Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome. Saccharomyces cerevisiae) as a model organism for research is largely due to its genetic tractability, a direct consequence of having highly active homologous recombination (HR) machinery. This enables the incorporation of DNA sequence changes that originate from exogenous DNA templates bearing 40 bp or more homology to the flanking genomic sequences to facilitate integration via HR. This is termed one- or single-step gene engineering [6−107 with 40 bp of flanking homology [URA3 marker cassette [The popularity of budding yeast  sequence, designed to target the desired region of the genome to be altered, directs Cas9 to make a DSB in the vicinity of the sequence homologous to the gRNA. To direct repair using HR machinery a linear PCR-generated dsDNA template, carrying both the desired mutation and homology to the native target locus, is co-transformed along with a plasmid encoding Cas9 and the gRNA ,12. The smid DNA , the selsmid DNA ,15. Furtsmid DNA ,16,17. While multiple methods of introducing Cas9 and gRNA into cells exist, including the direct transfection of a Cas9-gRNA ribonucleoprotein (RNP) into the nucleus of mammalian cells , such teE. coli, isolated from E. coli and validated, an unnecessarily time-consuming process [To avoid potential mutations resulting from PCR errors, other methods have been developed using restriction enzymes to clone the gRNA into the Cas9-expressing plasmid. A method based on traditional cloning with 2 restriction enzymes , while e process . Direct placement of type II-S restriction enzyme sites on either side of the gRNA sequence has been used to allow for a single Golden Gate cloning step to replace the gRNA with a new gRNA . A previhttp://n2t.net/addgene:60847 (accessed on 25 September 2022); RRID:Addgene_60847)), a vector which constitutively expresses Cas9 and the gRNA. We have inserted a pair of BsaI sites flanking the gRNA insertion site, enabling the directional ligation of annealed oligonucleotides encoding the gRNA after digestion with BsaI. The resulting plasmid, pCASB  dsDNA template containing the desired genetic mutation (discussed below). A schematic describing the steps involved when using pCASB for CRISPR-Cas9 site-directed mutagenesis is presented in The entire protocol takes approximately 2 weeks and can be completed in any laboratory set up for bacterial and yeast culture, subcloning, and PCR. Many portions of the method can be modified to suit the lab’s preferred methods, such as for yeast transformation and genomic DNA isolation.0.2 mL PCR tubes 1.7 mL microcentrifuge tubes Budding yeast strain to be genetically engineered pCASB Plasmid (Addgene ID: 190175)Oligonucleotides encoding desired gRNA  containing 50 mM sodium chloride ® v2 BsaI-HFT4 DNA Ligase and buffer Filtered water (such as Millipore), sterilized by means of autoclaving where indicatedE. coli, such as One Shot TOP10 Chemically Competent E. coli High-efficiency competent 14 mL culture tubes for bacterial culture growth . Autoclaved to sterilize.Kanamycin Sulfate , prepared to 400 mg/mL (1000× stock solution) in sterile water0.2-micron syringe filters , 50 mL conical tube vacuum filters , and 250–500 mL flask vacuum filters  for sterilization of reagents○LB Powder , 25 g/L in water. Autoclaved to sterilize.LB liquid media○Petri dishes ○Agar A ○Add 15 g/L Agar A to LB liquid media before autoclaving. Allow to cool to 55 °C before adding 1 mL/L of Kanamycin stock solution and pouring into Petri dishes.LB agar plates containing 400 μg/mL KanamycinGlass beads or equivalent for plating . Autoclaved to sterilize.Plasmid miniprep kit Oligonucleotides or other DNA encoding template sequence for HDR High-fidelity DNA polymerase enzyme and buffer, such as Expand10 mM dNTPs 100% ethanol 70% ethanol (diluted in filtered water)Sodium Acetate , 3 M solution in water○Tris Base ○Glacial acetic acid ○For 1 L of 50× solution, combine 242 g Tris Base, 57.1 mL glacial acetic acid, and 100 mL 0.5 M EDTA, pH 8.0. Add water to 1 L)TAE buffer ○w/v agarose in TAE buffer, microwave to boil and dissolve agarose powder. Cool slightly before adding SYBR safe DNA gel stain  and pouring into gel mold.To prepare DNA gel, mix 1% Quick dissolve agarose SYBR™ Safe DNA Gel Stain DNA loading dye 100 bp DNA ladder ○Tris Base ○EDTA ○2 • 2H2O. Add filtered water to under 500 mL, adjust pH to 7.8 with concentrated HCl , then fill to 500 mL with filtered water. Use a 0.2-micron filter to sterilize.For 500 mL of 100× solution, combine 60.55 g Tris Base with 1.86 g EDTA NaTE Buffer ○Lithium acetate , 1 M stock solution in water○For 50 mL, mix 5 mL of 10× TE, 5 mL 1 M lithium acetate, and 40 mL of filtered water. Use a 0.2-micron filter to sterilize.Yeast transformation buffer: 1× TE, 100 mM Lithium AcetateDeoxyribonucleic acid, single stranded from salmon testes, 10 mg/mL ○Poly(ethylene) glycol ○For 50 mL, combine 25 g poly(ethylene) glycol, 5 mL 1× TE, and 5 mL 1 M lithium acetate. Add warm (50–60 °C) filtered water to 50 mL. Mix until dissolved. Use a 0.2-micron filter to sterilize.PEG Solution: 40% Poly(ethylene) glycol in yeast transformation bufferDimethyl Sulfoxide (DMSO) ○20 g/L Peptone ○10 g/L Yeast Extract ○20 g/L Dextrose ○Mix in filtered water, autoclave to sterilize.YPD liquid mediaGlass tubes, 15 mL , and lids  for yeast culture. Autoclave to sterilize.Geneticin (G418) sulfate , prepared to 200 mg/mL (1000× stock solution) in autoclaved filtered water.○Add 15 g/L Agar A  to YPD liquid media before autoclaving.YPD Agar plates○After autoclaving, allow liquid agar to cool to 55 °C before adding 1 mL/L of G418 stock solution.YPD Agar plates containing G418Wooden dowels , toothpicks, or pipette tips, for picking colonies. Autoclave to sterilize.w/v SDS, 2% v/v Triton X-100, 100 mM NaCl, 10 mM Tris HCl pH 7.9, 1 mM EDTA)○SDS ○Triton X-100 ○Sodium chloride Yeast genomic prep buffer Phenol:chloroform:isoamyl alcohol, 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA Taq DNA polymerase , or similar enzyme for low fidelity PCRPCR purification kit 1 kb DNA ladder v/v in filtered water. Autoclave to sterilize.Glycerol , 80% Cryogenic tubes, 1.8 mL The materials used in this protocol include:ThermocyclerStandard horizontal agarose gel electrophoresis tank, gel tray, comb and power supplyAutoclave for sterilizing media, culture tubes, etc.37 °C incubator for bacterial growth37 °C shaking incubator for bacterial growth30 °C incubator with a tube rotator for yeast growthWater bath or heat block set to 42 °CMulti-tube vortex , kept at 4 °C in cold room or refrigeratorNanodrop Spectrophotometer600 of culturesSpectrophotometer to measure ODMicrowave for preparing agarose gelBenchtop microfugeAccess to a DNA sequencing facilityAccess to oligonucleotide synthesis for preparation of gRNA oligos, gRNA sequencing primer, HDR template DNA, and for PCR/sequencing of target geneThe equipment used in this protocol includes:https://benchling.com (accessed on 25 September 2022)), online resource for gRNA and HDR template design. Alternatively, another gRNA design software such as E-CRISP [Benchling (The software used in this protocol includes:Streptococcus pyogenes encoded by pCASB recognizes the protospacer adjacent motif (PAM) 5′ NGG 3′. This sequence must be present immediately downstream of the 20-bp gRNA target site [After identifying the target gene and desired mutation, design a gRNA that is as close to the mutation site as possible. gRNA design programs, such as the one integrated into the Benchling online lab notebook service, can identify a variety of potential gRNA sites in a genomic region and can provide scores based on predicted on- and off-target activity. The Cas9 from get site .Ideally, the gRNA should be contained within the sequence of the HDR template DNA that will be co-transformed along with the plasmid to create the desired mutation. This way, the template DNA can contain a silent mutation of the gRNA’s associated PAM site, preventing repeated cutting by Cas9 after the mutagenesis has taken place. Therefore, the gRNA should typically be no farther than 40–70 bp away from the desired mutation site.https://help.benchling.com/en/articles/670980-design-guide-rnas-grnas (accessed on 25 September 2022)).To use the Benchling gRNA design tool, use the following steps (further information can be found at YDR007W rather than TRP1) to facilitate gene import.Import the sequence of the gene to be edited as a DNA sequence. Search for the gene using the systematic open reading frame nomenclature .Set the parameters to design a 20-nucleotide guide, using the PAM sequence NGG, using the Select a region of the gene immediately adjacent to the desired mutation site (±50–100 bp is suggested).Identify the gRNA sequences which are considered good based on the on- and off-target scores: those with an on-target score  of 60 orSelect a gRNA sequence as close to the target mutation as possible .Make note of the chosen gRNA sequence and proceed to gRNA oligonucleotide design.The gRNA-encoding oligos must contain the following sequences, to match the BsaI overhangs in the pCASB vector.Top strand: 5′ CTTT 3′, followed by the 20 bp gRNA sequenceBottom strand: 5′ AAAC 3′, followed by reverse complement of the gRNA sequenceN-terminus of the yeast TRP1 gene [Once the gRNA sequence has been designed, design two complementary oligos that can be cloned into the pCASB vector after digestion with BsaI. Digestion will leave behind the gRNA site shown in RP1 gene , requireTop strand oligonucleotide: CTTTATGTCTGTTATTAATTTCACBottom strand oligonucleotide: AAACGTGAAATTAATAACAGACATAfter annealing, the paired oligonucleotides will form the double-stranded sequence shown in 8.Resuspend each oligonucleotide in filtered water to 100 mM.9.In a PCR tube, mix 2.5 µL of each 100 mM oligonucleotide with 20 µL of oligonucleotide annealing buffer.10.Using a thermocycler, heat oligonucleotides to 95 °C for 2 min then cool by 0.1 °C per second to 20 °C.11.Use annealed oligonucleotides within a few hours or freeze at −20 °C for future use. Their final concentration is approximately 160 ng/µL.12.Dilute annealed oligonucleotides 1:1000 in filtered water.13.Filtered water 3 µL of annealed oligonucleotides (~480 pg)75 ng of pCASB plasmid2 µL of T4 DNA Ligase buffer1 µL of T4 DNA Ligase enzyme1 µL of BsaIHF-v2 enzymeAssemble a 20 µL cloning reaction, adding reagents in the following order:14.In a thermocycler, incubate reaction for 30 