The dual-specificity phosphatases responsible for the inactivation of the mitogen-activated protein kinases (MAPKs) are designated as the MAPK phosphatases (MKPs). We demonstrated previously that MKP5 is regulated through a novel allosteric site suggesting additional regulatory mechanisms of catalysis exist amongst the MKPs. Here, we sought to determine whether the equivalent site within the phosphatase domain of a highly similar MKP family member, MKP7, is also important for phosphatase function. We found that mutation of tyrosine 271 (Y271) in MKP7, which represents the comparable Y435 within the MKP5 allosteric pocket, inhibited MKP7 catalytic activity. Consistent with this, when MKP7 Y271 mutants were overexpressed in cells, the substrates of MKP7, p38 MAPK or JNK, failed to undergo dephosphorylation. The binding efficiency of MKP7 to p38 MAPK and JNK1/2 was also reduced when MKP7 Y271 is mutated. Consistent with reduced MAPK binding, we observed a greater accumulation of nuclear p38 MAPK and JNK when the MKP7 Y271 mutants are expressed in cells as compared with WT MKP7, which sequesters p38 MAPK/JNK in the cytoplasm. Therefore, we propose that Y271 is critical for effective MAPK dephosphorylation through a mechanism whereby binding to this residue precedes engagement of the catalytic site and upon overexpression, MKP7 allosteric site mutants potentiate MAPK signaling. These results provide insight into the regulatory mechanisms of MKP7 catalysis and interactions with the MAPKs. Furthermore, these data support the generality of the MKP allosteric site and provide a basis for small molecule targeting of MKP7. The MAPKs are grouped as the growth factor–responsive extracellular signal-regulated kinases 1 and 2 (ERK1/2), the stress-responsive c-Jun NHresidues . As suchresidues , 6, 7, 8residues , targetiresidues .5RS (The MKPs have a conserved MAPK-docking site at the N-terminus designated as the kinase-interacting motif (KIM) which is essential for both substrate recognition and orientation , 12, in 5RS . The act5RS . Simulta5RS . This fi5RS , 16, 17.The active site cysteine has a low pKa that causes this residue to be negatively charged in the MKPs and in high throughput screens often leading to the identification of small molecule inhibitors that have reactivity to the cysteine thiol group and/or charged molecules with extremely poor membrane permeability . The difDUSP16 gene, which maps to human chromosome 12p12 and exhibits 44% sequence similarity within the PTP domain as compared with MKP5  . T. TA)  (apo-MKP7 using the AlphaFold protein structure database (B). The entire region constituting the allosteric site (C), including other key residues that impact MKP5 catalytic activity, are well-aligned structurally with MKP7 (D). Specifically, Y271 of MKP7 is overlayed with Y435 in MKP5 and is similarly distant from the catalytic cysteine (C244). The observation that there is a high degree of structural similarity within this region involved in forming an allosteric site for MKP5 raises the possibility that Y271 represents a critical residue involved in MKP7 catalysis.Inspection of the MKP5 allosteric region to other MKPs demonstrates that this site is similar, and Y435 is highly conserved amongst the MKP family. Although the MKP5 allosteric inhibitor has reduced efficacy on MKP7 catalytic activity, we focused on whether Y271 of MKP7 (corresponding to MKP5 Y435) is important for catalytic activity. To gain insight into the structural similarity between the allosteric pocket of MKP5 and the putative allosteric site of MKP7, we compared the crystal structure of this region  of the catalytic domain of the 5 (1ZZW)  with thedatabase , 26  as substrate, we found that Y271A, Y271S, and Y271W retained 66%, 60%, and 91% of their activity relative to that of WT, respectively (A). To investigate the effect of Y271 on the catalytic activity with a physiological MKP7 substrate, an 11 amino acid phospho-peptide of p38α MAPK with the regulatory threonine