 mL/min). The H2 consumption was monitored by a TCD detector.A Rigaku D/Max 2500/PC powder diffractometer was used to collect the X-ray diffraction (XRD) patterns. Cu Kα radiation at 40 kV was used as the X-ray source. Thermo Escalab 250Xi spectrometer with Al Kα was used to characterize the X-ray photoelectron spectroscopy (XPS) spectra. The sample powder was overspread on a double-faced adhesive tape on aluminum foil. The sample was pressurized to 8 MPa for 30 s and used for measurement. Before measurement, the chamber pressure was vacuumized to <1 × 103)2·6H2O (2.50 g) and (NH4)6H2W12O40·xH2O (1.05 g) were dissolved in water (10.00 g). Then Al2O3 was added to the solution. The mixture was stirred evenly to form a paste and kept standing for 24 h. Then, the paste was dried at 393 k overnight. Followed by calcined at 823 K in the air for 4 h. The obtained solid was ground to pass through 100 mesh sieve and reduced at 773 K in H2 to afford the NiWOx/Al2O3-0.5. The 0.5 referred to the mole ratio of W to Ni.In a typical procedure, Ni was added and the mixture was diluted to 10 mL by ethanol. After centrifugation, the liquid was used for analysis. The qualitative analysis was conducted by GC on a Shimadzu GC-2014 equipped with a SH-Rtx-1701 column (30 m × 0.32 mm × 0.25 µm). The oven temperature was started from 353 K for 2 min and raised to 523 K with 20 K/min heating rate. The oven was kept at 523 K for 1.5 min. The GC-MS was performed on Shimadzu GC/MS-TQ8040 equipped with an SH-Rxi-5Sil MS column (30 m × 0.25 mm × 0.25 µm). The oven temperature was started from 323 K for 1 min and raised to 473 K with 40 K/min heating rate and then raised to 553 K and kept at the temperature for 5 min.The hydrogenation reaction was conducted in a 20 mL stainless steel reactor. Typically, the HMF aqueous solution , catalyst (20.0 mg), and magneton were put into a glass lining. The lining was set in the reactor and sealed and purged with H


P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43− cells and derivative populations, but not that of CD34lowCD43− cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-β or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).Deficiency of  Cell differentiation, including development of the hematopoietic lineage, is closely related to the cell cycle and is regulated by multiple cell cycle factors6. Among those factors, cyclin-dependent kinase inhibitors (CKIs) can manipulate the cell cycle and induce cell cycle arrest7. CKIs can be divided into two families: the Cip/Kip family, including p21Cip1/Waf1/Sdi1 (P21), p27Kip1 (P27), and p57Kip2 (P57), and the INK4 family, including p16INK4a (P16), p15INK4b (P15), p18INK4c (P18), and p19INK4d.2 (P19). P15, P18, P19, and P27 are expressed in all cell lines8. Members of the INK4 family play important roles in hematopoietic differentiation, which is downstream of the TGF-β/SMAD signaling pathway and is also controlled by RUNX1, especially in adulthood11.Hematopoiesis can be divided into primitive and definitive hematopoiesis, which originate from the yolk sac (YS) and the aorta/gonad/mesonephros (AGM) region, respectivelyCDKN2C) is focally expressed during embryonic development13 and has important functions during hematopoiesis15. By interacting with the Cyclin D–CDK4/CDK6 complex, p18INK4c blocks the activation of the CDK kinases, thus playing a key role in controlling the G1 phase of the cell cycle. This interaction is associated with G1 arrest16.Among the members of the INK4 family, p18INK4c  is reduced in P18−/− mouse bone marrow, indicating that deletion of P18 has a positive effect on HPCs in vivo22. To date, however, the effects of P18 overexpression on hematopoiesis have not been systematically explored.Deletion or inhibition of the RUNX1b during early hematopoiesis prevents the generation of CD34+ cells1. Subsequent studies revealed that RUNX1b overexpression also changes the status of the cell cycle and increases the expression of some cell cycle regulators, including P18 (unpublished data). Hence, we sought to elucidate the function of P18 on different stages of hematopoiesis using a mature inducible expression system based on piggyBac transposon and the AGM-S3 co-culture system that we established previously24. Our findings provide the first evidence that P18 overexpression can promote hematopoiesis, providing insight into the molecular mechanisms underlying CKI activity during hematopoietic differentiation.In previous research, we found that overexpression of 5 cells per well and cultured in an incubator containing 5% CO2 at 37 °C. After the cells had grown to 80–100% confluence, they were irradiated with 13 Gy of X-rays. Undifferentiated hESCs (provided by Prof. Tao Cheng) were cut into small squares containing 0.5–1 × 103 cells each by 200 μl tips, which were inoculated into 12-well plates (25 pieces per well). hPSC maintenance medium (Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, F-12 nutrient mixture, 20% knockout serum replacement , 1% L-glutamine, 1% non-essential amino acid solution , and 5 ng/ml basic FGF) was used for the co-culture system for the first 3 days, and then replaced with hematopoiesis-inducing medium (Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal bovine serum , 1% NEAA (Gibco), 60 ng/ml ascorbic acid (Sigma), and 20 ng/ml vascular endothelial growth factor ); this day was defined as D0. The co-cultures were grown for up to 14 days with 5% CO2 at 37 °C, with a medium replacement once per day. The detailed procedure was defined previously24.This study was approved by the institutional ethics committee of Institute of Blood Transfusion, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC). AGM-S3 cells (provided by Prof. Tatsutoshi Nakahata) were plated in 12-well plates at 1 × 10P18 (CDKN2C) inducible hESC lines (referred as P18/hESCs) was described in “P18/hESCs co-cultured with AGM-S3 cells were treated without induction or with DOX from D0, D2, D4, D6, D8, D10, D12, or with DOX and 0.33 μM RepSox  from D0, as previously described1. An equal volume of DMSO was added to control samples. Treated co-cultures were subjected to cell-cycle analysis at D4 and flow cytometry using 7-AAD and anti-CD34/CD43 antibodies (D8) or 7-AAD and anti-CD34/CD43/CD45, CD71/GPA, or CD34/CD43/GPA/CD41a antibodies (D14). Untreated co-cultures were used as negative controls. The detail information of flow cytometry was described in “The establishment of P18/hESC co-cultured cells induced with DOX from D0, D6, or D10, or not induced, were dissociated into single cells with 0.25% trypsin–EDTA solution, centrifuged at 400g for 5 min, and resuspended in 400 μl of IMDM medium. After cell counting. 5 × 104 cells in suspension were mixed well on methylcellulose  containing 1% Antibiotic–Antimycotic (Gibco) and cytokines, as previously described25, and then 1.1 ml of the mixture was divided into each 35-mm Petri dishes at a final concentration of 5 × 104 cells per dish. The cells were incubated for 12–14 days at 37 °C in a 5% CO2/95% humidity incubator. Colony forming unit–erythrocyte (CFU-E) was determined after 7 days, and other types of colonies were counted after 12–14 days.D14 P18/hESC co-cultures at D2 or D6 were dissociated by treatment with 0.05% trypsin EDTA solution and stained with 7-AAD and anti-KDR antibodies (D2) or anti-CD34/CD43 (D6). KDR+ cells at D2 or CD34highCD43- and CD34lowCD43- cells at D6 were sorted using a BD FACSJazz Cell Sorter, and their purity was confirmed by flow cytometry analysis. 