min at 37 °C, followed by 5 min at 60 °C.  into 2 mL LB + Kanamycin (0.4 mg/mL diluted from 1000× stock) liquid cultures in 14 mL sterile plastic tubes.18.Grow cultures overnight at 37 °C with shaking.19.Isolate plasmid DNA from each culture using a Miniprep kit or similar method.20.Measure concentration of each plasmid using a NanoDrop spectrophotometer, and send 800 ng (or as directed by the sequencing facility) of pCASB-based vector for Sanger sequencing of the gRNA site using the following sequencing primer :21.After obtaining sequencing results, save the samples containing successfully cloned plasmids. This DNA will be directly used for yeast transformation (step 41-i) and can be stored for many months at −20 °C.Using any preferred software, design the desired mutant locus in the target gene. Designed mutations should include a silent mutation in the PAM sequence of the gRNA (or another mutation that disrupts gRNA binding) to prevent repeated cutting by Cas9 after recombination has taken place. For most gene edits such as deletions, short insertions, or point mutations in a small region, the HDR template DNA will be designed as three 60 bp oligonucleotides that are PCR-amplified to create a 160 bp template sequence. The resulting DNA can be concentrated and transformed into yeast along with the pCASB plasmid encoding your specific gRNA. The three oligonucleotides are designed in a variety of ways, a few examples of which are shown in Following the example of gene deletion methods such as by Longtine et al.  we prefeThis method has been used successfully to replace over 300 bp of a gene with an alternative sequence and to add 1 kb to a gene in our hands. Efficiency of all the forms of genetic editing shown in 22.Dilute the three oligonucleotides to 10 µM in filtered water23.76 µL of filtered water10 µL of Expand HF buffer2 µL of 10 mM dNTPs1 µL of “middle” template oligonucleotide5 µL of “forward” primer oligonucleotide5 µL of “reverse” primer oligonucleotide1 µL of Expand HF enzymeSet up PCR reaction .24.Split the reaction into two PCR tubes, 50 µL per tube.25.Initial denaturation: 2 min, 95 °C○Denaturation: 30 s, 95 °C○Annealing: 30 s, 55 °C * ○Elongation: 1 min *, 72 °C20 * cycles:Final elongation: 7 min, 72 °CParameters marked with an asterisk (*)  are recommended for all PCR enzymes. Other parameters may vary.Perform PCR with the following parameters:26.Combine all PCR reactions into one 1.7 mL PCR tube.Add 3× volume of 100% ethanol (300 µL for two PCR reactions).Add 1/10th volume of 3 M Sodium Acetate (10 µL for two PCR reactions).OPTIONAL STEP: Incubate at −20 °C for 30 min or more (freezing DNA facilitates precipitation from ethanol but is not required).Centrifuge at maximum speed in a room temperature benchtop microfuge for 10 min, aspirate and discard supernatant while being careful not to disturb the clear/white DNA pellet.Wash pellet with 1 mL of 70% ethanol.Centrifuge at maximum speed in a room temperature benchtop microfuge for 5 min, aspirate supernatant.Repeat steps vi and vii, for two washes in total.Let pellet air dry for 15 min or until no ethanol remains in the tube.Resuspend pellet in 12 µL of filtered water: 10 µL for transformation reaction (below), plus 2 µL to run on an agarose gel.Ethanol precipitate PCR product :27.Dilute 2 µL of resuspended PCR product in 8 µL of filtered water and 2 µL of 6× DNA loading dye.28.Confirm that the PCR product is the correct size by visualizing on a 1% agarose/TAE gel containing 1× SYBR Safe DNA stain . Include 6 µL of 100 bp ladder).29.Image the gel on a UV transilluminator. If the PCR product is brighter than the equivalently sized band of the 100-bp ladder, there is an adequate amount of PCR product. If the PCR band is noticeably dimmer, repeat PCR with more reactions. If the band is very bright (>10× the intensity of the ladder), consider diluting the remaining DNA with additional water or using less for each yeast transformation.PAUSE STEP: the HDR template DNA can be stored for many weeks at −20 °C.30.The night before the transformation, use a wooden stick to inoculate 2–5 mL of liquid YPD media  in a glass culture tube with a colony of the desired yeast strain. Grow overnight at 30 °C on a rotator.31.600 on a spectrophotometer and dilute the culture to an OD600 of 0.2 in at least 5 mL of YPD. Grow diluted culture at 30 °C on a rotator.The next morning, measure the OD32.600 0.8–1.0).Allow the culture to go through at least 2–3 more cell divisions, until it reaches mid-log phase (OD33.7) cells.In two 1.7 mL microcentrifuge tubes , harvest 2 OD .5–10 µL of prepared HDR template DNA PCR product, depending on brightness of visualized band.Add nothing else to the negative control tube. To the other add:42.Incubate tubes on the benchtop for 10 min.43.Add 270 µL of 40% PEG solution to each tube, pipette to mix.44.Place tubes in 30 °C incubator for 30 min (no shaking or rotating necessary).45.Add 50 µL DMSO to each tube.46.Heat shock cells for 15 min at 42 °C, in either a heat block or water bath.47.Centrifuge cells for 1 min at 13,000 RPM in a benchtop microfuge, aspirate off and discard the supernatant.48.Wash pellets once with 1 mL of sterile filtered water, centrifuge at 13,000 RPM in a benchtop microfuge. Aspirate off and discard the supernatant.49.Resuspend cell pellets in 250 µL of YPD media.50.Allow cells to recover for 2 h at 30 °C with rotating.51.Spread each 250 µL culture on a YPD plate containing 400 µg/mL G418, allow to grow for 2–3 days at 30 °C for colonies to form. After this time, confirm that the negative control plate has no colonies and discard it.The CRISPR-Cas9 gene editing will take place over the initial 2–3 days where cells are grown on YPD + G418 to select for presence of the pCASB-based plasmid (the KanR marker confers resistance to the drug G418 in yeast). After that point, the presence of highly expressed Cas9 is not desirable. Most yeast will naturally lose the plasmid if grown without drug selection, preventing unwanted lingering Cas9 activity and freeing the KanR selection marker to be used for future genetic manipulations of the same strain.52.Using a sterile wood stick, pick 12 individual colonies (isolates) from the transformation plate and re-streak (for single colonies) onto YPD plates.53.Grow plates for 2 days at 30 °C until single colonies form.54.YPD plates, for single coloniesnot grow on G418-containing media.G418 plates  to check for plasmid loss. Yeast that has lost the pCASB plasmid should now From the YPD plates, pick one colony from each of the 12 isolates, and re-streak again onto:55.Grow plates for 2 days at 30 °C.56.not grow on G418.Identify isolates which did 57.From the corresponding isolate on the YPD plate, use a wood stick to choose a single colony. Use the same stick to both inoculate a fresh YPD plate and a glass culture tube containing 2 mL of YPD liquid media. Streak each isolate on the fresh YPD plates for single colonies.58.Grow plates for 2 days at 30 °C and liquid cultures overnight at 30 °C on a rotator.59.Harvest 1–1.5 mL of a saturated overnight yeast culture in a 1.7 mL microcentrifuge tube by centrifugation 1 min at 13,000 RPM in a benchtop microfuge.60.Discard supernatant, resuspend cell pellet in 1 mL of filtered water.61.Centrifuge cells 1 min at 13,000 RPM in a benchtop microfuge, aspirate off and discard supernatant.62.1× TE bufferYeast genomic prep buffer0.5 mm zirconia/silica beadsPhenol:chloroform:isoamyl alcoholTo each tube, add 200 µL of the following:63.Vortex for 5 min at 4 °C.64.Centrifuge 10 min at 13,000 RPM in a benchtop microfuge.65.Tubes will have visible layers; transfer the top layer (should be 250–300 µL) to a fresh 1.7 mL tube. Discard other layers.66.Precipitate DNA from the top layer by adding 750 µL (~3× volume) of 100% ethanol and 25 µL (1/10th volume) of 3M sodium acetate. Mix tubes by inverting several times.67.OPTIONAL STEP: Freeze tubes at −20 °C for 30 min to facilitate precipitation.68.Centrifuge cells at 13,000 RPM in a benchtop microfuge to pellet DNA.69.Wash DNA pellets as described in steps vi–ix of step 15.70.Resuspend air-dried pellets in desired amount of water (~300 µL will yield a DNA concentration that can be used directly as a template in the subsequent PCR reaction for screening).71.Design PCR primers flanking the region of interest, to amplify a region of at least 250 bp around the site of the mutation.72.Design a sequencing primer at least 100 bp upstream of the desired mutation (Sanger sequencing will begin >50 bp downstream of the sequencing primer).73.Using any standard PCR polymerase enzyme (high-fidelity not necessary), amplify the desired site.74.OPTIONAL STEP: Run a small portion of each PCR product (~4 µL of a 25 µL reaction) on a 1% agarose/TAE gel with 1 kb ladder to visualize a product of the correct size).75.Use a PCR purification kit to isolate the PCR products76.Send PCR product from each isolate for Sanger sequencing to confirm gene editing success.77.Using a wood stick, pick a single colony of each desired isolate and inoculate 2 mL of YPD liquid media in a glass culture tube.Grow overnight at 30 °C on a rotator.The next day, in a cryogenic tube, combine 800 μL of the saturated overnight culture with 200 μL of 80% glycerol. Store at −80 °C.After obtaining sequencing results and identifying successful mutants, the yeast strains are complete. If desired, make glycerol stocks and store at −80 °C (Glycerol stocks should be made of multiple isolates due to the chance of off-target effects. While off-target mutations as a result of CRISPR-Cas9 in yeast have been found to be rare , we recoE. coli transformation (The ormation  should yormation  step 3, Yeast transformation  should yWe recommend picking twelve yeast colonies to yield a successfully genetically edited yeast strain. When editing is successful, efficiency is greater than 50%; sequencing twelve colonies yielded sufficient isolates to save at least three for further experiments. Similar to what was reported by Ryan et al. , mutatio