and tyrosine residues phosphorylated was used in a malachite green assay. Upon incubation of the p38α MAPK phospho-peptide with WT and mutant MKP7 proteins, we found that the mutant proteins Y271A, Y271S, and Y271W retained 57%, 39%, and 83% of their activity relative to that of WT, respectively (B). The differences in the activity of Y271 mutants are likely attributed to loss of intrinsic MKP7 activity as both WT and mutant proteins demonstrated similar protein folding properties (C). Thus, either using a small molecule phosphotyrosine mimetic (A) or the p38α MAPK phospho-peptide (B) as substrates demonstrated a requirement for Y271 for optimal catalysis. Furthermore, the Y271W mutant retained most of its activity relative to that of WT MKP7 likely due to the maintenance of aromaticity of this residue to that of tyrosine in WT MKP7. Taken together, these data support the interpretation that Y271 of MKP7 plays an essential role in regulating catalysis.To determine the effects of Y271 on MKP7 activity, we purified the catalytic domain of WT MKP7, along with proteins in this domain containing mutations Y271A, Y271S and Y271W. Using para-nitrophenyl phosphate . As expected, overexpression of WT MKP7 resulted in the dephosphorylation of both p38 MAPK and JNK1/2 following sorbitol-induced activation as compared with vector control transfectants (A–C). Consistent with the lack of substrate selectivity of ERK1/2, overexpression of WT MKP7 did not affect sorbitol-induced ERK1/2 phosphorylation levels (A and D). In contrast, overexpression of MKP7 Y271A, Y271S, and Y271W resulted in 7-, 6-, and 4-fold increase respectively, in p38 MAPK phosphorylation as compared with WT in sorbitol-treated cells (A and B). Similarly, sorbitol-treated cells exhibited a 6-fold increase in JNK1/2 phosphorylation when Y271A and Y271S were overexpressed and a 4-fold increase when Y271W was overexpressed as compared with WT (A and C). When compared to the catalytically inactive and substrate-trapping MKP7 C244S mutant, the MKP7 Y271 mutants were not as effective at inducing either p38α MAPK or JNK1/2 hyperphosphorylation as compared with WT. These results suggest that unlike MKP7 C244S, the MKP7 Y271 mutants do not efficiently trap and subsequently accumulate phosphorylated MAPKs in a dead-end complex. Together, these data demonstrate that Y271 is critical for MKP7 catalytic activity in a cellular context and functions as a dominant-interfering mutant.Given the identified contribution of Y271 toward MKP7 catalytic activity, we next investigated the effect of this residue on full-length MKP7 in a cellular context. FLAG-tagged full-length MKP7 WT, Y271A, Y271S, Y271W, and the catalytically inactive mutant C244S  were eacFig. 3A). Consistent with the substrate-trapping properties of the MKP7, C244S mutant p38α MAPK bound up to 3-fold higher levels, as compared with WT . Remarkably, binding to JNK1β was completely abrogated in MKP7 Y271S and Y271W as compared with WT MKP7, whereas the C244S mutant bound ∼3-fold higher levels of JNK as compared with WT . As expected, the C244S mutant bound increased levels of ERK2, but no differences in ERK2 binding were observed with the MKP7 Y271 mutants . These results demonstrate that Y271 in addition to contributing to the catalytic activity of MKP7 is also involved in mediating complex formation with its physiological substrates, p38α MAPK and JNK.It has been shown that MKP7 interacts with JNK through the FXF motif . FurtherFig. 4A). Similarly, the YS/CS mutant completely abrogated the binding capacity of the C244S mutation to trap JNK1, resulting in undetectable levels of JNK1 in MKP7 immunoprecipitants as compared with the Y271S mutant . Taken together, these data suggest that Y271, and the putative allosteric pocket, is a major determinant for both MKP7 catalytic activity and binding interactions for the MAPKs.Our previous structural studies with MKP5 and the allosteric inhibitor showed a collapsed catalytic pocket by ∼18%, which prevented the dephosphorylation of the bound MAPKs presumably by disrupting the proper orientation of the catalytic cysteine with the incoming MAPK phospho-tyrosyl and phospho-threonyl residues . This moFig. 