1 × 104 sorted KDR+ cells were re-plated on irradiated AGM-S3 cells in each well of a 24-well plate, and 1 × 104 sorted CD34highCD43− and CD34lowCD43− cells were re-plated in FLHD  medium in each well of a 48-well plate, further cultured with or without DOX induction for 11 or 14 days respectively. Half of the media was replaced every day, and flow cytometry analysis was performed. The detail information of flow cytometry and sorting was described in “Non-induced or induced P18/hESCs co-cultured with AGM-S3 cells were detected by qRT-PCR at D14 using primers for NFKB1 and NFKB2. QNZ, an inhibitor of the NF-κB signaling pathway (Selleck Inc) was dissolved in DMSO; siRNAs against NFKB1  were described previously24. D10-induced P18/hESC co-cultures were treated from D10 with 20 nM QNZ  or 20 nM siRNA against NFKB1 , and changed fresh media every day. At D14, the cells were evaluated by qRT-PCR, flow cytometry, and cell cycle analysis; untreated co-cultures were used as negative controls. qPCR primer pairs are listed in Table D10-induced https://www.flowjo.com/solutions/flowjo/downloads/) and GraphPad Prism5 (https://www.graphpad.com/scientific-software/prism/) were used for data analysis.All experimental data are described as means ± SD, and statistical significance was evaluated using Student’s t-test. p < 0.05 was considered statistically significant. FlowJo 10 , expression of P18 was upregulated at D4 . All of these effects can be counteracted by addition of 0.33 µM RepSox from D0  medium without DOX induction  were subjected to flow cytometry analysis at D8 to compare non-induced co-cultures and the GFP+ fractions of co-cultures treated with DOX. Overexpression of P18 at the early stage, especially from D0, significantly decreased production of CD34highCD43−, CD34−CD43+, and CD34+CD43+ populations at D8; these effects gradually weakened when P18 overexpression was initiated later, disappearing after D6  is a key CKI during the cell cycle. Knockout, knockdown or chemical inhibition of P18 in mouse models promotes the generation of HSCs21. To date, however, the effects of up-regulation of P18 on hematopoiesis have not been deeply explored. Previously, we reported that RUNX1b overexpression at the early stage blocks human hematopoiesis1. Here, we observed that in RUNX1b/hESC co-cultures, expression of P18 at D4 was upregulated when RUNX1b was induced from D0, and that this effect could be counteracted by addition of the TGF-β signaling inhibitor RepSox from D0  can severely decrease the production of these cells and their derivative populations, such as CD34+CD43+ and CD34−CD43+ cells at D8, which effect weakened when it was initiated after D2 and disappeared when it was initiated after D6. The CD34+CD43+ and CD34−CD43+ populations exhibited similar patterns  and not later than D4. Together, P18 overexpression in mesodermal populations (mainly KDR+ cells) at the early stage inhibited formation of the CD34high sub-population.In the cocultures with hESC and AGM-S3 at D8, CD34+CD43− cells can be produced and obviously divided into two sub-populations: CD34ion Fig. a. The CDRUNX1b1, and the function of P18 at the early stage involves TGF-β signaling, a point that warranted investigation. The inhibition of TGF-β signaling could counteract most effects of P18 overexpression from D0  might help to these prohibitory effects, but probably not be the main reason.In a previous study, we showed that inhibition of TGF-β signaling partially rescued inhibition of hematopoietic differentiation by  D0 Fig. a,b. The erm Fig. d. For alP18 from D6 or later significantly promoted the production of the aforementioned populations at D8  p18 overexpression reduced the percentage of apoptotic cells significantly28, which indicated that it effects on apoptosis seem distinctive in different context and cell type.From the view of apoptosis, the ge Figs. d, 6d. Itp18 overexpression from D0 can block transition from CD34lowCD43− cells to CD34highCD43− cells so as to prevent the emerging of hematopoietic endothelium and their derived hematopoietic stem/progenitor cells. p18 overexpression from D10 can promote the development of hematopoietic stem/progenitor cells and other progenitors of classic hematopoietic lineage. It is reasonable to speculate that both of them ought to be caused by similar effects that G1 arrest, proliferation blockage and apoptosis were increased, but they influence the production of hematopoietic stem/progenitor cells with contrary style, which detail mechanism need further exploration. The primary mechanisms and functions of p18 overexpression in hematopoiesis were summarized in Fig. P18 and P21 had opposing effects on the proportions of cells in S and G2/M, but similar inhibitory effects on hematopoietic differentiation. All of these effects could be counteracted by inhibition of TGF-β signaling, as in the case of RUNX1b29. Overexpression of any of these genes increased the proportion of cells in G0/G1, indicating that TGF-β signaling plays a key role in G1 arrest. Stimulation of TGF-β signaling mediates cell-cycle arrest and up-regulates expression of P1532.In our co-culture system, RUNX1b overexpression stimulated TGF-β signaling, which upregulates not only P15 (unpublished data) but also P18 and P21. P18, P15 and other members of the INK4 family contain repeated ankyrin motifs and can regulate G1 phase by inhibiting CDK4/6 and interfering with cyclin–CDK assembly, thereby inducing G1 arrest34. P21, which belongs to the Cip/Kip family, does not contain repeated ankyrin motifs and therefore cannot specifically bind to CDK4 and CDK6; consequently, its mechanism is distinct from that of P1819; however, the two proteins appear to have similar effects on hematopoiesis at the early stage. It is reasonable to speculate that with the help of TGF-β signaling, RUNX1 might coordinate and organize various CKIs  to control early hematopoietic differentiation via distinct mechanisms.At the early stage of AGM-S3 co-culture, overexpression of P21, P18 overexpression at the late stage efficiently promotes hematopoietic differentiation, potentially due to differences in protein structure34. Similar effects can be observed following overexpression of HOXA9 and HOXC4 at the same stage24. Overexpression of these factors changed the cell cycle status with different styles while the inhibition of NF-κB signaling can counteract all these changes except for apoptosis, which reveal it involved in the function of P18 on hematopoietic differentiation through the controlling of cell cycle status but not apoptosis. This study provides the first evidence that P18 overexpression promotes hematopoiesis, a property that is very rare among CKIs. The underlying mechanism, which requires further exploration, may have important clinical applications.In contrast to Supplementary Information 1.Supplementary Information 2.