TM version 15.2. The HCC spectral library supports quantification of 7,637 protein groups of triples 2-hour DIA analysis of HepG2 and discovering biological alteration. This study provides valuable resources for HCC cell lines and efficient DIA quantification on LC-Orbitrap platform, further help to explore the molecular mechanism and candidate therapeutic targets.Cell lines are extensively used tools, therefore a comprehensive proteomic overview of hepatocellular carcinoma (HCC) cell lines and an extensive spectral library for data independent acquisition (DIA) quantification are necessary. Here, we present the proteome of nine commonly used HCC cell lines covering 9,208 protein groups, and the HCC spectral library containing 253,921 precursors, 168,811 peptides and 10,098 protein groups. The proteomic overview reveals the heterogeneity between different cell lines, and the similarity in proliferation and metastasis characteristics and drug targets-expression with tumour tissues. The HCC spectral library generating consumed 108 hours’ runtime for data dependent acquisition (DDA) of 48 runs, 24 hours’ runtime for database searching by MaxQuant version 2.0.3.0, and 1 hour’ runtime for processing by Spectronaut  Hepatocellular carcinoma (HCC) represents approximately 90% of all primary liver cancer2. Studies on the proteomic landscape of HCC have advanced our knowledge at the molecular basis. Based on the label-free proteomic data of hepatocellular carcinoma patients of BCLC 0-A stage, we defined three subtypes, and found SOAT1 as a potential therapeutic target3. Gao et al. identified the tumour characteristics in HCC patients by isobaric tandem mass tags (TMT)-based proteomics, and identified two prognostic biomarkers, PYCR2 and ADH1A4. Cancer cell lines are the most extensively used model systems in tumour biology and development of therapeutics5, thus, a clear understanding at the proteome level may help us make better usage of HCC cell lines to analyse molecular mechanism and screen anti-tumour drugs. In 2014, Megger, D. et al. analysed the proteome of mixture of HepG2, Hep3B and SK-Hep-1 by label-free analysis and identified 2,757 protein groups and 13,744 peptides6. In 2020, the proteome of 375 cell lines of the Cancer Cell Line Encyclopedia were analysed using TMT-based proteomics, while did not cover the commonly used HCC cell lines including HepG2.2.15, PLC/PRF/5, MHCC97L, MHCC97H, HCCLM3 and HCCLM67. Recently, Goncalves, E. et al.8 identified 8,497 protein groups from 949 cell lines by data independent acquisition (DIA) method and identified 5,302 protein groups from Huh7 and 5,589 protein groups from Hep3B. However, whether HCC cell lines are identical or heterogeneous at proteome level was still not being revealed. Meanwhile, it remains unknown whether HCC cell lines are representative of primary HCC tumour at the proteome level. Thus, systemic exploration on the proteomic characteristics of HCC cell lines, and their comparison with primary HCC tumour is still necessary.Liver cancer ranks the sixth most common cause of cancer-related death world widely9. DIA quantification is based on the MS2 level through extraction of fragment ion chromatograms, which are less prone to be interfered than MS1 peak area10. DIA data can be analysed by the spectral library-based approach or the library-free approach. Both approaches could provide highly convergent identification and reliable quantification performance12. The spectral library is usually generated through data dependent acquisition (DDA) measurement of the peptides to be analysed by DIA13 and provides the precursor peptide-fragment connection14. Recently, it has been reported that the reproducibility, specificity, and accuracy of spectral library-based approach of DIA quantification is superior to DDA15. Thus, an HCC spectral library covering protein groups from HCC cell lines and primary tumour tissues could provide a valuable resource for DIA quantification, thus further support discovery of novel molecular mechanism and candidate therapeutic targets of HCC.DIA mass spectrometry is an emerging method for quantifying protein groups consistently and accurately across multiple samplesTM version 15.2  and a free software suite, DIA-NN version 1.8. In summary, our results obtained proteome and outstretched pathway overview of commonly used nine HCC cell lines, provided a valuable guide for the usage of these cell lines. The HCC spectral library generated was available for in-depth DIA quantification of HCC cell lines and could help to explore the molecular mechanism and candidate therapeutic targets of HCC. Our research provides a pipeline composed of sample choice, peptide pre-fractionation, spectral library generation and DIA quantification, which is universal and can be used for DIA quantification study in other tumours.Here, we present the proteomic overview of nine commonly used HCC cell lines covering 9,208 protein groups, and an in-depth HCC spectral library containing 253,921 precursors, 168,811peptides  and 10,098 protein groups. We revealed the poor consistency of proteome with transcriptome of these cell lines. Characteristic pathways of each cell line, and difference and similarity between HCC tissues were demonstrated. The HCC spectral library was used to analysis the differentially induced protein groups upon TGFB1 stimulation on HCCLM3 by Spectronaut16 (ssGSEA) were then implemented. For generation of the HCC spectral library, peptides of the HCC cell lines, or tumour tissues were mixed and fractionated by Hp-RP, respectively, and then analysed by LC-MS/MS   to six fractions and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using DDA mode Table . The gai2. All cell lines we used were proven to be free from bacteria, fungi and mycoplasma by PCR. To investigate the effect of TGFB1 on HCCLM3, HCCLM3 was stimulated with 10 ng/mL of TGFB1  for 48 hours.Cell lines were cultured in Dulbecco’s modified Eagle’s medium  containing 10% FBS , 100 U/mL penicillin and streptomycin mixture  in an incubator at 37 °C with 5% CO17. Each aliquot of 400 μg protein lysate was injected into a 30-kDa ultra-filter  followed by centrifugation at 14,000 g for 20 minutes at 25 °C. Then, 200 μL of UA solution  with 10 mM DTT was injected into each ultra-filter. All the ultra-filters were kept for 2 hours at 37 °C for denaturing and reduction reaction. The solution in ultra-filters was removed by centrifugation at 14,000 g for 15 minutes at 25 °C, then 100 μL UA solution with 50 mM iodoacetamide  was injected into each ultra-filter for alkylation. The ultra-filters were kept in dark for 30 minutes at 25 °C. After IAA incubation, the solution in ultra-filters was removed by centrifugation at 14,000 g for 10 minutes at 25 °C. Then, 200 μL of UA solution with 10 mM DTT was injected, and ultra-filters were kept at room temperature for another 15 minutes. The ultra-filters were centrifuged at 14,000 g for 15 minutes, and then washed with 200 μL UA solution once and 200 μL of ABC  three times by centrifugation at 14,000 g for 10 minutes at 25 °C. Then, 100 μL of ABC containing 8 μg trypsin  was injected into each ultra-filter. All ultra-filters were incubated at 37 °C for 12 hours, and then peptide mixtures were collected into new collecting tubes by centrifugation at 14,000 g for 15 minutes at 25 °C. All ultra-filters were washed twice times with 100 μL of ABC by centrifugation at 14,000 g for 15 minutes at 25 °C. The flow-through solution was collected into the same collecting tube. The peptide concentration was measured using a Nanodrop 2000C  at 280-nm absorbance. The peptide mixtures were acidized with 10 μL of 4% trifluoroacetic acid , heat-dried and then stored at −80 °C.Cell line samples were minced and lysed in Tissue Protein Extraction Reagent  followed by 3 minutes of ultrasonic  . The lysate was then centrifuged at 14,000 g for 15 minutes at 25 °C, and the supernatant was collected. The concentration of protein lysate was measured by the Bradford assay. The protein digestion was performed by filter-aided sample preparation (FASP)The peptide mixture was fractionated by Hp-RP with stepwise gradients manually. The C18 tip packed with 5 mg C18 reverse-phase media  was washed with 90 μL methanol  and then with 90 μL ammonia water (pH 10). Then, 50 μg peptide re-dissolved in 160 μL ammonia water (pH 10) was loaded. And the tip was centrifuged at 1,000 g for 8 minutes at 25 °C to remove the liquid followed by washed with 90 μL of ammonia water (pH 10). Peptides binding on the C18 reverse-phase packing was then sequentially eluted with different concentration of acetonitrile  in ammonia water (pH 10). These fractions were collected, and the 25%, 30%, and 50% fractions were mixed with 6%, 9%, 12%, respectively. The final six fractions were heat-dried stored at −80 °C.5 and the scan ranged from 300 to 1,400 m/z at a resolution of 120,000 and a maximum injection time of 50 ms. For the MS2 scan, a duty cycle of 3 s was set with the top-speed mode. Only spectra with a charge state of 2–6 were selected for fragmentation by higher-energy collision dissociation with a normalized collision energy of 35%. The MS2 spectra were acquired in the ion trap in rapid mode with an AGC target of 5,000 and a maximum injection time of 35 ms.For analysis of peptide mixture of each HCC cell line, the LC-MS/MS system consisted of a nanoflow high-performance liquid chromatograph (HPLC) instrument  coupled to a Orbitrap Fusion Lumos Tribrid MS mass spectrometer  with a nano-electrospray ion source . For data acquisition, each fraction of peptide mixture was re-dissolved in mobile phase A , 99.9% pure water), and 1/10 of which was loaded onto the trapping column  using mobile phase A and then separated on the analytical column  at a flow rate of 320 nL/min with following gradients: 0–8 min, 5–8% mobile phase B (0.1% FA in 99.9% acetonitrile); 8–58 min, 8–23% mobile phase B; 58–70 min, 23–32% mobile phase B; 70–71 min, 32–95% mobile phase B; and 71–80 min, 95% mobile phase B. The Orbitrap Fusion Lumos was set to the OT-IT mode. For the MS1 scan, the AGC target was 5 × 105. The maximum injection time for MS2 was set to auto. The normalized collision energy was set to 27, and the spectrum type was set to profile.For analysis of peptides of HCCLM3 and TGFB1-stimulated HCCLM3 using DIA, the LC-MS/MS system consisted of the EASY-nLC 1000 nanoflow LC  coupled to the Q-Exactive HF mass spectrometer . For data acquisition, 2 μg peptide mixtures re-dissolved in mobile phase A was loaded and separated on the analytical column at a flow rate of 500 nL/min with following gradients: 0~13 min, 6 ~ 10% mobile phase B (0.1% FA in 99.9% acetonitrile); 13 ~ 99 min, 10~23% mobile phase B; 99 ~ 120 min, 23 ~ 33% mobile phase B; 120~123 min, 33 ~ 90% mobile phase B; 123 ~135 min, 90% mobile phase B. For the MS1 scan, the AGC target value was set to 3E6, and the m/z