5A and C). Next, we assessed whether Y271 influenced the distribution of the MAPKs between the cytoplasm and nucleus given our earlier observations that this residue was involved in MAPK binding (A–D). The observed redistribution of p38 MAPK and JNK to the nucleus in cells expressing the MKP7 Y271 mutants supports the notion that this residue plays a critical role in MAPK binding and further participates in the compartmentalization of localized MAPK dephosphorylation.We next investigated whether Y271 in MKP7 exerts additional properties on the behavior of MKP7 beyond catalytic regulation and MAPK binding. First, we tested whether MKP7 Y271 mutants affected the subcellular localization of MKP7. The subcellular localization of MKP7 has been reported to be predominately cytoplasmic , 21. Con binding . It is c binding . To test binding , A–D. ThTo determine the contribution of Y271 on MKP7-mediated MAPK signaling, we utilized a serum-responsive element (SRE) that responds to JNK, p38 MAPK, and ERK activity . Cells opNPP or the p38α MAPK phospho-mimetic peptide by approximately 50%, whereas mutation to tryptophan reduced activity relative to WT by approximately 20%. These results suggested that maintaining the aromaticity at this site is a key factor for maintaining MKP7 activity. In agreement with the in vitro MKP7 activity data, we showed that overexpression of Y271 mutants of MKP7 in cells resulted in the hyperactivation of p38 MAPK and JNK1/2 but not ERK1/2 consistent with the substrate preference of MKP7   and usedY271A primers: 5′-TTAGATGAAGCTGCAAGATTTGTGAAAGA.3′TCTTTCACAAATCTTGCAGCTTCATCTAA.Y271S primers: 5′TTAGATGAAGCTTCTAGATTTGTGAAAGAA.3′TTCTTTCACAAATCTAGAAGCTTCATCTAA.Y271W primers: 5′TTAGATGAAGCTTGGAGATTTGTGAAAGA.3′TCTTTCACAAATCTCCAAGCTTCATCTAA.Renilla vector for purposes of normalization was purchased from Promega.Full-length FLAG-tagged MKP7 WT (1–665 aa) in pCDNA3.1 was ordered from Genscript (Clone ID OHu20957) and was used for mutagenesis to generate Y271A, Y271S, and Y271W using the same primers for MKP7 PTP mutants with QuickChange II (Agilent) site-directed mutagenesis protocol and transformed into XL-GOLD Ultracompetent cells (Agilent cat#200317). FLAG-MKP7 C244S mutant was generated using primers: 5′ATGTGTTCTAGTGCACTCTTTAGCTGG and 3′ CCAGCTAAAGAGTGCACTAGAACACAT. Double mutant, Y21S/C244S was generated using FLAG-tagged MKP7 C244S as a template using QuickChange II (Agilent) site-directed mutagenesis protocol and the use of the same Y271S primers used to generate the single mutant. HA-tagged p38α MAPK (1–360 aa) was cloned into pcDNA3.1, and HA-tagged ERK2 (1–360 aa) was cloned into pCG5 for mammalian cell expression. GFP-tagged JNK1 in pEGFP-C2 vector was a gift from Rony Seger (Addgene plasmid # 86830). The SRE luciferase reporter vector (5XSRE-luciferase) was used as described previously  and the pet28a vector, His-MKP7 PTP-WT, Y271A, Y271S, and Y271W were transformed into BL21 GOLD DE3 (Agilent cat#230132). Cells were grown in LB with kanamycin (50 μg/ml) at 37 °C. For expression of WT, 800 μM IPTG was added, while for Y271 mutants, 400 μM IPTG was added to induce protein expression and cells were harvested after overnight incubation at 16 °C. Cells were lysed and purified by His-tag affinity purification in a gravity flow column using TALON Metal Affinity Resin (Takara cat#635502) as previously described .Evaluation of protein folding was carried out using circular dichroism. MKP7-PTP constructs were diluted to a final volume of 400 μl in storage buffer (20 mM tris (pH 7.4), 150 mM NaCl, 5% glycerol, and 5 mM DTT) and were analyzed for circular dichroism absorption by an Applied Photophysics Chirascan circular dichroism spectrophotometer . Curves for each construct were normalized so that the minimum ellipticity