Bloom's syndrome (BLM) protein is a known nuclear helicase that is able to unwind DNA secondary structures such as G-quadruplexes (G4s). However, its role in the regulation of cytoplasmic processes that involve RNA G-quadruplexes (rG4s) has not been previously studied. Here, we demonstrate that BLM is recruited to stress granules (SGs), which are cytoplasmic biomolecular condensates composed of RNAs and RNA-binding proteins. BLM is enriched in SGs upon different stress conditions and in an rG4-dependent manner. Also, we show that BLM unwinds rG4s and acts as a negative regulator of SG formation. Altogether, our data expand the cellular activity of BLM and shed light on the function that helicases play in the dynamics of biomolecular condensates.  DNA or RNA G-quadruplexes (dG4s or rG4s) are nucleic acid secondary structures that are formed in guanine (G)-rich sequences . ThousanComplex biological processes involving G4s rely on the involvement of specific G4-binding proteins. The cellular localization of these proteins, their target specificity, binding affinity, and enzymatic activity contribute to defining their function. In this context, G4 helicases unwind dG4 and/or rG4 structures ,14. Sincvia the formation of stress granules (SGs)  ,15–17. Ses (SGs) ,18–20. Tes (SGs) .The connection between SGs and rG4s is further substantiated by the fact that several rG4 binding or unwinding proteins have been found in SGs ,15,25,26Bloom's syndrome helicase (BLM) is one of the first human helicases reported to resolve dG4, requiring a 3′ single-stranded overhang for enzyme loading ,29. The Here, we demonstrate that BLM is enriched in SGs under several stress conditions and also binds rG4s, thus expanding its reported functionality beyond dG4s. Moreover, we show that BLM is recruited to SGs in an rG4-dependent manner and negatively regulates their formation. These observations provide new insights into the cellular regulation of the stress response and more broadly into the functions of G4 helicases in biomolecular condensates.MgCl2 , 100% Glycerol , RNase/DNase-free UPW , 1 M Tris–HCl pH 8.0 , 1 M Tris–HCl pH 7.5 , 0.5M EDTA , 1M DTT , Tween-20 , TBE ×10 . Stock solutions of 4 M of NaCl , 3 M KCl , 3 M LiCl , 50mM ATP  or 50 mM ATPgS  were prepared by dissolving the powders in RNase-free UPW or in UPW, treated with DEPC  before use.Chemically synthesized DNA or RNA oligonucleotides  or in 10°C intervals (between 50°C to 30°C) as follows: 85 - 70°C for 5 min each, 65–50°C for 15 min each, 40–30°C for 30 min each and 25°C for 2 hr. All samples were stored at 4°C.We performed CD experiments at 25°C using Chirascan™-plus ACD spectropolarimeter with a quartz cuvette with a 1 mm path length. We collected CD spectra from 360 to 210 nm. The bandwidth was 1 nm, and the response time was 1 s. CD spectra signal corrected to background (buffer only) and represented the average of 3 runs.A truncated BLM 636–1298  was expressed in E. coli and purified as described previously ,34 with 2 and 0.1% Tween-20. For RNA oligonucleotides we also added Ribolock , with or without recombinant human core-BLM protein (50 or 150 nM). For the reactions with QUMA-1 , we added to the samples 1 uM QUMA-1. We incubated the binding reactions at 37°C for 30 min and then loaded them onto a 5% native non-denaturing polyacrylamide (acrylamide:bis-acrylamide 29:1 (30%); Bio-Lab, UN3426) gel consisting of (for 12.5 ml) 9 ml DEPC-DDW, 1.25 ml TBE 10×, 2.075 ml 30% polyacrylamide, 125 ul 10% Ammonium Persulfate  and 12.5 ul TEMED . We performed electrophoresis at 70 V for 70 min in TBE 1× buffer on ice and in the dark. After 70 min, we performed gel scanning using ImageQuant LAS4000  gel imager at cy2 channel (488 nm).We prepared 20 ul reaction mixtures, which contained 80 nM 5′-6FAM-labelled DNA or RNA oligonucleotides of G4-forming sequences , and binet al. (A mixture of 1 uM Dabcyl-labelled oligonucleotide (Dabcyl-S-rG4-VEGFA-U15) and 0.85 uM 6FAM-labelled oligonucleotide (F-rShort- 6FAM) was prepared in 20 mM Tris–HCl buffer (pH 7.5) containing 5 mM MgCl2, 1 mM KCl, 99 mM NaCl and RNase-free UPW. The 1.2-fold excess of the dabcyl-labelled strand ensured complete hybridization of the 6FAM-labelled strand and maximal quenching of the fluorescent signal. The mixture was annealed by PCR program as follows: 90°C, 5 min; 80°C, 10 min; 70°C, 10 min; 60°C, 1 h; 50°C, 1 h; 40°C, 1 h; 30°C, 1 h; 25°C, 2 h; 4°C to end. The annealed samples were then stored at -80°C in aliquots. A competitor oligonucleotide  was heated at 90°C for 5 min and then cooled on ice for 15 min before the reaction. Oligonucleotides described in et al. , with ad2. BJ fibroblasts were cultured in RPMI medium  supplemented with 10% fetal bovine serum, at 37°C, with 5% CO2. For live imaging and staining experiments, we used G3BP1-GFP stably expressing U2OS cells. For proteomics and validation experiments for APEX proximity labelling, we used tetracycline-inducible G3BP1-APEX or NES-APEX expressing U2OS cells, as described previously  and human Retinal Pigment Epithelial (RPE) cells were used, either BLM knockout (BLM KO) or wild-type (wt) . Cells weviously . For SG-siGenome siRNAs against Dhx36 (M-013167-00) and Blm (M-007287-02) genes, as well as siGENOME non-targeting siRNA #5 control  were from Dharmacon (Dharmacon siGENOME Human/Mouse/Rat SMARTpool). For transfection, we mixed siRNAs with Lipofectamine 2000  and incubated them in Opti-MEM  for 20 min. Then, the cells were incubated with the solution in transfection media  for at least 4 h up to over-night (o.n) and afterward, we replaced the transfection media to complete medium for 48 h.2O2 30%  was added for 1 min. APEX activity extinguished with quenching solution , sodium ascorbate  and Trolox  in PBS 1× twice for 1 min each time. Then, the cells were washed two more times with PBS 1×, 1 min each time, and for the last wash with quencher solution as mentioned before. After the fifth wash, the cells were scraped in quencher solution, centrifuged at 800 × g for 10 min at 4°C, pelleted and lysed in ice-cold RIPA lysis buffer supplemented with cOmplete Protease Inhibitor Cocktail  and PhosSTOP . Lysates were centrifuged at 14 000 × g for 10 min at 4°C. Protein concentration was quantified with Bio-Rad Protein Assay Dye Reagent . Streptavidin-coated magnetic beads  were incubated for pulldown experiments with the ratio between extract: beads as 1 mg:200 ul, respectively  with rotation overnight at 4°C. Then, the beads were washed twice with RIPA containing quencher solution, once with 1 M KCl solution, once with 0.1 M Na2CO3 solution , once with 2 M Urea solution  and for proteomics, each wash was done for 3 min and the biotinylated proteins were transferred to on-bead digestion process by trypsinization.16 M Tetracycline-induced G3BP1-APEX or NES-APEX expressing U2OS cells were seeded per flask, in 180T flasks (90–100% confluency) with a medium which was supplemented with tetracycline for 24 h (50 ng/ml for each of the APEX baits) for inducing the APEX-bait gene expression. On the day of the experiment, the cells were incubated for 3 h with 1 uM QUMA-1, or an equivalent volume of DMSO prior to 150 uM Sodium arsenate  stress for 2.5 h. Labelling activity was induced by supplementing Biotin-phenol  for the last 30 min of the stress and then H2O/acetonitrile + 0.1% formic acid. Each sample was loaded and analyzed using split-less nano-Ultra Performance Liquid Chromatography . The mobile phase was: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a Symmetry C18 reversed-phase trapping column . The peptides were then separated using a T3 HSS nano-column  at 0.35 ul/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4 to 25%B in 155 min, 25 to 90%B in 5 min, maintained at 90% for 5 min and then back to initial conditions.ULC/MS grade solvents were used for all chromatographic steps. Dry-digested samples were dissolved in 97:3% HFor MS, the nanoUPLC was coupled online through a nanoESI emitter  to a quadrupole orbitrap mass spectrometer  using a FlexIon nanospray apparatus (Proxeon).m/z), a mass range of 375–1650 m/z, AGC of 3e6, and maximum injection time was set to 60 ms. MS2 resolution was set to 17500, quadrupole isolation 1.7 m/z, AGC of 1e5, dynamic exclusion of 45 s, and maximum injection time of 60 msec.Data was acquired in data-dependent acquisition (DDA) mode, using a Top10 method. MS1 resolution was set to 70 000  >0, for pairs of APEX-On and corresponding APEX-Off samples for each group of condition/ treatment . Proteins that passed all QC filters were separated for each condition (DMSO or QUMA-1). Within each condition, the LFQ intensities of G3BP1-APEX samples were compared to the LFQ intensities of NES-APEX samples to characterize the stress-granule associated proteins under each condition, as follows: Data were filtered to retain only proteins with at least two LFQ values in at least 1 group. Importantly, through the analysis, one repeat from the G3BP1-APEX samples under DMSO treatment (sample 1) was excluded because of suboptimal correlation with the other samples from this group. Missing data were imputed by creating an artificial normal distribution with a downshift of 1.5 standard deviations and a width of 0.4 of the original ratio distribution. Student's t-test called enriched SG proteins with S0 = 0.1 and FDR P-value ≤0.05 and a minimum of two-fold enrichment of proteins in G3BP1-APEX samples versus NES-APEX (log2(SG-APEX – NES-APEX) > 1). Following that, after filtered valid values of at least 20% in total, we imputed the LFQ intensities by creating an artificial normal distribution with a downshift of 1.8 standard deviations and a width of 0.3, and the G3BP1-APEX values per each condition (DMSO or QUMA-1) were normalized by the mean of their corresponding NES-APEX values. Then, we categorized the normalized G3BP1-APEX for two groups: DMSO and QUMA-1. We compared these two conditions by student's t-test with FDR correction by using R as mentioned above. Enrichment of proteins in SGs for DMSO or QUMA-1 treatment was determined by FDR p-value ≤ 0.05 and a minimum of two-fold enrichment (for DMSO: log2(QUMA-1 – DMSO) < –1; for QUMA-1: log2(QUMA-1 – DMSO) > 1). Principal component analysis (PCA), Volcano plot and Heatmap for comparison between DMSO and QUMA-1 SG proteomes were generated by Perseus and R.ProteinGroups output table was imported from MaxQuant to Perseus environment version 1.6.2.3  and analow value . StudentE. coli K12 protein database was downloaded from Uniprot.org and an in-house list of 128 common lab contaminants.Shifted G4 bands were cut from the gel and subjected to in-gel tryptic digestion followed by a desalting step. The resulting peptides were subjected to nanoflow liquid chromatography (nanoAcquity) coupled to high resolution, high mass accuracy mass spectrometry . Raw data was processed using Proteome Discoverer