Energy homeostasis is maintained through multiple pathways; therefore, CRTC2 may interact with other transcription factors, particularly under metabolic stress. CRTC2 liver-specific KO mice were created, and the global proteome, phosphoproteome, and acetylome from liver tissue under high-fat diet conditions were analyzed using liquid chromatography-tandem mass spectrometry and bioinformatics analysis. Differentially regulated proteins (DRPs) were enriched in metabolic pathways, which were subsequently corroborated through animal experiments. The consensus DRPs from these datasets were used as seed proteins to generate a protein-protein interaction network using STRING, and GeneMANIA identified fatty acid synthase as a mutually relevant protein. In an additional local-protein-protein interaction analysis of CRTC2 and fatty acid synthase with DRPs, sterol regulatory element binding transcription factor 1 (SREBF1) was the common mediator. CRTC2-CREB and SREBF1 are transcription factors, and DNA-binding motif analysis showed that multiple CRTC2-CREB–regulated genes possess SREBF1-binding motifs. This indicates the possible induction by the CRTC2–SREBF1 complex, which is validated through luciferase assay. Therefore, the CRTC2–SREBF1 complex potentially modulates the transcription of multiple proteins that fine-tune cellular metabolism under metabolic stress.  •LKO revealed changes in metabolic-related pathways.Global proteome analysis of CRTC2•Animal model also replicated altered glucose, fat deposits, and TG levels.•Computational analysis showed that the altered metabolism is influenced by SREBF1.•Knockout and luciferase validated the cooperation of CRTC2 and SREBF1 in metabolism. CRTC2 (with CREB1) in glucose metabolism is well studied; however, further studies are required to delineate the metabolic regulatory aspects. Here, using various biochemical and computational tools, it has been identified that CRTC2 along with SREBF1 regulates targeted genes to fine-tune the metabolic response under various conditions. This should further highlight the tight regulation of glucose metabolism and could be therapeutically used for various diseases. Metabolic disorders, such as insulin resistance and type 2 diabetes, are on the rise and pose a risk of life-threatening challenges. Among the various factors, genetic predisposition, overnutrition, and sedentary lifestyle worsen the current disease landscape . In addiCRTC2 functions mainly as a transcriptional coactivator of CREB and controls hepatic glucose metabolism . RecentlCrtc2 KO (Crtc2LKO) mice were generated and fed a high-fat diet (HFD), and global proteome, phosphoproteome, and acetylome changes in the liver were studied using LC-MS/MS analysis and protein-protein interaction (PPI) network. The diverse effects of Crtc2LKO should involve other factors that in different stoichiometric combinations drive the expression of metabolic-related proteins as the derivative of this process is directly linked to a plethora of cellular functions.The functional diversity of CRTC2 in energy homeostasis prompted the use of global proteomic data to underline the combination of key factors involved in energy metabolism. In this study, liver-specific LKO and Crtc2f/f tissues were used, and each pair of replicates (Crtc2LKO and Crtc2f/f) were labeled with tandem mass tag (TMT) and pooled. From the pooled samples, 5% of labeled samples were used for global proteome analysis, and 95% were used for enrichment of phosphopeptides and acetylated peptides. Samples for global proteome analysis were divided into 12 fractions by high pH fractionation, while phosphopeptides and acetylated enriched peptide samples were divided into 12 and 6 fractions, respectively, using mid pH fractionation. All the fractions were subsequently injected into the LC-MS/MS system, and the raw mass spectra were processed by Proteome Discoverer v2.1 (Proteome Discoverer Software | Thermo Fisher Scientific - KR). The statistical significance of differential peptide expression has been measured using Student’s t test.Three biological replicates of Crtc2Crtc2 liver-specific KO (Crtc2LKO) mice were described previously  was used.eviously . All anig at 20 °C for 90 min to remove SDS. This procedure was performed thrice to completely remove SDS; subsequently, 100 μl of iodoacetamide buffer (0.05 M iodoacetamide in 8 M urea) was added to the filter and incubated for 30 min at room temperature in the dark, followed by centrifugation at 14,000g at 20 °C for 30 min. The buffer on the membrane filters was diluted with 200 μl of 8 M urea and concentrated thrice. Finally, 100 μl of 100 mM triethylammonium bicarbonate (pH 8.0) was added to the filter and centrifuged thrice at 14,000g at 20 °C for 30 min. Trypsin  was added at an enzyme to protein ratio of 1:50 (w/w), and the proteins were incubated at 37 °C for 12 h. The digested peptides were eluted through centrifugation at 14,000g and 20 °C for 30 min. To collect the residual peptides on the filter, the filter was washed thrice with 70 μl of 100 mM triethylammonium bicarbonate and centrifuged at 14,000g and 20 °C for 10 min. The eluents were dried and stored at −80 °C.Proteins (500 μg) from each tissue type were digested using the filter-aided sample preparation method. Proteins were reduced using SDT buffer  at 37 °C for 45 min and boiled for 15 min. The protein samples were sonicated for 15 min in a bath sonicator. The supernatant was transferred to a membrane filter  and centrifuged at 14,000f/f and 129 for Crtc2LKO. All TMT-labeled peptides were pooled and concentrated using vacuum centrifugation. The labeled sample was concentrated to 200 μl and subjected to high-pH reverse-phase liquid chromatography fractionation. The TMT-labeled peptides were fractionated and concatenated into 12 fractions. Peptide samples were loaded on an analytical column . The concatenated 12 fractions were vacuum-dried and stored at −80 °C until LC-MS/MS analysis.The digested peptides were labeled with TMT isobaric tags  according to the manufacturer’s protocol. The two peptide samples from filter-aided sample preparation digestion were labeled with 126 and 129 for TMT reagents, where 126 for Crtc2+, the beads were treated with 1.2 ml of 100 mM EDTA (pH 8.0) for 30 min using an end-over-end rotator . After removing EDTA, the beads were rinsed thrice with 1.2 ml of water and treated with 1.2 ml of ice-cold 10 mM FeCl3 solution for 30 min with constant rotation. The resulting Fe3+-NTA beads were rinsed thrice with 1.2 ml of water, resuspended in 1.2 ml of 1:1:1 ACN/MeOH/0.01% acetic acid, and divided into 12 microcentrifuge tubes containing 100 μl bead solution each. Fe3+-NTA beads were washed with 400 μl binding buffer (80% ACN/0.1% TFA). TMT-labeled peptide samples of the 12 fractions were resuspended in 500 μl of binding buffer and transferred to a tube containing aliquoted beads. The binding reaction was allowed to proceed for 30 min with rotation; the reacted beads were rinsed with 500 μl binding buffer thrice; the phosphopeptides were eluted from the beads through incubation in 125 μl of 1:1 ACN/2.5% ammonia in 2 mM phosphate buffer (pH 10) for 1.5 min. The eluted phosphopeptides were acidified immediately with 10% TFA to pH 2.5 − 3.0 before vacuum drying.Immobilized metal affinity chromatography beads were prepared from Ni-NTA magnetic beads ; 1.5 ml of Ni-NTA bead slurry was washed thrice with 1.2 ml of HPLC-grade water. To remove Ni2g for 10 min at 4 °C; the supernatant was removed, and this step was repeated thrice. The peptides (700 μl) were diluted to 1.3 ml with MOPS buffer, added to the antibody, and incubated for 12 h in a rotator. After centrifugation at 2000g for 10 min at 4 °C, the supernatant was collected. The peptides were washed thrice with 1 ml of MOPS buffer. Next, 100 μl of 0.15% TFA was added to elute the acetylated peptides. After incubation for 5 min, peptides were centrifuged at 2, 500g for 10 min at 4 °C. Eluted peptides were dried and resuspended in 0.1% formic acid for LC-MS/MS analysis.For the analysis of acetylated peptides, flow-through peptides from the phospho-enrichment experiment were loaded into acetyl lysine antibody–enriched agarose beads . The antibody with captured peptides (130 μl) was diluted to 1.2 ml and centrifuged at 2000To avoid any acetylation to carbamylation site modification, the experiments have been performed with caution such as (1) freshly prepared urea was used, and urea reagent was added to the mixture just before the experiment to avoid longer exposure and carbamylation, (2) urea was used only in alkylation step and immediately removed before the tryptic digestion, (3) heating the samples were avoided to prevent carbamylation, and finally, triethylammonium bicarbonate buffer (pH 7.6) was used before adding urea, which is a known buffer to prevent carbamylation reaction as reported previously .The prepared samples  were resuspended in 0.1% formic acid in water and analyzed using a Q Exactive Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific) and an Easy-nLC1000 system (Thermo Fisher Scientific). The linear gradient was set up as follows: 5 to 40% of solvent B in 100 min, 40 to 80% of solvent B in 5 min, 80% of solvent B for 20 min, 80 to 5% of solvent B in 5 min, and equilibrating the column with 5% solvent B for 20 min. A trap column (2 cm × 75 μm packed with 2 μm C18 resin) and an analytical column (50 cm × 75 μm packed with 2 μm C18 resin) were used to separate the hydrophobic peptides. The data-dependent acquisition method was adopted, and the top 10 precursor peaks were selected and isolated for fragmentation. Ions were scanned at resolution of 70,000 in MS1 and 17,500 in MS2. The mass scan range was 400 to 2000 m/z at both MS1 and MS2 levels. The precursor ions were fragmented with an NCE of 30, and dynamic exclusion was set to 30 s.LKO and Crtc2f/f group. For highly confident quantifications of proteins, the protein ratios were calculated from two or more unique quantitative peptides in each replicate. The detailed list of identified peptides and criteria for inclusion from global proteome, phosphoproteome, and acetylome have been provided in The raw data were preprocessed with post-experiment monoisotopic mass refinement to increase peptide identification sensitivity and accuracy by selecting a unique mass class. The refined data were analyzed using Proteome Discoverer version 2.1 (Thermo Fisher Scientific), where the mass spectral files were queried using the Sequest HT search engine against the UniProt mouse reference dataset  with two missed cleavages that allowed using trypsin. While searching the database, carbamidomethylation, alkylation of S-bonds in cysteine (+57.021 Da), TMT 6-plex modification of lysine, and N-terminal of the peptide (+229.163 Da) were added as static modifications; oxidation of methionine (+15.995 Da) and phosphorylation of serine, threonine, and tyrosine (+79.966 Da) were added as variable modifications. The mass tolerance of precursor ions was chosen as 10 ppm, and the mass tolerance of MS/MS was set as 0.02 Da. The false discovery rate at the peptide level was kept at 1% for removing false positive peptide identification. To quantify each sample’s reporter ion, a “reporter ion quantifier” with a TMT 6-plex was used. Abundances of the TMT reporter ions were used at default parameters to quantify Crtc22 fold change (log2FC) as follows:The average value of triplicate protein intensity was used to calculate the log2 (protein intensity in Crtc2LKO/protein intensity in WT)log2FC = logp-value of < 0.05 were considered as differentially abundant proteins (DAPs), differentially phosphorylated peptides (DPPs), and differentially acetylated peptides (DAcPs), respectively, and were used for gene ontology (GO) analysis and PPI generation. For GO enrichment analysis, either the DAPs from the independent dataset or all the differentially regulated proteins (DRPs) (a merge of DAPs from the independent dataset) were merged and used as input in EnrichR  were inputted, and the interactions were retrieved based on a confidence level of 0.4 with no additional interactors. STRING can create a PPI based on a variety of interaction types, such as physical interactions, localization, occurrences, and text mining, which are above the threshold.For the PPI, the Search Tool for the Retrieval of Interacting Genes/Proteins database (STRING v11.0)  was usedThe GeneMANIA tool , 20 can p-value was corrected using the Benjamini–Hochberg method. There were cases in which none of the pathways crossed the default p-value threshold (0.05); therefore, a lower value  was used to analyze the top hit pathways.For functional class scoring, ClusterProfiler (v3.14.3) in R (v3.6.2) was used . A list Signaling pathway impact analysis (SPIA) was performed to analyze pathway activation/inhibition based on upregulated or downregulated proteins . Only prIdentification of motifs was performed in Rv3.6.2, using BioStrings (v2.58.0). The position frequency matrix for the CRE and E-box was retrieved from the mouse database, while the SRE1 human database was used, and the JASPAR2018 model  was usedLKO and Crtc2f/f tissues were obtained from Korea University. The weight of each tissue sample was measured; the tissues were pulverized with 500 μl of amino acid extraction buffer  using a centrifuge tube sample pestle. Standard branched-chain amino acid (BCAA) with heavy isotope labeling was spiked into each sample and incubated at −20 °C for 1 h. After incubation, samples were centrifuged at 16,000g for 10 min at 