is equal.2. Cells were maintained in Dulbecco’s modified eagle’s medium (DMEM) (cat#11965092) supplemented with 10% fetal bovine serum (FBS), 1% sodium pyruvate (Gibco cat#11360070), and 1% penicillin-streptomycin (Gibco cat#15140122).Cell-based assays utilized COS-7 or 293 cells grown at 37 °C at 5% CO2O and filtered through a 0.2 μM unit. Ammonium Molybdate solution consisted of 4.2% Ammonium Molybdate (Sigma A-7302) in 4 M HCl per 100 ml. A 2.5× dilution of the Ammonium Molybdate solution into the malachite green solution created the working assay Malachite green solution. For pNPP assays, experiments were performed in 384-well plates in 25 μl. Five microliters of MKP7 PTP at 10 μM were added to wells with 20 μL of pNPP solution comprised of 10 mM pNPP, 24 mM Hepes (pH 7.4), 120 mM NaCl, and 5 mM DTT. The reaction mix was incubated at 37 °C for 30 min and was quenched with 75 μl NaOH at a concentration of 0.2 M. The absorbance at 405 nM was taken immediately after terminating the reaction.For malachite green assays, a phospho-p38α MAPK peptide  was used. Experiments were performed in a 384-well plate in 10 μl solution. Five microliters of MKP7 PTP at 2.5 μM were added to wells with 5 μl of pTpY peptide (p38α MAPK phospho-mimetic) at 100 μM for 30 min at 30 °C. After incubation, 40 μl of malachite green was added to all wells and incubated at room temperature for 10 min followed by absorbance measurement at 620 nM. Proteins were diluted in a PTP buffer composed of 50 mM Tris pH 7.2, 1 mM EDTA, 0.1% 2-beta-mercaptoethanoll, 0.01% TritonX-100. Malachite green solution was composed of 0.045% Malachite green (Sigma #M-9636) in H6 in 100-mm dishes and maintained in DMEM supplemented with 10% FBS for 24 h prior to transfection. Cells were transfected with 8.5 μg DNA of either pcDNA3.1 or cFLAG-MKP7 (WT or mutants) using FuGENE transection reagent as per manufacturer’s instructions. Twenty four hours after transfection, cells were washed once with PBS and starved overnight in 0.1% FBS in DMEM. Next day, cells were washed with PBS followed by incubation with 500 mM sorbitol in 0.1% FBS in DMEM for 30 min before lysis followed by immunoblot analysis for detection of endogenous p38 MAPK, JNK1/2, and ERK1/2. COS-7 cells were seeded at a density of 8.5 × 105 in 100-mm dishes and maintained in DMEM supplemented with 10% FBS for 24 h before transfection. Cells were cotransfected with 6.8 μg DNA of either pcDNA3.1 vector or cFLAG-MKP7 (WT or mutants) expression vector and 3.4 μg of either pcDNA3-HA-p38α MAPK, pEGFP-JNK1β, or pCG-HA-ERK2 using FuGENE transection reagent as per manufacturer’s instructions. Cells were transfected for 48 h in 10% FBS DMEM before harvest. Cells were lysed on ice using Pierce IP lysis buffer (cat# 87787). Cell lysates were precleared with protein-G Sepharose (Sigma cat# GE-17-0618-01) followed by overnight incubation with anti-FLAG M2 monoclonal antibody (Sigma cat# F3165). Lysates were then incubated with protein-G beads for 1 h followed by boiling of samples in 1× sample buffer for immunoblotting detection of p38α MAPK-HA or ERK2-HA using anti-HA (12CA5) monoclonal antibody (Roche cat#11583816001), JNK-GFP using anti-GFP monoclonal antibody , and anti-FLAG polyclonal antibody (Sigma cat#7425).COS-7 cells were seeded at a density of 1 × 105 on coverslips in 12-well dishes maintained in DMEM supplemented with 10% FBS for 24 h before transfection. Cells were transfected with 1.5 μg DNA of either pcDNA3.1 vector or cFLAG-MKP7 (WT or mutants) expression vector and FuGENE transfection reagent as per manufacturer’s instructions. Twenty four hours after transfection, cells were washed once with PBS and placed in 0.1% FBS in DMEM for starvation overnight. Next day, cells were washed with PBS followed by cell permeabilization and fixation using ice cold methanol/acetone (1:1). Cells were blocked for 1 h in 5% normal goat serum (Gibco cat#16210064) followed by overnight incubation with anti-FLAG M2 monoclonal antibody (Sigma