Ammonia is the most typical neurotoxin in hepatic encephalopathy (HE), but the underlying pathophysiology between ammonia and aberrant glycosylation in HE remains unknown.2+) homeostasis by overexpression of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in astrocytes.Here, we used HBV transgenic mice and astrocytes to present a systems-based study of glycosylation changes and corresponding enzymes associated with the key factors of ammonia in HE. We surveyed protein glycosylation changes associated with the brain of HBV transgenic mice by lectin microarrays. Upregulation of Galβ1-3GalNAc mediated by core 1 β1,3-galactosyltransferase (C1GALT1) was identified as a result of ammonia stimulation. Using in vitro assays, we validated that upregulation of C1GALT1 is a driver of deregulates calcium (Ca2+ activity, thereby C1GALT1 and IP3R1 may serve as therapeutic targets in hyperammonemia of HE.We demonstrated that silencing C1GALT1 could depress the IP3R1 expression, an effective strategy to inhibit the ammonia-induced upregulation of CaThe online version contains supplementary material available at 10.1186/s13578-022-00751-4. Hepatic encephalopathy (HE) is a complex neurological syndrome of patients with liver failure, cirrhosis or hepatitis due to severe liver disease or portal-systemic shunts, causing a wide spectrum of neuropsychiatric disorders . HE signGlycosylation, the enzymatic process is controlled with the glycosyltransferases and glycosidases by which glycans are attached to proteins and lipids, is the most abundant and functionally important type of post-translational modification . Differe2+ concentration is the major component of astrocytic signaling [2+ events in astrocytes [2+ signaling, including metabotropic glutamate receptor 5 (mGluR5) and inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), have been reported to deregulates Ca2+ homeostasis in astrocytes [Studies of CNS have suggested that neurons transmit electrical and chemical impulses, while glial cells, including astrocytes, microglia, oligodendrocytes and ependymal cells, provide protection, immune defense and support neurons . Astrocyignaling . The mettrocytes , 15. Of trocytes .2+ homeostasis. We found that the expression levels of galactose type, fucose type, N-acetylglucosamine and mannose type glycans in HBV transgenic mice were remarkably different than those in control mice. Moreover, we showed that C1GALT1, upregulated in NH4Cl treated astrocytes associated with Ca2+ signaling by targeting IP3R1, demonstrating that aberrant glycosylation can drive neurological dysfunction.In the present study, we examined the alternations of protein glycosylation in the brain of HBV transgenic mice and in astrocytes stimulated by ammonia and the effects of glycosyltransferase on astrocytic CaHBV transgenic mice overproduce the hepatitis B virus (HBV) large envelope polypeptide and accumulate toxic quantities of hepatitis B surface antigen (HBsAg) in hepatocyte that develop a severe and persistent hepatocellular injury, leading to precancerous proliferation reaction and unrestrained cell growth . To idenThe H&E staining of mice liver tissues showed that the livers of control mice aged 4 to 18 months were normal, while hepatocellular overexpression of HBV protein leaded to severe and persistent hepatocellular injury in HBV transgenic mice  and control mice  were measured by lectin microarrays, which included 37 lectin probes, providing specific information for the glycan repertoire of tissue glycoproteins . The lay2–4 binder LEL, the α-D-Man, Fucα-1,6GlcNAc, α-D-Glc binder PSA, Fucα-1,6GlcNAc, α-D-Man, α-D-Glc binder LCA and the High-Mannose, Manα1-6Man binder NPA, exhibited significantly altered NFIs in the olfactory bulb of HBV transgenic mice compared with control mice  for each lectin are shown in Additional file n, Galβ1-4GlcNAc binder DSA, the α-D-Man, the High-Mannose, Manα1-6Man binder NPA, the Fucα1-2Galβ1-4GlcNAc, Fucα1-3GlcNAc, anti-H blood group specificity binder LTL and the Fucα-1,6GlcNAc, α-D-Glc binder PSA, exhibited significantly altered NFIs in left hemisphere of HBV transgenic mice compared with control mice , N-acetylglucosamine (GlcNAc) and mannose type glycans in HBV transgenic mice and control mice, the fluorescence-based lectin histochemistry was performed using three lectins selected randomly , according to our previous protocol . The negThese results demonstrated that the protein glycopatterns identified by 12 lectins  exhibited significantly changed NFIs in the brain of HBV transgenic mice compared with control mice. Notably, a total of 10 lectins  showed significant differences in the cerebral hemispheres and midbrain, where astrocytes were widely expressed. The sugar-binding specificities of the 10 altered lectins in HBV transgenic mice are shown in Additional file 4Cl on protein glycosylation in astrocytes . After serum-free for 24 h, astrocytes were left untreated for 72 h or treated with 0.5, 2, 5 or 10 mmol/L NH4Cl for 72 h. The medium was changed daily to ensure the concentration of NH4Cl. The NFIs for 37 lectins in astrocytes are summarized in Additional file 4Cl treated astrocytes and untreated astrocytes are shown in Fig. 4Cl treated compared with untreated SVG p12, SW1088 and CCF-STTG1 cells  for 72 h followed by another incubation for 72 h in an NH4Cl-free culture medium. The results showed that Galβ1-3GalNAc returned to basal level when NH4Cl containing medium was removed after 72 h  induced glutamine formation on the expression of Galβ1-3GalNAc, astrocytes were treated with NH4Cl and the glutamine synthetase inhibitor . In liver cirrhosis, HE is accompanied by oxidative stress due to the activation of NADPH oxidase [4Cl  induced NADPH on Galβ1-3GalNAc, astrocytes were exposed to NH4Cl and NADPH oxidase inhibitor . To determine whether NH4Cl-induced intracellular pH changes trigger increased Galβ1-3GalNAc, astrocytes were treated with CH3NH3Cl (5 mmol/L) for 72 h, which is a compound that produces the same intracellular pH changes as NH4Cl [4Cl mediated upgrade of Galβ1-3GalNAc, and exposure to CH3NH3Cl did not affect the expression of Galβ1-3GalNAc.Ammonia accumulated in the blood can cross the blood–brain barrier into astrocytes, metabolized into glutamine by glutamine synthetase . Ammonia oxidase . The syn oxidase . To testas NH4Cl , 27. As 4Cl treated SVG p12, SW1088 and CCF-STTG1 cells  are responsible for the synthesis of Galβ1-3GalNAc . To gain2+ signaling is an important regulator of many functions of astrocytes [2+ signaling might be subjected to ammonia regulation, so we used Fluo-4 AM to detect the Ca2+ in SVG p12 and SW1088 cells. The results demonstrated that after NH4Cl treatment, the intensity of Ca2+ was significantly stronger  or a non-targeting control (siNTC). The transfection efficiency was determined by negative control FAM  to analyze the roles of IP3R1in Ca2+ dysbiosis induced by C1GALT1. This result suggested that AdA significantly increased intracellular Ca2+ in astrocytes, and AdA plus NH4Cl induced a slight increase in Ca2+ compared with AdA alone 2–4 recognized by LEL, Fucα1-2Galβ1-4GlcNAc, Fucα1-3GlcNAc, anti-H blood group specificity recognized by LTL, Fucα-1,6GlcNAc, α-D-Man, α-D-Glc recognized by LCA, Galβ1-3GalNAc recognized by MPL, High-Mannose, Manα1-6Man recognized by NPA, α-D-Man, Fucα-1,6GlcNAc, α-D-Glc recognized by PSA, Bisecting GlcNAc, biantennary complex-type N-glycan recognized by PHA-E, Terminal in GalNAc and Gal, anti-A and anti-B human blood group recognized by SJA, α-/β-linked terminal GalNAc, n, GalNAcα1-3Gal, blood-group A recognized by SBA, and Terminating in GalNAcα/β1-3/6Gal reginized by WFA exhibited significantly changed in the brain protein of HBV transgenic mice compared with control mice.Although it has long been verified that protein glycosylation plays an important role in CNS, few glycomic analyses of HE. Here we have taken a systematic approach to identify protein glycosylation changes in the brain of HBV transgenic mice associated with chronic liver disease. Our analysis revealed the expression level of β-D-GlcNA, (GlcNAcβ1-4)n-butyric acid systems in the pathogenesis of