4 °C. The supernatants were collected, dried, and subjected to LC-MS/MS analysis using an Agilent Triple Quadrupole 6490. An XBridge BEH amide column was used for chromatographic separation with Sol A and Sol B . A total of 30 min gradient with a flow rate of 0.25 ml/min was applied as follows: 85% of Sol B for 2 min, 15% to 20% of Sol A in 3 min, 20% to 25% of Sol A in 7 min, 25% to 30% of Sol A in 8 min, 30% to 50% of Sol A in 10 min, 50% to 75% of Sol A in 13 min, 75% to 100% of Sol A in 18 min, and 100% to 15% of Sol A in 24 min. The BCAAs transition was applied as follows: leucine light, 132.1/86; leucine heavy, 138.1/91.1; isoleucine, 132.1/86.1; isoleucine, 138.1/91; valine light, 118.1/76; and valine heavy, 123.1/72.Crtc2For the liver disease mouse model maintained according to protocols approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC-2018-0031), male mice were fed a 60% HFD  from 9 weeks of age for 8 weeks before being sacrificed under 4-h fasting conditions (from 9 AM to 1 PM) to eliminate any potential variations that are associated with differential feeding. The mice were euthanized by cervical dislocation to avoid any chemical contamination. For the glucose tolerance test, 16 h-fasted mice were intraperitoneally injected with a bolus of glucose (2 g/kg body weight). Blood glucose was measured in the tail vein blood using an automatic glucose monitor . The livers were isolated from the mice and fixed using 10% formalin. Histological changes were examined by H&E staining. For Western blot analysis, antibodies against CRTC2  and HSP90  were used.Liver tissues were homogenized in 5% NP 40 solution. The concentration of triglyceride (TG) was measured using triglyceride quantification kit (Sigma) according to the manufacturer’s protocol. TG contents were normalized by the protein concentration .293T cells were seeded on 24 well-plate, and then each expression plasmid  100 ng, pcDNA3-Srebp1c (1–436) 10 ng, pcDNA3-Crtc2 50 ng) was transfected into cells using TransIT-LT1 transfection reagent (Mirus) according to the manufacturer’s protocol. After 48 h of transfection, cells were treated with Forskolin 10 μM to activate CRTC2 for an additional 4 h before being harvested. The luciferase activity was measured by Luciferase Assay System (Promega) and normalized to the amount of β-galactosidase activity.AML12 hepatocyte cells were plated in 6-well plates. After 4 h of seeding, siRNA/liposome mixes containing 25 nM siRNA targeting mouse SREBP1 (Dharmacon) and 5 μl of Lipofectamine RNAiMAX (Invitrogen) were transfected for 72 h. Cells were harvested and mRNA and protein levels were analyzed.f/f mice or Crtc2LKO mice as described previously and were cultured for 72 h before being harvested for the measurement of mRNA and protein levels.Primary hepatocytes were prepared from either Crtc2t test was used to determine the significance between the differences in Crtc2LKO and WT; a p-value < 0.05 was considered significant.Student’s t test p-value <0.05) and subsequently evaluated for differential abundance (log2FC >±0.26). The abundance levels remained unaffected by Crtc2LKO for most of the identified proteins; however, 56 proteins were upregulated, and 59 proteins were downregulated. For phosphoproteome analysis, we identified 11,449 phosphopeptides from triplicate experiments  that the number of DRPs was less than that of the total proteins, and a higher number of proteins were downregulated across these datasets. The PPI of DRPs was created using STRINGv11.0 with a 0.4 confidence score and without additional interactions . Most proteins have mutual interactions, indicating their similar roles in cells. EnrichR was used for enrichment analysis; proteins were enriched in metabolic pathways  are shown in Fig. 2pathways  16). Th. ThA) thpathways . The degpathways . The molpathways  and S5. To confirm the role of CRTC2 in metabolism, functional class scoring and SPIA were performed. The functional class scoring was calculated using the gene set enrichment analysis tool in R for all three proteomic datasets . In the LKO mice; however, SPIA revealed that, based on the protein abundance trends, these pathways are inhibited , kynurenine/α-aminoadipate aminotransferase (protein metabolism), NADPH-dependent 3-keto-steroid reductase Hsd3b5 , 1,4-α-glucan-branching enzyme , solute carrier organic anion transporter 1A1 , and ACLY (fatty acid metabolism), were identified in more than one dataset and were termed seed proteins. Among these, SLCO1A1 and ACLY were identified in proteome and phosphoproteome, while the CYP1A2, kynurenine/α-aminoadipate aminotransferase, HSD3B5, GBE1 and ACLY were identified in proteome and acetylome, with ACLY is the only protein that was identified in all the datasets. Almost all these proteins are involved in cellular metabolism in various forms except SLCO1A1, which highlights the pivotal role of CRTC2 in metabolism regulation. Moreover, this set of proteins offers an opportunity to narrow down the proteome to a limited number of targets to explore the regulatory mechanism in detail.A); however, GeneMANIA integrated all proteins into a single network (B). The newly identified proteins were queried against our datasets; only fatty acid synthase (FASN) was identified in both networks, as well as in our dataset  and GeneMANIA  and was called local-PPI (LPPI). In STRING, the LPPI consists of three subnetworks, and no interaction was identified for SLCO1A1 A; howeve network B. The ne dataset . In addiA and B, Fig. 3A); however, in GeneMANIA, there were only two direct interaction partners (SREBF1 and ACLY) . SREBF1 was the common interacting partner in both networks  −5.126). Both CRTC2-CREB (CRE) and SREBF1 (sterol regulatory element (SRE-1) and E-BOX1) are DNA-binding proteins; therefore, binding motifs were identified in the promoter regions (±5kb from the transcription start site) of genes from LPPI-CRTC2 . There were multiple genes with SRE-1–binding motif in their promoters; this included Fasn, Acly, Gbe1, Cyp2e1, and Crtc2  was created, including FASN and CRTC2, using STRING and GeneMANIA  A and B, . In STRIFig. 3D,  and S7; Fig. 3D, , 32, SREpathways .Fig. 3CRhttps://chip-atlas.org) provides the relationship between transcription factors and their target genes using ChIP-seq data (M.musculus [mm10] with region spanning ±10kb from the transcription start site). To remove the low confidence target genes, the data were filtered using average Model-based Analysis of ChIP-seq (MACS2) score  < 50 resulting in 11,854 and 2177 target genes for CREB1 and SREBF1, respectively. CREB1 and SREBF1 shared 1820 induced genes (15.35% with respect to CREB1 and 83.6% with respect to SREBF1); of those, 671 were detected in Crtc2LKO dataset and 10 were differentially expressed with no significantly enriched pathway . This further point out the plausibility that CRTC2 and SREBF1 can coregulate a subset of these genes . CREB has a long helical structure, whereas SREBF1 is divided into two helices connected by a loop, with the second part at an angle from the first. Twelve CREB residues interacted with CRTC2; among those, four (33.3%) were identical in SREBF1  (B). Following the alignment, the similarity of the interacting residues was calculated using Sneath for comparison (C), where the most dissimilar residue pair was Asp-Trp with a value of 45. In addition, the bound DNA in each complex was poorly aligned and tilted at an angle of ∼35 − 40°. The transcription complex was large and contained multiple proteins; therefore, adjustment of relative and spatial orientation was expected. Based on structural and functional similarities, there is strong evidence of PPIs between CRTC2 and SREBF1 in transcriptional complex formation, where CRTC2 could modulate the transcriptional landscape of SREBF1 for a subset of genes in a certain metabolic environment. For instance, the altered gene expression of 33 genes (2.12%) is mostly from the proteomics dataset (16/33), indicating that loss of expression rather than alteration of posttranslational modification reinforces the transcriptional landscape modulation.CRTC2 may interact with SREBF1 similarly to CREB1. To analyze this, SREBF1 (PDB ID:1AM9 ) was supM9 (5ZKO ). The C-M9 (5ZKO A. CREB hom CREB) B. Followmparison   or siRNA-mediated knockdown of Srebf1 significantly reduced the expression of lipogenic genes such as Fasn, acetyl-CoA carboxylase (Acc), and Stearoyl CoA desaturase 1 (Scd1) . Moreover, when a reported construct containing a natural SRE-1 motif . Here, the coregulation has been studied for a limited number of genes to prove the hypothesis; however, it should note that many other genes can also follow a similar mechanism that warrants further studies. A schematic representation of these steps has been shown in Figure 4D outlining the motives, methods, and outcome of the study.Finally, the proposed computational model has been validated using knocking out and luciferase assay in hepatocytes, where either Crtc2-1 motif  was usedLKO altered metabolic pathways; therefore, we validated this phenotype alteration and the impact of Crtc2LKO in an animal model. Western blotting analysis indicated that CRTC2 was not expressed in the liver tissue of Crtc2LKO compared with that in the control, confirming the successful knockout of CRTC2 in the liver (A). To verify whether changes at the protein level reflected that at the mRNA level, the mRNA levels of selected target genes of CRTC2 were analyzed. The mRNA levels of Pck1, Fasn, acetyl-CoA carboxylase α (Acaca), and ATP citrate synthase (Acly) were significantly downregulated in Crtc2LKO mice (B). In the absence of CRTC2, these changes were expected and in line with proteomic data, indicating the downregulation of FASN (log2FC in prot/phospho/acetyl = ND/-0.61/NS), ACACA (log2FC = −0.35/−0.67/ND), and ACLY (log2FC = −0.658/−0.69/−0.81) (B). Although PCK1 was not detected in the LC-MS/MS data, it is an expected interactor of CRTC2 as shown in Figure 5A. The absence of PCK1 from the proteomics data might indicate the different cellular conditions when the cells were processed for proteomics data versus the cells were harvested for the RNA analysis. This further highlights the tight regulation of the metabolic environment.The proteome data set indicated that Crtc2he liver A. To verLKO mice B. In the9/−0.81) B. Althoude novo lipogenesis, and their reduction due to Crtc2 ablation causes a sharp drop in liver fat deposits and liver TG levels. For instance, Crtc2LKO mice with HFD had significantly lower body weight and blood glucose levels in both glucose tolerance test  in right panel) and fasting conditions , owing to the disruption of gluconeogenesis, compared to that in the control. Biological analysis reinforced that the liver-specific depletion of Crtc2 could significantly disturb glucose and lipid biosynthesis under HFD conditions, which is in line with earlier reports , liver TG levels , and liver fat deposits . The level of BCAA was downregulated in the Crtc2LKO model under HFD conditions ; the reduction in glucose concentration triggered alternative energetic pathways such as BCAA degradation through kruppel-like factor 15 (KLF15)   38). Th. Thde no (KLF15) .LKO mice to study the impact of CRTC2 on energy homeostasis under metabolic stress (A and B). The differentially abundant proteins were enriched in the metabolic pathways of glucose, proteins, and fatty acids . SREBF1 is a key transcription factor that regulates the expression of genes that modulate cholesterol biosynthesis and lipid homeostasis . Genes with a higher number of SRE-1 motifs included Crtc2, Fasn, Cyp2e1, and Gbe1, which are involved in glucose and lipid metabolism at various levels  (A and B). Similarly, the luciferase activity was significantly increased when Crtc2 was added (C), indicating a cooperative regulation of critical genes. As the glucose and lipid metabolisms are linked at various points, it is rational that these proteins cooperate and tightly regulate the flow and appendages of the metabolic network at multiple levels.The presence of binding motifs of CRTC2-CREB (CRE) and SREBF1 (SRE-1 and E-box motif) was identified in the promoter region of interacting proteins C. Genes Fig. 3D, . The motFig. 3D, , 44, 45.Fig. 3D, ; from a Fig. 3D, , A–C 47C. Genes 3D, A–C) , 49. Thi3D, A–C) , A and Bas added C, indicaCoactivator binding is transcription factor-dependent; the CREB-binding domain (CBD) of CRTC2 does not bind to DNA independently; however, it strongly binds to DNA in the presence of CREB . HoweverLKO, transcription by either factor is hampered, resulting in downregulation. This dual regulation may be an evolutionary measure to ensure the conducive levels of glucose and lipids in cells , which culminated in the disruption of glucose tolerance, body weight, lipid storage, liver weight, and TG levels (C) . Gl. GlB), wvels (C)  Fig. 5DDB, whichs (C) D, . Howevers (C) D, ; whereass (C) D, . Activats (C) D, . TherefoIn summary, liver-specific knockout of Crtc2 in mice revealed that it is vital in glucose, lipid, and protein metabolism and may influence BCAA metabolism indirectly under metabolic stress in the liver. These diverse functions are executed by interacting with more than one transcription factor such as CREB and SREBF1, and the relative interplay between the induction of target genes can fine-tune cellular homeostasis under various metabolic conditions.via the PRIDE (The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner This article contains The authors declare no competing interests.