cat#F3165) (1:500) and anti-p38 MAPK  (1:50) or anti-FLAG polyclonal antibody (Sigma cat#7425) (1:500) and anti-JNK1 monoclonal antibody (Santa Cruz biotechnology cat#398989) (1:50). Cells were fixed on slides using VECTORSHIELD mounting medium with DAPI (cat#H-1500-10) to stain for nuclei.COS-7 cells were seeded at a density of 1 × 103VO4, 10 mM NaF, 1 mM PMSF, 1 mM benzamidine, 1 μg/ml pepstatin A, 5 μg/ml leupeptin, and 5 μg/ml aprotinin. Cell lysates were clarified by centrifugation at 20,000g at 4 °C for 15 min. Protein concentration was determined using Pierce BCA kit. Protein samples were boiled in 1× sample buffer and separated using SDS-PAGE and transferred to nitrocellulose membranes by semi-dry transfer for 1 h. Membranes were incubated at room temperature in 1% Casein in TBS (BioRad cat #1610782) blocking buffer followed by overnight incubation with primary antibodies (1:1000 in 5% BSA in TBST). Proteins were visualized after incubation with fluorescence secondary antibodies and image processing using LI-COR image Studio Software. Primary antibodies used from Cell Signaling were p38 MAPK (Thr180/Tyr182) (3D7) (cat# 9215), pERK1/2 (cat#9101), ERK1/2 (cat#9107), pSAPK/JNK (cat#4668), JNK1 (cat#3708), GFP (cat#2955) and from Sigma flag M2 (cat#F3165) and M2 (cat#F7425), and Roche: HA 12CA5 (cat#11583816001).For Western blot analysis, cells were lysed on ice in lysis buffer containing 25 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 5% glycerol, 0.5% Tween-20, 1 mM NaAnalysis of immunofluorescence images of COS-7 cells stained for MKP7 (FLAG), p38 MAPK or JNK1/2, and DAPI (cell nuclei) required the single channel of each image to be uploaded to the program to quantify pixel/color intensity. The CellProfiler Program used DAPI (blue) images to identify all cells/nuclei in each image frame and used the red channel images to identify all FLAG-MKP7–positive cells as well as individual cell boundaries. Thus, both DAPI and FLAG images allowed for individual cell nuclei and cytoplasm to be identified. The green channel intensity was measured and quantified in cells and the ratio of nuclear/cytoplasmic (green) intensity for either p38 MAPK or JNK were measured in cells.Renilla (2 ng), and either pcDNA3.1 FLAG-MKP7 WT, Y271A, Y271S, Y271W, or C440S (1.0 μg). After 24 h, cells were serum-starved overnight followed by stimulation for 4 h with 10% FBS. Cells were lysed and luciferase activity measured according to the manufacturer’s instructions.SRE luciferase activity was measured using the Luciferase assay system kit from Promega as previously described . 293 celin vitro experiments were performed at least three times independently. Statistical analysis and graphing were performed using GraphPad Prism 9.4.1 software. We did not estimate variations in the data. The variances are similar between the groups that are being statistically compared. All data represent the means ± SEM. For p value determinations, we used one-way or two-way ANOVA with multiple comparisons.No statistical methods were used to predetermine sample size. The number of samples (n) used in each experiment is shown. All anton.bennett@yale.edu).All data are included in this article. Further information and requests for resources and reagents should be directed to and will be fulfilled by Anton M. Bennett (The authors declare that they have no competing interests with the contents of this article.