HE, the exact underlying mechanisms associated with glycosylation remain poorly understood [4Cl treated astrocytes. Interestingly, except Galβ1-3GalNAc, the expression levels of other glycans did not alter consistently after ammonia stimulation. These data suggest that Galβ1-3GalNAc, mediated by glycosyltransferases of C1GALT1, has increased in ammonia-treated astrocytes within a glutamine synthesis-dependent manner. There is a single linear relationship between glycosyltransferase and glycan expression in some specific glycans coding pathways. However, due to the complementary coding of some glycosyltransferases, there is not a simple linear relationship between a single glycosyltransferase and its synthesis pathway, especially in the case of multiple factors, the regulation of glycosyltransferase is more complex.The biosynthesis of glycans requires the synergism of glycosyltransferases and glycosidases . There iderstood . We spec2+, suggesting that astrocytes play a more active role in brain function [2+ signaling might be subjected to ammonia regulation in astrocytes, and our results demonstrated that the intensity of Ca2+ was significantly stronger after ammonia treatment in astrocytes. Various plasma membrane receptors trigger Ca2+ signaling, and the key components of mGluR5 and IP3R1 are responsible for deregulates Ca2+ homeostasis in astrocytes [2+ homeostasis in astrocytes according to the overexpression of IP3R1. There is no established role for Galβ1-3GalNAc and neurotoxin ammonia in HE, and the importance of C1GALT1 to function of astrocytes is unknown. So, we next focused our functional studies on C1GALT1 as a candidate regulator of Ca2+ homeostasis in astrocytes. The results indicated that C1GALT1 silencing in astrocytes down-regulated the Ca2+ activity through the lower expression of IP3R1 in astrocytes, and C1GALT1 silencing could suppress ammonia induced up-regulation of Ca2+ activity.Astrocytes release chemical messengers of Cafunction . Moreovefunction . We nexttrocytes . Our dat2+ concentration due to the overexpression of IP3R1, and a proposed molecular network is shown in Fig. Together our data point toward that increased Galβ1-3GalNAc mediated by C1GALT1 in hyperammonemia and C1GALT1 facilitates CaAll mice experiments were performed in compliance with the People's Republic of China Legislation Regarding the Use and Care of Laboratory Animals and approved by the Laboratory Animal Ethics Committee of Northwest University and Fourth Military Medical University. HBV transgenic mice C57BL/6 J-TG(ALB1HBV)44BRI/J were provided by The Jackson Laboratory  . The neg2. SW1088 cells were maintained in Leibovitz L-15 containing 10% FBS and 1% Penicillin–Streptomycin-Gentamicin Solution  in a 37 ℃ incubator under humidified atmosphere.Human brain astrocytes (SVG p12) and human astrocytoma cells  were verified by STR and maintained in mycoplasma-free conditions. SVG p12 cells were maintained in Dulbecco’s Modified Eagle’s Medium  and CCF-STTG1 cells were maintained in RMPI 1640 medium  supplemented with 10% fetal bovine serum  and 1% Penicillin–Streptomycin-Gentamicin Solution  in a 37 ℃ incubator under 5% COThe formalin-fixed paraffin-embedded (FFPE) tissues were prepared as previously described . BrieflySerum ammonia concentrations in all mice at 4 time points were determined using ELISA kits , according to the manufacture’s protocols. Serums from mice at each time point were pooled respectively, and each sample was independently performed four times. The standard curve was obtained according to the standard substance, then the absorbance value of each sample was substituted to get the corresponding serum ammonia concentration.g for 15 min at 4 °C, then protease inhibitors  were added. Cells were lysed in cold RIPA buffer supplemented with protease inhibitors, and then the proteins were obtained by collecting the supernatant after ultracentrifugation at 120,000×g for 15 min at 4 °C.The olfactory bulb, left hemisphere, right hemisphere, midbrain and cerebellum of each mouse were placed in glass tubes containing 1 mL of ice-cold tissue protein extraction reagent . The tissue was fragmented by homogenate in a homogenate instrument  until solid materials were not visible. The proteins were obtained by collecting the supernatant after ultracentrifugation at 120,000×P-values less than 0.05 were considered statistically significant.The proteins were labeled with Cy3 fluorescent dye  and purified using Sephadex G-25 columns . Lectins were purchased from Sigma or Vector Laboratories. The lectin microarrays were produced as previously described . BrieflyThe lectins were labeled with Cy5 fluorescent dye  and purified using Sephadex G-25 columns . Dewaxed and rehydrated tissue sections were microwaved in 10 mM citrate buffer (pH 6.0) at 100 °C for 10 min and cooled at room temperature to eliminate endogenous peroxidase activity, and 5% bovine serum albumin and 0.08% Triton X-100 was used to block nonspecific staining at 25 °C for 1 h. The tissue sections were incubated with Cy5-labelled lectin at 4 °C overnight. Finally, sections were stained with 1 μg/ml of DAPI  for 10 min. Cells were pre-inoculated into the dish, and 4% paraformaldehyde was used to immobilize the cells for 10 min. Then 5% bovine serum albumin and 0.01% Triton X-100 were used to block nonspecific staining at 25 °C for 1 h. Cells were incubated with Cy5-labelled lectin at 4 °C overnight. Finally, sections were stained with 1 μg/ml of DAPI for 10 min. A laser scanning confocal microscope FV 1000  was used to obtain the images. The images were acquired using the same condition and shown on the same scale in the Cy5- and DAPI-merge channel. The average fluorescence intensities of the images were obtained by total fluorescence intensities of lectins binding with cells divided by the total area of cells.A protein microarray was produced by 72 individual tissue samples of olfactory bulb divided into 8-, 10-, 12-, 14-, 16- and 18-months old HBV transgenic mice and control mice. The tissue samples were dissolved to a concentration of 1 mg/ml. Each tissue sample was spotted twice on the homemade epoxy silane-coated slides with Stealth micro spotting pins (SMP-10B) by a Capital smart microarrayer. The layout of the protein microarray is shown in Additional file Equal amounts of protein were resolved by SDS-PAGE, transferred onto PVDF membranes, and blocked in Carbo-Free Blocking Solution  for 1 h at room temperature. Cy5-labeled lectins were diluted in blocking buffer with gentle shaking overnight at 4 °C in the dark. The membranes were then washed twice each for 10 min with TBST and scanned by fluorescence channel (635 nm excitation/650LP emission) with the voltage of 800 PMT using a phosphorimager .Equal amounts of protein were resolved by SDS-PAGE, transferred onto PVDF membranes, and blocked-in buffer . Primary antibodies were diluted in blocking buffer, as follows: C1GALT1 , IP3R1 , mGluR5 , β-actin . Secondary antibodies were mouse-HRP or rabbit-HRP . Blots were developed using Western ECL Blotting Substrate  and imaged in Qinxiang imaging system .https://pga.mgh.harvard.edu/primerbank/). Gene-specific primers were purchased from Sangong Biotech, China  and quantified using Nanodrop 8000 . 