Appropriate charge compensation was targeted by co-substitution of Gd3+, Ca2+, Al3+, or Cr3+, on the Th or Ti site. U L3 edge X-ray Absorption Near Edge Spectroscopy (XANES) and High Energy Resolution Fluorescence Detected U M4 edge XANES evidenced U5+ as the major oxidation state in all compounds, with a minor fraction of U6+ (2–13%). The balance of X-ray and Raman spectroscopy data support uranate, rather than uranyl, as the dominant U6+ speciation in the reported brannerites. It is considered that the U6+ concentration was limited by unfavourable electrostatic repulsion arising from substitution in the octahedral Th or Ti sites, which share two or three edges, respectively, with neighbouring polyhedra in the brannerite structure.ThTi Although typically metamict due to self-radiation damage, natural brannerite specimens retain significant fractions of their actinide inventory under geochemical conditions over geological timescales (107–108 Ma)6. Indeed, brannerite is known to be amongst the most refractory of uranium minerals, highly resistant to leaching under acidic conditions, but potentially less resistant to dissolution under alkaline conditions8. Consequently, UTi2O6 is of interest as a host phase for the immobilisation of long lived actinides, such as plutonium, in tailored ceramic and glass–ceramic composite materials, as radioactive wasteforms21.The mineral brannerite, prototypically UTi2O6, the monoclinic brannerite structure, characteristic of UTi2O6, comprises corrugated sheets of edge sharing BO6 octahedra connected by chains of edge sharing AO6 octahedra, in space group C2/m2. This structure is also adopted by the high temperature polymorph of ThTi2O6, synonymous with the mineral species thorutite22. Several U5+ dominant brannerites have been previously reported, typically stabilised by substitution of trivalent lanthanides on the U site (i.e. (U5+0.5Ln3+0.5)Ti2O6, Ln3+ including Gd3+, Ce3+, Dy3+, Tb3+, etc.)14. Recently, we reported a novel U5+ dominant brannerite, U1.09(Ti1.29Al0.71)O6, stabilised by an alternative crystal chemical strategy, involving Al3+ substitution on the Ti site17. Whilst the stability of U4+ and U5+ species within the brannerite structure is well-established, the stability of U6+ species is less clear, but evidently important to fully understand the geochemical stability of brannerite minerals, and the design and performance of their synthetic wasteform counterparts.Taking the general formula AB. examined the compounds (Th0.85U0.10Ca0.05)Ti2O6 (targeting U5+ only) and (Th0.90U0.05Ca0.05)Ti2O6 (targeting U6+ only), based on ThTi2O6, with the brannerite structure16. The incorporation of uranyl species, (UO2)2+, was inferred based on the observation of weak bands at 780.9 and 807.8 cm−1, in the respective Raman spectra (attributed as the ν1 symmetric stretch). However, no direct determination of uranium oxidation state, for example by X-ray absorption or photoelectron spectroscopy, was reported. Finnie et al. investigated (Th0.55U0.30Ca0.15)Ti2O6, and established U5+ as the dominant oxidation state (75%), with minor U4+ (15%) and U6+ (10%), using X-ray photoelectron spectroscopy (XPS)18. The U6+ contribution was considered to be a consequence of surface oxidation of the brannerite material, combined with the surface sensitivity of XPS. Interestingly, a higher fraction of U4+ was determined from XPS data acquired from a polished surface of (Th0.55U0.30Ca0.15)Ti2O6, compared to a fracture surface, which was attributed to enrichment of Ca2+ and U5+ and/or U6+ at grain boundaries.Previous investigation by Zhang et al6+ in the brannerite structure, we designed three solid solutions based on ThTi2O6, targeting U5+ or U6+ speciation, with charge compensation by co-substitution on the Th or Ti site:0.85U5+0.10Ca0.05)Ti2O6 and (Th0.90U6+0.05Ca0.05)Ti2O6(Th0.80U5+0.10Gd0.10)Ti2O6 and (Th0.85U6+0.05Gd0.10)Ti2O6(Th0.95U5+0.05)(Ti1.95M0.05)O6 and (Th0.95U6+0.05)(Ti1.90M0.10)O6; with M3+  = Al3+, Cr3+(ThTo further elucidate the incorporation and stability of U0.95U0.05)Ti2O6 and (Th0.90U0.10)Ti2O6Ti2O6 and (Th0.90U6+0.05Ca0.05)Ti2O6 are nominally identical to those previously investigated by Zhang et al., enabling a direct comparison, in principal, with this earlier study16.Recognising that ThTi6+ (2–13%) was indeed stabilised within the synthesised brannerite compositions, by High Energy Resolution Fluorescence Detected (HERFD) X-ray Absorption Near Edge Spectroscopy (XANES) at the U M4 edge. However, both U L3 edge XANES and HERFD U M4 edge XANES evidenced U5+ as the major oxidation state in all compounds. The evidence in support of uranate and uranyl speciation, and the factors limiting the concentration of U6+, are discussed.Herein, we show that a minor fraction of U2O6; PDF #04-007-2825) was the major phase formed in all compositions. Trace quantities of TiO2 (rutile) and ThO2 were also present in many compositions; trace ThO2 was differentiated from UO2 by electron microscopy observation of the microstructure and EDX analysis. No reflections characteristic of UO2, U3O8 or UO3 were observed; the estimated limit of detection of these oxides is around 0.5 wt% as determined by simulation of X-ray diffraction patterns. The diffraction patterns of compositions targeting Al3+ or Cr3+ substitution on the Ti4+site, exhibited reflections characteristic of trace ThO2. In contrast, the diffraction patterns of compositions targeting Ca2+ or Gd3+ substitution on the Th4+site did not generally exhibit reflections characteristic of trace ThO2.X-ray diffraction was used to characterise the phases present in each product, as shown in Fig. w) were estimated using appropriate Shannon radii (assuming as-batched compositions with U present as U5+ only)25. When compared to previously reported unit cell parameters for ThTi2O626, the synthesised brannerites produced in this work had smaller overall unit cell volumes, in accordance with their reduced weighted average cation radii, as summarised in Table e.g. 228.96(1) Å3 for nominal composition (Th0.80U0.10Gd0.10)Ti2O6 with rw = 0.8506 Å; compared to 231.21 Å3 reported for ThTi2O6 with rw = 0.8567 Å). The unit cell parameters b and c, and the angle β, generally decreased with the weighted average cation radii, as shown in Fig. a-parameter was comparably small and hence a clear trend was not apparent. These observations are consistent with previous systematic studies of the response of the brannerite crystal structure to substitution on the U/Th and/or Ti sites21.The unit cell parameters of synthesised brannerite phases were determined by LeBail analysis of XRD data Table ; the wei3 edge XANES data were acquired to assess the bulk U oxidation state. The position of the U L3 edge is dependent on the average U oxidation state, with some contribution from the local coordination environment of the U absorber. Initial examination of the acquired spectra, and U L3 edge positon . The presence of a high concentration of Th in all compositions examined, with the Th L3 edge at 16,300 eV, combined with the low U concentration , resulted in very small edge steps and incomplete normalisation of monochromator glitches in the pre-edge regions.U L3 edge positions of the reference compounds  was used to estimate the average bulk U oxidation state of the brannerite materials reported here. All estimated average U oxidation states were in the range 4.8(2) + to 5.1(2) + , as shown in Table 0.85U0.05Gd0.10)Ti2O6, with sufficient Gd3+ to charge balance 0.05 f.u. U6+ (6Gd), and (Th0.95U0.05)(Ti1.90Cr0.10)O6, with sufficient Cr3+ to charge balance 0.05 f.u. U6+ (6Cr), both of which had estimated U oxidation states of 5.1(2) + .Linear regression of the U L4+, U5+ and U6+ cannot be deconvoluted by conventional U L3 edge XANES, due to the core–hole lifetime broadening of the spectra, HERFD U M4 edge XANES spectra were also acquired to ascertain, conclusively, the contributions of the different U oxidation states to the overall average speciation.As the individual contributions of U4 edge XANES allows for a more definitive determination of average U oxidation state due to greater relative separation between the edge positions of U4+, U5+ and U6+ and reduced core–hole lifetime broadening28. As with the U L3 edge spectra discussed above, the low U and high Th concentrations, in the materials examined, resulted in low intensities of the HERFD U M4 spectra. The spectra of U4+Ti2O6, CrU5+O4 and CaU6+O4 reference compounds were also acquired for comparison and to support further quantitative analyses. Consistent with analysis of U L3 edge spectra, initial examinations of the HERFD U M4 edge XANES supported an average oxidation state close to U5+, evidenced by the principle feature at ca. 3726 eV, as shown in Fig. 4 edge XANES of (Th0.90U0.10)Ti2O6 and (Th0.95U0.05)Ti2O6, also identified a small but distinct contribution at ca. 3725 eV, Fig. 4+. Inspection of the HERFD U M4 edge XANES of some compositions targeting U6+, most obviously (Th0.90U0.05Ca0.05)Ti2O6 and (Th0.95U0.05)(Ti1.90Al0.10)O6, evidenced a small contribution at ca. 3727.5 eV, Fig. 6+28. The HERFD U M4 edge XANES of materials with a uranyl speciation exhibit additional post-edge features, observed at ca. 3730 eV for CaU6+O4, see Fig. 29 These features were not observed in any of the HERFD U M4 spectra of the materials studied here, demonstrating that, although detectable fractions of U6+ were present  + to 5.12(12) + (detailed in Table 0.90U0.10)Ti2O6 and (Th0.95U0.05)Ti2O6 were the least oxidised, with average U oxidation states of 4.93(12) + and 4.91(12) +, and U4+ fractions of 14.9(20)% and 13.3(32)%, respectively. The most oxidised materials were: (Th0.85U0.05Gd0.10)Ti2O6 and (Th0.95U0.05)(Ti1.90Al0.10)O6, with average U oxidation states of 5.12(11) + and 5.10(13) +, respectively. Fitting of the spectra of these compositions also necessitated a small but significant contribution from the U6+ reference compound (CaU6+O4), of 14.6(11)% and 10.2(14)% respectively. These observations are in keeping with the oxidation states estimated from the U L3 edge spectra, as well as previous reports of a fraction of retained U4+ in air-synthesised charge compensated brannerites21.Linear combination fitting (LCF) was utilised to estimate the proportions of Unge 4.911 + to 5.14+, U5+ and U6+ to the final spectra and average U oxidation state. Initial principal component analysis of the brannerite and reference compounds determined that only three spectral-like components were necessary to reproduce all experimental spectra . This suggested that the spectra of the materials studied here can be well described by the three reference compounds.Iterative Target Transformation Factor Analysis (ITFA) was also performed to evaluate the individual contributions of UFollowing the principal component analysis, the ITFA procedure was continued, with each of the three theoretical components accurately describing one of the reference compounds . As such, each component was assigned a given U oxidation state, with the relative fractions of each component utilised to calculate an average U oxidation state for the materials under examination. The relative fractions of the synthetic spectra and the calculated U oxidation states were both in excellent agreement with those calculated from linear combination fitting of the spectra  are in excellent agreement (see Table 0.85U0.05Gd0.10)Ti2O6, contained 13.3(1)% U6+ as determined by ITFA of the HERFD U M4 edge spectrum (detailed in Supplementary Information Table S1). Assuming target stoichiometry, this corresponds to only 0.0133 f.u. of U6+. No features relating to the presence of uranyl speciation were observed in the spectra of any of the materials produced here.The average U oxidation states determined by all methods (linear regression of the U L16. An earlier investigation of (Th0.85U0.10Ca0.05)Ti2O6 and (Th0.90U0.05Ca0.05)Ti2O6  determined the presence of uranyl species, from analysis of Raman spectra. This assessment was made by assignment of the ν1 symmetric stretch, apparent as a weak and broad band in the range 780–820 cm−115, deconvoluted from the comparatively strong and intense band at 765–770 cm−1 attributed to the Ag symmetric stretch of the TiO6 octahedra. The reported ν1 stretch modes are within the wavenumber range previously determined for a diverse suite of uranyl compounds31. The ThTi2O6 brannerite compounds investigated here all exhibited evidence of a weak and broad band, centred at 780 cm−1, apparent as a shoulder on the comparatively intense and sharp band, centred at 765 cm−1, which can confidently be assigned as the Ag symmetric stretch of TiO6 octahedra. However, we