G93A mice following onset had no effect on either the accumulation of misfolded SOD1 or the neuroinflammatory response in the spinal cord and, consequently, failed to extend the survival of SOD1G93A mice or to improve their motor symptoms. Finally, pharmacokinetic (PK) studies demonstrated that high concentrations of C4 and C5 reached the brain and spinal cord but only for a short period of time. Thus, our findings suggest that use of such chemical chaperones for ALS drug development may need to be optimized for more effective results.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the degeneration of motor neurons. Mutations in the superoxide dismutase (SOD1) gene, causing protein misfolding and aggregation, were suggested as the pathogenic mechanisms involved in familial ALS cases. In the present study, we investigated the potential therapeutic effect of C4 and C5, two derivatives of the chemical chaperone 4-phenylbutyric acid (4-PBA). By combining in vivo and in vitro techniques, we show that, although C4 and C5 successfully inhibited amyloid aggregation of recombinant mutant SOD1 in a dose-dependent manner, they failed to suppress the accumulation of misfolded SOD1. Moreover, C4 or C5 daily injections to SOD1 Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disease characterized by the degeneration of the upper and lower motor neurons (MNs) in the brain and spinal cord . About 1Although the exact mechanisms underlying MN degeneration remain to be elucidated , many stMany strategies have been investigated as therapeutic targets for ALS and other neurodegenerative diseases. Among them are chemical chaperones, which have the potential to restore protein homeostasis and improve cellular function . A well-N-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenylbutanamide  and 2-isopropyl-4-phenylbutanoic acid   , exhibitG93A mouse model of ALS, combining both in vitro and in vivo studies. We hypothesized that C4 and C5 chaperone activity could reduce the levels of mutant SOD1 misfolding and/or aggregation, which may result in improved motor function as well as the prolonged survival rate of the transgenic mice. Although C4 and C5 successfully suppressed recombinant mutant SOD1G93A amyloid aggregation levels in vitro, C4 or C5 daily injections to adult mice commencing after disease onset did not significantly affect disease progression or survival of the treated SOD1G93A mice.In the current study, we investigated the potential therapeutic effect of C4 and C5 in the mutant SOD1G93A aggregation, we purified and incubated recombinant SOD1G93A protein under aggregation-promoting conditions , with increasing concentrations of C4 (1–20 mM)  A and C5 1–20 mM) C. We meagregates A,C. Thisgregates B,D. In cce of C4 A,B or C5ce of C4 C,D reducce of C4 B,D. The ce of C4 A,C.G93A model. We alleged that C5, as a α-isopropyl derivative of 4-PBA, would pass the BBB, as is the case with 4-PBA [19F-labeled C4 in acetate buffer, and 30 min later the brains were harvested and analyzed by nuclear magnetic resonance (19F-NMR). Two clear 19F-NMR signals were observed in injected but not in the noninjected mouse brain tissue  were IP injected daily, starting at postnatal day 104 (following disease onset), with either C4 , C5 , or 200 µL acetate buffer (pH 5.6) as a control. Disease progression was monitored until the end stage by the neurological scoring system NeuroScore (NS), the inverted screen test, and the routinely used mice weight loss rate  and microglia (Iba1) activation . Immunoprecipitation with a conformational antibody that specifically recognizes misfolded SOD1, B8H10 ,41, was 93A mice A. Mutantnal cord B,C and inal cord  comparedG93A mice . Immunoprecipitation produced by the B8H10 antibody showed no difference in the accumulation of misfolded SOD1 following incubation with C4 . We separated the soluble and insoluble fractions 72 h post-transfection and compared SOD1 aggregation levels in the insoluble fraction. Immunoblotting revealed no difference in SOD1G93A aggregation levels following incubation with C4 at the tested concentrations  was conducted, as described in “Materials and Methods”. The obtained tectively . The maxectively . Moreoveectively . C5 reacectively . Moreoveectively .G93A model of ALS. We found that C4 and C5 strongly inhibited the formation of mutant SOD1G93A amyloid aggregates in a dose-dependent manner in vitro by changing the aggregation pattern from amyloid aggregation to an amorphous less toxic one [G93A mouse model, starting after disease onset, failed to significantly affect disease