1 μg of RNA was reverse transcribed to cDNA using PrimeScript™ RT Master Mix  with the manufacturer’s recommendations. Transcripts were quantified by real-time PCR using Power SYBR Green PCR MasterMix . Gene-specific primers were designed using PrimerBank . Cells were incubated with 5 nM Fluo-4 AM for 30–40 min at 37 °C. Then, cells were washed with PBS buffer and incubated at 37 °C for another 30 min to allow complete esterification of the probe. A laser scanning confocal microscope FV 1000  was used to obtain the images. The images were acquired using the same condition and shown on the same scale in the Fluo-4 AM channel. The calcium fluorescence intensity of each cell was corrected by the average background intensity and then divided by the cell area to obtain the fluorescence intensity of each cell.Cytosolic free Ca4Cl for 72 h. During this period, the media was changed daily to ensure the concentration of NH4Cl. AdA  is the most potent known agonists of IP3R1. After loading with Fluo-4 AM, cells were stimulated with 3/6 μmol/L AdA. Then, Ca2+ was determined by a laser scanning confocal microscope.Astrocytes were incubated with transfection complexes for 12 h, then changed fresh media containing 1% FBS and 5 mmol/L NH2+ of the brain from mice was determined by a Calcium Ion Assay Kit . According to the manufacture’s protocols, the absorbance value was determined at 575 nm by colorimetric comparison with calcium standard samples.CaAdditional file 1: Table S1. Sugar-binding specificities of the 10 altered lectins in the HBV transgenic mice. Table S2. Related to real-time PCR methods: primers sequence. Table S3. Related to transfection of siRNA methods: siRNA primers sequence. Fig. S1. H&E staining of mice. a–b, The H&E staining of olfactory bulb (a) and cerebellum (b) from HBV transgenic mice and control mice at 10 to 18 months old. Scale bar, 100 μm. Fig. S2. Layout of the lectin microarrays. The lectin microarrays included 37 lectin probes, and each lectin was spotted in triplicate per block, with quadruplicate blocks on one slide. Cy3-labeled BSA as a marker and BSA as negative controls. Fig. S3. Glycopatterns of olfactory bulb and cerebellum in HBV transgenic mice and control mice. a, The glycopatterns of Cy3-labeled olfactory bulb samples bound to the lectin microarrays and their NFIs. b, Heat map and hierarchical clustering of the 37 lectins in glycopatterns of olfactory bulb. c, Four lectins revealed significant differences glycopatterns in olfactory bulb between HBV transgenic mice and control mice. d, The glycopatterns of Cy3-labeled cerebellum samples bound to the lectin microarrays and their NFIs. e, Heat map and hierarchical clustering of the 37 lectins in glycopatterns of cerebellum. The NFIs for each lectin were summarized as the mean values ± SD. In the heat map and hierarchical clustering, the samples were listed in columns and the lectins were listed in rows, and the color of each square represented the expression levels relative to the other data . Fig. S4. Normalized fluorescent intensities (NFIs) for each lectin in mice. a-c, The NFIs of lectin microarrays in left hemisphere (a), right hemisphere (b) and midbrain (c) from HBV transgenic mice and control mice. The NFIs for each lectin are summarized as the mean values ± SD. Fig. S5. Protein microarrays and lectin blotting analyses. a–b, The results of protein microarrays from olfactory bulb of HBV transgenic mice and control mice (a) and their fluorescence intensity (b). c, The results of SDS-PAGE from olfactory bulb of HBV transgenic mice and control mice. d-e, LEL bond to protein of olfactory bulb from HBV transgenic mice and control mice (d) and their fluorescence intensity (e). Data were presented as mean ± SD. Fig. S6. The images of fluorescence-based lectin histochemistry. a, Cy-5 labeled BSA as negative controls showed no positive signal. Scar bar 80 μm. b–c, The High-Mannose, Manα1-6Man recognized by NPA exhibited strong binding to the nuclear and cytoplasmic regions of granulosa cells in olfactory bulb (b), while NPA mainly bond to the cytoplasmic regions in left hemisphere (c). d–e, The α-D-Man, Fucα-1,6GlcNAc, α-D-Glc recognized by PSA showed strong binding to the cytoplasmic and membrane areas in the olfactory bulb (d) and left hemisphere (e). f–g, Galactose type recognized by SBA showed strong binding to the cell membrane, and little binding to cytoplasmic regions in the left hemisphere (f) and midbrain (g). Scar bar, 40 μm. Fig. S7. The normalized fluorescent intensities (NFIs) for each lectin in astrocytes. a–c, The NFIs for 37 lectins in three astrocytes are summarized as the mean values ± SD in NH4Cl treated compared with untreated SVG p12 (a), SW1088 (b), and CCF-STTG1 (c) cells. The average background was subtracted, and values less than the average background as negative data. The median of the effective data for each lectin was globally normalized to the sum of medians of all effective data points for each lectin in a block. Fig. S8. The images of fluorescence-based lectin cytochemistry and lectin microarrays. a, Cy-5 labeled BSA as negative controls showed no positive signal. Scar bar 80 μm. b–c, The glycopatterns of a Cy3-labeled SVG p12 bound to the lectin microarrays (b) and their NFIs (c). SVG p12 cells were either left untreated or exposed to NH4Cl for 72 h, followed by another incubation for 72 h in NH4Cl free culture medium. The NFIs of MPL are summarized as the mean values ± SD. The average background was subtracted, and values less than the average background as negative data. The median of the effective data for each lectin was globally normalized to the sum of medians of all effective data points for each lectin in a block. Fig. S9. The fluorescence-based lectin cytochemistry. a–b, The fluorescence-based lectin cytochemistry (a) and average fluorescence intensity (b) of Galβ1-3GalNAc binder MPL in CH3NH3Cl treated and untreated SVG p12 cells. The images were acquired using the same condition and shown on the same scale in the Cy5- and DAPI-merge channel. Data were presented as mean ± SD. Scar bar 80 μm. Fig. S10. The transfection efficiency in SVG p12 cells. The transfection efficiency of siRNA and transfection reagent was determined using negative control FAM. Scale Bar, 100 μm.


We show thatthe argyrodite structure is equivalent to the cubic antiperovskitesolid electrolyte structure through anion site and vacancy orderingwithin a cubic stacking of two close-packed layers. Construction ofmodels that assemble these layers with the combination of hexagonaland cubic stacking motifs, both well known in the large family ofperovskite structural variants, followed by energy minimization identifiesLi6SiO4Cl2 as a stable candidatecomposition. Synthesis and structure determination demonstrate thatthe material adopts the predicted lithium site-ordered structure witha low lithium conductivity of ∼10–10 S cm–1 at room temperature and the predicted hexagonal argyroditestructure above an order–disorder transition at 469.3(1) K.This transition establishes dynamic Li site disorder analogous tothat of cubic argyrodite solid electrolytes in hexagonal argyroditeLi6SiO4Cl2 and increases Li-ion mobilityobserved via NMR and AC impedance spectroscopy. The compositionalflexibility of both argyrodite and perovskite alongside this newlyestablished structural connection, which enables the use of hexagonaland cubic stacking motifs, identifies a wealth of unexplored chemistrysignificant to the field of solid electrolytes.A hexagonal analogue,Li The argyrodite family is compositionally flexible, withthe general formula An–y)/m,1 where the A cation content can vary from 3.25 to 9 to maintaincharge neutrality. This family of compounds contains examples of fastAg+ and Cu+ ion conductors  as well as materials interestingfor their nonlinear optical properties  and thermoelectricproperties .Compoundsrelated to the mineral argyrodite .7 Inthe lithium-containing argyrodites, the introduction of a halide aniontransforms the room-temperature orthorhombic polymorph : space group Pna21) into cubic symmetry : space group F4̅3m), while charge compensation occurs via reduction of the lithiumcontent. The latter cubic phase exhibits an order–disorderphase transition from static order (LT cubic phase) to an extendeddynamic disorder (HT cubic phase), with delocalization of the lithiumdistribution.8 In cubic oxide analoguessuch as Li6PO5Br, this phase transition doesnot occur, and lithium localization within the F4̅3m structure is maintained over a wide temperature range(173–873 K).9 In the ordered F4̅3m structure of Li6PO5Br, lithium ions fully occupy a single crystallographic24g position. In the dynamically disordered HT F4̅3m structure of the lithium sulfideargyrodites, the additional available lithium sites (Wyckoff position48h) and extended diffusion of Li+ arelinked to a dramatic increase in ionic conductivity by six ordersof magnitude.11 The most highly conducting Li-containingargyrodites are based on mixed sulfide–halide compositions,e.g., Li6PS5Br6 andLi6.35P0.65Si0.35S5Br,in which