version 2.4, and searched with SequestHT  and MS A, the treatments and stress conditions as well as the APEX proximity labelling protocol were performed as described above. For BLM signal from pull-down of biotinylated proteins by APEX proximity labelling under DMSO or QUMA-1, we seeded 8–9 M tetracycline-inducible NES-APEX or G3BP1-APEX expressing U2OS cells with tetracycline in 80T flasks a day before the experiment. The wash steps were done except for the last steps, as follows: washes were done without prolonged incubation, and in addition to the wash steps above, the beads were washed twice more with RIPA buffer again and the biotinylated proteins were eluted from the beads by boiling (95°C) with 5× sample buffer supplemented with 2 mM free biotin for 10 min. Based on protein quantification with Protein Assay Dye Reagent, we loaded the beads 360 ug with 40 ul beads per sample, before the washing steps. The supernatant of each solution was taken for loading on the gel after the beads were magnetized.For WB analysis of biotinylated proteins, we seeded 500 K from the cell lines above in 6-well plates a day before the experiment. However, whole cell lysate was obtained by RIPA lysis buffer without pull-down and wash steps with Streptavidin beads. Based on protein quantification as mentioned, the protein was loaded 50 ug of total protein per well.For general pattern of biotinylated proteins by APEX proximity labelling under DMSO or QUMA-1, we seeded 100 K G3BP1-GFP U2OS cells in 6-well plates and were transfected with siRNA control or siRNA against BLM in triplicates, as described above (see ‘siRNAs’ section). Then, we lysed the cells with RIPA lysis buffer, and based on protein quantification, the protein was loaded with 50 ug of total protein per well.For WB analysis of BLM signal under siRNA treatmentAfter lysis and preparation steps of the samples, for all the WB experiments, we resolved the proteins by 10% SDS–PAGE at 100 V for 10 min and then 120 V up to 80 min. After gel electrophoresis, proteins were transferred to nitrocellulose membranes  at 250 mA for 70 min. Membranes were blocked for 1 h at R.T. with 3% bovine albumin fraction V  in PBS containing 0.05% Tween-20 (PBST) and for each experiment above, the procedure was done differently.For WB analysis of BLM signal under siRNA treatment or in SGs after APEX proximity labelling and pull-down, we incubated the membranes with primary rabbit polyclonal antibody anti-BLM  overnight at 4°C with rocking in antibody solution . Specifically for WB analysis of BLM signal under siRNA treatment, we used also incubated the membranes with primary monoclonal mouse antibody anti-Tubulin  as a control overnight at 4°C with rocking in antibody solution. Following primary antibodies incubation, membranes were washed three times for 5 min at R.T. with 0.05% PBST and were incubated for 1 hr at R.T. with horseradish peroxidase-conjugated species-specific secondary antibody. Specifically for WB analysis of biotinylated proteins, Streptavidin-HRP  was used for 1 h at R.T in the dark.For all the experiments we then washed the membranes three times for 5 min each in 0.05% PBST at R.T. and visualized them using EZ-ECL Chemiluminescence  by ImageQuant LAS 4000 . Densitometric analysis was performed using Fiji software (NIH) and representative bands are presented.50 K G3BP1-GFP expressing U2OS cells, BJ fibroblasts or iPSC-derived neurons seeded per well in 24-well plates on coverslips 24 hr prior to stress. After the stress induction, we fixed the cells with 4% PFA  for 15 min at R.T. and washed them with RNase-free PBS 1X three times. Then, we treated the cells with 0.1% Triton-X for 15 min at R.T., blocked with CAS-Block reagent  for 10min at R.T., incubated with primary rabbit polyclonal anti-BLM antibody (1:100). Primary mouse monoclonal anti-G3BP1 antibody  incubated at cold room o.n. A day after, we washed the cells with RNase-free PBS three times, 5 min each, and then incubated with secondary Cy5-conjugated anti-Rabbit antibody (1:200) or also Cy2-conjugated anti-Mouse antibody  for 1 h at R.T. Plates kept in the dark, washed with RNase-free PBS three times, 5 min each, dried and mounted with DAPI . SG induction, performed with NaAsO2  or Thapsigargin , Puromycin  or by heat shock for 90 min at 43°C. A similar procedure was done for BLM staining in SGs as a result of DMSO versus QUMA-1 treatments (U2OS cells). Specifically for this experiment, the cells were incubated with DMSO or QUMA-1 (1 uM) for 3 hr prior to sodium arsenate stress .via a Zeiss LSM900 laser scanning confocal microscopy system equipped with a Zeiss Axiovert microscope and using a 63 × 1.4 NA oil immersion lens. Similar steps after fixation as well as image processing and analysis were done also for RPE cells (wt versus BLM KO).For QUMA-1 staining in fixed cells, we seeded 12 K G3BP1-GFP expressing U2OS cells per well (in 96-well plates) 24 h prior to transfection, incubated the cells with 1 uM siRNAs  against Dhx36, Blm, or siControl for 4-16 hr. 48 hr later, we fixed the cells with 4% PFA for 15 min and washed them with RNase-free PBS three times. Then, we incubated the cells with 2 uM QUMA-1 and Hoechst 33342  for 10 min at 37°C. We kept the plate in the dark from this point. Next, we washed the cells with RNase-free PBS three times, 5 min each. We acquired the fixed cells without (BLM stainings) or with (QUMA-1 staining) taking z-stacks 2O2 for 1 min. Next, the media was removed and the cells were washed three times with quencher solution (as mentioned above) and then fixed with 4% PFA for 15 min. After we washed them with PBS three times, the cells were treated with 0.1% Triton-X for 15 min at R.T., blocked with CAS-Block reagent for 10 min at R.T., and incubated with primary monoclonal anti-V5 tag , which represents the AEPX-bait proteins and goat polyclonal anti-TIA1  in a cold room o.n. A day after, we washed the cells with PBS three times, 5 min each, and then incubated them for 1 h at R.T. in the dark with secondary Cy5-conjugated anti-goat antibody, Cy2-conjugated anti-mouse antibody and NeutrAvidin-TexasRed conjugate  to stain the biotinylated proteins . We washed the cells with RNase-free PBS three times, 5 min each, dried them and mounted them on slides with DAPI. We acquired the fixed cells via a Zeiss LSM800 laser scanning confocal microscopy system equipped with a Zeiss Axiovert microscope, and using a 63 × 1.4 NA oil immersion lens.For APEX proximity labelling validation in fixed cells, 50 K tetracycline-induced G3BP1-APEX or NES-APEX expressing U2OS cells were seeded 24 hr prior to stress. We incubated the cells with or without 400 uM sodium arsenate stress supplemented with or without 500 uM biotin-phenol for 30 min, and then the APEX proximity labelling was induced by the presence of HFor analysis of the BLM enrichment signal, every particular enrichment value was analyzed per single SG and was determined as the signal-to-background ratio of the BLM intensity  in the SG  compared to the fixed surrounded cytoplasmic area of the same SG in the U2OS cells. The analysis was done with Fiji software.QUMA-1 enrichment signal, analyzed per single SG as the signal-to-background ratio of the rG4  intensity in the volume of the SG (G3BP1-GFP (U2OS cells) or cy2, anti-G3BP1 (RPE cells); green) compared to the fixed surrounded cytoplasmic volume of the same SG. The analysis was done with the Arivis software.We cloned DHX36 isoform 1 CDS or BLM cDNA into mCherry-containing pUltraHot vector by using a restriction-free (RF) procedure with Q5 Hot start High-Fidelity DNA polymerase (NEB). The source of BLM cDNA was from a pTRIP-CMV-puro-2A-BLM plasmid . The original plasmid for DHX36 CDS was a kind gift from Dr Daniel Benhalevy, Prof. Marcus Hafner and Prof. Katrin Paeschke.vs. the intensity of the DNA staining . Cells positive for GFP expression were selected by plotting the intensity vs. Max Pixel  of the GFP channel (ch02). To eliminate out-of-focus cells, cells were further gated using the Gradient RMS and contrast features . Single cells were selected by plotting again the area of Hoechst staining, versus the aspect ratio normalized for the intensity of the Hoechst staining (Aspect Ratio Intensity M07_Hoechst). Flat cells were further selected according to the intensity vs. Max Pixel values of the Hoechst staining. The normalized levels of QUMA-1 were calculated by dividing the total intensity (Intensity_MC_QUMA) by the cell area of the bright-field image (Area_M01).Levels of QUMA-1 signal under knockdown of DHX36 and BLM. 800 K G3BP1-GFP expressing U2OS cells were seeded. A day after, the cells were transfected with siRNAs against DHX36, BLM, or siControl as described above (see ‘siRNAs’ section). Next, we incubated the cells with or without 150 uM sodium arsenate stress for 2.5 h. We fixed the cells with 4% PFA for 15 min at R.T. and washed them three times with PBS 1×, 5 min each time. Then, the cells were incubated with 0.5 uM QUMA-1 and Hoechst (1:8000) for 15 min at 37°C and were washed three times with PBS 1×, 5 min each. The cells were scraped and collected with PBS supplemented with 1% BSA. The cells were centrifuged gently  and were suspended by quick vortex in the volume of 20–50 ul of PBS 1× with 1% BSA. Cells were imaged by an Imaging Flow Cytometer . Data were acquired using a 40× lens, and the lasers used were 405 nm (20 mW), 488 nm (100 mW), 561 nm (20 mW) and 785 nm (1 mW). Data were analyzed using the manufacturer's image analysis software IDEAS 6.3 (AMNIS corp.). Images were compensated for spectral overlap using single-stained controls. Cells were selected for Hoechst positive cells by plotting the area of the DNA staining  versus BLM KO), we performed the same fixation procedure as above, but we incubated the cells with 0.5 uM QUMA-1 3 hr prior to the fixation without any other treatment before fixation. After the fixation, we incubated the cells with Hoechst (1:8000) for 15 min at 37°C and were washed three times with PBS 1X, 5 min each. ImageStream analysis and the settings were the same as above.For RPE cells  to dye the nuclei). Quantification of SGs was taken into account only in mCherry overexpressing cells. The laser settings were the same as above, and mCherry was collected in channel 4. Cells positive for mCherry were selected by plotting the intensity We seeded 12–15 K G3BP1-GFP expressing U2OS cells per well 24 or 48 h prior to the experiment in a 96 well plate .For experiments of SG formation under knock-down conditions, we transfected the G3BP1-GFP expressing U2OS cells with 1 uM siRNAs  against Dhx36, Blm, or siControl, and incubated them for 4 h to on and then replaced the transfection medium to complete medium for 48 h.For experiments of SG formation under overexpression conditions, we transfected the G3BP1-GFP expressing U2OS cells with pUltraHot-mCherry or