also observed the Raman spectrum of ThTi2O6 to present such a shoulder on the Ag symmetric stretch of TiO6 octahedra at 765 cm−1, in agreement with earlier investigation15. It is evident that this band, also observed in the spectrum of ThTi2O6, must have at least some contribution from other Raman active modes of the brannerite structure . We believe caution should be exercised in attributing uranyl speciation in brannerite compounds by assignment of the weak and broad band deconvoluted at 780–820 cm−1 as the ν1 symmetric stretch. This is further evidenced by comparison of the Raman spectra of (Th0.85U0.05Gd0.10)Ti2O6 and (Th0.80U0.10Gd0.10)Ti2O6. These compositions were determined to incorporate 13.3(1)% and 6.8(1)% of U6+ respectively  relevant to this work.As expected from the near single phase nature of the materials produced here, determined by XRD, the Raman spectra of the brannerite compounds, Fig. 6+ in the brannerite structure, in four ThTi2O6 solid solutions, targeting U5+ or U6+ speciation, stabilised by co-substitution and charge compensation on the Th or Ti site. The materials studied here were produced to examine the possibility of stabilising a significant fraction of U6+ within the brannerite structure. HERFD U M4 edge spectra provided direct and unambiguous evidence for a small fraction of U6+ in all materials, however, U5+ was the major U oxidation state present . U5+ was dominant whether sufficient charge balancing species were present to permit oxidation of all U present to U6+ or not, or indeed when no charge balancing species were present (e.g. (Th0.90U0.10)Ti2O6, 15.7(1)% U4+, 77.3(6)% U5+, 6.9(1)% U6+, from ITFA). However, it is interesting to note that the average U oxidation states of materials targeting U6+ were generally characterised by a greater fraction of U6+ speciation, compared to those targeting U5+ speciation; for example, (Th0.85U0.05Gd0.10)Ti2O6 and (Th0.80U0.10Gd0.10)Ti2O6, which were determined to incorporate 13.3(1)% and 6.8(1)% U6+ respectively from ITFA. Compositions designed to target U6+ speciation were determined to have a lower average oxidation sate, closer to U5+, which is likely to be realised by a low concentration of cation and/or oxygen vacancies, consistent with the detection of trace ThO2 and TiO2 impurities, and the known defect chemistry of the brannerite structure33. An earlier XPS study of (Th0.55U0.30Ca0.15)Ti2O6, with sufficient Ca2+ to charge balance U5+, evidenced a higher contribution of U4+ to the U 4f7/2 peak when collected on a polished surface, compared to an unpolished fracture surface10. This was attributed to apparent Ca2+ and U5+ and/or U6+ enrichment at the grain boundaries. Such enrichment of charge compensating elements in the grain boundaries of the materials studied here could also result in the determined bulk average oxidation state being lower than targeted, in addition to any cation and/or anion vacancies.The aim of this study was to further investigate the stability of U3 edge and HERFD U M4 edge XANES are effectively bulk techniques, giving insight into the average U oxidation states and environments throughout a material. HERFD U M4 edge spectra provided direct and unambiguous evidence for a small fraction of U6+ in all materials, however, no features characteristic of uranyl speciation were observed, even in the most oxidised material, (Th0.85U0.05Gd0.10)Ti2O6, with 13.3(1)% U6+ from ITFA. Although Raman spectra evidenced a weak and broad band centred at 780 cm−1, potentially characteristic of a ν1 symmetric stretch of uranyl species16, the evident presence of this band in the spectrum of ThTi2O6 means that caution must be exercised in its diagnostic attribution to the presence of uranyl species in the brannerite structure. In comparison with X-ray spectroscopies, Raman spectroscopy using a 532 nm laser has a relatively low penetration depth in polycrystalline opaque materials such as those examined in this work, and may be more sensitive to uranyl speciation formed by surface oxidation. The data presented here directly and conclusively demonstrate the stabilisation of a small fraction of U6+ in the brannerite structure, but the balance of evidence tends to support uranate, rather than uranyl, as the dominant U6+ bulk speciation. Raman spectroscopy has proven more conclusive in substantiating the presence of uranyl speciation in metamict mineral brannerite, however, this may reflect past aqueous alteration of the specimen consistent with the observation of U-OH bending vibrations34.U L6+ concentration in the ThTi2O6 compounds reported here35. The unit cell volume and weighted average cation radius of these compounds were shown to be well within the actinide brannerite stability field, as shown by Fig. 6+ concentration. The brannerite structure comprises corrugated sheets of BO6 octahedra, in which each BO6 octahedron shares three edges, with the sheets connected by chains of edge sharing AO6 octahedra, in which each AO6 octahedron shares two edges. In ThTi2O6, across the shared octahedral edges, the A…A cation distance is 3.823 Å, and the B…B cation distances are 3.039 and 3.161 Å22. Substitution of U6+ at either site would be expected to give rise to highly unfavourable electrostatic repulsions, as a consequence of the short approach distance to neighbouring cations, which we consider may be limiting on the concentration of U6+ incorporation.A wide range of uranium (VI) oxometallates may be synthesised at high temperature in either air or oxygen atmosphere, so the partial pressure of oxygen is not thought to be the limiting factor in stabilisation of only a modest U2O6 compounds with the brannerite structure were designed to incorporate U5+ or U6+, at dilute concentration of 0.10 or 0.05 formula units, by appropriate charge compensation involving co-substitution of Gd3+, Ca2+, Al3+, or Cr3+. Near-single-phase compounds were produced in each case. X-ray absorption spectroscopies evidenced a majority U5+ speciation in all compounds, regardless of the nature and relative fraction of the charge compensating species. All compositions exhibited a small contribution from a U6+ oxidation state, as evidenced from HERFD U M4 edge XANES, and in many cases also a small contribution of U4+. Compositions targeting only U6+ were, in general, determined to contain a greater contribution of U6+. Although Raman spectra presented a weak and broad band plausibly indicative of uranyl speciation, the observation of this band from ThTi2O6, means that caution must be exercised in its diagnostic attribution to the presence of uranyl species in the brannerite structure. In contrast, no spectroscopic signatures of uranyl speciation were apparent in the HERFD U M4 edge XANES, although the signal to noise ratio of the data presented here mean that a minor contribution of uranyl speciation cannot be ruled out. Therefore, the balance of our evidence tends to support uranate rather than uranyl, as the dominant U6+ speciation in the brannerites reported here.ThTi2O6 brannerite structure can incorporate a small fraction of U6+, alongside a more significant inventory U5+, by charge compensation on the Th or Ti site. Stabilisation of U6+ was not thought to be limited by the weighted average cation radii, which were within the stability field of the actinide brannerites, or conditions of synthesis, which were conducive to formation of U6+ oxometallates. It is considered that the U6+ concentration may be limited by unfavourable electrostatic repulsion arising from substitution in the octahedral Th or Ti sites, which share two or three edges, respectively, with neighbouring polyhedra in the brannerite structure.This investigation has demonstrated that the ThTi2, ThO2 (produced by decomposition of Th(NO3)4·5H2O at 550 °C), TiO2 (anatase), CaTiO3, Gd2O3, Al2O3, and Cr2O3 were homogenised by high energy ball milling  utilising yttria-stabilised zirconia mill pots and milling media, with isopropanol as a carrier fluid. The milled slurries were dried at 85 °C, and the resulting powder cakes broken up by hand in a mortar and pestle. The milled powders were then pressed into 10 mm pellets under 2 t (approx. 250 MPa). Pellets were heat treated in alumina crucibles at 1400 °C for 24 h in air.Materials were prepared by solid state reaction and sintering. Stoichiometric amounts (see Table α radiation). Phase analysis was conducted by matching the reflections observed to phases in the PDF-4 + database36. Unit cell parameters of the brannerite phase in each composition were derived using LeBail method refinements, utilising the Topas37 and JEdit38 software packages. The background of each diffraction pattern was modelled with an eight term shifted Chebyshev polynomial; peak shapes resulting from instrumental and sample-based contributions were modelled using modified Thompson-Cox-Hastings (TCHZ) pseudo-Voigt functions.X-ray diffraction (XRD) patterns of each sample were collected on powdered material  microscope with Bruker Quantax 70 EDX system.Scanning electron microscopy with coupled energy dispersive X-ray spectroscopy (SEM/EDX) was used to confirm incorporation of the dopant cations. Due to the significant overlap between the Th M−1 band of a Si wafer.Raman spectra were collected on polished materials prior to carbon coating. Spectra at various points across the polished surfaces were collected using a Horiba XploRa PLUS Raman microscope with a 532 nm laser at 100 × magnification. The instrument was calibrated using the 520.7 cm3 edge XANES were acquired at Diamond Light Source (DLS) beamline B18. The incident energy was selected using a Si (111) monochromator, with data collected in transmission mode from 16,970 to 17,560 eV. The intensities of the incident and transmitted beams were measured using ionisation chambers, operating in a stable region of their I/V curve. The K edge of an Y foil was used to calibrate the spectra, with the first peak in the first derivative of the Y foil spectrum set to 17,038 eV. The spectra of well-characterised specimens of UTi2O6 , U0.5Yb0.5Ti2O6 , and CaUO4  were collected to act as reference compounds of known U oxidation state. The energy position, E0, of each spectrum was determined by the energy position of the maximum in the first derivative.U L4 edge XANES were collected at ESRF beamline BM2039. The incident energy was selected using the (111) reflection from a double Si crystal monochromator. XANES spectra were measured in HERFD mode using an X-ray emission spectrometer40. The sample, analyser crystal, and photon detector (Si drift detector) were arranged in a vertical Rowland geometry. HERFD U M4 edge spectra were obtained by recording the maximum intensity of the U Mβ emission line (ca. 3337 eV) as a function of the incident energy. The emission energy was selected using the (220) reflection of five spherically bent striped Si crystal analysers (1 m bending radius) aligned at a 75° Bragg angle. The paths of the incident and emitted X-rays were minimised, and the sample was maintained under He atmosphere, in order to avoid losses in intensity due to absorption. Spectra of well-characterised specimens of UTi2O6, CrUO4, and CaUO4 were also acquired to act as reference compounds of known U oxidation state .High Energy Resolution Fluorescence Detected (HERFD) U M3 and HERFD U M4 edge spectra were processed, and linear combination fitting of the U M4 spectra performed, in Athena, part of the Demeter software package42. For Iterative Target Transformation Factor Analysis (ITFA), data were normalised using PyMca43 and were analysed using the ITFA software package45, to further define the oxidation state of U within the samples48. Recent work has suggested that the errors on this analysis have not been fully quantified50, and therefore these values are considered to have a margin of error of 5%. Principal component analysis showed three components were generally necessary to describe the HERFD U M4 edge spectra , and the initial fractions of the three components were set to unity for one of each of the reference compounds. The iterative target test procedure was then utilised to find the relative concentrations of the three components in the spectra of the materials studied here, with each component representing U4+, U5+ or U6+ according to comparisons with the reference compounds used. For validation, the procedure was then repeated twice, once including only U4+Ti2O6, CrU5+O4 and CaU6+O4 reference compounds and once including a wider range of reference compounds . In both cases principal component analysis showed that three components were necessary to describe the spectra.Both U LSupplementary Information.