scan ranged from 400 to 1,200 Da at a resolution of 120,000 and a maximum injection time of 80 ms. For the MS2 scan, the isolation window range was set to 26 m/z at resolution of 30,000, and the AGC target was set to 5 × 10The DDA raw files were searched against the human UniProt database  with MaxQuant version 2.0.3.0. The digestion mode was set to specific, and trypsin/P was chosen. Oxidation of methionine and acetylation of N-term of peptides were set as variable modification, and Carbamidomethyl of cysteine was set as fixed modification. False discovery rate (FDR) was set to 0.01 on both PSM and protein groups level. The max peptide mass range was set to 4,600 Da, and the peptide length range was set from 7 to 25, and the missed cleavage was set to 2. The MS/MS match tolerance was set to 20 ppm, and MS/MS de novo tolerance was set to 10 ppm. The proteinGroups.txt file generated by MaxQuant version 2.0.3.0 was then imported into Perseus v1.5.2.6 to extract the iBAQ value of each protein group of each cell line.TM version 15.2 against the HCC spectral library. Trypsin/P was chosen for digestion. Maximum intensity was used for intensity extraction of MS1 and MS2. Both correction factor for MS1 and MS2 mass tolerance were set to 1. XIC RT extraction window was set to dynamic and correction factor was set to 1. The calibration mode was set to automatic, and the iRT-RT regression was set to local (non-linear regression), and used Biognosys iRT Kit was chosen. Decoy method was set to Mutated, and decoy limit strategy were set to dynamic. Kernel density estimator was chosen to estimated p value. The precursor and protein group q-value cut-off was both set to 0.01. Proteotypic sequences and the MS2-level peak area were used for protein quantification and the same human UniProt database used for MaxQuant version 2.0.3.0 was set as the reference database. The top 3 precursors were used for peptide quantification, and the top 3 peptides were used for protein quantification. For DIA-NN version 1.8, Trypsin/P was chosen for digestion, and the miss cleavage site number was set to 2, and modifications including oxidation of methionine, acetylation of N-term and Carbamidomethyl of cysteine were chosen. Peptide length range was set to 7 to 25, and precursor charge range was set to 1 to 4, and precursor m/z range was set to 400 to 1,200, and fragment ion m/z was set to 200 to 2,000. The generated HCC spectral library was used as spectral. Single-pass mode neural network was chosen, and high accuracy was selected for quantification. RT-dependent cross-run normalization was selected. Precursor FDR was set to 1%.The DIA raw files were analysed by SpectronautPeptide mixture of cell lines and HCC tumour tissues was fractionated by Hp-RP with stepwise gradients manually. The C18 tip packed with 5 mg C18 reverse-phase media  was washed with 90 μL methanol  and then with 90 μL ammonia water (pH 10). Then, 50 μg peptides dissolved in 160 μL ammonia water (pH 10) was loaded. And the tip was centrifuged at 1,000 g for 8 min at 25 °C to remove the liquid followed by washed with 90 μL of ammonia water (pH 10). Peptides were then sequentially eluted with 8 different concentrations of acetonitrile  in ammonia water (pH 10). These fractions were collected, heat-dried and stored at −80 °C.The LC-MS/MS detection system consisted of the EASY-nLC 1000 nanoflow LC coupled to the Q-Exactive HF mass spectrometer . For data acquisition, 1/8 of each of the Hp-RP fractions re-dissolved in mobile phase A was loaded and separated with the analytical column at a flow rate of 500 nL/min with following gradients: 0~13 min, 7 ~ 13% mobile phase B (0.1% FA in 99.9% acetonitrile); 13 ~ 99 min, 13~28% mobile phase B; 99 ~ 120 min, 28 ~ 42% mobile phase B; 120~123 min, 42 ~ 95% mobile phase B; 123 ~135 min, 95% mobile phase B. For the MS1 scan, the target value was set to 3E6 and the m/z scan ranged from 300 to 1,400 Da at a resolution of 120,000 and a maximum injection time of 80 ms. Only spectra with charge states of 2~ 6 were selected for fragmentation with a normalized collision energy of 27%. Precursor ions with top 20 intensities were selected for fragmentation. For the MS2 scan, the AGC target value was 5E4 and the resolution was 15,000 with a maximum injection time of 45 ms. The iRT peptide standards  were spiked into all runs of spectral library generation.The obtained DDA raw files were searched against the human UniProt database  by MaxQuant version 2.0.3.0. The digestion mode was set to specific, and trypsin/P was chosen. Oxidation of methionine and acetylation of N-term of peptides were set as variable modification, and Carbamidomethyl of cysteine was set as fixed modification. FDR was set to 0.01 on both PSM and protein groups level. The max peptide mass range was set to 4,600 Da, and the peptide length range was set from 7 to 25, and the missed cleavage was set to 2. The MS/MS match tolerance was set to 20 ppm, and MS/MS de novo tolerance was set to 10 ppm.TM version 15.2 to generate the HCC spectral library. The missed cleavage site number for peptide was set to 2. The m/z range was set as 400 to 1,200 Da, and the best N fragments per peptide was set as 3 to 6. B and y fragments were chosen, and modifications including oxidation of methionine, acetylation of N-term of peptides and Carbamidomethyl of cysteine was kept during library generation. The empirical iRT database was set as the iRT reference, and the minimum square cutoff was set to 0.8. FDR was set to 0.01 on both precursor and protein level. For calibration and main search, the tolerance was set to dynamic.The search results of MaxQuant version 2.0.3.0 were imported into Spectronaut18 was used for data normalization and quantile function was used. Differently expressed protein groups between cell lines or tissues were identified by R package limma (v3.46.0)19. GO analysis was implemented using R package clusterProfiler (v3.18.1)20 using enricher function. The ssGSEA analysis was implemented using R package GSVA (v3.18.2)21 using GSVA function and ssGSEA method. Gene sets recorded in the Molecular Signatures Database (MSigDB) v7.5.122 was used as reference gene sets for all the analysis. Protein groups whose fold-change value was greater than 1.5 and adjust p-value was less than 0.01 was chosen as differently expressed protein groups. All the analysis was operated on R software (v4.0.3).The R package NormalyzerDE (v1.8.0)23 with the dataset identifier PXD03664324. The 48 DDA raw mass spectrometry data (.raw) for library generation had been deposited to the ProteomeXchange Consortium via PRIDE with the dataset identifier PXD03502825.The 162 DDA raw mass spectrometry data (.raw) had been deposited to the ProteomeXchange Consortium via PRIDE26 and QC reports generated by DIALib-QC, the search result of DDA raw files of HCC cell lines and spectral library generation by MaxQuant version 2.0.3.0, DIA raw mass spectrometry data (.raw) and all search results of HepG2, HCCLM3 and TGFB1 stimulated HCCLM3 by DIA-NN version 1.8 and SpectronautTM version 15.2 had been deposited to the ProteomeXchange Consortium via PRIDE with the dataset identifier PXD03715927.The HCC spectral library at Figshareet al. and Nusinow, D.P. et al., our proteomic data still uniquely identified 1,800, 896 and 911 protein groups from HepG2, Hep3B and Huh7  with their transcriptome, including SOAT1 and NPC1, two core molecules for cholesterol metabolism30, and AFP, an important biomarker of HCC31, and SRC, an important tyrosine protein kinase for cancer proliferation and metastasis32. However, we also found that 9.4% of protein groups showed negative correlation (Pearson correlation coefficient < −0.4) with their transcriptome, including DOCK6, a molecule which could promote chemo- and radio-resistance in cancer33, and YTHDF1, a key regulator of m6A methylation34  and principal component 2 (16.4%): MHCC97L, MHCC97H, HCCLM3 and HCCLM6 almost overlapped, and were far from other cell lines of principal component 1; Hep3B and PLC/PRF/5 displayed the maximum distance of principal component 2  and 10,098 protein groups. Evaluation by DIALib-QC41 showed a high quality of the HCC spectral library. About 14.5%  protein groups were exclusively provided by DDA files of HCC cell lines, while 17.7%  provided by tumour tissue DDA files only  of nine HCC cell lines. Then, for combinations with a specific number, we select the combination with the max covered protein groups number. We found that the covered protein groups number of combination three HCC cell lines, HCCLM3, HepG2 and PLC/PRF/5, could cover 97%  of protein groups covered by all the nine HCC cell lines Fig. . Thus, pnly Fig. . About 9nly Fig. . Precursnly Fig. . Proteinnly Fig. . Statistnly Fig. . Comparedes Fig. .Fig. 5Ov44, and 94.2%  of protein groups and 73.4%  of peptides were quantified three times. Meanwhile, 6,845 protein groups and 73,599 peptides could be identified from the same raw files by SpectronautTM version 15.2, and 95.7%  of protein groups and 69.6%  were quantified three times . We observed down-regulation of CDH1, the main initiation signals of EMT45, and up-regulation of THBS146 and CDH647, two proteins whose up-regulation could represent the activation of EMT by both DIA-NN version 1.8 and SpectronautTM version 15.2  higher than S-I or S-II (Fig. We could identify 7,637 protein groups and 82,243 peptides in triples 2-hour DIA analysis of HepG2 peptides using the HCC spectral library and DIA-NN version 1.8 with optimized parameters for LC-MS/MSmes Fig. . High qumes Fig.  was reve5.2 Fig. . In 121 5.2 Fig. . Based o-II Fig. . The 101-II Fig. . These r