pUltraHot-mCherry-DHX36 or with pUltraHot-mCherry-BLM by using jetOPTIMUS DNA transfection reagent  and incubated them for 4 h and then replaced the transfection medium to complete medium. A day later, we induced overexpression with tetracycline.48 hr after the transfection (both siRNAs or overexpression plasmids), we replaced the medium with a 150 uM NaAsO2-added medium and immediately took them to the microscope to monitor SG formation. We took SG live imaging by a PCO-Edge sCMOS camera controlled by VisView installed on a VisiScope Confocal Cell Explorer system  and an inverted Olympus microscope . We analyzed SG and cell areas using surface features in Imaris software 9.5.1.To validate the efficiency and the function of the siRNAs, we performed rtPCR on cDNA from G3BP1-GFP expressing U2OS cells after transfection with the siRNAs. Whole cell RNA extract was isolated from the cells by TRI reagent  and RNA isolation kit . Next, cDNA was generated from the extracted RNA by qScript cDNA synthesis kit . We performed a real-time PCR procedure using KAPA SYBR Fast qPCR kit Master Mix (2×) Prism ABI  and measured the amplification cycles per tested gene and control gene  compared to negative control samples by the StepOne Plus machine .2-transformed unless it is not written. Normal distribution was tested after this transformation. We used an unpaired t-test or Welch's test for pairwise comparisons. We analyzed multiple-group comparisons using one-way ANOVA with Dunnett's correction. For statistical analysis for proteomics see the specific section above. We used a repeated-measure two-way ANOVA test for helicase activity assay (with Tukey's correction) and for the analysis of live-imaging experiments. For the latter, we tested the normal distribution of the residuals of the data (by histograms) and used the Levene test to compare variances between the treatments within the data. Statistical tests were considered significant if adjusted p-values or FDR-corrected P-values ≤0.05. We show data as means ± SD (or ± SEM for helicase activity assay).We performed statistics with Prism software 9.3.1 or with R (version 4.0.5) . Most ofWe placed and organized all the figures by using Adobe Illustrator software. We generated all the graphs by Prism or R (version 4.0.5)  softwareBLM is often thought of as a nuclear DNA helicase ,30,46. HUnder basal growth conditions, without stress, BLM was mainly located in the nucleus. However, BLM was detected within cytoplasmic G3BP1-GFP expressing SGs under all the stress conditions tested in U2OS cells Figure . The enrSince SGs are composed of RNAs and RNA-binding proteins, we hypothesized that BLM binds rG4s. We thus performed electromobility shift assays (EMSA) with a recombinant, truncated, BLM variant , which retains the translocation and unwinding activities of the full-length BLM . A serieE.coli peptides  bound to cBLM were extracted and analyzed by mass spectrometry, to reveal the nature of the predominant proteins in these bands . Gel areP-value = 0.0034 or 0.0262, unpaired t-test, respectively), while it barely affected cBLM binding to rG4-VEGFA-U15 . Forty-eight hours after transfection, the levels of BLM were decreased by approximately 50% . We thens Figure . This inIn addition, because rG4s accumulate and regulate SG formation ,15,17,59Finally, we quantified the SG-enriched rG4 signal as a result of BLM KO in RPE cells Figure . G3BP1-srG4 interactions contribute to SG formation . In thisP-value < 0.05, FDR correction, log2FC > 0). Of these, 90 proteins were enriched and 20 depleted when rG4s were sequestered by QUMA-1 . Clustering of experimental repeats between the proteomes affected by QUMA-1 versus control treatment was satisfying (Figure Following the pull-down of biotinylated proteins, shotgun mass spectrometry identified proteins that are enriched or depleted in SGs, in response to the availability of cellular rG4s Figure . Overallg Figure  and the g Figure .BLM was relatively depleted from SG proteome in cells treated with QUMA-1, suggesting that rG4 levels and/or their accessibility affect its recruitment (adjusted p-value = 0.03; Figure in vitro and in cells, Figure This wealth of data led us to postulate that BLM may regulate SG formation, as rG4s affect SG formation , recruitvia unwinding of rG4s (Figure Orthogonally, we detected lower percentages of cells that harbor visible SG in cultures that express mCherry-BLM or mCherry-DHX36, relative to cultures that expressed mCherry alone Figure . Altogets Figure .In this study, we demonstrate that BLM, a known nuclear DNA G4 helicase, also localizes to cytosolic SGs, under a variety of stress conditions and in different cell types. In SGs, BLM binds to and unwinds endogenous RNA G4s. Furthermore, BLM was found to be recruited into SGs in an rG4-dependent manner and regulates their formation.via rG4 unwinding, which is in line with our recent observation that rG4-protein interactions and rG4 availability contribute to SG formation (in vitro cBLM helicase activity and previous cellular results showing that rG4 accumulation in SGs is prevented by DHX36, which then leads to SG reduction (We propose that cellular BLM levels alter SG formation ormation . This hyeduction .Nuclear breakdown as a result of oxidative stress, leads to the presence of fragmented DNA in the cytoplasm. Some of these DNA fragments form dG4s that accumulate within SGs . In thisin vitro, and inhibits SG formation (rG4s were suggested to trigger and/or maintain RNA-driven phase separation ,64. As ain vitro (It is important to better understand the mechanisms by which BLM regulates SG formation, as well as, the mechanisms underlying BLM recruitment to SGs, in addition to rG4 interaction. Intriguingly, BLM protein undergoes phase separation in vitro , supportin vitro , and we in vitro . Therefoin vitro  unwind Sin vitro ,25. To dvia the targeting and unwinding of endogenous rG4s. The study emphasizes the crucial regulatory role of rG4s and related helicases in SG dynamics and suggests that additional explorations of RNA-associated BLM functions are warranted in the context of G4-associated genetic diseases and specifically in Bloom's syndrome.In summary, our study reveals a new role for BLM in RNA biology in general, and under stress conditions in particular, which likely occurs gkad613_Supplemental_FilesClick here for additional data file.


Xenopus. Visual deprivation (VD) for 2 days shifts the neurogenic balance toward proliferation at the expense of differentiation, which is accompanied by a reduction in nuclear-accumulated β-catenin in SOX2-positive neurons. The knockdown of β-catenin decreases the expression of SOX2 and increases the number of progenitor cells. Coimmunoprecipitation studies reveal the evolutionary conservation of strong interactions between β-catenin and SOX2. These findings indicate that β-catenin interacts with SOX2 to maintain homeostatic neurogenesis during thalamus development.In the vertebrate brain, sensory experience plays a crucial role in shaping thalamocortical connections for visual processing. However, it is still not clear how visual experience influences tissue homeostasis and neurogenesis in the developing thalamus. Here, we reported that the majority of SOX2-positive cells in the thalamus are differentiated neurons that receive visual inputs as early as stage 47  Xenopus  within a 20 °C incubator. For experimental manipulations, tadpoles were anesthetized in 0.02% MS-222 . The stages of tadpoles were characterized according to the developmental changes in the anatomy [All animal procedures were performed according to the requirements of the ‘Regulation for the Use of Experimental Animals in Zhejiang Province’. Tadpoles were obtained by mating adult male/female  anatomy .C57BL/6 laboratory mice were kept in a controlled environment with a regulated temperature of 22 ± 1 °C and a 12 h light/dark cycle. The mice’s overall health was monitored daily throughout the study . For theXenopus SOX2  and Xenopus β-catenin . The control tadpoles were transfected with a control MO . All morpholinos were tagged with Lissamine for fluorescent visualization. The plasmid of pSOX2::GFP  was used to visualize SOX2-positive cells in the thalamus. For whole–brain/thalamus electroporation, tadpoles were anesthetized, and morpholinos (10 μM) or plasmids (0.5 μg/μL) were injected into the midbrain ventricle/third cerebral ventricle. The two parallel platinum electrodes were placed on the skin above the tectum/thalamus, and current pulses were applied by a stimulator. The transfected tadpoles were screened for further experiments.To knock down endogenous protein expression, we used translation–blocking morpholinos  against w/o morpholinos filled with a glass electrode was slowly injected into the third ventricle to observe the thalamic progenitor cells. The tadpoles were incubated in Steinberg’s solution for 2 days, which was followed by being anesthetized and fixed in PFA overnight at 4 °C. Brain sections were treated with 2 N HCl for 45 min at 37 °C to denature the DNA and rinsed 3 times. All sections were immunostained with anti-BrdU  antibody and secondary antibodies for image collection by a confocal microscope .BrdU  Tadpoles were anesthetized and fixed in 4% paraformaldehyde  at 4 °C overnight. Tadpoles were rinsed with 0.1 M phosphate buffer  and immersed in 30% sucrose overnight for dehydration. On the second day, animals were embedded in optimal cutting temperature (OCT) media and cut into 20 µm cryostat sections with a microtome . Sections were rinsed with PB for 2 × 20 min, permeabilized with 0.3% Triton X-100 in PB for 4 × 10 min, and blocked with goat serum for 30 min before incubating with primary antibodies at 4 °C overnight. For primary antibodies, we used the antibodies of anti-SOX2, anti-SOX9, anti-HuC/D, anti-tubulin, anti-Nkx2.2, anti-β-catenin, anti-PCNA, anti-BLBP, and anti-vimentin . SectionFixed tadpoles were washed with 0.1 M PB for 2 × 20 min and permeabilized with 0.3% Triton  X-100 in PB for 4 × 10 min. Tadpoles were placed in blocking buffer  for 1 h and incubated for 2 days at 4 °C with an anti-SOX2 antibody , which was followed by additional washes and detection with secondary antibodies for 2 days at 4° C. Embryos were washed and mounted for photography using a confocal microscope.Animals were anesthetized in 0.02% MS-222. The brain was exposed by peeling off the covered skin. The dissected optical tecta or thalamus (approximately 30–50 brains for each group) were homogenized in radioimmune precipitation assay (RIPA) buffer with a protease inhibitor cocktail (Sigma Aldrich) and phenylmethylsulfonyl fluoride  at 4 °C. Protein concentrations were measured by BCA assay using a Nanodrop . Protein homogenates were separated by SDS–PAGE  and transferred to PVDF membranes. Membranes were blocked in 4% nonfat milk for 1 h with TBS buffer