Three approaches of partial substitution of O with F, reducing particle size and C coating on the particle surface are used simultaneously to improve realisable capacity, rate capability and stability. We rationalise that the improved electrochemical performance is due to the improved short and long range Li+ diffusion kinetics, electrical conductivity and reduced O loss. These strategies can also be applicable to a variety of DRX materials to improve performance.Disordered rocksalt (DRX) materials are an emerging class of cathode materials for Li ion batteries. Their advantages include better sustainability through wider choices of transition metal (TM) elements in the materials and higher theoretical capacities due to the redox reaction contributions from both the TM and O elements compared with state-of-the-art cathode materials. However, the realisable capacities of the DRX materials need to be improved as their charge transport kinetics and cycling stability are still poor. Here, Li Disordered rocksalt material containing abundant transition metal elements only and C coating with improved capacity, rate capability and cycling stability for battery cathodes. Within the LIB cell, the cathode material is one of the main factors that limits the capacity and dominates the battery costs.2 Two prevalent cathode materials, LiNi0.8Co0.15Al0.05O2 (NCA)3 and LiNixMnyCozO2 (NMC),4 adopt a layered crystal structure that has an ordered arrangement of Li and transition metal (TM) atoms where Li+ ions diffuse in the alkali layer with a 2D pathway upon battery (dis)charge.5 Another widely used cathode material LiFePO4 (LFP) has an olivine crystal structure that still exhibits a clear 1D Li+ diffusion pathway.6 For decades, cation disordering, i.e., mixing of the Li and transition metals (TMs) atoms in the lattice sites, has been considered to limit Li+ ion diffusion.1Electrochemical energy storage systems such as Li ion batteries (LIBs) are attractive for electric transportation and storing electrical energy generated by intermittent renewable sources.xTMx2−O2 (0 ≤ x ≤ 2)7 that can offer high specific capacities of >300 mA h g−1 at 10 mA g−1 at 50 °C and energy densities of ∼1000 W h kg−1 in the voltage range of 1.5–4.8 V.8 It was found that Li+ ions are able to diffuse in the DRX crystal structure through a mechanism called ‘o–t–o diffusion’, which means the Li+ located in the octahedral (o) site diffuses to another octahedral (o) site via a tetrahedral (t) site.9 There are five types of Li+ ion diffusion environments in DRXs according to different cations mismatches: 0-TM, 1-TM, 2-TM, 3-TM and 4-TM local environments which correspond to Li4, Li3TM, Li2TM2, LiTM3 and TM4 tetrahedral clusters.9 However, only two types of the o–t–o channel allow efficient Li+ ion diffusion: 0-TM and 1-TM. The energy barrier of 1-TM is higher than that of 0-TM as the octahedral site in 1-TM is occupied by TM, which has a stronger electrostatic repulsion against Li+, reducing the mobility of Li+ ions. Therefore, effective Li+ diffusion in DRXs is mainly through the 0-TM channel with a diffusion barrier that is almost independent of the average tetrahedron size and the charge of face-sharing species.9 Monte Carlo simulations show that Li-rich cathode materials can achieve the interconnected 0-TM channels and form microscopic diffusion pathways.9 This mechanism has provided valuable insights for the design of DRX materials with Li+ percolation.10Pioneering work demonstrated the feasibility of a disordered rocksalt (DRX) crystal structure for cathode materials Li1.25Nb0.25Mn0.5O2,11 Li1.2Ti0.4Fe0.4O2,11 Li1.2Ti0.4Mn0.4O2 12 and Li1.25Nb0.25V0.5O2.13 Almost all of these DRXs contain a type of TMs such as Ni, Mn and V with an unfilled outer shell electron orbital, and another type of d0 elements that are TM elements with a filled outer shell electron orbital such as Ti4+, Nb5+, Ru5+, Mo6+.14–19 The TMs with an unfilled outer shell electron orbital are redox centres that contribute to capacities, whereas the d0 elements do not contribute to the overall capacity, but play a crucial role in stabilising the crystal structures to accommodate distorted lattice sites due to their smaller sizes and higher charges. DRX materials present flexible compositional space, their stoichiometry is not limited to the explored compositions so far. The high capacities of DRXs reply on redox reactions of both TM and O in the cathode materials.7,20,21 On the other hand, O redox leads to irreversible O loss, structural changes, and ultimately poor capacity retention.7,20,22 Previous studies on the DRX materials have performed detailed simulations23,24 and characterisation such as operando X-ray total scattering and X-ray absorption spectroscopy-based techniques to study the changes of structures and oxidation states of TM.25,26 These studies have identified that the challenges of current DRX cathodes include limited realisable capacities due to restricted Li+ diffusion in both short and long ranges, limited electrical conductivity and rapid capacity losses of nearly 40% after just 30 cycles.7,27 As a result, these limitations hinder the commercial viability of the DRX materials.Most studied chemical compositions of DRX materials include Li0 element are selected for the DRX materials as both Mn and Ti are significantly more abundant 28 than Ni and Co  present in the conventional layered cathode materials,29 and Ti has more favourable effects on Li+ percolation when coupled with Mn due to its size for the DRX materials.1 Three scalable experimental approaches of F substitution with O, reducing particle size and C coating on the particle surface are used to improve the electrochemical performance of the DRX materials. Replacing some of O2− with F− allows charge compensation with more freedom to tune the stoichiometry of cations such as minimising the level of redox inactive Ti4+ and increasing the Li+ content to increase capacity.30 Although there have been studies on substituting O with F,31 the amount of substitution is usually relatively low , e.g., Li1.2Mn0.55Ti0.25O1.85F0.15.32 There have also been studies on F substitution for phosphate-based Na ion batteries Na4MnCr(PO4)3.33 Here, we synthesise Li1.2Mn0.4Ti0.4O2 (LMTO), the novelty of this work is to increase both Li and F amounts and synthesise Li1.3Mn0.4Ti0.3O1.7F0.3 (LMTOF). + diffusion kinetics, electrical conductivity, and reduced irreversible O loss.Here, Mn as the redox centre and Ti as the d22.1Six types of cathode materials were prepared , as summarised in 34,35 and the lattice parameters of LMTO and LMTOF were 4.1703 Å and 4.1811 Å, respectively. Table S3e.g. NASICON Na3Cr0.5V1.5(PO4)3 (ref. 4)3.37 The LMTO and LMTOF patterns corresponded well to the space group Fm3̄m, indicating single phase DRX materials were synthesised.19 The XRD patterns after C coating show the diffraction peaks at the same 2θ positions, but the peaks were broadened compared with those before C coating, indicating the DRX crystal structure was maintained and the particle size was reduced. Inductively coupled plasma - atomic emission spectroscopy (ICP-AES) results 3 ref.  and Na4M7,20 Fluorination led to particle growth, one of the reasons of using the two-stage sintering during the LMTOF synthesis was to prevent F from being released during heating.30 Ball-milling with and without C were then used to reduce the particle sizes. The scanning electron microscopy (SEM) images of as-synthesised LMTO and LMTOF in 43 indicating a thin layer of C was coated on the particles. Electron diffraction of the LMTOF@C particles in †2.2+ diffusion in the large-size (up to ∼40 μm) particles as the Li+ diffusion coefficient DLi in Li-rich DRXs (10−16 to 10−17 cm2 s−1) is still lower than DLi in the conventional layered cathode materials (10−11 to 10−8 cm2 s−1) at room temperature.43–45 To investigate the effects of F substitution on the O site, −1 higher than that of LMTO@C at 0.05C, and the difference increased to 33 mA h g−1 at 0.5C. The reversible capacity retention of LMTOF@C (81%) was higher than LMTO@C (65%) as the C rate increased, indicating a higher rate capability. These results can be attributed to (i) particle size reduction improved long range Li+ diffusion kinetics; and (ii) F substitution improved short range Li+ percolation kinetics because the more electronegative F− exhibits a stronger bonding tendency with Li+ than O2– charge current of 30 mA g−1 .32 The capacity of LMTOF@C was also competitive with 210 mA h g−1 at 0.1C achieved by Li2Mn0.5V0.5O2F (LMVOF),47 but again LMVOF was tested at 1/5th of the (dis)charge C rate compared with LMTOF@C. The comparisons show LMTOF@C exhibited faster Li+ percolation kinetics due to the higher amount of Li+ and electronegative F−. Electrochemical impedance spectroscopy (EIS) was used to further investigate the effects of C coating. R1 is the intercept of the Nyquist plot and the Z′ axis, and represents the internal resistance of the cell such as the ohmic resistance from electrolyte and other components.48R2 is the diameter of the semi-circle and relates to the charge transfer resistance of the electrode whereas Q1 is the constant phase element of the porous electrodes, and the linear part of the Nyquist plot is the Warburg diffusion impedance W1.49–51 Using the equivalent circuit model, R1 was calculated to be 2–4 Ω for both samples, LMTOF@C exhibited a significantly smaller R2 of 25.3 Ω compared with 75.4 Ω for LMTOF-B. The reduced charge transfer resistance of LMTOF@C suggests the C coating on the particle surface increased electronic conductivity of the material.Fig. S3a and b at 0.5C . Table 2 at 0.5C . The cap3+/Mn4+ redox reaction. Increasing UCV to 4.5 V triggered a new anodic peak at 4.5 V, indicating partial activation of O redox.17 During the cathodic scan, only one peak was observed, indicating the reductions of Mn and O were coupled. Further increasing UCV to 4.8 V largely increased the intensity of the O oxidation peak, indicating O redox contribution increased.30,32,52 A similar set of CV scans for LMTOF@C is shown in 30,53 Hence, the peak at ≥4.5 V refers to O redox that contributes to the higher capacity of Li-rich DRX materials.54 The O redox peak intensity at ≥4.5 V reduced significantly at the 3rd cycle for LMTO@C due to oxygen loss. Although the same peak intensity also reduced, the current of the peak was still higher for LMTOF@C (4.31 A) than that of LMTO@C (2.59 A).Cyclic voltammetry (CV) was performed to understand the cation and anion redox processes. −1 over 100 cycles for LMTOF@C (capacity retention 74%) whereas the capacity reduction was 84.2 mA h g−1 for LMTO@C (capacity retention 51%). The discharge capacity retention of LMTOF@C was also higher than other DRX oxides with similar Li stoichiometry and without F substitution, e.g., 43% after 50 cycles for Li1.3Fe0.4Nb0.3O2 (ref. 1.2Ti0.4Mn0.4O2 after 50 cycles.30 The results corroborate with the (dis)charge profiles in −1 for LMTO@C which was reduced to 20.2 mA h g−1 for LMTOF@C, showing both higher capacity and reversibility for LMTOF@C than LMTO@C. The results suggest that fluorination suppresses O loss, while the oxidation state of lattice O is 2−, the oxidation state of F is 1−, so F substitution allows TM to have more d electrons by reduction.55 In general, the charge compensation for the Li+ ion extraction preferentially happens through