progression or survival of treated mice.In this study, we evaluated the potential therapeutic effect of the chemical chaperones C4 and C5, two 4-PBA derivatives which exhibited positive outcomes when tested in several models of protein aggregation diseases ,34, on toxic one , as examG93A mice lumbar spinal cord. Correlating with these in vivo findings, was the observation that the accumulation of soluble misfolded SOD1 was not reduced in the spinal cord and brain of C4- and C5-injected mice. Moreover, SOD1 aggregation in end-stage spinal cords was only slightly (but not significantly) reduced by C4 and C5 daily injections, as revealed by a soluble–insoluble assay and confirmed by immunofluorescence staining. Finally, our treatment had no effect on the neuroinflammatory response in SOD1A key question in the field of protein misfolding-related disorders is whether the toxic species is the soluble misfolded form of the protein or the formation of insoluble cytoplasmic inclusions. Specifically, the benefits and harmful processes associated with the formation of insoluble aggregates are still being investigated . TraditiG93A pathogenesis, it is worth considering the possibility that C4 or C5 injection starting at an earlier time point, prior to disease onset, might have been more beneficial. The 4-PBA was already tested in the SOD1G93A ALS mouse model [In an attempt to explain the lack of in vivo effect of the compounds on SOD1se model ,54,55. Tse model ,55. SuppG93A amyloid aggregates during incubation with relatively high concentrations of the compounds, suggesting that the synthesis of these new 4-PBA derivatives failed to achieve therapeutic outcomes at low dosages, as we had expected. Supporting these findings was a soluble–insoluble assay, where the incubation of SOD1G93A-expressing human neuronal cells with lower concentrations of C4 failed to reduce SOD1 aggregation levels.We hypothesized that, when compared to 4-PBA, C4 and C5 may be effective in inhibition of aggregation at lower, more therapeutically relevant, concentrations. However, our Th-T results, accompanied by TEM imaging, revealed a complete suppression of SOD1G93A mice show that an effective therapeutic dosage was achieved by the administration of 200–400 mg/kg per day of 4-PBA [G93A. Thus, increasing C4 and C5 stability might have resulted in slower disease progression.Furthermore, previous findings from SOD1of 4-PBA ,54,55. HG93A model of ALS [In addition, we tested the potential therapeutic effect of C4 and C5 on the early onset and very aggressive SOD1l of ALS . Other SG93A mice [Riluzole ,58 and e93A mice ,64,65,6693A mice . Combini93A mice  or with 93A mice  was repo93A mice ,55. ThusIn conclusion, our findings suggest that the use of such chemical chaperones alone may not be realistic due to their high and barely tolerable active doses and bad PK parameters. Thus, the chemical chaperone-based strategy for ALS drug development may need to be optimized for more effective results.19F-C4 were synthesized as described [C4 and its fluorinated derivative escribed . C5 was G93A) female and male mice were used for the experiments. The treatment protocol was approved by the Animal Care and Use Committee of Ben-Gurion University of the Negev, as required by Israeli legislation. Mice received 200 µL final volume of intraperitoneal (IP) injections every 24 h. C4-treated mice received 100 mg/kg/day diluted in acetate buffer, pH 5.6. C5-treated mice received 25 mg/kg/day diluted in PBS, pH 7.3. The control group included noninjected mice and mice injected only with acetate buffer.Altogether, 48 B6 background , 2O and the 19F-NMR analysis was conducted as described [A solution of compound 4  at 4 °C, the pellet was washed in 50 mM Tris-HCl buffer (pH 8), followed by an additional 20 min of centrifugation. After 30 min incubation on ice in sonication buffer , 1 mg/mL lysozyme), the cells were disrupted by sonication for 3 min . To remove the DNA, the crude extract was incubated on