Herein, we demonstrate low-defect and sodium-enriched nickel hexacyanoferrate nanocrystals synthesized by a facile low-speed co-precipitation technique assisted by a chelating agent to overcome these problems. As a consequence, the prepared high-quality nickel hexacyanoferrate (HQ-NiHCF) exhibited a high specific capacity of 80 mA h g−1 at 15 mA g−1 , maintaining a notable cycling stability (78 mA h g−1 at 170 mA g−1 current density) without noticeable fading in capacity retention after 1200 cycles. This low-speed synthesis strategy for PBA-based electrode materials could be also extended to other energy storage materials to fabricate high-performance rechargeable batteries.Prussian blue analogs (PBAs) are attractive cathode candidates for high energy density, including long life-cycle rechargeable batteries, due to their non-toxicity, facile synthesis techniques and low cost. Nevertheless, traditionally synthesized PBAs tend to have a flawed crystal structure with a large amount of [Fe(CN) A low-speed synthesis strategy was designed to fabricate Prussian blue analog based electrode materials for high-performance rechargeable batteries. Latterly, the cost of lithium has been escalating rapidly, owing to the irregular circulation of worldwide lithium reserves.6 To address this challenge, sodium-ion batteries (SIBs) have become a progressively more appealing option to LIBs in EESs because of their low cost and elongated lifecycle.7,8 Besides, the chemical attributes of sodium show similar properties and comparing its source, sodium has more plentiful sources than lithium, and thus different types of cathode materials have been contemplated for SIBs.Lithium-ion batteries (LIBs) are well known in application and are gaining significant interest for energy-storage applications.9–11 Because of their moderately low capacity value,12,13 including poor rate performance,14 and competition with their anode counterparts,15–17 cathode materials are of immense significance in hastening the commercialization of SIBs. However, a significant distortion of and strain on the crystal lattice may be induced due to the broad radius of Na+ during the incorporation phase, and this also poses challenges in finding relevant electrode materials for SIBs.18–21 Some cathodes, P2–Na0.67Cox1−TixO2,22 Na3V2(PO4)3,23,24 NaTi2(PO4)3,25,26 and Na0.7[Mnx1−Lix]Oy2+,27 demonstrated significant performance in terms of variable capacity, including rate efficiency, but due to certain phase transitions, and large changes in volume during cycling, they have failed to meet long cycling-life requirements. Prussian blue analogs (PBAs), a kind of metal–organic framework with the formula AxMy[M′(CN)6]z·nH2O, where A refers to an alkaline metal and M, M′ refer to transition metals, have been widely studied as attractive cathodes for SIBs in the past decade due to their high theoretical capacity and long-cycle life. As a significant cathode source for Na-ion batteries, PBAs have been given a lot of attention, not only for their rigid open framework  but also since they present a volume difference between Na+ insertion and extraction.28–31 Also, their facile synthetic procedure makes them suitable for large-scale applications, besides their nontoxicity and low cost, and they have become more widespread.32,33 The open crystal structure could effectively tolerate flexibility in the lattice volume and facilitate the passage of Na+.34–40In recent years, metal–organic frameworks (MOFs) have been prevalent in considering this purpose.34,41,42 Earlier it was proved that the electrochemical performance of PBAs is firmly identified with their inherent crystal structure.43,44 In our research group, synthesized CeHCF as a cathode material performed with a specific capacity of ∼60 mA h g−1 at 0.25C (with capacity retention of about ∼80% at 8.3C) in LIBs, and similarly ∼55 mA h g−1 at 0.25C (with capacity retention of nearly 55% at 8.3C) in SIBs, due to an open network with large lattice spaces.45 Additionally, recently some innovative work has reported on PBAs based upon long-term cyclability and improved electrochemical kinetics as well efficiency.46–49 Guo's group revealed that the suitability of K0.09Ni[Fe(CN)6]0.71·6H2O as a cathode for SIBs was attributed to its zero-strain characteristics, where there was less than 1% change in lattice parameter distortion during the Na+ insertion/extraction phase50 and it achieved excellent cycling stability and a highly stable structure. Jiang's group also reported remarkable cycling stability from synthesized Na1.014Ni[Fe(CN)6]0.818·3.53H2O via a simple co-precipitation method, but the specific capacity was as low as 68 mA h g−1.51 Nevertheless, in practical applications, the tested AxNi[Fe(CN)6]y cathodes could not carry more sodium content and presented a high number of defects in crystallization. To increase the content of sodium in the framework, Liu's group firstly came up with the idea where different amounts of sodium citrate were employed in the synthesis process as a cathode material for SIBs.52 Recently, another study has been done on highly stable monoclinic sodium-rich nickel hexacyanoferrate by introducing tri-sodium citrate to control its crystallization.53 Henceforth, when designing a new cathode, it is important that the sodium content should be high with fine crystallization and meet the requirements of applications, such as temperature adaptability, steadiness and more. The preparation of nickel–iron Prussian blue analogs (Ni–Fe PBAs) has often shown a significant amount of vacancies within the crystal structure due to quick precipitation via the conventional synthesis process.54However, the theoretical value of specific capacity for PBAs is still unreachable, apart from their poor cycling stability and low coulombic efficiency, due to defects and crystal water in the framework of PBAs.6]4− openings in the crystal structure. For these reasons, the ion storage ability was significantly improved and there were adequate transportation behaviors for Na+ and e−, as well as effective preservation of the integrity of the crystal structure during cycling. Hence, in terms of its good specific capacity of nearly 80 mA h g−1, which is very close to the theoretical value, remarkable cycling stability of 78 mA h g−1 around 1200 cycles, and superb rate capability of 62 mA h g−1 at 10C with high coulombic efficiency, it has shown persuasive electrochemical performance as a cathode for SIBs. For comparison purposes, low-quality nickel hexacyanoferrate (LQ-NiHCF) was also synthesized by the conventional procedure without any additive, and the relative performances are offered in our report.In this research, we have established low-defect and sodium-rich nickel hexacyanoferrate nanocrystals synthesized through a simple co-precipitation mechanism with the assistance of a chelating agent called tri-sodium citrate. Because of the moderate development process for crystallization, HQ-NiHCF displayed a small zeolite water content with few  happened immediately, later causing a large amount of Fe(CN)6 defects, and synchronized water molecules in the crystal framework. However, in the chelating agent-assisted co-precipitation method, tri-sodium citrate (Na3C6H5O7·2H2O) was co-precipitated with a solution of sodium ferrocyanide, which basically suppressed the rate of growth and achieved a superbly crystallized Na2Ni[Fe(CN)6] sample. The formulae of LQ-NiHCF PBAs and HQ-NiHCF PBAs were determined to be Na0.79Ni[Fe(CN)6]0.74, and Na1.67Ni[Fe(CN)6]0.87, corresponding to 26% and 13% defect contents, respectively.According to the XRD results in 58,59 There was less weight loss for HQ-NiHCF because of its synthesis method where water coordination was reduced. Furthermore, the final material degradation for both materials at high temperature (>500 °C) showed that the newly fabricated samples were almost 10% more efficient than the generally synthesized LQ-NiHCF. The chemical bonds were examined in the structures and the presence of contamination was determined using Fourier-transform infrared (FT-IR) spectra.60 From the position of the absorption peaks for these two samples in −1 corresponded to the elongating vibrations of the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.tbd" format="PBM" resolution="300" x-size="17" y-size="11" xml:space="preserve">0000000000000000000000000000000000000000000000000000111111111111111000000000000000000011111111111111100000000000000000001111111111111110000000000000000000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="23.636364pt" height="16.000000pt" viewBox="0 0 23.636364 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N ligands coordinated with Fe2+ and Ni2+.61 The absorption points at 1622 cm−1 and the area between 3300 and 3500 cm−1 could be ascribed to the O–H bending vibrations and stretching O–H modes, respectively, due to interstitial water.62The amount of water content and the material degradation process were studied by TGA, as shown in −1 for HQ-NiHCF and the peaks at 2112 and 2148 cm−1 for LQ-NiHCF, are attributed to the vibrations of the cyanide group and show that Fe and Ni ions exhibited divalent ions and almost two Na ions existed in the materials (FeII–CN–NiII).63 According to the increased intensity of the peaks, it can be speculated that the number of Na ions increased with the slow reaction rate in the synthesis of HQ-NiHCF.Raman spectroscopy was used to explain the change in valance state of iron in the compound, as shown in 44 In contrast, due to the deceleration and segregation of crystal growth, HQ-NiHCF in 34 The TEM images are shown in + and e− transportation in the electrochemical reaction phase.SEM was used to explain the size of the nanoparticles of the as-prepared samples. Here, the different magnification images of the LQ-NiHCF sample shown in 2+ cations responded to [Fe(CN)6]4− anions in the aqueous solution. Here, the nucleation and growth stage happened instantly, and therefore the particles accumulated together, with large amounts of [Fe(CN)6]4− flaws also corresponding to water inside the framework. Citrate, by playing the main role in controlling the speed of agitation and segregating molecules, helped form perfectly shaped nanocubes with prominently inhibited [Fe(CN)6]4− defects. In the chelating agent-assisted method, the Ni2+ ions firstly linked with citrate ions and formed a nickel-citrate chelate, and afterward co-precipitated in aqueous solution with hexacyanoferrate ions. The nickel-citrate chelate served as a buffer for gradually releasing Ni2+ ions to interact with hexacyanoferrate ions to form initial nucleuses. Due to the high multifaceted potential of citrate ions and their carrying a negative charge, they accumulated uniformly on the nucleus surfaces, thereby overturning the rate of growth and suppressing the aggregation of crystals. Eventually, the slow growth and constant release of Ni ions helped these nucleuses to develop well-formed monodispersed Prussian blue nanocubes with high crystallinity.Based on the aforementioned experimental data, a probable mechanism for the growth of HQ-NiHCF nanocubes is suggested and shown in −1 within 2.0 and 4.2 V (vs. Na+/Na) where a pair of distinct reduction/oxidation peaks appeared at 3.47/3.14 V, correlating with the