d orbitals of TMs as they are closer to the Fermi level than O.56 As a result, F reduces O loss without inhibiting significantly redox reactions that contribute to capacities.Fig. S531.2Mn0.4Ti0.4O2 (LMTO) and Li1.3Mn0.4Ti0.3O1.7F0.3 (LMTOF) with abundant transition metal (TM) elements of Mn and Ti are synthesised. Three scalable experimental approaches of F substitution with O, reducing particle size and C coating on the particle surface were used together to improve the electrochemical performance of the disordered rocksalt (DRX) crystal structured cathode materials. The discharge capacity was increased from 45 to 215 mA h g−1 at 0.5C for LMTOF@C after using these approaches together. Furthermore, the capacity increase became higher as the (dis)charge rate increased, showing LMTOF@C also exhibited a higher rate capability. The capacity retention after 100 cycles at 0.5C was increased from 51% for LMTO@C to 74% for LMTOF@C. We rationalise the improved performance was due to improved Li+ diffusion kinetics in both short and long range, electrical conductivity and reduced irreversible O loss. In particular, the role of F was to maximise Li+ content around F− to achieve a greater number of 0-TM clusters in the crystal structure for the short range Li+ percolation and to allow TM to undergo more redox reactions and suppress O loss.Li4.4.11.2Mn0.4Ti0.4O2 (LMTO), stoichiometric amounts of Mn2O3 , TiO2 , Li2CO3  were thoroughly mixed by wet ball milling with ethanol for 24 h at 400 rpm. To synthesise Li1.3Mn0.4Ti0.3O1.7F0.3 (LMTOF), stoichiometric amounts of Mn2O3 , TiO2 , Li2CO3  and LiF  were thoroughly mixed by wet ball milling with ethanol for 24 h at 400 rpm. The ball-milled mixtures were dried overnight in air and pressed into pellets at 5 tonnes for 5 min. The LMTO pellet was calcinated at 900 °C for 10 h in an Ar atmosphere tube furnace followed by natural cooling. The LMTOF pellets were heated at 600 °C for 3 h as a pre-calcination step followed by sintering at 1000 °C under for 6 h in an Ar atmosphere. The pre-calcination step for synthesising the fluorinated material LMTOF was to prevent the loss of reactive LiF starting material during sintering.57 All the samples were sintered at a ramp rate of 5 °C min−1. The annealed pellets were finally grounded into powder and stored in an Ar-filled glovebox until use. To prepare LMTO@C and LMTOF@C, the as-synthesised materials were mixed with 10 wt% conductive agent C65 using dry ball milling at 400 rpm for 12 h. For comparison, the as-synthesised LMTO and LMTOF powders were also ball milled at the same condition without C to make LMTO-B and LMTOF-B.To synthesise Li4.2θ range of 10–90°, step size of 0.02°, and a scan rate of 0.5° min−1. Rietveld refinement of XRD was performed using GSAS II software. Scanning electron microscopy (SEM) images were obtained on Zeiss Gemini Sigma 300 with an energy-dispersive X-ray spectroscopy (EDS) detector and JEOL JSM-6010PLUS/LA. EDS was also obtained from JEOL STEM 2100Plus. Transmission electron microscopy (TEM) was carried out on a JEOL-2100F microscope operating. X-ray photoelectron spectroscopy (XPS) was conducted on Thermo Scientific K-Alpha+ X-ray photoelectron spectrometer equipped with a MXR3 Al Kα monochromatic X-ray source (hν = 1486.6 eV). All spectra were first calibrated using the C1s peak at 284.6 eV as a reference, the XPS peak fitting and analysis were performed using the standard Avantage software. Average compositions of samples were measured by inductively coupled plasma - atomic emission spectroscopy (ICP-AES) using a Thermo Scientific iCAP 6000 series instruments. Li, Mn and Ti calibration standard solutions (TraceCERT@) were diluted by water into 0, 5, 10, 15, and 20 ppm. Linear calibration curves were achieved for all elements. LMTO and LMTOF were dissolved in the high purity concentrated nitric acid (HNO3) (69%) first and then the solution were diluted with water for the ICP-AES characterisation.Phase purity of the synthesised materials was checked by X-ray diffraction (XRD) (Bruker AXS D2 Phaser) operating with Cu K-alpha in the range of 2N-methyl-2-pyrrolidone solvent  and the slurry was cast using a doctor blade method on Al foils followed by drying at 85 °C for 2 h under vacuum. The cathode was then cut into disks with a diameter of 16 mm, the average electrode thickness was 200 μm. Coin cells were assembled using 1 M LiPF6 (in 1 : 1(v/v) ethylene carbonate (EC)–ethyl methyl carbonate (EMC)) as the electrolyte. Polyethylene membrane (Celgard 2500) and Li chip were used as the separator and anode. Coin cells were assembled in the Ar-filled glovebox and tested on Arbin cycling instruments in different voltage ranges and at different C rates. Cyclic voltammetry (CV) tests were performed between in different voltage ranges on BioLogic Science instruments. Electrochemical impedance spectroscopy (EIS) was performed using Biologic Science Instruments over the frequency range from 10 mHz to 100 kHz with a voltage amplitude of 10 mV. All tests were performed at room temperature.To prepare the CR2032 type coin-cell, 70 wt% cathode active materials were mixed with 20 wt% conducting agent (C65), and 10 wt% polytetrafluoroethylene . The mixture was dissolved in There are no conflicts to declare.RA-013-D3RA05684H-s001


V) channel subtype NaV1.7 plays a critical role in pain signaling, making it an important drug target. Here we studied the molecular interactions between μ-Conotoxin KIIIA (KIIIA) and the human NaV1.7 channel (hNaV1.7). We developed a structural model of hNaV1.7 using Rosetta computational modeling and performed in silico docking of KIIIA using RosettaDock to predict residues forming specific pairwise contacts between KIIIA and hNaV1.7. We experimentally validated these contacts using mutant cycle analysis. Comparison between our KIIIA-hNaV1.7 model and the cryo-EM structure of KIIIA-hNaV1.2 revealed key similarities and differences between NaV channel subtypes with potential implications for the molecular mechanism of toxin block. The accuracy of our integrative approach, combining structural data with computational modeling, experimental validation, and molecular dynamics simulations, suggests that Rosetta structural predictions will be useful for rational design of novel biologics targeting specific NaV channels.The voltage-gated sodium (Na V) channels play a key role in the action potential generation in excitable cells (V channel α-subunits (named NaV1.1-NaV1.9) are differentially expressed throughout tissues, and are targets of therapeutics for pain, cardiac arrhythmias, and epilepsy (V1.7 (hNaV1.7) channel is important for pain signaling and its mutations have been linked to severe pain disorders ranging from complete lack of pain sensation to extreme sensitivity to pain   to TTX-sensitive NaV channels (The search for novel Naotoxins) , which tepilepsy . μ-Conotchannels   for their apparent coupling to residues R10 and R14 on KIIIA  and DIII, which exposed a partially open ion conduction pathway that may explain the incomplete blocking characteristic of the toxin. While many KIIIA mutations have been previously characterized on Nav1.2 and Nav1.4 channels, only a limited number of KIIIA mutants had been tested on Nav1.7. We independently characterized many of the KIIIA mutations on Nav1.7 in our laboratory, to confirm previously published data and to validate affinities that serve as the basis of mutant cycle calculations. Our study for the first time tested effects of the following mutations: 1) KIIIA W8A and D11A on the wild-type hNav1.7; 2) hNav1.7 Y362C, E919Q, D923A with the wild-type KIIIA; 3) paired toxin—channel mutations K7A—E919Q, D11A—E919Q, and H12A—D923A. We identified several unique contacts between KIIIA and extracellular loops on hNaV1.7, providing key structural insights into binding specificity for different NaV channel subtypes. We used mutant cycle analysis to validate representative pairwise contacts between specific residues on KIIIA and hNaV1.7 identified from our structural model of the KIIIA—hNaV1.7 complex. Remarkably, the published cryo-EM structure of KIIIA—hNaV1.2 complex    channel approach  to genertructure  and the tructure  to predi-helices . The pos-helices  and lant-helices  studies.-helices . Mappinge cavity . The mosby KIIIA . NotablyIA  were reported in the arbitrary Rosetta Energy Units (R.E.U). Our analysis revealed the active surface of KIIIA with residues K7, W8, R10, H12, and R14 each having significant contribution to the binding energy   . In addie noted) . D11 is e noted) . The othe noted) . The R10eviously . While Kine scan . N3 has channels .V1.7 model, we first confirmed the activity of the wild-type KIIIA on the wild-type hNaV1.7 using whole-cell voltage-clamp recordings. To estimate the KIIIA binding affinity, we performed concentration-response experiments and obtained an IC50 of 410 ± 160 nM when fit to a Hill equation assuming a single binding site (V channels retain 5%–10% of their conductance when blocked by KIIIA (kon) was determined based on single exponential fits to the kinetics of block after toxin addition . KIIIA substitutions K7A and H12A both had nearly 100-fold decreases in affinity for the wild-type hNaV1.7 channel, in agreement with previously published data , and 6- and 21-fold relative to the single mutations, E919Q and D11A   ; Table 3ctively) ; Table 4ctively) . In contctively) ; Table 3ctively) ; Table 4ctively) ; Table 3ctively) ; Table 4ctively) . These rctively)  and the  complex , which bV channel isoforms likely arise from variations in sequence in the P2 helices and extracellular loop regions  of the KIIIA—hNaV1.7 model and the KIIIA—hNaV1.2 structure over KIIIA and the P2-helices is ∼1.0 Å  on the P2-helix in DIII in hNaV1.2 removes this interaction, and a new hydrogen bond is formed between D11 with the nearby residue Y1429  or Y1443 (hNaV1.2) on the extracellular P2-S6 loop in DIII  on the extracellular S5-P1 loop in DII  and E1417 on the extracellular P2-S6 loop in DIII  simulations of our KIIIA—hNaV1.7 complex model. The 3 independent 1.5 μs MD simulations revealed highly dynamic KIIIA binding with the backbone RMSDs between KIIIA and hNaV1.7 interface fluctuating between 3.5 and 4.5 Å . Similar to the simulations of our KIIIA—hNav1.7 model, the KIIIA—hNaV1.2 complex structure was dynamic during simulations with the RMSDs at the toxin—channel interface between 3–5 Å  were more limited compared to that in the simulations of KIIIA—hNaV1.7 model (see site A3 in V1.7) were less dominant compared to that in hNaV1.7 , substituted by a hydrophobic residue, I1399 may facilitate interactions of positively charged K7 and R10 with the nearby acidic residue D1690 on the P2-helix in DIV in hNaV1.7  on the P1-P2-helix region in DII of hNaV1.2 quickly diffused out to the extracellular bulk, in agreement with our MD simulation of the KIIIA—hNaV1.7 model . InDB: 3RVY ]. In thiV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, and NaV1.8 channels  may contribute to KIIIA binding on hNaV1.7 by influencing the interactions of K7 and R10 on KIIIA with acidic residues on the channel. A recent study focused on optimizing KIIIA analogs guided by KIIIA—hNav1.7 model based on KIIIA—hNav1.2 structure, revealed reduced KIIIA potency for hNav1.7 when substituting R10 on KIIIA to hydrophobic amino acids  of KIIIA block observed in both hNaV1.7 and hNaV1.2, which