Li+ is delocalized across 48h and 24g positions.12The reported crystal structures of argyroditesare related to thehigh-temperature F4̅3m argyrodite structure has previously been described in terms of tetrahedralclose-packing1 or as localized pseudo-octahedralcages of A cations surrounding one anion and separated by the otheranions.11 In this work, we establish a new relationshipbetween the structures of argyrodites and those of cubic antiperovskites,which are themselves good conductors of lithium ions .13 We extend this relationship,which provides a framework for designing lithium-ion conductors, fromcubic to hexagonal antiperovskites and thus propose the lithium hexagonalargyrodite family, computationally identifying several new compositionaltargets within this family. Li6SiO4Cl2 is then successfully synthesized following these predictions basedon mixed cubic and hexagonal stacking and shows analogous dynamicalcation site disorder to cubic argyrodites, which is required for Litransport.The high-temperature 22.114 All calculationswere performed with the projector-augmented wave method,15 a plane-wave cutoff energy of 700 eV, and a k-point spacing of 0.15 Å–1. Geometryoptimization of both atomic positions and unit cell parameters wasterminated once all forces fell below 0.001 eV Å–1. The PBE functional16 was used to calculaterelative energies and mechanical properties, and the PBEsol functional17 was used for direct comparison between computationaland experimental crystal structures. The convex hull of energies forall reported phases in the Li–Si–O–Cl–Brand Li–Si–O–Cl–I phase fields was calculatedusing pymatgen.18 Normal mode calculationswere performed using the harmonic approximation, with finite displacementsof 0.01 Å and including distortions of the unit cell. This allowedthe calculation of elastic constants, including the bulk and shearmoduli.19 For comparison with existingcompounds, the elastic constants of Li3OBr (Pm3̅m structure)20 and Li6PS5Br (Cc structureof Li6PS5I)21 werecomputed using the same method.Periodic plane-wave-baseddensity functional theory (DFT) calculations were performed usingthe VASP code (version 5.4.4).2.22CO3 (99.99%),SiO2 , and LiCl (>99.0%) were purchased from Sigma-Aldrich.Li2CO3  andSiO2 (0.5013 g) were weighed according to the stoichiometric2:1 ratio. The powders were ground with an agate pestle and mortarfor 15 min, placed into an alumina crucible and heated in air to 1073K at a ramp rate of 5 K min–1, held at 1073 K for12 h, and cooled at a ramp rate of 5 K min–1. Theresulting product was ground to obtain a fine powder, which was thenused as a precursor in the final synthesis step.Precursors were dried overnight at473 K before weighing. Li4SiO4, LiCl, and LiBr were vacuum-dried overnight (under10–4 mbar) before placing them in an Ar-filled glovebox.All precursors and resulting powders were then handled in an Ar-filledglovebox. LiCl, LiBr, and Li4SiO4 were mixedin the stoichiometric ratio, ground with an agate pestle and mortarfor 15 min, and transferred to an alumina crucible. The crucible wasplaced in a silica tube, which was sealed under vacuum (<10–4 mbar). The tube was heated to 798 K at a ramp rateof 5 K min–1, held at 798 K for 12 h, and cooledat a rate of 5 K min–1. Once cooled, the silicatube was opened inside the Ar glovebox, and the powder was groundin a pestle and mortar for further characterization.Li6SiO4Cl2 were grownby mixing LiCl and Li4SiO4 in the stoichiometricratio, heating the mixture to 883 K at a ramp rate of 5 K min–1, annealing it for 3 h, and slow cooling at 3 K h–1 to room temperature.Singlecrystals of Li2.31, λ = 1.54056 Å) in a Debye Scherrer transmission geometrywith sample powders loaded into 0.5 mm borosilicate glass capillaries.Routineassessment of sample purity was carried out using a Bruker D8 Discoverdiffractometer with monochromatic Cu radiation  detector. The sample was introduced into a 1.0 mm diameterborosilicate glass capillary. The experiment was performed at roomtemperature.Synchrotron X-ray diffraction (SXRD) was performed at Diamond LightSource U.K., on high-resolution beamline I11,Synchrotron variable temperature X-ray diffraction(VT-XRD) wasperformed in transmission mode using a position-sensitive detector on a sample, which was introducedinto a 1.0 mm diameter silica capillary. The experiment was performedin the temperature range of 298–798 K in 25 K steps on heatingand then cooled directly to room temperature to assess reversibility.24 Initially, Pawley fits were performed on SXRDdata, refining the lattice parameters and the background using a Chebyshevfunction with 12 parameters. The peak shape was modeled using a pseudo-Voigtfunction (high-temperature patterns) and a Thomson–Cox function(room-temperature patterns). Refined parameters from final Pawleyfits were then used as starting points for Rietveld refinements wherethe following parameters were refined: (1) scale factor, (2) atomiccoordinates, (3) isotropic  and anisotropic (Cl) displacementparameters, and (4) atomic occupancies: the occupancies of Cl andO, and Li were refined, while the occupancy of the Si site was setto the nominal value and the Li occupancies were set to ensure chargecompensation.Rietveld refinements were carried out using TOPAS Academic.2.425 The volume ratio of the twin components was about 1:1. The degreeof overlap of two components is around 1%. The twin operator is 92.82°rotation around the −0.59 0.37 −0.72 reciprocal axis.The detwinned HKLF4 format file was used for structure solution usingOlex2.26 The crystal structure was solvedwith the intrinsic phasing method provided by the ShelXT27 structure solution program. Refinements werecarried out with the ShelXL28 refinementpackage using least-squares minimization.A twinned crystal was examined with a Rigaku MicroMax-007 HF X-raygenerator equipped with a Mo Kα rotating-anode microfocus sourceand a Saturn 724+ detector. The data were collected at 100 K. Refinementof the cell parameters, indexing of twin components, and reductionof the data were performed on the obtained diffraction images withthe use of the software package CrysAlisPro.2.52, H2O) using a Netzsch DSC 404F1 differential scanning calorimeter. Data were recorded on heatingto 823 K and then cooling to 323 K using heating and cooling ratesof 10 K min–1 under a constant 50 mL min–1 flow of helium. The transition temperature is the average of thevalues obtained from both the heating and cooling curves, which wereextracted through peak fitting.Heat flux profiles were measuredfrom 15.1 mg of powdered samplein a 40 μL aluminum crucible cold-welded under an Ar atmosphere. A sputteredgold coating of 300 nm thickness was used as the ion blocking electrodes.Sputtering was achieved under an argon atmosphere using a Q150R sputtercoater, and temperature-dependent conductivity measurements were performedunder an argon atmosphere over a frequency range of 2 MHz to 20 Hz(with an amplitude of 100 mV). Measurements were performed in thetemperature range of 333–575 K in 20 K steps. The ZView2 program6SiO4Cl2|Au symmetric cellby applying constant potentials of 0.05, 0.1, 0.5, and 1 V for 7200s and monitoring the current variation with time. Once a constantcurrent was achieved, the current was recorded and plotted againstthe applied voltage allowing for the electronic conductivity (σe) to be extracted from the following equationU refersto the polarization voltage, l refers to the pelletthickness, A refers to the Au electrode area, and I refers to the current.A pellet of 84% relative density was usedfor DC potentiation polarizationmeasurements. DC polarization data was collected at 300 °C ona Au|Li2.729Si magic-anglespinning (MAS) NMR spectra were recordedwith a 4 mm HXY MAS probe in double resonance mode on a Bruker 9.4T Avance III HD spectrometer. 29Si NMR data was obtainedusing a pulse length of 5 μs at a radio frequency (rf) amplitudeof 50 kHz and at an MAS rate of νr = 10 kHz. Thesample was packed into a rotor in an Ar-filled glovebox to eliminateexposure to air and moisture. 