balance potential of Fe2+/Fe3+;64 Ni2+ remains electrochemically inert for the sodium reaction.31,65 From −1, respectively, at a current density of 15 mA g−1, which is consistent with a coulombic efficiency of almost 99% while LQ-NiHCF delivers lower charge/discharge capacities of 67.43 and 66.55 mA h g−1, respectively. Here, Na+ storage sites were utilized more in HQ-NiHCF, and thus it can be ascribed to the high crystallinity and reduced water content inside the crystal framework together with a lower number of [Fe(CN)6] vacancies.34,66 In the fast charge/discharge process, HQ-NiHCF achieves an enhanced rate capability. −1 from 85 to 850 mA g−1, which remained incredibly stable without any obvious capacity loss over 90 cycles once the current changed back to 85 mA g−1. However, LQ-NiHCF could not hold any capacity and only maintained ∼47 mA h g−1 at 425 mA g−1. In practical applications, the cycling performance and the coulombic efficiency play an endlessly significant role.67 Moreover, HQ-NiHCF showed a dramatically improved cycling performance compared to LQ-NiHCF. −1 and LQ-NiHCF was also tested for comparison. Although LQ-NiHCF performed with excellent cyclic stability but a low discharge capacity of 57 mA h g−1, it exhibited a declining drift with a very poor coulombic efficiency. Remarkably, the HQ-NiHCF cathode carried a high discharge capacity of 78 mA h g−1 and capacity retention of ∼98% after 1200 cycles. While charging, a side reaction happened with zeolite water, and therefore the coulombic efficiency was a little lower for only the first few cycles, but later on, it jumped to more than 98%, which is considerably more than that of LQ-NiHCF, as shown in −1 at 10C but LQ-NiHCF retained only 13.7 mA h g−1.Electrochemical performances were studied by galvanostatic charge/discharge and cyclic voltammetry (CV). Such an improvement in the efficiency of Na-storage could be associated with Na-ion intercalation habits, the lower amount of water and the resulting improved crystallinity of HQ-NiHCF. These findings specify that the physical steadiness of the PBAs can be considerably improved by enriching the quality of the crystal structure. The abovementioned finding also verifies that the advent of PBAs with high-quality crystal structures will lead to progress in their applications.6]4− vacancies, including water molecules, in the framework, the HQ-NiHCF PBA exhibited a high specific capacity of 80 mA h g−1 at 15 mA g−1, and the cycling stability after 1200 cycles still remained at 99%, and 78 mA h g−1 of the capacity delivered at a current density of 170 mA g−1 and an excellent rate capability of 63 mA h g−1 at 850 mA g−1. Although it showed a lower specific capacity, considering its excellent electrochemical performance, simple method of synthesis and low cost, the application of HQ-NiHCF is a promising cathode material for SIBs in the large-scale energy storage systems.In summary, we have effectively fabricated low-defect and Na-rich nickel hexacyanoferrate nano-particles by introducing a chelating agent. Here, tri-sodium citrate acted to slow down the crystallization speed and achieved perfectly shaped crystalline particles. Due to the suppressed amount of [Fe(CN)There are no conflicts to declare.


Z1456467176 exhibited a stable and significant P2X7R inhibitory effect. Importantly, in MSU crystal-induced gout, the presence and involvement of ATP were confirmed. Z1456467176 blocked ATP-induced activation of the NLRP3-caspase-1-IL-1β pathway and exerted promising effects in reducing gouty joint inflammation in rats. In addition, molecular docking and molecular dynamics simulation studies showed that the P27XR protein conformation was remodeled by Z1456467176 binding. Collectively, our results provide a potent P2X7R allosteric inhibitor that facilitates the remission of MSU crystal-induced gout inflammation by inhibiting NLRP3 inflammasome activation, suggesting that allosteric inhibition of P2X7R represents a new direction in gout treatment.NLRP3 inflammasome activation is a central process in initiating gout flares. The unique conformational rearrangement of the P2X7 receptor (P2X7R) upon ATP binding is critical for the activation of the NLRP3 inflammasome. However, studies on allosteric modulation of P2X7R in gout treatment are limited. Here, we aimed to investigate the therapeutic implications of targeting P2X7R in gout by designing a P2X7R allosteric inhibitor and validating the inhibitory function on NLRP3 inflammasome activation. Through virtual screening, we identified Z1456467176 (N-{3-[(2-aminoethyl) sulfamoyl] phenyl}-2-methyl-3-[3-(trifluoromethyl) phenyl] propanamide hydrochloride) bound to the drug-binding pocket as a potential antagonist of P2X7R. In functional assays, ATP- or BzATP-induced P2X7R function was assessed  Gout is a common cyclical, recurrent and transient “autoinflammatory” disease in adults, characterized by rapidly developing monoarticular synovitis of peripheral joints, manifesting as joint redness, swelling, pain, and restricted movement . For theP2X7R (hP2X7R) gene polymorphisms and gout susceptibility was confirmed in our previous study (Purinergic receptor P2X7 (P2X7R) is an ATP-gated ion channel receptor that regulates NLRP3 inflammasome activation by inducing intracellular and extracellular ion flux . The assus study . In addius study , suggestus study . As an aus study . Howeverus study , and P2Xus study . This cous study , which iAlthough the involvement of ATP-P2X7R in the pathogenesis of gout is gradually being recognized, the evaluation of P2X7R for the treatment of gout is still limited. In the present study, we demonstrated that allosteric regulation of P2X7R to inhibit the NLRP3 inflammasome effectively alleviates gout inflammation, providing theoretical and data support for P2X7R as a therapeutic target for gout.Docking studies were performed using the Glide module of Schrodinger software to initiate virtual screening of P2X7R antagonists in a commercial compound library . The crystal of PDB number 5u1x was identified as the docking model. A small-molecule Z1456467176 (N-{3-[(2-aminoethyl) sulfamoyl] phenyl}-2-methyl-3-[3-(trifluoromethyl) phenyl] propanamide hydrochloride) was obtained that binds to the drug antagonist pocket. The purity of the compound was verified by mass spectrometry to be over 90% .2 atmosphere for 24 h (2 × 105 cells/ml). Transfection was performed with a lentivirus overexpressing hP2X7R (Hanheng Biotechnology Co (China)) or empty lentivirus in the presence of polybrene. On the following day, Z1456467176 (10 μM) or equivalent amounts of DMSO were incubated with HEK-293T cells overexpressing hP2X7R for 30 min. Cells were suspended in 0.5 ml of HEPES buffered medium followed by the addition of 25 μM ethidium bromide  and 1 mM ATP (Sigma A6559). Cells were collected using a CytoFLEX flow cytometer, counting 1,000 cells each time. The average fluorescence intensity of each sample was read using CytExpert. The IC50 value of Z1456467176 (concentrations ranging from 1 nM to 100 μM) was obtained from concentration-response curves for the EB uptake assay (HEK-293T cells were maintained in DMEM (Thermo 11965118) supplemented with 10% fetal bovine serum  and incubated at 37°C under a 5% COke assay .5 cells/ml) were cultured in RPMI 1640 medium (HyClone AG29775810) containing 10% FBS and stimulated overnight with 100 ng/ml phorbol 12-myristate 13-acetate  to obtain THP-1-derived macrophages. Bone marrow cells were isolated and collected from bilateral femurs and tibiae of 6-week-old C57BL/6J mice. Mouse bone marrow-derived macrophages (BMDMs) were obtained by culturing for 1 week in DMEM (2 × 106 cells/ml) supplemented with 10% FBS, 20% L929 cell supernatant, and penicillin/streptomycin. Peripheral blood mononuclear cells (PBMCs) from stable gout patients were isolated with lymphocyte isolate and erythrocyte lysate  and inoculated in RPMI 1640 medium (2 × 106 cells/ml) and cultured overnight. The supernatant was removed the next day to obtain crude extracted macrophages.THP-1 cells (2 × 10The above cells were then activated with 50 ng/ml LPS (Invitrogen LPS-EK) for 3 h. Furthermore, Z1456467176 at different concentrations was incubated for 30 min before 100 μM BzATP (Sigma B6396) treatment for 15–20 min 10 μM pyroptosis inhibitor  was incubated for 30 min before 100 μg/ml MSU crystal treatment for 4 h. MSU crystals were obtained by dissolving uric acid  in 500 ml of double-distilled water containing NaOH (0.01 M) at 70°C, and the pH was maintained at 8.9. The solution was filtered through a 0.22 μM filter and stored overnight at room temperature with continuous stirring. After centrifugation, the supernatant was discarded, and the precipitate was obtained and dried. The crystals were autoclaved and resuspended in sterile PBS .ELISA was performed to measure IL-1β, IL-6, and TNF-α levels in cell culture supernatants and IL-1β levels in rat serum according to the manufacturer’s instructions . Rat liver and kidney functions were detected by Jian Cheng C0009-2-1, C0010-2-1, C011-2-1, and C013-2-1 kits (China).IL-1β and Actβ in the peripheral whole blood cells of rats. Samples were processed with a total RNA extraction kit (Solarbio R4161-02) and a cDNA synthesis kit (BIOER BSB40M1) according to the manufacturer’s instructions. qPCR was conducted using SYBR Green PCR Master Mix (Muma A4004M). The primer sequences for the rat IL-1β gene were F-seq (5′-3′): TGGCAACTGTCCCTGAACTC and R-seq (5′-3′): AAGGGCTTGGAAGCAATCCTTA. The primer sequences for the rat Actβ gene were F-seq (5′-3′): ACCCGCCACCAGTTCGC and R-seq (5′-3′): CACGATGGAGGGGAAGACG.qPCR was performed to detect the gene expression of Western blotting was performed to detect protein levels in PBMC lysates from gout patients and in joint grinds from rat gout models. Segmented constant voltage was chosen for electrophoresis, and the concentrated gel was run at a constant voltage of 80 V. After running out of the concentrated gel, the voltage was increased to 120 V until bromophenol blue reached the bottom of the gel. The membrane was rotated at a constant flow of 200 mA in an ice bath and then blocked with 5% skim milk powder for 1 h. The membrane was incubated overnight at 4°C with the following primary antibodies: IL-1β , caspase-1 , NLRP3 , N-GSDMD  and β-actin . HRP, goat anti-rabbit IgG, and goat anti-mouse IgG  were incubated for 1 h at room temperature and then developed.Sprague–Dawley (SD) male rats