containing 0.1% Tween-20 (Sigma Aldrich) (TBST) and incubated with primary antibodies overnight at 4 °C. Antibodies were diluted in 1% nonfat milk. We used the following antibodies: anti-Phospho-β-catenin, anti-β-catenin, anti-SOX2, anti-HuC/D, and anti-GAPDH . Blots wThe whole brains or thalamus were harvested and lysed in 100 µL of RIPA buffer with protease inhibitors for 1 h at 4 °C. Samples of 20 µL were taken from the lysate for the input control and mixed with 2 X sample buffer. The remaining 40 µL was added to protein A/G agarose beads  for 3 h and incubated with 0.5–2 µg of specific antibody. The samples were collected by centrifugation, washed 3 times, boiled for 10 min, and subjected to Western blot analysis.2, 1.5 MgCl2, 5 HEPES, 10 glucose, and 0.01 glycine, pH 7.2, osmolality 255 mOsm). Excitatory postsynaptic currents were recorded by holding the membrane potential at −60 mV with intracellular solution containing . Recording micropipettes were pulled from borosilicate glass capillaries and had resistances in the range of 9–11 MΩ. The liquid junction potential was adjusted during recording. Signals were filtered at 2 kHz with a MultiClamp 700B amplifier . Data were sampled at 10 kHz and analyzed using ClampFit 10  or MiniAnalysis 6.03 .Tadpole preparation for patch clamp was performed as described previously . All recThe tadpoles were incubated with IWR-1-endo , a Wnt signaling inhibitor, or TDZD-8 , a GSK-3β inhibitor . If not t–test. Unless noted, multiple group data were tested with ANOVA followed by a post hoc Tukey’s test. Data are represented as the mean ± SEM. Experiments and analysis were performed blind to the experimental conditions unless noted.Two groups were tested with Student’s


In this review, we report recent progress on the experimental and theoretical studies of AV3Sb5 and provide a broad picture of this fast-developing field in order to stimulate an expanded search for unconventional kagome superconductors. We review the electronic properties of AV3Sb5, the experimental measurements of the charge density wave state, evidence of time-reversal symmetry breaking and other potential hidden symmetry breaking in these materials. A variety of theoretical proposals and models that address the nature of the time-reversal symmetry breaking are discussed. Finally, we review the superconducting properties of AV3Sb5, especially the potential pairing symmetries and the interplay between superconductivity and the charge density wave state.The quasi-two-dimensional kagome materials AV 3Sb5, which opens a new route towards unconventional orders in kagome metals.In this review, the authors report recent progress on the experimental and theoretical studies of kagome superconductor AV Unveiling new physics from simple lattice models plays a vital role in modern condensed matter physics. For instance, the exact solution of the two-dimensional 2D) Ising model on a square lattice by Onsager revolutionized our view of phase transitions in statistical physics ,2; honeyD Ising mRecently, fermionic models on kagome lattices have also become an important platform for studying the interplay among electron-electron correlation effects, band topology and lattice geometry . The poiau level , tunableau level ,16, Cherau level  and the au level ,18, etc.au level .d + id chiral superconductor (SC) [f-wave spin-triplet SC [3Sb5 [cT ≈ 2.3 K [3Sb5 (cT ≈ 0.93 K) [3Sb5 (cT ≈ 0.75 K) [In addition to these phenomena, superconductivity in kagome lattice materials has also been widely discussed. It has been argued that the kagome lattice can host a variety of unconventional pairing superconducting states, including the tor (SC)  and f-waiplet SC , among oSC [3Sb5  was foun ≈ 2.3 K . Subsequ 0.93 K)  and RbV3 0.75 K) . This di 0.75 K) .3Sb5 kagome family. This paper is organized as follows. We first discuss the crystal structure and the electronic properties of AV3Sb5 . Second, we review both the experimental evidence and theoretical understanding of the unconventional charge density wave order that forms and reports of accompanying time reversal symmetry breaking. Third, we report the current status of understanding the SC properties of AV3Sb5. Finally, we address other unconventional features in these compounds, such as pairing density wave order, and provide future research perspectives.In this review, we discuss the recent progress in studying this newly discovered AV3Sb5 materials crystallize into the P6/mmm space group and exhibit a layered structure of V-Sb sheets intercalated by K/Rb/Cs, as shown in Fig. 3Sb5, as discussed later. Above and below the V-Sb plane, out-of-plane Sb atoms form two honeycomb lattice planes respectively with lattice sites located above and below the centers of the V triangles in the kagome plane. A-site atoms form another triangular lattice above or below these Sb honeycomb or antimonene planes.The AV3Sb5 from the transport measurements. The low-temperature electrical resistivity ρ(T) and its field dependence are plotted in Fig. 3Sb5 [T) shows a broad transition towards the SC ground state with cT ≈ 2.3 K, which is continuously suppressed by applying a magnetic field. The magnetization data in Fig. 3Sb5 becomes the first example of quasi-2D kagome SCs. The critical field cH for CsV3Sb5 is relatively small with the c direction Hc2 ≈ 0.4T [T) of KV3Sb5 drops to zero with cT ≈ 0.93 K shown in Fig. 3Sb5 has a cT ≈ 0.75 K [3Sb5 compounds within the material family are superconducting at low temperature.We can first understand the electronic properties of AVig. 3Sb5 . One fin2 ≈ 0.4T ,41. Simi≈ 0.75 K . Hence, 3Sb5 also show quite different behavior. The temperature-dependent resistivity of KV3Sb5 can be modeled by a Fermi-liquid formula ρ(T) = ρ0 + aT2 [c/ρab ≈ 600 in CsV3Sb5, as shown in Fig. 3Sb5, where the V kagome layers play a dominant role in the electronic properties. Hence, the AV3Sb5 is a quasi-2D metal. The resistivity ρ(T) also contains a kink behavior around 94 K, which is related to the long-range charge-density wave (CDW) order discussed later [3Sb5, and the nature of the transition merits further study in other compounds.Above the SC ground state, the normal states of AVρ0 + aT2 , which sed later . A sharped later , where ted later ,43. It i3Sb5, density functional (DFT) calculations and angle-resolved photoemission spectroscopy (ARPES) measurements have been performed [FE) in CsV3Sb5, as shown in Fig. zp orbital. The bands around the Brillouin zone (BZ) boundaries are mainly attributed to the V d orbitals. Note that there are two van Hove (VH) points close to FE around the M point, which play an important role in the symmetry breaking observed in AV3Sb5. The upper VH point is further connected with the Dirac cone around the K point, which reflects a typical feature of the kagome model described above. ARPES measurements show that the electronic band structure of CsV3Sb5 qualitatively agrees with DFT calculations [3Sb5 systems. Note that there are still discrepancies between quantum oscillations and DFT calculations [To reveal the electronic nature of AVerformed ,42,44–53ulations , as showulations ,54, whic3Sb5, the three-dimensional Fermi surface (FS) of CsV3Sb5 is calculated in Fig. 3Sb5 materials are effectively modeled as weakly correlated systems [3Sb5 find excellent matching between the measured and calculated FSs [To confirm the quasi-2D nature of AV systems . For exaated FSs , as plot3Sb5 also carries a non-trivial Z2 topological index [Z2 topological invariant can be obtained from time-reversal invariant momentum points with their inversion operator eigenvalues [Z2 invariant is non-trivial for band numbers 131, 133, 135 enumerated in DFT calculations. The parity index for 133, 135 bands at the M point is different, which gives rise to a band inversion at M. Therefore, the normal state of CsV3Sb5 is a Z2 topological metal, and this Z2 topological property leads to a surface state embedding around the bulk FS at the M point. ARPES experiments have resolved this feature, as shown in Fig. Besides the above electronic structures, CsVal index . For invenvalues . As list3Sb5 materials ranging from 78 to 103 K  [3Sb5 crystal growth, elastic neutron scattering measurements ruled out the possibility of long-range magnetic order [3Sb5, scanning tunneling microscopy (STM) measurements were performed on the Sb and K surfaces of KV3Sb5, revealing that the transition is a CDW transition with 2 × 2 superlattice modulation [3Q in addition to those from the primary lattice structure [TCDW [As discussed in the previous section, a CDW phase transition occurs for all AV KV3Sb5) . In the ic order . The absic order . Soon afdulation ,60–66. Fdulation . By Fourtructure . STM furre [TCDW ,60–66. Are [TCDW , which icK between V(I) and V(II) sites shows a sudden jump at TCDW. Beyond the surface sensitive measurements, the CDW state is found to be three dimensional and be modulated along the c axis. This modulation is either 2 × 2 × 2 or 2 × 2 × 4 for AV3Sb5 materials with 2 × 2 × 2 reported for KV3Sb5 and both 2 × 2 × 2 and 2 × 2 × 4 reported for CsV3Sb5 [c axis impacts crystallinity in the direction of the out-of-plane modulation and potentially accounts for this discrepancy. The 3D modulation is also confirmed by the STM data collected across surface step edges [133Cs NMR spectrum study [c-axis periodicity of the superlattice. On the clean surface regions of CsV3Sb5 and RbV3Sb5, STM detects real-space modulations of the CDW gap, as shown in Fig. Nuclear magnetic resonance (NMR) measurements further support the absence of magnetic order and confirm that the CDW transition is indeed a first-order transition . From th CsV3Sb5 ,42,43,52ep edges  and a 13um study . Future 3Sb5 exhibit diverse CDW gap structures, as shown in Fig. zp band of the in-plane Sb, the zp orbital does not participate in the CDW formation [3Sb5. In addition to the gaps resolved around the FSs, ARPES data in KV3Sb5 have also observed a large CDW gap opening away from FE [3 at 20 K, as shown in Fig. zp orbitals is hardly changed.In order to determine the gap structures in momentum space, several high-resolution ARPES measurements have been performed ,49–51,53ormation ,51. In cormation ,51. Quanormation . Hence, 3Sb5 compounds. Since charge is a quantity to preserve time-reversal symmetry, the emergence of this TRSB becomes one of the more intriguing phenomena in these otherwise non-magnetic AV3Sb5 materials. The first evidence for TRSB was found in magnetic-field-dependent STM measurements [3Q from the STM topographic spectrum. However, the intensities of these three pairs of vectors are different in the clean regions for all AV3Sb5 materials [c axis. Owing to the Onsager reciprocal relation, the response functions of a time-reversal preserving system under +B and −B must relate to each other by a time-reversal operator. This non-reciprocal