29Si chemical shifts wereexternally referenced to the lowest-frequency signal of octakis(trimethylsiloxy)silsesquioxaneat −109 ppm,30 relative to tetramethylsilaneprimary reference at 0.0 ppm.7Li NMR experiments were recorded with a 4 mm HX High-TemperatureMAS Probe on a 9.4 T Bruker Avance III HD spectrometer under staticconditions with the X channel tuned to 7Li at ω0/2π (7Li) = 156 MHz. The sample was sealedin a glass ampoule, and the spectra were recorded with a pulse lengthof 1.5 μs at an rf field amplitude of ω1/2π= 83 kHz and referenced to 10 M LiCl in D2O at 0 ppm.Variable temperature 7Li nuclear spins d7Li7Li (in Hz) canbe calculated via the following expression0 is the permeabilityof free space, ℏ is the reduced Planck’s constant, γ7Li is the gyromagnetic ratio of 7Li, and LiLir is the Li–Li distance.The homonuclear dipolar coupling constant between two 3)2 using 207PbNMR31 and by monitoring the phase transitionof CuI and CuBr using 63Cu NMR.33 The errors associated with this method were calculated using theisotropic peak line broadening and range from 5 to 20 K.Temperaturecalibrations were performed using the chemical shiftthermometers Pb analysis was performed on SXRD and VT-XRD datausing the software Jana200633.16PO5Br 0.5Br0.5)(O0.5□0.5)Li3]2, where □ is a vacancy. As such, Li6PO5Br could be considered as an inverse double perovskite.When viewed as a cubic antiperovskite, the lithium ions in Li6PO5Br replace the anion positions in the conventionalcubic perovskite 3– anion is directed toward the vacant B-sites, coupling the rock-saltorder of the two A-site anions to the anion vacancy defect orderingon the B-site. Rock-salt order of 50% B-site vacancies occurs in thestructure of well-known K2PtCl638 close-packed stacking, they are stacked in a −h–c–h–c–(−a–b–a–c−) manner,as would occur in a hexagonal antiperovskite. Different close-packedlayer stacking motifs are known within the antiperovskites.44 The argyrodite/cubic antiperovskite structuralrelationship suggests that Li-containing materials based on the hexagonal,rather than cubic, antiperovskite stacking of these layers, related to the hexagonalstructures of Cu8GeSe6 and Ag5PS4I2, would be analogous to the lithium-containingargyrodites, where the number of Li sites and extent of Li orderingamong them control ionic conductivity.Although closely related in compositionto argyrodite, the compoundCu6PO5X  argyrodites, the phosphate (PO4)3– (P–O bond length: 1.54 Å) was replaced with silicate(SiO4)4– (Si–O bond length: 1.65Å) to increase the A-site cation radius and thus drive a cubicto hexagonal transition through the perovskite tolerance factor (rP(V) = 0.17 Å, rSi(IV) = 0.26 Å).45 The isolated oxideion was replaced by a halide ion  to balance charge.Structures were built with compositions Li6SiO4XX′  with the –h–c–h–c–(–a–b–a–c−) stacking pattern, followingCu8GeS6, in the P63mc space group. This affords a hexagonal antiperovskitein which half of the A-sites are occupied with silicate polyanionsand half with halide anions. Half of the B-sites are then occupiedin an ordered manner by the remaining halide anions, with the vacantB-sites chosen to avoid interactions between the halide anions andthe corners of the silicate tetrahedra ; 3 layers to the cubic argyrodites but they are stacked in a–h–c–h–c– (–a–b–a–c−)pattern and not a −c− (–a–b–c−)stacking pattern  (Table S1). Through phonon calculations, lower-energystructures than the high-symmetry hexagonal structures were found,arising from the rotation of lithium atoms off the mirror plane of P63mc through activation ofdisplacive Γ2, Γ3, Γ5, M2, and M3 modes .Periodic DFT calculationsshowed that the composition LiM3 modes 3b, leadind LiBr) S1. Throu6SiO4Cl2 and Li6SiO4ClBr(with the larger Br ordered on the A-site) predicted to be the moststable and chosen as targets for experimental synthesis.These results suggest that a new lithium hexagonalargyrodite familyof compounds may be experimentally accessible, with Li6SiO4Cl2 in the Pna21 structureresult in a computed bulk modulus, BDFT, of 53 GPa and a computed shear modulus, GDFT, of 31 GPa. This material is considerably softer than oxidesolid-state lithium electrolytes such as Li7La3Zr2O12 19 and has mechanical properties closer to that of the lithiumantiperovskites . Li6SiO4Cl2 is stiffer than related sulfide compounds and consequentlyeasily meets the Monroe–Newman criterion for the preventionof dendrite growth.47 Future electrolytesbased on these mixed oxide/halide compounds may thus be able to overcomesome of the processing challenges and mechanical stability issuesinherent in the use of pure oxide and sulfide ceramics.48DFTcalculations of the elastic constants of Li3.26SiO4Clx2–Brx  to explore the computationally predicted targets. Synthesisof powders was attempted at varying reaction temperatures (50 K stepsfrom 723 to 873 K) and reaction times , and startingmaterials and resulting powders were handled under an argon atmosphereand annealed in alumina crucibles in evacuated silica ampoules  and displays increased lattice parameters as a functionof bromine content . Despitethis clear indication of anion substitution, all compositions with x > 0 contained impurities; only Li6SiO4Cl2 was synthesized as a phase-pure white powder,andas such, all further discussion therefore concerns Li6SiO4Cl2. Single crystals of Li6SiO4Cl2 were synthesized by annealing at 883 K for 3 h beforeslow cooling (3 K h–1) to room temperature. High-resolutionsynchrotron XRD data (SXRD) were collected on the powder from 298to 798 K in 25 K steps. At temperatures 473–498 K, convergenceof some peaks and disappearance of other small peaks indicate a phasetransition to a high-temperature, higher-symmetry phase (denoted HTphase)  Å, b = 6.0756(4) Å, and c = 9.9530(7) Å. Theassignments of Li, Si, O, and Cl were determined based on interatomicdistances and relative displacement parameters. All atomic positionswere refined with fixed fully occupied sites. Lithium atoms exhibitednonpositive definite anisotropic mean square displacements, so ISORrestraints were applied to atoms Li1–6 during the final refinementand also to atoms O2–4 as a result of distorted thermal ellipsoids.The final anisotropic atomic refinement converged to R1 = 0.0530, wR2 = 0.1189 forreflections with I ≥ 2σ (I) and R1 = 0.0750, wR2 = 0.1279 for all reflections. Crystallographic dataand structural refinements for Li6SiO4Cl2 are summarized in Table S2. Theasymmetric unit contains two distinct crystallographic Cl positions,four distinct O positions, one Si position , and six Li positions. The finalrefined atomic positions, and isotropic and anisotropic displacementparameters of each atom are given in Tables S3 and S4, and the selected bond lengths and angles are givenin Tables S5 and S6. Orthorhombic superlatticereflections characteristic of the Pna21 ) symmetry are clearly visible in the diffraction image,ruling out possible C-centered orthorhombic or hexagonal (as a ≈ √3b) supergroups.The crystal structure was solvedin the space group Pna21 symmetry  Å, b = 6.07657(3) Å, and c = 9.960255(5)Å from a Pawley fit. The model obtained from SC diffraction provedto be a good starting point for the Rietveld refinement with valuesof Rwp = 4.61 and χ2 =2.81 after the initial refinement of site occupancies and displacementparameters. Cl and O site occupancies were refined, and Li site occupancieswere fixed to achieve charge neutrality. Refinement of atomic positionsimproved Rwp from 4.61 to 4.27 and χ2 from 2.81 to 2.61 .Small impurity phases of LiCl (1.95%) and Li2SiO3 (2.35%) were observed . Themaximum entropy method (MEM) analysis did not show any additionalelectron density, indicating that all lithium sites are accountedfor in the structural model described above . The final Rietveld fit is shown in Tables S7 and S8. The refinedcomposition is Li5.957(2)Si1.00O3.986(2)Cl1.985(1), and the RT phase will therefore be denotedRT-Li6SiO4Cl2 hereafter for simplicity.The observed systematic absences inthe SXRD data were consistentwith the  to 2.61 S3.Small3.2.2P63mc through activation of Γ5 and M2 modes, stabilizing the Pna21 symmetry.This leads to a change in anionic and cationic coordination environmentscompared to the hexagonal defect antiperovskite parent.As described in 3SiO4 and Li3Cl