weighing 200 g were purchased from the Laboratory Animal Center of the First Hospital of USTC and housed in a clean environment free of pathogens. A rat model of gout was established according to the classical modeling method (Coderre method ) and ranSerum was collected from gout model rats as well as from patients with acute phase gout and normal population. LDH levels were quantified using the LDH Cytotoxicity Assay Kit (Jian Cheng A020-2) according to the manufacturer’s instructions. Patients with acute-phase gout were defined as positive joint redness, swelling, and pressure pain in the metatarsophalangeal or ankle or knee joint; elevated joint skin temperature; history of hyperuricemia or gout; and duration of this joint symptom for 2 days and less. The people in the control group were age- and sex-matched to the gout group and had no local or systemic inflammatory symptoms.ATP (%) values in cell cultures of PBMCs from rats with MSU crystal-induced gouty arthritis and patients with stable gout stimulated with MSU crystals (100 μg/ml) were determined using a luminescent ATP assay kit (Abcam ab113849) according to the manufacturer’s instructions.Rats were anesthetized with intraperitoneal injection of chloral hydrate  in a gouty arthritis model, and magnetic resonance imaging (MRI) examination was performed after the rats had decreased muscle strength and stabilized respiration. MRI of the ankle joint was performed using a 3.0T MR scanner (GE Discover 750 W) and an eight-channel mouse coil (Medciol MS80-3T) with the following sequence parameters: T2 FLAIR; TE: 101/Ef; TR: 3,800 ms; EC:1/1 50 Hz; slice thickness: 2 mm; FoV = 12*12 cm; matrix size = 256*256.The isolated rat ankles were fixed in tissue fixative  for 24 h, and subsequently placed in decalcification solution  with shaking for 4-6 weeks to ensure adequate decalcification. The endpoint of decalcification is indeed considered smooth penetration of the needle through the entire bone tissue. After the washing, dehydration, and decalcification steps, the ankle tissue was fixed in paraffin and sectioned for HE staining. Cell count quantification was performed using ImageJ software (NIH).https://pubchem.ncbi.nlm.nih.gov), and Chem3D16 was applied for structure optimization. Global docking was used, with the docking box wrapping the entire 5u1x protein and other parameters kept as default. Molecular dynamics simulations of 100 ns were performed, and the binding free energy of the protein and ligand was calculated using g_mmpbsa. The conformation with the lowest energy in the molecular docking results was selected as the initial structure for molecular dynamics. Molecular dynamics (MD) simulations were performed using Gromacs 2019.4 software at constant temperature and pressure as well as periodic boundary conditions. The Amber99sb-ildn force field, TIP3P water model, was applied. The force field parameters for small molecules were generated by the acpype.py script in AmberTools. During the MD simulations, the hydrogen bonds involved were constrained using the LINCS algorithm with an integration step of 2 fs. The electrostatic interactions were calculated using the particle‒mesh Ewald (PME) method with the cutoff value set to 1.2 nm. The nonbonded interaction cutoff value was set to 10 Å. The simulation temperature was controlled to 300 K using the V-rescale temperature coupling method, and the NVT and NPT equilibrium simulations were performed at 300 K for 100 ps. Finally, 100 ns of finished MD simulations were performed for the protein and protein-ligand complex systems, and the pure protein system was used as a control group for the experiments. The GROMACS embedded program and PyMOL 2.4 were used to visualize the simulation results.The trimeric conformation of 5u1x was used as the target protein. Molecular docking was performed using AutoDock 4.2 software. The structure of Z1456467176 was downloaded from the database -041) and Medical Research Ethics Committee(2021 KY No.162) of the First Hospital of the University of Science and Technology of China , respectively. All persons gave their informed consent before their inclusion in the study.t tests and one-way analysis of variance (ANOVA) were used to compare differences between two groups and multiple groups, respectively. Significant differences revealed by one-way ANOVA were validated by Dunnett’s t test. Statistical significance was set at p < 0.05. All bar graphs were plotted using GraphPad Prism 8.0 software (GraphPad Software).Data were analyzed using IBM SPSS Statistics for Windows, version 17.0 (IBM). Quantitative data were tested for normality. Independent sample 50: 3.416 μM) was able to inhibit ATP-induced P2X7R pore formation, exhibiting a potential P2X7R inhibitory effect.P2X7R is an ATP-gated channel receptor. Pore formation upon sustained stimulation of ATP allows the influx of extracellular macromolecules. To investigate the inhibitory effect of Z1456467176 on ATP-induced P2X7R pore formation, we constructed a lentivirus overexpressing hP2X7R to transfect HEK-293T cells. ATP-induced uptake of EB by HEK-293T cells was assayed in the presence or absence of Z1456467176. Flow cytometric analysis showed that the uptake of EB by HEK-293T cells overexpressing hP2X7R was significantly increased upon ATP stimulation, whereas Z1456467176 treatment inhibited the uptake of EB by the cells . This fiin vitro macrophage system including THP-1 cells and BMDMs. BzATP is a synthetic ATP analog that acts only on P2X1R and P2X7R in the P2XR family and exhibits a more potent and specific induction of P2X7R activation than ATP ‒ligand (Z1456467176), providing data on receptor and ligand affinity. During the 100 ns kinetic simulations, the RMSD values of the protein‒ligand systems were kept in equilibrium, with mean values of 0.3664 ± 0.0137 nm and 0.3413 ± 0.016 nm, respectively, suggesting that the systems reached stability . The radTo determine whether Z1456467176 binding resulted in structural rearrangement of the P2X7R protein, we compared the secondary structures of the P2X7R protein and protein‒ligand. The results showed that the open region at the upper end and the TM region at the lower end of P2X7R expanded outwards after ligand binding compared with the pure protein, leading to a change in secondary structure , which wOn this basis, we further analyzed the protein‒ligand binding data. The total binding free energy of the protein‒ligand is -60.493 kJ/mol, of which the van der Waal energy is -196.508 kJ/mol; the electrostatic energy is -56.783 kJ/mol; the polar solvation energy is 214.035 kJ/mol; and the SASA energy is -21.236 kJ/mol. The binding free energy of the two shows that the van der Waals force and the electrostatic force are dominant, which is beneficial to the combination. The contribution of amino acid residues at different positions to the binding free energy was obtained by decomposing the binding free energy .Protein‒ligand binding pattern analysis showed that the pi-cation electrostatic interaction exists between the amino groups of Arg316 (C), Asn100 (A), Asn100 (B), Asn100 (C) and the ligand, and the interaction distance is 5.0 Å, 4.7 Å, 4.8 Å, 5.5 Å. In addition, there is an pi-anion electrostatic interaction between the F atom of the ligand and Phe102 (A) with a distance of 5.2 Å. Pi-alkyl hydrophobic interactions exist between Val61 (B), Ile319 (A), Leu320 (B) and the ligand, with distances of 4.0 Å, 6.0 Å, and 3.6 Å, respectively. Hydrogen bonds are formed between Asn100 (A) and the ligand at distances of 1.8 Å and 2.0 Å, respectively .vivo and in vitro studies, Z1456467176 inhibited ATP- or BzATP-induced P2X7R activation to block the NLRP3-caspase-1-IL-1β pathway, alleviating the clinical manifestations of gouty arthritis and confirming P2X7R as an effective therapeutic target. Importantly, we herein found that the binding of Z1456467176 induces a conformational change in the P2X7R protein, suggesting that allosteric inhibition of P2X7R is a promising strategy for gout treatment.In this study, we identified a potent P2X7R antagonist through virtual screening and functional validation. In In the past, several competitive and noncompetitive antagonists against P2X7R have been developed. Oxidized ATP is an irreversible competitive P2X7R antagonist but simultaneously exhibits nonspecific modulation of NF-κB signaling pathway expression . NotablyP2X7R gene polymorphisms and gout susceptibility (P2X7R gene polymorphisms in gout patients imply a more sensitive ATP response in these populations, as evidenced by the rapid opening function of P2X7R channels and the massive release of IL-1β (Currently, we have witnessed the clinical application of several P2X7R antagonists. However, the expected therapeutic effect has not been achieved in the treatment of inflammation-associated diseases , suggesttibility . Functioof IL-1β . As illuof IL-1β , NS-SNPsof IL-1β . Therefoof IL-1β . Thus, sin vivo, although it did not completely inhibit BzATP-induced IL-1β secretion in vitro. IL-1β is the main proinflammatory cytokine in initiating gout; however, the IL-1β-mediated inflammatory response is also critical in resolving infections to some extent, contributing to the body’s timely response to danger and self-recovery. In tissue recovery after injury, low levels of IL-1β and TNF-α promote wound healing by increasing growth factor production (Z1456467176 achieved significant therapeutic effects in gout rats oduction . In infloduction . In addioduction . Thus, toduction . TherefoIn conclusion, the study identified a potential P2X7R antagonist, Z1456467176, that can inhibit ATP-induced NLRP3-caspase-1-IL-1β pathway activation by allosterically modulating P2X7R, effectively alleviating MSU crystal-induced gouty joint inflammation in rats . These d