relation under magnetic field breaks the Onsager relation indicating the TRSB in this non-magnetic kagome system [Interestingly, accumulated evidence for time-reversal symmetry breaking (TRSB) signals was found in the CDW phases of AVurements . As discaterials ,61,66, te system . Howevere system ,68, whic3Sb5 single crystals. The muon spin will rotate and relax under the influence of local magnetic fields. The μSR technique is highly sensitive to the extremely small magnetic fields, capable of detecting of the order of 0.1 Gauss fields experienced by the implanted muons. As shown in Fig. 3Sb5 start to increase below the CDW transition temperature TCDW, which strongly suggests the emergence of a local magnetic field owing to TRSB [3Sb5 also found TRSB signals [TCDW ≈ 90 K. We return to discuss the physical origin of this TRSB in the next section.The straightforward evidence for TRSB comes from the zero-field muon spin relaxation/rotation (μSR) spectroscopy ,70. The  to TRSB . Similar3Sb5 [AHE and longitudinal σxx conductivity, both the intrinsic Berry curvature and the impurity-induced skew scattering contribute to the giant AHE in KV3Sb5 and CsV3Sb5. However, compared to conventional spontaneous AHE with ferro- or ferrimagnetic ordering, the AHE in AV3Sb5 exhibits σAHE(B → 0) = 0 without a hysteresis behavior. The σAHE(B → 0) = 0 feature might originate from the anti-phase TRSB between adjoining kagome layers or domain walls [Moreover, a giant anomalous Hall effect (AHE) has also been observed in AV3Sb5 ,39, and 3Sb5 . As showin walls . The ori3Sb5 is still lacking. Besides the above μSR, and magnetic-field-dependent STM measurements, results from other TRSB sensitive techniques like the polarized neutron diffraction and Kerr effect are highly desired.We want to emphasize that the conclusive proof of TRSB in AV3Sb5P6/mmm space group is Dh6, which can be generated by the C6 rotation, inversion operator x about the y–z plane [Besides the translation symmetry breaking and time-reversal symmetry breaking associated with the CDW state, an interesting question is: what are the remaining symmetries within the CDW state? The point group of the AV–z plane . Althoug3Sb5 [0 is the vacuum permittivity. Since (2) is only finite when parity is broken. Only negligibly small SHG signals (likely originating from the surface) were detected from 120 K down to 6 K. Hence, inversion symmetry 3Sb5 at all temperatures, which constrains the CDW order and will also be important for the superconducting pairing possibilities discussed in the following section.To test the inversion symmetry  CsV3Sb5 . SHG mea3Sb5, low-temperature STM data above SC cT at zero field showed that the CDW peak intensities at Q3Q show a C6 rotation broken feature [3Sb5 also reveal the nematic nature of the CDW state persisting into the superconducting phase [C2 rotation symmetry at low temperature. Note that the z-direction-modulated CDW reduces the point group symmetry from Dh6 down to Dh2 [3Sb5. More than that, the signature of this nematic transition can also be found in μSR, coherent phonon spectroscopy and Raman spectroscopy [T = 30 K in addition to the onset of the primary TRSB transition [3Sb5.Rotational symmetry breaking without translational symmetry breaking, namely nematicity, is another important issue for understanding unconventional electron liquids ,74. For  feature ,43,63, ang phase ,41, as sn to D2h . Howevern to D2h . Hence, troscopy ,75,76. Tansition . Opticalansition , as plotansition . Hence, 1Q) appearing alongside the structural Bragg peaks (QBragg) and 2 × 2 CDW peaks (Q3Q) [Additionally, STM experiments show an in-plane 1 × 4 charge modulation below 50 K ∼ 60 K ,62,64, aks (Q3Q) . Since sks (Q3Q) , this 1 ks (Q3Q) . As it dks (Q3Q) ,43,52,67ks (Q3Q) . On the 3Sb5 at low temperature is Dh2. Since Dh2, Ch2 and iC, which calls for further experimental investigations to determine the remaining symmetries, especially the bulk sensitive measurements.For the mirror symmetry, there is still a lack of conclusive evidence for its existence or absence at low temperatures. For example, the STM data in  breaks a3Sb5 materials, especially their unconventional CDW states, becomes a crucial question. As discussed above, DFT calculations qualitatively agree with the electronic structures of AV3Sb5 from ARPES measurements. Therefore, DFT calculations could provide a reasonable starting point for the understanding of AV3Sb5. Since the structural transition is found to play a vital role in the CDW formation, the most stable structural distortion can be probed by DFT. For example, in CsV3Sb5, phonon dispersion relations are calculated from the ab initio DFT calculations shown in Fig. TCDW in a single-layer model. To accomplish the 2 × 2 × 2 structure modulation, a π shift between the adjacent kagome layer TrH distortions is needed [Theoretically, how one models and describes the AVs needed ,75, as is needed , which c3Sb5 is important. DFT calculations and ARPES measurements show that multiple bands cross the Fermi level [zp orbital forms one electron pocket around the Γ point and the V d orbitals form multiple FSs around the M points, as illustrated in Fig. 3Sb5 is widely believed to be dominated by the quasiparticles around the VH points based on the following facts. First, the VH points are very close to the Fermi level as obtained from DFT calculations and ARPES measurements [a, Qb, Qc) related to three M points as well as the Γ-point FS-induced q1 scattering [3Sb5 [zp orbital is proposed, as shown in Fig. Beyond the structural transition, a model that captures the electronic properties of AVmi level ,31. As durements ,51. Secoattering ,62, as iattering ,51. Therng [3Sb5 . Followi3Sb5 CDW is its TRSB. However, neutron scattering, NMR and μSR experiments have already ruled out the possibility of long-range magnetic order with conventional moments in the resolution of the measurements [et al. [The most intriguing property of the AVurements ,37,69,70urements . Moreoveurements ,88. Geneurements  proposed [et al.  proposed [et al. ,89–92.d+id SC, etc. Based on the minimal model and the 3Q electronic instabilities, several TRSB flux states have been proposed to explain the TRSB. The most promising candidate is the chiral flux phase among the 18 flux classes [For kagome lattices and other hexagonal lattices, the 3Q electronic instabilities at VH filling have been widely discussed ,29,93–99 classes ,100,101. classes .3Sb5 can be constructed by projection [g2 > 0. There are three possible phases, I, II and III. Although both the leading and subleading instabilities have been discussed in this work, we only focus on the leading one. Among these three phases, the leading instability of phase II is the ‘imaginary charge-density wave’, which is the low-energy version of the flux phase. In this case, we find that the TRSB phase can be stabilized if the bare interaction g1 is negative and g2, g3, g4 are positive. But how to achieve attractive interactions needs to be further explored [Microscopically, how to stabilize the flux state is still under debate. Starting from the VH points, the low-energy effective theory of AVojection ,82, as iexplored ,100. Howexplored .3Sb5 materials. We focus on discussing the superconducting mechanism and pairing symmetry. Whether an SC is driven by electron-phonon coupling, or unconventionally driven by electron-electron correlation, is the central issue we need to address. To find clues for this hard-core question, we first focus on the superconducting pairing symmetries of AV3Sb5. Since the inversion symmetry 3Sb5, as found in SHG measurements [Superconductivity remains an important property of AVurements , the spiz-direction Knight shift of 121Sb drops below the SC transition cT in CsV3Sb5[cT [3Sb5 belongs to a spin-singlet SC. Additionally, the μSR measurements fail to detect any additional TRSB signals below cT, compared to the distinct increase in the Sr2RuO4 SC [3Sb5 belongs to the time-reversal preserved spin singlet.To reveal the pairing properties, multiple experimental techniques have been applied. The first task is to determine whether the Cooper pairs form a singlet or triplet, which can be determined through the temperature-dependent spin susceptibility. From the NMR spectrum shown in Fig. n CsV3Sb5. The KniV3Sb5[Tc . Therefo2RuO4 SC , suggest2RuO4 SC ,70,104. cT in CsV3Sb5 from the spin-lattice relaxation measurement of the 121/123Sb nuclear quadrupole resonance [3Sb5 [3Sb5 is a conventional spin-singlet SC. This feature is also consistent with the weakly correlated nature of AV3Sb5 and remarkable electron-phonon coupling of the V-derived bands found from ARPES [The superconducting gap structure can also provide information about the pairing symmetry. A Hebel-Slichter coherence peak appears just below esonance , as showesonance ,106. Morce [3Sb5 ,107, as ce [3Sb5 . Hence, om ARPES .0 at T → 0 has been found in CsV3Sb5, which suggests a nodal feature of the pairing order parameter [0 also shows a similar magnetic field dependence found in a d-wave cuprate, as shown in Fig. 3Sb5, both the U-shaped and V-shaped suppression of the density of states have been observed at the Fermi level with a relatively large residual density of state that can hardly be explained by thermal excitations [However, this simple picture is complicated by experimental observation of nodes or deep minima in the superconducting gap. From thermal transport measurements, a finite residual thermal conductivity κarameter ,108. Thiitations ,64. Thes3Sb5. However, if we take the TRSB normal states into account, the gapless excitations arise within a fully opened superconducting gap [ck, ↑ck, ↓− − ck, ↓ck, ↑−), the system at least contains time-reversal symmetry k, ↑〉 state to a | −k, ↓〉 state. Similarly, the odd-parity, spin-triplet pairing needs inversion symmetry I maps a |k, ↑〉 state to a | −k, ↑〉 state. These two symmetry conditions are known as Anderson’s theorem [3Sb5 SC cases, the normal state before the SC transition breaks the This leads to a seeming dichotomy between gapless excitations in the SC state and a conventionally gapped s-wave SC for AVting gap . There aocument} . For the theorem . For AV33Sb5 could lead to non-trivial excitations. Based on Fu-Kane’s seminal proposal, if the helical Dirac surface states of a topological insulator are in proximity to an s-wave SC, Majorana zero modes (MZMs) may arise inside the vortex cores of the superconducting Dirac surface states [2Te3/NbSe2 heterostructures, and in the iron-based SC Fe, (Lix1 − Fex)OHFeSe, etc. [3Sb5 hosts Dirac surface states near the Fermi energy [c. Therefore, MZMs are theorized to emerge inside the vortex core. Using STM, zero-bias states with spatial evolution similar to the zero-bias peaks in Bi2Te3/NbSe2 heterostructures have been resolved in the vortex cores of CsV3Sb5 [Although