layers. Comparison of the experimentallydetermined Pna21 structure to the computedstructure shows almost identical atomic positions . The chloride ions that occupy 50% of the antiperovskiteB-site positions (Cl1) are found in an octahedral environment withbond lengths ranging from 2.415(3) to 2.621(3) Å to neighboringLi. Compared to the idealized octahedral environment expected forB-site anions in antiperovskites, the octahedra are distorted slightlyas a result of the nonrigid rotation of Li ions and the displacementof chlorine toward neighboring vacant B-sites. The chloride ions thatoccupy 50% of the A-site (Cl2), typically forming cuboctahedral (12-fold)coordination environments in hexagonal perovskites such as 4H-BaMnO3, occupy a distorted octahedral environment in the orthorhombicpolymorph with bond lengths from 2.446(3) to 2.798(7) Å . All lithium atoms are found in tetrahedralLiCl2O2 coordination environments. Due to thedifference in ionic radii ,45 lithium atoms are displaced toward the oxygenatoms (6PO5Cl (1.93 Å)9 and shorter thanLi–O bonds in Li4SiO4 (1.84–2.51Å).49 Li–Cl bond lengths rangefrom 2.415(3) to 2.798(3) Å. These values are larger than expectedfor typical LiCl4 bond lengths, e.g., 2.38 Å in Li2MgCl4.50 The differencein bond lengths compared to homoleptic LiCl4 and LiO4 tetrahedral environments is expected in heteroleptic LiCl2O2 and optimizes the bond valence sum for Li+ (BVS: 0.939(8)–1.12(1)). The Li–Cl distancesin Li6SiO4Cl2 are shorter than inthe cubic oxide argyrodite Li6PO5Cl (2.910(4)Å), in which Li atoms have a trigonal bipyramidal coordination(LiO3Cl2) with Cl occupying the axial positions.9Theexperimentally determined RT structure is shown in ositions S7a. The 2O2 tetrahedra areconnected through the corner-sharing of Cl and O vertices and thesharing of Cl–Cl edges; they are referred to using the centrallithium atom, i.e., Li(1)O2Cl2 as a Li1 tetrahedron.Edge-sharing between Li1–Li3 and Li4–Li6 tetrahedrais facilitated by the lithium-ion displacements away from the sharedCl–Cl edges -networkof corner- and edge-shared tetrahedra.The Liescribed 6a as alt-sharing 6b,c. Cl2irection 6b. The L-sharing 6c,d. The3.2.3a = 6.11Å and c = 10.02 Å. Systematic absencesin the powder pattern were consistent with the P31c and P63mc space groups, agreeing with the computationally stable hexagonalstructures (a = 6.110805(12) Åand c = 10.02068(3) Å for P63mc . The higher-symmetry computed P63mc structure gave a reasonable starting point for the Rietveldrefinement. The unit cell contains two distinct crystallographic Clpositions (Wyckoff position 2b), two O positions(Wyckoff positions 6c and 2a), oneSi position (Wyckoff position 2a), and three Li positions(Wyckoff positions 6c and 12d).Occupancies of Cl and O sites were refined, and the occupancies ofLi sites were fixed to ensure charge neutrality. In the computationalmodel, Li2 is located on the mirror plane in special position 6c . Inspection ofthe Fourier density map (FDM) reveals extra electron density aroundthis position and indicates displacement of Li2 away from the mirrorplane onto the general position 12d . Replacing the 6c site with the 12d position improved Rwp from 3.61 to 2.98%. The FDM also revealed electrondensity around the Li1 position along the c-direction. Splitting the Li1 site intotwo distinct Li1a and Li1b sites along c further improved Rwp to 2.75%. The computed P31c model . The final refinement is shownin Table S7. The refined composition is Li5.82(4)Si1.00O3.934(6)Cl1.956(4), and the HT phasewill therefore be denoted HT-Li6SiO4Cl2 hereafter for simplicity.Indexing of the HT phase (above523 K) with TOPAS resulted in ahexagonal unit cell with lattice parameters ructures 3c. Veryor P63mc S4b. The or P63mc S4b. The 3.2.4P63mc structurecan be described as a 4H-hexagonal antiperovskite of general formulaABX3, with structural similarities to 4H-BaMnO3  with lithium atoms displacedtoward the oxygens due to its smaller ionic radius compared to chlorine.Li–Cl bond lengths vary from 2.366(3) to 2.908(8) Å, andLi–O bond lengths range from 1.847(4) to 1.942(4) Å (The HT H-BaMnO3 2b in whi942(4) Å 5d.6SiO4Cl2 are disordered in the higher-symmetry HT structure. Similar to theresults from normal mode calculations described in 2 andΓ5 modes, with a small contribution from displaciveΓ5 modes that predominantly involve displacementof the Li1 atoms within the close-packed Li3SiO4 layer . This disorder ofthe A cation positions is well known in An*–y)/m, in whichLi partially occupies both tetrahedral and trigonal planar coordinationenvironments  and isone of the reasons for reduced energy barriers for bulk Li-ion transportin these materials.8As shownin 6SiO4Cl2  and a disordered structure at 298 K in which lithium isdelocalized across the 24g and 48h positions  temperature range . A typical set ofdata measured at 533 K in an inert atmosphere are shown in Z*, consist of a high-frequencyarc with the presence of a small low-frequency inclined spike. Thelarge single arc is attributed to the sample bulk, as shown by overlappingpeaks in the combined Z″/M″ spectra . The associated capacitance of the high-frequencyarc, 0.6 pF cm–1, corresponding to a permittivityof ∼6.8, is also consistent with the grain response . The low-frequency spike is attributedto the double-layer capacitance at the blocking sample–electrodeinterface, where Li ions cannot pass. To a first approximation, thehigh-frequency arc could be modeled with an equivalent circuit consistingof a resistor in parallel with a constant phase element (CPE). Impedancedata were fitted to the equivalent circuit using ZView software . From the low-frequency intercept of the impedance arc on the Z′ axis, the values of the total resistance wereobtained and are shown in an Arrhenius format in The ionic conductivityof Li spectra S12a. The spectra S12a. The7Li solid-state NMR. The temperaturedependence of the static 7Li NMR spectra of Li6SiO4Cl2 over the temperature range of 331–593K is shown in 7Li–7Li homonuclear dipolar coupling. ForLi6SiO4Cl2, this region is between350 and 450 K, where the line width of the central transition is ∼5.5kHz. As the temperature is increased above 450 K, the line width decreasesdrastically over a small temperature range  Å), the absolutevalue of the dipolar coupling constant can be calculated  Å) for HT-Li6SiO4Cl2. Due to the increase in dipolar coupling after thephase transition, it would be expected that 7Li spectrademonstrate increased line widths at higher T in the absence of anincrease in Li-ion mobility. However, the line narrowing observed in hexagonalLi6SiO4Cl2 , leadingto lower activation energies for ion mobility. Local jumps of Li+ between these tetrahedral sites separated by the trigonalanion windows are the most favorable for ionic diffusion in argyrodites.8 Occupational disorder via chalcogenide–halidemixing on the anion position that forms part of the trigonal windowthat mobile cations traverse 45 increases the size of this window,further lowering the activation energy of ion transport compared toLi6PO5Cl.The distinction betweenthe oxide materials Lin window 5f and thtraverse 5f is an 46SiO4Cl2. Here, the resulting layer stacking gives accessto a disordered Li distribution, which is observed in fast lithium-ionconductors, a distribution not accessible to its nearest known cubiccompositional counterpart, Li6PO5Cl. Explorationof the many accessible mixed layer sequences and associated site andchemical orderings afforded within this family opens new routes tothe tuning and creation of ion transport pathways through variationof both composition and structure, as well as broader functional outcomesbased on the importance of the cubic argyrodite materials.The connection between argyrodite and antiperovskite structuresis established, thus identifying close-packed layers that can be assembledthrough the diversity of combined hexagonal and cubic stacking operationsknown to give the perovskite material family its breadth of scientificand technological importance. We exemplify these mixed stackings inthe hexagonal argyrodite material family through the prediction andthen isolation of the nontoxic and earth-abundant Li