Jm, ranging from ≈ 38 to 107 MPa√m, compared to a monolithic W toughness of ≈ 8 MPa√m. In most cases, the fracture of WHAs occurs through stable crack tearing. However, the 97W WHA has the lowest toughness and fracture elastically in all but the smallest specimens. As lower Ni contents are desirable for fusion application, we explore the potential for improving the ductility and KJm of WHAs using vacuum annealing at 1300 °C for 24 h. The microstructural observations reveal negligible changes in the WHA microstructure and constituent compositions. While annealing reduces the Vickers microhardness (HV), it does not significantly change the RT yield (σy) and ultimate (σu) strengths but results in beneficial increases in total elongation in the 95 and 97W WHAs by a factor of 2. RT tests on the precracked three-point-bend (3PB) bars show that annealing increases the KJm of these WHAs, and in the case of the 97W WHA, the increase is from 42 to 92%, depending on the size of the specimen. Toughening is due to enhanced crack tip process zone microcracking and dilatation. Tungsten heavy alloys (WHAs) are candidates for use in fusion reactor divertors. Here, we characterize liquid-phase sintered WHAs with 90, 92.5, 95, and 97 (wt.%) tungsten (W), with a balance of a 0.7Ni–0.3Fe ductile phase. These WHAs show remarkable room temperature (RT) fracture toughness at the maximum load, K The advantages and open questions about WHAs related to fusion service are described elsewhere [Tungsten heavy alloys (WHAs) are a class of bi-phasic metallic composites composed of tungsten (W) powders consolidated by liquid-phase sintering (LPS) with lower melting point ductile phase (DP) metals or alloys consisting of Ni, Fe, Cu, and Co constituents. WHAs are known for their good room- to high-temperature tensile strength and ductility ,2,3,4. Ilsewhere ,13,14,15lsewhere . For varlsewhere ,20,21,22Jm) was similar in alloys with 90 to 95W but was lower at 97W [Jm are desirable, we explore the potential for improving the KJm of a series of WHAs, including a lower Ni content 95 and 97W, using vacuum annealing at 1300 °C for 24 h.Sufficient fracture toughness is critical to qualify a structural material for fusion divertor applications. Our literature search shows no studies on the use of high-temperature annealing heat treatments to improve the fracture toughness of as-sintered WHAs. Indeed, fracture toughness evaluations on the precracked WHA specimens for any compositions or conditions following ASTM standards is very rare. Note, others work on the heat treatment on WHAs mostly involves the impurity phase or tensile property characterization and can be found in ,24,25,26r at 97W . As lowey) of all the WHAs and the total elongation (ε) of the 90 and 92.5W WHAs were only weakly dependent on annealing, with the most significant effect for the 97W. However, annealing the 95 and 97W alloys nearly doubles their respective tensile ductility.The detailed microstructural observations show annealing has little effect on the WHA microstructure—W particle size, contiguity, DP phase compositions, area fraction, and thickness. Note, unless otherwise stated, here, annealing (AN) indicates a 1300 °C/24 h condition. Vickers microhardness (HV) was used to probe individual W, the ductile phases, and the WHA composite. Annealing decreases HV in all cases, especially at higher W contents. The RT tensile 0.2% yield strength (σJm decrease between the 1× and 3× specimens (up to 45% drop), with minimal KJm variations between the 3× and 8× specimens (less than 5%) [Im and elastic–plastic KJm toughness [Jm of all WHAs, especially the 97W alloy, in this case by ≈42%. The enhanced toughening is primarily due to enhanced process zone microcracking and dilatation. Annealing results in an increased amount of more widely distributed microcracking. Annealing also reduced the high fraction of cleavage fracture peculiar to the 97W AS condition. Finally, a lower temperature and shorter time anneal of 1100 °C/1 h (labeled 111) and 1200 °C/1 h (labeled 121) did not affect KJm but slightly improved KIm compared to the AS 3× 97W condition.The as-sintered (AS) RT fracture toughness of 3PB bars with nominal length/width/thickness dimensions of ≈ 16/3.3/1.65 mm (with a designated size of 1×) to 110/25.4/12.7 mm (8×) . Note, tthan 5%) . Note, toughness . AnnealiDetails of material acquisition, specimen fabrication, microstructural observation, precracking, and mechanical testing procedures can be found elsewhere ,27. Briew), W-W contiguity, DP composition, DP area fraction, ligament thickness (t), ligament thickness to W-particle diameter ratio (t/DW), and local fracture modes were observed and analyzed using imageJ64 software. Vickers microhardness (HV) was probed on both individual W and ductile phases at low load (10gf) and the composites at 500gf load, both for the AS and AN conditions. Uniaxial tensile tests at RT were carried on EDM-fabricated flat dog-bone-shaped sub-sized SSJ2 specimens with a nominal gauge section length/width/thickness dimension of 5.0/1.2/0.5 mm [−3/s in accordance with the ASTM E8 standard [Microstructural characterization, including optical, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and electron backscatter diffraction (EBSD) was carried out on the pre-and post-annealed conditions. The W-particle diameter  and load-point displacements (d) were measured, and KJm was defined as the maximum load (Pm) based on the ASTM E1921 standard practice method of estimating the J-integral elastic–plastic fracture toughness: Jm = Je + Jp; KJm = √{EJm/(1−ν2)}; Je = KIm2(1−ν2)/E. Here, KIm is the elastic stress intensity factor at maximum load, Jp = 2AP/Bbo [o is the initial unbroken ligament dimension, and Ap is the plastic area under the load-displacement curve [o –d/S curves by dividing P by the plane strain limit load Po [The RT fracture toughness tests on the WHAs were conducted on fatigue precracked 3PB bars, with nominal dimensions (length/width/thickness) of 1× ≈ 16/3.3/1.65 mm, 3× ≈ 50/10/5 mm, and 4× ≈ 60/12.7/6.35 mm. All the specimens were fatigue-precracked to a/W ≈0.4 to 0.5 at 20 Hz at a maximum ∆K 2AP/Bbo , B is spnt curve ,31,32. I load Po ,31 and d load Po . At leasThe selective EBSD on the 90 and 97W alloys was characterized to observe possible texture development after annealing. The inverse pole figure (IPF) maps shown in f load was used to probe the DP and W particles. f/mm2, respectively. After annealing, the HV decreases to 421 ± 20 kgf/mm2 (≈ 12%) and 299 ± 22 kgf/mm2 (≈ 14%) in the W particles and the DP, respectively. A 500 gf load was used to measure the WHA composite HV. While the average composite HV increases with W from 321 ± 8 kgf/mm2 for the 90W to 344 ± 9 kgf/mm2 for the 97W in the AS conditions, the opposite trend was observed for the AN conditions, decreasing from 319 ± 14 kgf/mm2 for the 90W to 289 ± 18 kgf/mm2 for the 97W alloy, as shown in Room temperature Vickers microhardness (HV) measurements were performed on individual W particles, DP regions, and the composite in both the AS and AN conditions. A low 10gp) curves, while y but has a larger effect on σu due to lower initial strain hardening  between 0 and 0.1 true plastic strain. Note that the flow stress (σfl) curves are linearly extrapolated for tests that fractured at less than 0.1 true fracture strain. The average σfl for all the WHAs decreases ≈ 6.4% following annealing. HV depends on, and can be correlated with, σfl, rationalizing the observed HV decreases, while σy is less affected by annealing.ardening d. The con σt see e. Table u between 90 and 92.5W but nearly doubles this measure of tensile ductility for the 95 and 97W alloys. Notably, in all cases, the tensile data in the AN condition have lower standard deviations, suggesting that annealing might help to homogenize the microstructure and heal processing damage, which is also an issue for the toughness properties of the as-sintered condition [The total elongation decreases systematically with increasing W in both AS and AN conditions. Annealing has little effect on εondition .o–d/S, for the 3-point bend bar tests are plotted in o is the limit load, d is the displacement, and S is the bend bar span. As reported previously [o–d/S trend with stable crack growth; however, the maximum P/Po is higher than for the AS condition. This is partly due to the decreased limit load (Po) for annealed specimens with lower flow stress, which, in this case, is taken as the average of σy and σu [o, dm, also slightly increased in the AN condition, except at 92.5W.Representative normalized load displacement curves, P/Peviously  and showy and σu ,31,34 . As expected, the P/Po for larger specimens is lower than for the 1× specimens due to the larger crack length. Note that in all AN cases, the maximum P/Po is higher than their AS counterparts, and the d/S at maximum P/Po is also larger for cases with stable fracture.fracture . The AN Im and KJm values are summarized in Im in the 1× specimens  from values of 17.6% for 1× to 57.1% for 3× 97W WHAs, as shown in f/mm2 for AN 90W, while for 97W, the HV are 344 ± 9 for AS and 289 ± 18 kgf/mm2 for AN 97W). However, while annealing has little effect on the corresponding yield strength (≈2 to 5% lower), the uniform and total elongation doubles in 95W (εt ≈ 8 ± 1% vs. 16 ± 0.3%) and 97W (εt ≈ 4 ± 1% vs. 8.8 ± 1.0%). With the exception of the 92.5W WHA, annealing increases the 97W elastic–plastic fracture toughness for the smaller WHA up to 42% (69 ± 12 MPa√m vs. 98 ± 17 MPa√m). Annealing also improves the KIm and KJm for the larger specimens, again especially at higher W contents (from 75 ± 4 MPa√m to 125 ± 6 MPa√m for 95W and 38 ± 4 MPa√m to 73 ± 29 MPa√m for 97W). The size effect between the 1× and 4× specimens is minimal in the AN condition. Given the lower allowable Ni contents with higher RT fracture toughness and tensile ductility, the 1300 °C/24 h annealed 95 and 97W WHAs would be more suitable for the nuclear fusion divertor application.High-temperature 1300 °C/24 h annealing of 90 to 97W WHAs has a minimum effect on the as-sintered (AS) microstructure. The HV decreases in the annealed (AN) condition at higher W contents (HV ≈ 321 ± 8 for AS and ≈319 ± 14 kgThe microcracking pattern is very different in the AS versus the AN condition. The AS microcracking is more localized to the near-tip process zone of the macro-fatigue crack. In contrast, after annealing, the microcracking is much more widely distributed in the large plastic zone principal stress lobes formed by the loaded fatigue crack. The enhanced microcracking leads to additional dilatational toughening. The local fracture surface damage modes are similar in both the AS and AN conditions, except at 97W. However, the microcracking mechanisms in both the tensile and fracture specimens shift from WC to WW and WD mechanisms in the AN condition. Given the similarity in the AS and AN conditions’ microstructures and strength properties, it seems likely that the effects of annealing are due to increased ductility and a reduction in residual stresses. Thus, XRD studies will be pursued in the near future.