SC seems to be conventional, the non-trivial band structure of AVe states . The proSe, etc. . Similari energy  that can CsV3Sb5 , as show3Sb5 may host an intriguing electronic state, known as the pair density wave (PDW), in which the Cooper-pair density modulates spatially at a characteristic wave vector. A low-temperature STM study on CsV3Sb5 found that both the height of the superconducting coherence peak and the zero-energy gap depth show spatial modulations with a distinct periodicity of 4a/3, suggesting a PDW state [a4/3 modulation peaks were found in addition to the 2 × 2 CDW peaks Q3Q, 1 × 4 CDW peaks Q1Q and Bragg peaks shown in Fig. a4/3 vectors cannot be obtained by linear combinations of Q3Q and Q1Q peaks, which provides evidence for the PDW in AV3Sb5 [In addition, CsVDW state . In the n AV3Sb5 .3Sb5 arises within the pre-existing CDW states, exploring the correlation between these two states can help to reveal the underlying physics [3Sb5, CDW order becomes destabilized quickly and vanishes at 2 GPa, while the SC state shows a double-peak behavior with a maximum of 8 K around 2 GPa [3Sb5 materials, while the double-peak behavior is clearest in CsV3Sb5 [3Sb5 materials, as shown in Fig. cT calculated from the McMillan-Allen-Dynes formula qualitatively agrees with the experimental values obtained above 20 GPa [cT calculated based on electron-phonon coupling in the low-pressure range is far above the experimental values, which cannot give rise to a reliable conclusion. The underlying pairing mechanism for AV3Sb5 needs more experimental exploration and theoretical analysis.As the superconductivity in AV physics . By applnd 2 GPa ,123, as  CsV3Sb5 . Hence, e 20 GPa , as show3Sb5. Owing to tremendous efforts during the past years, we have achieved considerable understanding of AV3Sb5, which can be summarized as follows.In this article, we have reviewed the physical properties of the newly discovered kagome materials AV3Sb5 is a quasi-2D electronic system with cylindrical Fermi surfaces, where the electronic properties are dominated by the V-Sb kagome layers.AV3Sb5 is a multi-band system with at least four bands crossing the Fermi level. The FS around the Γ point is attributed to the Sb zp bands, while FSs around the BZ boundary mainly consist of V d orbitals. The VH points at the M points play an important role in the unconventional properties of AV3Sb5.AV3Sb5 is a Z2 topological metal with unconventional surface states.Owing to band inversions at M points, AV3Sb5 is weak based on DFT calculations and ARPES measurements.The correlation strength of AV3Sb5 undergoes a first-order phase transition into charge density wave order around 80 to 104 K, depending on the A-site cation. Within the kagome layer, the CDW enlarges the unit cell to 2 × 2 accompanied by a c-axis modulation.AVC6 rotation symmetry is broken.There is evidence for the emergence of time-reversal symmetry breaking inside the CDW state. Besides translational symmetry breaking and time-reversal symmetry breaking, inversion symmetry perseveres while 3Sb5 SC is a spin singlet with cT around 1–3 K, depending on the A-site cation. The SC appears to be a conventional s-wave with unconventional excitations inside the vortex core. The CDW order is intertwined with the SC in an unconventional way, inducing multiple SC domes under pressure.The superconducting order parameter of the AV3Sb5 SC opens a new route towards realizing unconventional orders within 2D kagome metals, which brings us a new platform to investigate the interplay between correlation, topology and geometric frustration. We hope that this review provides a broad picture of the recent progress on AV3Sb5 kagome materials and stimulates new research frontiers within kagome-related physics.The discovery of the AV


Nucleic acid is the carrier of genetic information and the keymolecule in life science. It is important to establish a simple and feasible method for nucleic acid quantification in complex biological samples.Four kinds of hydrogen bond acceptors (choline chloride (ChCl), L-carnitine, tetrabutylammonium chloride (TBAC) and cetyltrimethylammonium bromide (CTAB)) were used to synthesize deep eutectic solvents (DESs) with hexafluoroisopropanol (HFIP). DESs based manganese dioxide (MnO2) nanosheets composites was synthesized and characterized. DNA concentration was determined by a UVVis spectrometer. The mechanism of DNA-DES/MnO2 colorimetric system was further discussed. The composite composed of DES/MnO2 exhibited excellent oxidase-like activity and could oxidize 3,3’,5,5’ -tetramethylbenzidine (TMB) to produce a clear blue change with an absorbance maximum at 652 nm. When DNA is introduced, the DNA can interact with the DES by hydrogen bonding and electrostatic interactions, thereby inhibiting the color reaction of DES/MnO2 with TMB. After condition optimization, ChCl/HFIP DES in 1:3 molar ratio was used for the colorimetric method of DNA determination. The linear range of DNA was 10–130 µg/mL and exhibited good selectivity. A colorimetric method based on DES/MnO2 was developed to quantify the DNA concentration. The proposed method can be successfully used to quantify DNA in bovine serum samples.The online version contains supplementary material available at 10.1186/s13065-023-00922-5. Deep eutectic solvents (DESs), an emerging class of environmentally friendly solvents are formed by hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) in an appropriate ratio. The formation of strong hydrogen bonds leads to a melting point lower than that of each individual component . DESs poNucleic acid, the carrier of genetic information, is a crucial molecule in life sciences. High-purity nucleic acids are the foundation of studies in clinical trials, genomics, food safety and other fields , 13. How2) is a functional transition metal oxide and its nanosheets have unique properties, such as a high specific surface area and oxidase-mimicking activity [2 nanosheets with oxidase-like activity [2 nanosheets/TMB has been reported for the detection and quantification of target compounds and biomacromolecules, including glucose, pesticides, metal ions, antibacterial agents, and nucleic acid [Manganese dioxide  was selected as the HBA to synthesize the DES with HFIP. The DNA quantification procedure was based on the colorimetric reaction between DES/MnO2 and TMB. We demonstrated that this method could accurately quantify DNA from bovine serum samples.Hexafluoroisopropanol (HFIP) is a perfluorinated alcohol with a high density and strong hydrophobicity . HFIP ha4, NaOH, (NH4)2SO4, K2HPO4, KH2PO4, Na2CO3, Na2HPO4 and Na2SO4 were purchased from Sinopharm Chemical Reagent Co., Ltd. . Salmon sperm DNA sodium salt, morpholine ethanesulfonic acid (MES), HFIP, ChCl, L-carnitine, tetrabutylammonium chloride (TBAC), and TMB were purchased from Macklin . Bovine serum was purchased from Haoyang Biological Manufacture Co., Ltd. . All other reagents were of analytical grade and were commercially available. Deionized (DI) water (18.25 MΩ) was used in all the experiments.Cetyltrimethylammonium bromide (CTAB), sodium acetate (NaAc), anhydrous acetic acid, KMnO2 was investigated using a Nicolet 470 fourier transform infraed (FT-IR) spectrometer  in a KBr pellet at room temperature. Nuclear magnetic resonance (1 H NMR) spectra were obtained using an Avance III 400 MHz spectrometer  and the morphology of the MnO2 nanosheets was observed using a JEM-2100 transmission electron microscope (TEM) . Thermal gravimetric analysis (TGA) was performed using TG 209F1 . A PHI5000 VersaProbe  was used for X-ray photoelectron spectroscopy (XPS) analysis. The zeta potential and dynamic light scattering (DLS) were analyzed using a Zeta sizer Nano ZS90 . Agitation and extraction were performed using an UXI orbital shaker . The concentration of the DNA solution was determined using a UV-1600PC ultraviolet-visible (UV-Vis) spectrophotometer . The obtained MnO2 nanosheets were dried using an XMTD-8222 vacuum dryer . The obtained DES-MnO2 was dried in a ZX-LGJ-1 A freeze dryer .The surface modification of the obtained DES and DES/MnOFour types of DESs  were synthesized by stirring a designed amount of HBAs and HFIP in a 150 mL thick-walled pressure-resistant flask at an appropriated temperature until a homogeneous transparent liquid was formed. After optimization, a DES composed of ChCl/HFIP at a 1:3 molar ratio was prepared.2 nanosheets were synthesized according to a previous reported method [4 was accurately weighed and transferred to a 50 mL conical flask, dissolved in 18 mL of DI water, and stirred for 1 h at room temperature. Subsequently, 60 mg of CTAB was added to the flask and stirred continuously until a stable emulsion was formed. Next, 2 mL of 0.1 mol/L MES was poured into the mixture and reacted for 6 h. Finally, the MnO2 nanosheets were washed three times with DI water. After centrifuging at 12,000 rpm for 5 min, the MnO2 nanosheets were dried at 60 °C under vacuum.MnOd method , 36. 20 2 was prepared using a previously reported method [2 nanosheets were dispersed in 2 mL of methanol and 0.5 mL of synthesized ChCl/HFIP DES and the mixture was ultrasonicated for 2 h at room temperature. The resulting solution was centrifugated at 5000 rpm for 10 min and washed three times with methanol. Finally, the DES/MnO2 solid was collected by vacuum freeze-drying.DES/MnOd method  with som2 of different concentrations were added to this above mixed solution and shaken on an incubator shaker for 30 min at room temperature. Finally, the resulting solution was measured at 652 nm by UV-Vis spectrometer.50 µL of TMB (2 mg/mL) was dissolved in 1800 µL NaAc (pH 4.0). Subsequently, 150 µL DES/MnO2 (0.1 mg/mL) was added to 1750 µL NaAc (pH 4.0) aqueous solution. Therefore, 50 µL of DNA solutions with different concentrations was added to the mixed solution. After the addition of 50 µL TMB (2 mg/mL), the mixture was shaken for 30 min at room temperature. Finally, the absorbance of the resulting solution was measured at 652 nm using a UV-Vis spectrometer.Next, 150 µL of DES/MnO2 (0.1 mg/mL), 50 µL TMB (2 mg/mL), and 1850 µL NaAc (pH 4.0) was prepared to conduct selectivity experiments. Various non-specific proteins, carbohydrates, and salts were selected to replace DNA and were added to the prepared aqueous solution for the DNA selectivity test. The mixture was then shaken for 30 min at room temperature. Finally, the absorbance of the resulting solution was measured at 652 nm using a UV-Vis spectrometer.An aqueous solution of 150 µL DES/MnO4)2SO4, K2HPO4, KH2PO4, Na2CO3, Na2HPO4, and Na2SO4) were used as phase separation inducers. A system of 0.5 mL DES  and 0.8 g inorganic salts ((NH4)2SO4, K2HPO4, KH2PO4, Na2CO3, Na2HPO4, and Na2SO4) were prepared in 5 mL of aqueous solution. The molar ratio of HBAs to the HFIP was 1:2. DNA (10 µg/mL) was added to investigate