implied such re-binding events are rare and may not be observed in our conventional 1.5 μs MD simulation trajectories. Our results agree with the functional data showing that KIIIA is a highly efficient but incomplete pore blocker of NaV channels. Study of the full binding and blocking mechanism of KIIIA on NaV channels is beyond the scope of this study and will require much longer simulation times or enhanced sampling techniques, and a structure of KIIIA bound to an open and conductive state of NaV channel, which is not currently available. Furthermore, our homology model of hNaV1.7 channel based on hNaV1.4 structure may have uncertainties in NaV1.7 channel—KIIIA residue-residue interactions.We also observed escapes of sodium ions initially located in the selectivity filter in both KIIIA—hNaV channels  (PDB ID: 5XSY)   was usedsoftware . Initialprotocol  and pickosettaCM  was theneviously . Visual V1.7 model using Rosetta protein-protein docking approach   was usedapproach . By defaapproach . In stagV1.7 and the cryo-EM structure of hNaV1.2 (PDB ID: 6J8E) were used to setup systems for MD simulations. For the hNaV1.2 structure, Rosetta density refinement protocol was applied as described above for the hNaV1.7. The missing region on DI extracellular loop was modeled using Rosetta loop modeling. We placed one sodium ion in the selectivity filter and one in the cavity of the channels as initial setup for both simulations. CHARMM-GUI (V1.7 model and the KIIIA—hNaV1.2 structure (PDB ID: 6J8E) in a lipid bilayer of POPC with explicit TIP3P water molecules at a concentration of 150 mM NaCl. CHARMM36 forcefield was used for proteins, lipids, ions, and waters in both systems. Each system contains approximately of 164,000 atoms. Protonation state is assigned at neutral pH. N-epsilon nitrogen of H12 on KIIIA is protonated instead of N-delta nitrogen as suggested in both the Rosetta model of hNaV1.7—KIIIA and the hNaV1.2—KIIIA structure. The C-terminal of KIIIA is amidated to be consistent with the KIIIA variant used in our experiments.The docking complex of KIIIA—hNaARMM-GUI  was used2) were then applied only to protein backbone atoms, and the systems were equilibrated further for 20 ns with a timestep of 2 fs. All bonds to H atoms were constrained using the SHAKE algorithm in order to use a 2 fs timestep. Simulations were performed in NPT ensemble with semi-isotropic pressure coupling to maintain the correct area per lipid, and constant temperature of 303.15 K. Particle Mesh Ewald (PME) method was used to compute electrostatic interactions. Non-bonded pair lists were updated every 10 steps with a list cutoff distance of 16 Å and a real space cutoff of 12 Å with energy switching starting at 10 Å. Independent simulation systems were created by using different seed numbers in the equilibrations.Equilibrations were run on our local GPU cluster using NAMD version 2.12 . After 1V1.7—KIIIA model and 3 independent simulations of 1μs for the hNaV1.2—KIIIA structure. Simulations were performed in the NPT ensemble at 303.15 K A with 2 fs timestep. Non-bonded long-range interactions computed every 6 fs using the RESPA multiple time step algorithm. The multi-integrator algorithm was used for temperature and semi-isotropic pressure coupling and the u-series algorithm was used for long-range electrostatic interactions. A long-range Lennard-Jones (LJ) correction (beyond cutoff) was not used as was suggested for CHARMM36 lipid force field.We used the Anton 2 software version 1.31.0 for production runs of each system on the Anton 2 supercomputer. We ran 3 independent simulations of 1.5 μs for our hNaV channels based on bacterial NaV channel structure before any eukaryotic NaV channel structures were published. We first generated a model of hNaV1.4 pore using x-ray structure of the bacterial Nav channel NaVAb (PDB ID: 3RVY) as a template using Rosetta homology modeling .We previously performed homology modeling of human Namodeling . We selelidation . The extmodeling . We used channel  to guidestraints . We thenstraints  to buildmodeling . To creaxPepDock  and the V1.7 (gift from Chris Lossin). Cells were grown at 37°C, 5% CO2 in DMEM with 4.5 g/L D-glucose, L-glutamine, and 110 mg/L sodium pyruvate (Gibco cat# 11995-065) with 10% FBS, and 100 units/mL Penicillin/Streptomycin (Gibco cat# 15140-122). The stable cell line was raised in the same conditions with 500 μg/mL G418 as a selection agent. Cells were grown to 70% confluency in 35 mm dishes and passaged every 2–3 days for tsa-201 and 3–4 days for the stable-cell line. Cells were washed with divalent-free DPBS (Gibco cat# 14190-144) and dissociated with 0.05% Trypsin-EDTA (Gibco cat# 25300-054) and seeded to fresh dishes with pre-warmed media. tsa-201 cells were transfected via Lipofectamine 2000 24–48 h prior to experiments with 1 μg pCMV6-SCN9A (gift from Dr. Christoph Lossin) and 0.5 μg pMaxGFP (Lonza) for identification of transfected cells. Mutant constructs were purchased from, and coding sequences verified by Mutagenex. Prior to experiments, cells were washed with DPBS and dissociated in 1 mL Versene (Gibco cat# 15040-066) and scraped from the dishes and transferred to a 14 mL conical tube with 3 mL DMEM. They were centrifuged at 1,000 × g for 2 min and resuspended in a microfuge tube in 1 mL extracellular solution (described below) with 10 mM D-glucose and rotated at RT until use.Electrophysiology experiments were performed on transiently transfected tsa-201 cells  and a HEK 293T cell line stably expressing hNas) was typically 1–4 MΩ. 60%–80% Rs compensation (10 μs lag) and prediction was used to reduce voltage error to less than 10 mV as determined from the product of the peak current and Rs with compensation. P/5 leak subtraction protocol was used during recording. Signals were pre-filtered with a low-pass Bessel filter at 5 or 10 kHz before digitizing at 20 kHz and recorded with Patchmaster  on a Windows 7 PC. The solutions were as follows in mM: External 3.5 KCl, 155 NaCl, 10 HEPES, 1 MgCl2, 1.5 CaCl2 adjusted to pH 7.4 with NaOH, and 315 mOsm; Internal: 35 NaCl, 70 CsCl, 50 CsF, 1 EGTA, 10 HEPES adjusted to pH 7.4 with CsOH at 310 mOsm. After break-in, cells were held at −120 mV and tested for stable Na+ current with depolarizing 35 ms voltage steps to −10 mV from −120 mV collected every 5 s for up to 5 min to allow for a stable level of current prior to vehicle addition. Once stable current levels were achieved, 150 μL of vehicle was manually added to the bath with displaced solution removed via an overflow vacuum line. After approximately 5 min, whole cell parameters were checked, and toxin (described below) was added by the same method as vehicle. Once apparent block plateaued, whole cell parameters were checked and adjusted as necessary, and pulsing resumed. To measure dissociation, gravity-fed perfusion with fresh external solution was started at a rate 1–2 mL/min during recording. Cells with stable leak and Rs allowing fitting to a single-exponential function (see below) throughout the experiment were included for analysis.Whole-cell voltage-clamp recordings were performed at RT (21–22°C) in an RC-24N recording chamber fixed to a glass coverslip (Warner Instruments), mounted on a Zeiss Axiovert 35 microscope illuminated with a Zeiss HBO 100W AttoArc lamp and filter set for epifluorescent detection of GFP expressing cells. Approximately 40 μL of cell suspension was added to the pre-filled chamber and allowed to adhere to the glass bottomed chamber for 2–10 min. Fresh external solution was perfused through the chamber prior to patching. Borosilicate pipettes  were pulled, fire-polished, coated with Sylgard. Tip resistances were 1–2 MΩ, when filled with the internal recording solution. GFP expressing cells were patched and signals were amplified with an Axon Axopatch 200-B (Molecular Devices) and acquired with an Instrutech LIH 8 + 8 ADC board (HEKA). GΩ seals were obtained, and pipette capacitance was corrected for prior to break-in achieved by suction. Access resistance , reconstituted in water and stored as 100 μM stock aliquots at −80°C prior to use. Toxin variants were produced by solid state synthesis as described previously  and storStructural modeling data were analyzed using Rosetta and rendered using UCSF Chimera , VMD Hu was used: We used CAVER (version 3.0) (Tunnel detection for KIIIA block : We usedion 3.0)  to detec: Fractional contact is defined as probability of finding two residues, one on the KIIIA and one on the channel forming contacts over time course of simulation. We considered two residues are in contact if any heavy atoms of one residue is within 4 Å of any heavy atoms of the other residues. Only contacts that have probability greater than 0.25 are shown for clarity.Fractional contacts : Fractio: We used 10 Å as a cutoff for interface calculation between KIIIA and the channels. The interface is comprised of the KIIIA itself and channel residues that are within 10 Å of KIIIA heavy atoms, defined at the beginning of the simulations. Backbone heavy atoms of the interface were used for RMSD calculation.Interface RMSD , peak currents during depolarizing voltage steps were plotted by time, and data were fit with a single exponential function:Electrophysiology data were analyzed and plotted in IGOR 7 Pro (Wavemetrics). Geometric means of kinetic parameters were determined using Excel (Microsoft) and plotted in IGOR 7 Pro. Curve fitting was performed in IGOR Pro 7 as described previously . To detekon was determined by Eq. Fblock) was already known and koff could not be determined independently:koff was determined by Eq. The association rate Kdvia Eq. Affinity was determined kinetically as the dissociation constant V1.7 made measurement of koff difficult due to limited recovery during the experiment, thus values shown here are estimates assuming eventual full recovery of the maximal current before toxin association. The resulting affinity values are consistent with previous reporting of kinetic determination of affinity for this channel  for other channel and toxin variants, except where noted, according to Eq. Maximum fractional block at saturating concentrations was determined from kinetic data and observed block  were calculated from the dissociation constants of the four conditions for each cycle according to Eq. Fblock, while logarithmically scaled kinetic parameters were summarized with geometric means and standard deviations. Standard errors of kinetic parameters were obtained for the tables as the dividend of the standard deviation and the square root of the sample size for each toxin-channel pair, as noted in parentheses in each table. Errors for coupling coefficients and coupling energies were calculated by linear propagation of error from fractional standard deviations of the reported Kd values for the toxin—channel mutant pairs used to calculate the coupling coefficients.Descriptive statistics: Arithmetic means and standard error were calculated for