the extraction performance of the two-phase system. After separating into two phases, the bottom phase was removed and detected at 260 nm using a UV detector. The extraction results are summarized in Table ChCl, L-carnitine, TBAC, and CTAB were selected as HBA, and HFIP was selected as the HBD. To determine the extraction potential of the proposed DESs for DNA extraction, six inorganic salts , 4.01 , 3.52 , 3.21 . These results verified that the HFIP/ChCl DES was successfully synthesized.FT-IR spectra and 2 nanosheets . Figure 2 nor a significant absorption peak for TMB from 400 to 800 nm (blue). However, owing to the oxidase-like activity of the MnO2 nanosheets, a deep blue color (characteristic absorption peak at 652 nm) was observed upon the binding of DES/MnO2 with TMB owing to the oxidation of the colorless TMB.TMB was selected as the substrate to investigate the oxidase activity because DES/MnO2 further, different concentrations of DES/MnO2 (0–30 µg/mL) were reacted with TMB. The absorbance gradually increased with increasing DES/MnO2 concentration .To verify the catalytic activity of DES/MnOS/MnO2 0–0 µg/mL wS/MnO2 0–0 µg/mL w2 composites with TMB under different pH conditions was thereafter evaluated   and after (A) the addition of DNA, exhibited a good linear relationship with DNA concentration in the range of 10–130 µg/mL, and the linear equation was y = 2.019x + 0.004 (R2 = 0.996). The adsorption of DNA onto the surface of DES/MnO2 was mainly attributed to electrostatic interactions and hydrogen bonding between the phosphate group of DNA and the cationic part of the DES. With the addition of DNA adsorbed on the surface of DES/MnO2, the colorimetric reaction of DES/MnO2 with TMB was inhibited [To explore the utility of the DES/MnOor) Fig. . Furthernhibited .2-TMB system to various interfering substrates  was studied , hemoglobin, and cytochrome C had a weaker effect. This is primarily because RNA has a structure similar to that of DNA, resulting in a similar inhibitory effect. Consequently, when testing samples containing both DNA and RNA, masking or pre-treatment steps are required.To investigate the specificity of this method for DNA detection, the absorption spectral response of the DES/MnOied Fig. . The fir2 was used to detect DNA in bovine serum. Different concentrations of DNA standard solution were spiked into the bovine serum samples to examine the recovery. The analytical results are summarized in Table To evaluate the viability of our designed assay for practical applications, DES/MnO2 composite that efficiently catalyzes TMB. The composition and molar ratio of DESs were evaluated and DES composed of ChCl and HFIP with molar ratio of 1:3 was suitable for DNA extraction. The addition of DNA to the system significantly inhibited the colorimetric reaction and reduced the absorbance of DES/MnO2-TMB owing to hydrogen bonding and electrostatic interactions between DNA and the DES. Consequently, a colorimetric method based on DES/MnO2 was developed to quantify the DNA concentration. This method exhibited good linearity and specificity and could be used to determine DNA concentration in a simple and rapid manner. Consequently, it exhibits potential for application in DNA detection.Herein, we report the synthesis of a DES/MnOBelow is the link to the electronic supplementary material.1H NMR spectra of ChCl/HFIP DES. Table S2 The data for Fig. 6. Table S1 The extraction efficiency of DESs with inorganic salts for the DNA extraction. Fig. S1 The effect of ChCl:HFIP molar ratio on DNA extraction. Fig. S2 FT-IR spectra of ChCl/HFIP DES. Fig. S3


There are few early biomarkers to identify pregnancies at risk of preeclampsia (PE) and abnormal placental function. In this cross-sectional study, we utilized targeted ultra-performance liquid chromatography-ESI MS/MS and a linear regression model to identify specific bioactive lipids that serve as early predictors of PE. Plasma samples were collected from 57 pregnant women prior to 24-weeks of gestation with outcomes of either PE (n = 26) or uncomplicated term pregnancies (n = 31), and the profiles of eicosanoids and sphingolipids were evaluated. Significant differences were revealed in the eicosanoid, (±)11,12 DHET, as well as multiple classes of sphingolipids; ceramides, ceramide-1-phosphate, sphingomyelin, and monohexosylceramides; all of which were associated with the subsequent development of PE regardless of aspirin therapy. Profiles of these bioactive lipids were found to vary based on self-designated race. Additional analyses demonstrated that PE patients can be stratified based on the lipid profile as to PE with a preterm birth linked to significant differences in the levels of 12-HETE, 15-HETE, and resolvin D1. Furthermore, subjects referred to a high-risk OB/GYN clinic had higher levels of 20-HETE, arachidonic acid, and Resolvin D1 versus subjects recruited from a routine, general OB/GYN clinic. Overall, this study shows that quantitative changes in plasma bioactive lipids detected by ultra-performance liquid chromatography-ESI-MS/MS can serve as an early predictor of PE and stratify pregnant people for PE type and risk. Preeclampsia (PE) is a hypertensive disorder and a serious complication in pregnant women that affects 5–7% of all pregnancies in the United States. PE is diagnosed based on a constellation of symptoms and clinical features including arterial hypertension, proteinuria, edema, hepatic dysfunction, and hypercoagulation, which can be reflected by thrombocytopenia and abnormal serum hepatic enzyme levels. These clinical symptoms of PE begin after 20 weeks of pregnancy, and PE can be further subclassified into early-onset PE (manifesting before 34 weeks of pregnancy) and late-onset PE (manifesting at or later than 34 weeks of pregnancy). The disease is thought to be caused by placental dysfunction and only occurs in the presence of the placenta or placental tissue , 7. PE i2) and prostacyclin , VLDLs, and LDLs in serum samples from pregnant women with PE (18:1/20:0 and de18:1/14:0 CERs) and SM species (SM de16:0 and SM de18:1) may be early biomarkers of PE  in the plasma of pregnant women linked to later PE development , total CER, and specific CER species linked to complicated pregnancies and PE examining plasma samples after 24 weeks  at risk of developing PE , 5-HETE, 8-HETE, 12-HETE, and 15-HETE are present in the sera at 20 weeks of gestation in pregnancies complicated by PE . A signi with PE . Sphingors of PE . A studyelopment . In the 24 weeks , 46, 47.oping PE .Even with accumulating evidence that additional bioactive lipids are predictive of later PE development, there is no accepted method for predicting PE risk based on lipid analysis , 50, 51.Whole blood (6 ml) was collected in K2 EDTA 10.8 mg tubes (BD Vacutainer) from uncomplicated term pregnancies (n = 31) and preeclamptic pregnancies (n = 26) from women . The mead4) 6keto-prostaglandin F1α, (d4) prostaglandin F2α, (d4) prostaglandin E2, (d4) prostaglandin D2, (d8) 5-HETE, (d8) 12-HETE, (d8) 15-HETE, (d6) 20-HETE, (d11) 8,9 epoxyeicosatrienoic acid, (d8) 14,15 epoxyeicosatrienoic acid, (d8) AA, (d5) EPA, (d5) DHA, (d4) prostaglandin A2, (d4) leukotriene B4, (d4) leukotriene C4, (d4) leukotriene D4, (d4) leukotriene E4, (d5) 5(S),6(R)-lipoxin A4, (d11) 5-iPF2α-VI, (d4) 8-iso prostaglandin F2α, (d11) (±)14,15- dihydroxyeicosatrienoic acid (DHET), (d11) (±)8,9-DHET, (d11) (±)11,12-DHET, (d4) prostaglandin E1, (d4) thromboxane B2, (d6) dihomo gamma linoleic acid, (d5) resolvin D2, (d5) resolvin D1 (RvD1), (d5) maresin2, and (d5) resolvin D3. Samples and vial rinses  were applied to Strata-X solid phase extraction (SPE) columns (Phenomenex), previously washed with methanol (2 ml) and then dH2O (2 ml). Eicosanoids eluted with isopropanol (2 ml) were dried in vacuo and reconstituted in EtOH:dH2O  prior to UPLC ESI-MS/MS analysis.Eicosanoids were extracted and analyzed by UPLC ESI-MS/MS as previously described by us and others , 57, 58 Eicosanoids were separated using a Shimadzu Nexera X2 LC-30AD coupled to a SIL-30AC auto injector, coupled to a DGU-20A5R degassing unit in the following way. A 14 min, reversed phase LC method utilizing an Ascentis Express C18 column  was used to separate the eicosanoids at a 0.5 ml/min flow rate at 40°C. The column was equilibrated with 100% solvent A [acetonitrile:water:formic acid ] for 5 min and then 10 μl of sample was injected. Hundred percent solvent A was used for the first 2 min of elution. Solvent B [acetonitrile:isopropanol:formic acid ] was increased in a linear gradient to 25% solvent B at 3 min, to 30% at 6 min, to 55% at 6.1 min, to 70% at 10 min, and to 100% at 10.10 min. Hundred percent solvent B was held constant until 13.0 min, where it was decreased to 0% solvent B and 100% solvent A from 13.0 min to 13.1 min. From 13.1 min to 14.0 min, solvent A was held constant at 100%.2 gas for collisionally induced dissociation. Analysis used multiple-reaction monitoring in negative-ion mode. Eicosanoids were monitored using precursor → product multiple reaction monitoring (MRM) pairs. The mass spectrometer parameters used were as follows: curtain gas, 20 psi; collisionally activated dissociation, medium; ion spray voltage, −4,500 V; temperature, 300°c; gas 1, 40 psi; gas 2, 60 psi; declustering potential, collision energy, and Cell Exit Potential vary per transition  was added to the plasma, and the mixture was sonicated to disperse plasma clumps. Samples were then incubated for 6 h at 48°C. Extracts were then centrifuged at 5,000 rpm for 20 min, transferred to a new glass tube, dried down, and reconstituted in methanol (500 μl) by sonicating. Extracts were again centrifuged at 5,000 rpm for 20 min and transferred to injection vials for mass spectrometric analysis.Plasma (50 μl) was extracted using a modified Bligh Dyer Extraction and analyzed by UPLC ESI-MS/MS as previously described by us and others , 60, 61 Sphingolipids were separated using a Shimadzu Nexera X2 LC-30AD coupled to a SIL-30AC auto injector, coupled to a DGU-20A5R degassing unit in the following way. An 8 min, reversed phase LC method utilizing an Ascentis Express C18 column  was used to separate the eicosanoids at a 0.5 ml/min flow rate at 60°C. The column was equilibrated with 100% solvent A [methanol:water:formic acid  with 5 mM ammonium formate] for 5 min and then 10 μl of sample was injected. Hundred percent solvent A was used for the first 0.5 min of elution. Solvent B [methanol:formic acid  with 5 mM ammonium formate] was increased in a linear gradient to 100% solvent B from 0.5 min to 3.5 min. Solvent B was held constant at 100% from 3.5 min to 6 min. From 6 min to 6.1 min, solvent B was reduced to 0%, and solvent A returned to 100%. Solvent A was held constant at 100% from 6.1 min to 8 min.Sphingolipids were analyzed via mass spectrometric means using an AB Sciex Triple Quad 5500 Mass Spectrometer. Q1 and Q3 were set to detect distinctive precursor and product ion pairs. Ions were fragmented in Q2 using N2 gas for collisionally induced dissociation. Analysis used MRM in positive-ion mode. Sphingolipids were monitored using precursor → product MRM pairs. The mass spectrometer parameters used were as follows: curtain gas, 30 psi; collisionally activated dissociation, medium; ion spray voltage, 5,500 V; temperature, 500°c; gas 1, 60 psi; gas 2, 40 psi; declustering potential, collision energy, and Cell Exit Potential vary per transition .t test with Welch’s correction. These data were analyzed for proper data distribution by the Shapiro-Wilk Test. Some of the group data failed this test , and thus, nontransformed data were also analyzed by the Wilcoxon Sum Rank Test. For depicted heat maps, data were scaled and then plotted. All data are reported as mean ± SD, and a P value < 0.05 was considered statistically significant in both statistical analyses employed. Only those bioactive lipid mediators found statistically significant in a group comparison by both methods are discussed in the results section.Raw data generated via UPLC ESI-MS/MS were evaluated and plotted using the R statistical computing program [R version 4.1.0 (2021-05-18) -- "Camp Pontanezen"]. Subject data that had a value ≤0.0001 for any specific lipid were excluded due to being below the level of detection via the UPLC ESI-MS/MS method used. For violin plots portrayed in the figures, raw data were log transformed to promote normality and differences between the two groups and then examined using Students Demographic data for 31 normal pregnant and 26 preeclamptic subjects are presented in 18:1/14:0, de18:1/16:0, de18:1/18:0, de18:1/20:0, de18:1/24:0, de18:1/26:0 , and de18:1/26:1 (increased); C1P : de18:1/16:0, de18:1/22:0, de18:1/24:0, and de18:1/24:1; SM : de18:1/16:0, de18:1/18:1, de18:1/20:0, de18:1/24:0, de18:1/26:0, and de18:1/26:1; MonHex : de18:1/14:0, de18:1/16:0, de18:1/18:0, de18:1/18:1, de18:1/20:0, de18:1/22:0, de18:1/24:0, de18:1/24:1, and de18:1/26:0 prior to 24 weeks of gestation, which were associated with the later development of PE (2 and one SM species (de18:1/16:0 SM) (data not shown). Each lipid was determined to be an independent predictor of future PE onset with a P ≤ 0.05 using two separate statistical analyses. Therefore, these data show that the levels of specific eicosanoids and sphingolipids are significantly different at early gestational times between subjects with normal pregnancies and subjects subsequently presenting with PE with severe features.Targeted lipidomic analysis revealed significant differences by two different statistical analyses in the eicosanoid, (±)11,12 DHET (decreased), prior to 24 weeks of gestation, which were associated with the later development of PE   Tables  and 4 s. Targetent of PE , Fig. 5 nt of PE  and 6  versus PE patients with preterm (birth occurred prior to 37 weeks) or term births. In contrast to the nonstratified PE comparisons, patients later diagnosed with PE, but also had a preterm birth, showed decreased plasma levels of 12-HETE and 15-HETE with RvD1 levels observed to be increased . In PE s2 levels were now significantly lower in PE pregnancies and (±)11,12 DHET as well as several sphingolipid species were no longer significantly different between the groups , the lipid profile of PE patients was similar to the nonracially stratified data, although TXBe groups  and S10.e groups  and S12.ancestry . Patientancestry . Interes18:1/14:0 CER, de18:1/26:1 CER, and increase in de18:1/18:1 MonHex, which were only observed when comparing general OB/GYN clinic subjects with uncomplicated pregnancies to all subjects who later developed PE .Because approximately half of our study subjects were recruited from a high-risk clinic, we were able to examine whether the plasma levels of bioactive lipids can stratify patients at higher risk for PE development. In this regard, subjects with normal pregnancies from the general OB/GYN clinic  were initially compared to combined PE patients (26 patients). In contrast to the nonstratified findings, significant increases in 20-HETE and RvD1 and significant decreases in 12-HETE were observed . When coloped PE  and S18.loped PE . A simily groups . These f2 as expected before aspirin treatment. In contrast, we found no significant differences in plasma TXB2 levels as well as substantial variability. We attribute both the high variability and lack of increased TXB2 plasma levels associated with later PE development to the initiation of aspirin treatment, as well as variation in aspirin therapy compliance. This conclusion is supported by our findings that TXB2 levels were not associated with PE development when comparing uncomplicated term pregnancies from high-risk clinic  to total PE patients (84.6% of patients prescribed aspirin). One limitation to note, we did not have a measure of compliance in women prescribed aspirin, a reported efficacy factor in previous studies (EPHX2) promotor or polymorphisms in this gene are associated with PE development. Hence, the reduced levels of (±)11,12 DHET early in pregnancy may be indicative of an inability of the placenta to convert EETs to DHETs leading to elevated EETs in the placenta. These elevated EETs may inadvertently enhance neutrophil function and chemoattraction over time, which could promote neutrophil aggregation and a chronic sterile inflammatory response as observed in the spiral arteries of PE patients and in maternal subcutaneous and omental vessels in which neutrophils extensively infiltrate causing inflammation . Notably, we did not observe increases in TXB studies . Based o studies , which iammation , 65. Intammation , and thu18:1/14:0 CER (decreased), de18:1/16:0 SM (increased), and de18:1/18:0 SM (increased)) could serve as early biomarkers for PE development  and gestational age. For other early gestational age studies of PE utilizing plasma as the biomatrix, only the prescription of aspirin was a major variable, although differences in diet, geographical location, and socioeconomic status cannot be ruled out as confounding variables. Furthermore, the levels of most sphingolipid species were significantly reduced in patients with normal pregnancies recruited from a high-risk clinic. Many of the reported studies on blood, plasma, and serum levels of sphingolipids in PE do not state the venue at which subjects were recruited , which may also explain the differences in some of our findings compared to those of others.Significant differences in many sphingolipid species in the plasma of patients who develop PE were also observed in our study. These findings are both congruent and in contrast to recent studies where mass spectrometric analysis was utilized to create a plasma sphingolipid panel. One example is a study analyzing plasma from seven normal and seven PE patients. Although the study was not well powered statistically, the authors concluded that the first trimester maternal plasma sphingolipids . In contrast, pregnant women with PE had lower RvD1 levels at 30–34 weeks than those in normotensive pregnant women suggesting that RvD1 levels are modulated in a multiphasic fashion during gestation, becoming dysregulated as PE evolves. As with our findings with DHETs linked to possible increases in placental EETs, we surmise that the increased levels of RvD1 are an attempt by the body to reduce systemic inflammation associated with PE development, and increased levels of RvD1 were observed in the plasma levels from patients at risk for pregnancy complications regardless of PE development suggesting a plausible systemic inflammation in these patients. Furthermore, RvD1 levels have been noted to be higher in septic patients with lower survival, opposite to the hypothesized expectation of a decrease, since resolvins are linked to the resolution of a hyper-immune response . Therefo18:1/26:1 CER and de18:1/18:1 MonHex along with decreased levels of de18:1/C14:0 CER, but these differences were not observed in patients later diagnosed with PE with a term birth. Congruent with Johnstone et al., our study did not find a correlation between S1P and So in regard to preterm PE and term PE  that developed PE showed significant decreases in the anti-inflammatory omega-3 polyunsaturated fatty acid, EPA, and DHA. Decreases in EPA and DHA levels have also been observed in the plasma from PE patients of Asian descent as well as associated with preterm births in Danish women , 82. The2 observed for subjects of Hispanic ancestry represents a conundrum as PGA2 is a hypotensive agent. Hence, increased levels of PGA2 would be expected to repress PE development. As with RvD1, we surmise that early in gestation, these eicosanoids are modulated in an attempt by the body to prevent placental dysfunction. Although our study does support the conclusions that there are significant differences in the plasma levels of specific lipid mediators linked to PE development between self-determined ancestry, there are limitations in our study that should be noted. For example, we did not characterize our study subjects with ancestry-informative genetic markers. Additionally, our study did not take into account regional diet, urban versus rural environment, and other social determinants of health, all of which could affect levels of lipid-based biomarkers in the plasma.From a causation standpoint, the decrease of anti-inflammatory EPA and DHA levels in Caucasian subjects developing PE is logical due to links between PE development and inflammation as well as high omega-3 fatty acid diets reported to reduce the incidence of severe PE . On the 2 (cPLA2α), a key gene in the establishment and maintenance of pregnancy . Due to the statistical power of this study and employment of multiple statistical analyses, new knowledge as to the stratification of patients into later PE diagnosis with preterm or term birth was also obtained, suggesting specific lipid signatures should be examined for these different subclasses of PE. Furthermore, our study serves as a foundation to reexamine past reports and for future studies to determine the effect of ancestry-linked differences in lipid-based biomarkers for precision-based analyses of later development of PE. Lastly, our study suggests that additional bioactive lipids may be therapeutic targets for prevention of pregnancy-related complications .Although metabolic biomarkers for PE are not yet widely used in clinical practice, this study and the existing literature shows that metabolomics can potentially become a clinical tool for predicting and diagnosing PE, as well as clarifying the etiology and pathogenesis of the disease , 93. OurAll data are contained within the article.This article contains All authors of this article declare that they have no competing financial interests.