One acyl hydrazide, adipic acid dihydrazide (ADH), was tested on a mouse model of breast cancer and showed nice contrast in the tumor region. We also prepared a derivative, acyl hydrazone, which showed the furthest shifted labile proton (6.4 ppm from water) and excellent contrast properties. Overall, our study expands the catalog of diaCEST agents and their application in cancer diagnosis.Chemical exchange saturation transfer (CEST) MRI is a versatile molecular imaging approach that holds great promise for clinical translation. A number of compounds have been identified as suitable for performing CEST MRI, including paramagnetic CEST (paraCEST) agents and diamagnetic CEST (diaCEST) agents. DiaCEST agents are very attractive because of their excellent biocompatibility and potential for biodegradation, such as glucose, glycogen, glutamate, creatine, nucleic acids, et al. However, the sensitivity of most diaCEST agents is limited because of small chemical shifts (1.0–4.0 ppm) from water. To expand the catalog of diaCEST agents with larger chemical shifts, herein, we have systematically investigated the CEST properties of acyl hydrazides with different substitutions, including aromatic and aliphatic substituents. We have tuned the labile proton chemical shifts from 2.8–5.0 ppm from water while exchange rates varied from ~680 to 2340 s Chemical exchange saturation transfer (CEST) MRI is an alternative method based on contrast mechanisms, which is “switched on” by applying selective saturation using radiofrequency pulse(s) at the frequency of labile protons on solute molecules which exchange with water +) 999.5384, found 999.5369.terature , 1.72 g,1) from 1.8, 2.4, 3.0, 3.6, 4.2, and 4.8 μΤ. One hundred two offsets between ±10 ppm were acquired with a 0.2 ppm stepsize to produce saturation images, plus one at +40 ppm. Other imaging sequence parameters: matrix size 64 × 64; slice thickness 1.5 mm; TR/TE 6000/4.6 ms. For B0 inhomogeneity calculation, water saturation shift referencing (WASSR) images were acquired using B1 = 0.5 μΤ with 31 offsets from −1.5 to +1.5 ppm with a 0.1 ppm stepsize.Acyl hydrazides and acyl hydrazone were dissolved in 0.01M phosphate-buffered saline (PBS) at different concentrations and titrated to various pH values ranging from 5.7 to 7.5. The samples were kept at 37 °C during imaging. In vitro CEST MRI experiments were performed on a horizontal bore 9.4 T Bruker Biospec system . For phantoms, a transmit-receive volume coil with an inner diameter of 40 mm was used. CEST images were acquired using a continuous-wave RF saturation pulse of 3s followed by a RARE imaging sequence. CEST data were acquired using six different saturation power . Prior to imaging, the tail vein was cannulated, permitting intravenous injection in the MRI scanner. Mice were warmed, and their respiration rate was monitored throughout the duration of the imaging experiment. A T2w image was obtained using a multi-slice RARE sequence. RARE factor = 8, TR/TE = 2 s/20 ms, field of view = 28 mm2 × 20 mm2, matrix size = 256 × 256, slice thickness 1.5 mm. For CEST data acquisition, 4 s long cw saturation pulses were applied with B1 = 3.6 μΤ using 31 offsets from −7.0 to +7.0 ppm with stepsize 0.2 ppm and one offset of +40 ppm for 72 offsets. WASSR images were collected for generating B0 maps using 31 offsets from −1.5 to +1.5 ppm with stepsize 0.1 ppm and B1 = 0.5 μT. After completion of the initial Z-spectrum, the mouse was injected with 100 μL of 500 mM (~435 mg/kg) ADH, and the line was flushed with 100 μL saline. No adverse events were noted in any of the mice receiving ADH injections. Post-contrast Z-spectra were acquired sequentially up to 34 min post-injection. The time taken for each set of 72 offsets was 8 min and 24 s, and the other sequence parameters were as follows: TR/TE 7000/4.6 ms; number of averages = 1; matrix size = 64 × 64; field of view 28 mm2 × 20 mm2.All animal experiments were performed under a protocol approved by the Jinan University Animal Care and Use Committee. Tumor xenografts of murine 4T1 breast cancer were established in three 4-week-old female Balb/c nude mice (Charles River Laboratories) through the subcutaneous injection of 2 × 10asym = (S(−Δω) − S(+Δω))/S0, where S+Δω and S−Δω were the MRI signals with RF irradiation at particular offsets +Δω and –Δω, respectively, and S0 is the signal acquired without RF saturation. Both S+Δω and S−Δω were corrected pixel-wise for B0 inhomogeneity using the WASSR method as described previously [All post-processing was performed using custom-written script implemented in MATLAB . Saturation transfer (ST) contrast was calculated by applying magnetization transfer ratio asymmetry analysis with MTReviously .asym values at the maximum CEST offsets for the set of 6 saturation field strengths were fit numerically using the Bloch equations to estimate the exchange rates (kex).The exchange rates for the labile protons were estimated using the quantifying exchange using saturation power (QUESP) method as described previously . In brie4 cells per well for 24 h at 37 °C and in an atmosphere of 5% CO2. Then, the medium was removed and the cells were washed with PBS (×2). Afterward, the cells were exposed to different concentrations of ADH  in each well. The cells were grown for a further 24 h. Then a standard CCK-8 assay was carried out to evaluate the cell viability. Briefly, 10 μL of the CCK-8 solution was added to each well of the plate, and the cells were allowed to grow for another 3 h. The absorbance ) was measured at 450 nm using a microplate reader . The measured absorbance value (OD) of the wells was then used to calculate the cell viability via the formula4T1 cells were cultured in completed RPMI 1640 media  and seeded into 96-well plates (100 μL/well) at a density of 1× 10In conclusion, we have identified a number of compounds from the acyl hydrazise–hydrazone scaffold, which produces strong CEST contrast from 2.8–6.4 ppm from water. We have also demonstrated that one compound from this scaffold (ADH) allows the detection of altered perfusion in a mouse model of breast cancer. Our study expands the catalog of compounds suited as CEST agents, which will accelerate the clinical translation of CEST MRI in the future.


Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T0 plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein (Cas) is the predominant RNA-guided nuclease technology for inducing targeted DNA double strand breaks (DSBs) in living organisms. CRISPR-Cas and its derived technologies have been widely used for genome editing, transcriptional regulation, epigenetic modifications, genomic region visualization and isolation, etc . Cas12a CRISPR-Cas12a is a powerful tool for genome editing in many plant species . RecentlTo broaden the application of the CRISPR-Cas12a system, seventeen novel Cas12a orthologs have been identified and investigated in this study. To fully compare their performance in genome editing, these novel Cas12a orthologs were tested in various plant species and plant systems (protoplasts and stable transgenic lines), as well as at different temperatures. Our results demonstrated that Ev1Cas12a and Hs1Cas12a enable efficient genome editing in various plant species and hold great potential as robust genome editing tools in a wider range of plant species.Since genome editing in plants is mostly conducted at ambient temperatures, we are interested in identifying Cas12a nucleases that are more tolerant to lower temperatures. We identified new Cas12a ortholog candidates using LbCas12a, FnCas12a, ErCas12a, Mb2Cas12a as queries to BLAST  against the Joint Genome Institute Microbial Genomes and Microbiomes database, and selected Cas12a nucleases originated from bacteria with ambient living temperatures and capability to reproduce in soil. The novel Cas12a nucleases all have estimated length of more than 1,000 amino acids and less than 80% sequence homology to queries . FinallyTo test the editing efficiency of these 17 Cas12a orthologs, we first conducted rice protoplast transformation targeting four sites with TTTV and VTTV PAMs at 32°C. Screening of 17 Cas12a orthologs showed variable genome editing activities across these target sites. Seven Cas12a nucleases  showed comparable or higher genome editing efficiencies than LbCas12a and Mb2Cas12a at the CC1-TTTC target site . At thisTo further investigate the editing activity of Ev1Cas12a and Hs1Cas12a at lower temperatures, including 22°C, 25°C, and 28°C, multiplexed genome editing was performed with Ev1Cas12a and Hs1Cas12a comparing to LbCas12a and Mb2Cas12a in rice protoplasts . Three cThe analysis of NGS data revealed the editing profiles of these two novel Cas12a orthologs. As expected, the majority of edits were deletions . DeletioBlc site  frequencies at two SVP target sites, respectively , simpler multiplexing, and generating double strand break with staggered ends . In the Arabidopsis and maize  database, using LbCas12a, FnCas12a, ErCas12a, and Mb2Cas12a as queries. Cas12a nucleases derived from bacteria with ambient living temperature and soil reproductive capability were specifically chosen. The novel Cas12a orthologs were identified based on their estimated length of over 1,000 amino acids and less than 80% sequence homology to the query sequences. A phylogenetic tree was constructed using the Neighbor-Joining method with MEGA11. Ultimately, 17 Cas12a orthologs were selected for genome editing evaluation, namely, Ba1Cas12a, Ca1Cas12a, Cb1Cas12a, Cc1Cas12a, Cu1Cas12a, Ev1Cas12a, Hs1Cas12a, Pa2Cas12a, Pc1Cas12a, Sc1Cas12a, Bb1Cas12a, Cd1Cas12a, Cw1Cas12a, Mb1Cas12a, NbCas12a, PbCas12a, and UaCas12a .To assess the genome editing efficiency of all 17 novel Cas12a orthologs in rice, Cas12a genes were synthesized with rice codon optimization and cloned into pYPQ230 , lycopene ε-cyclase (LCY-E), beta-lycopene cyclase (Blc), lycopene β-cyclase1 (LCY-B1), and lycopene β-cyclase2 (LCY-B2). A tandem ribozyme multiplexing system was used as previously described  in tomato, the same Cas12a entry clones for rice genome editing were used. A multiplexed crRNA entry clone was generated containing six crRNAs targeting five genes involved in lycopene synthesis and catabolism, including escribed . The 2 ×escribed .Pt4CL1, PtPII, and PtSVP, using the tandem ribozyme multiplexing system as described previously  plasmid, and then mixed with 230 μL polyethylene glycol (40% PEG). The mixture was set at room temperature for 30 min and then 900 μL W5 were added to stop the transformation. Protoplasts were collected and resuspended in 1 mL W5 buffer and incubated in 24-well culture plates at 32°C  in dark for 2 days. Protoplasts transformed with water were used as the wild type (WT) control for all genome editing experiments.Rice protoplasts were isolated and transformed as previously described . BrieflyTomato seedlings (M82) were grown with 16h/8 h light/dark photoperiod for 7–9 days until the cotyledons were fully expanded. Cotyledons were cut off and placed in the same enzyme solution used for rice. After incubation at 28°C for 8 h in dark, tomato protoplasts were filtered through a 75 μm cell strainer and collected by centrifugation. To purify the viable protoplast, 6 mL 0.55 M sucrose solution was added to the protoplasts followed by 2 mL W5 on top. After centrifuge for 30 min at 200 g, protoplasts were collected at the interface. Protoplasts were moved to new tubes and washed with W5 buffer twice. Protoplasts were then resuspended in the MMG buffer and used for transformation based on the same method for rice.Agrobacterium-mediated transformation was carried out following the previously described method . For high-throughput next-generation sequencing (NGS), target sites were amplified with two rounds of PCR using barcoded primers . Pooled