ice for 30 min in the presence of 25 µg/mL bovine pancreas DNaseI  and 5 mM MgSO4, followed by 30 min centrifugation (1486× g) at 4 °C. The supernatant was loaded on a 5 mL HisTrap FF column , using ÄKTA pure protein purification system  equilibrated with binding buffer . The column was washed with washing buffer , and the protein was eluted by a linear 20–400 mM imidazole gradient. The peak fractions were dialyzed (8–12 h each) at 4 °C against dialysis buffer 1 , followed by dialysis against dialysis buffer 2  three times, followed by dialysis against dialysis buffer 3  twice. Afterwards, the fraction was centrifuged at 100,000× g at 4 °C for 1 h using ultracentrifuge , and the supernatant was stored at −80 °C until use. Protein concentration was measured by the Bradford method using bovine serum albumin as standard.Recombinant mutant SOD1escribed . Briefly1 vector , and expG93A protein (50 µM) with or without C4 (10–50 mM) was incubated in 200 µL HEPES buffer ), 5 mM Ethylenediaminetetraacetic acid , and 1 mM Tris(2-carboxyethyl)phosphine hydrochloride  in the presence of 2 mM thioflavin T  in a black 96-well plate at 37 °C with fast continuous shaking. All samples were performed in triplicates and the fluorescence  was measured at 15 min intervals for 70–80 h by Infinite M200 pro .Recombinant mutant SOD1TEM imaging was performed by the Nano-Fabrication Center team at Ben-Gurion University of the Negev, as described previously . Briefly2 incubator in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% tetracycline-free fetal bovine serum (FBS), 2 mM L-Glutamine, penicillin (100 units/mL), and streptomycin (0.1 mg/mL). Cells were split every two or three days (when they reached ~90% confluency) using Trypsin . SH-SY5Y cells were maintained at 37 °C in a humidified, 5% COTM transfection reagent  according to manufacturer’s protocol. Briefly, cells were seeded ~0.8 × 105 in 2 mL media  in a 60 mm petri dish. Keeping a ratio of DNA:TurboFectTM (1:2), 3 µg of DNA and 6 µL TurboFectTM were dissolved in 200 µL of DMEM, mixed by vortex, and incubated for 25 min at room temperature. The mixture was added dropwise to the preseeded cells and incubated for 48 h at 37 °C in a humidified, 5% CO2 incubator.Cells were transfected using TurboFectg for 30 min at 4 °C. The supernatant (soluble fraction) was collected and stored at −20 °C until use. The pellet was resuspended in 1 mL ice-cold soluble buffer  and centrifuged at 17,000× g at 4 °C for 30 min. Next, the pellet (insoluble fraction) was resolved using 400 µL of 8 M urea and sonication for 1 h at 4 °C. Protein concentration of soluble fractions was measured by the Bradford method using bovine serum albumin as standard, and the protein concentration of insoluble fractions was measured by the protein determination (BCA) kit .Protocol was performed on ice. Briefly, media was removed, and cells were washed twice with 0.1 M PBS, and then lysed in 1 mL of ice-cold soluble buffer  with 20 min incubation at 4 °C. Cells were then detached using a cell scraper, collected into an Eppendorf, homogenized at 4000 RPM for 30 s, and centrifuged at 17,000× g at 4 °C for 30 min, the supernatant (cytosolic fraction) was collected, and then stored at −80 °C until use. Protein concentration was measured by the Bradford method using bovine serum albumin as standard.Brain/spinal cord (SC) tissues were dissected out, cut in half, and homogenized on ice in 3 volumes of ice-cold homogenization buffer , 0.5% sodium deoxycholate, 0.1% SDS). Homogenates were centrifuged at 5000× G93A incubated with C4 (1–50 mM) or C5 (0.25–12.5 mM) for 1 h at 37 °C, were solubilized in IP buffer , 0.5% Nonidet P-40) and incubated at 4 °C overnight with B8H10 antibody , previously crosslinked to DynabeadsTM protein G  according to the manufacturer’s instructions. The beads were magnetically isolated and, after crosslinking with the antibody, were washed three times with PBST (0.1 M PBS with 0.02% Tween20). After magnetic separation, unbound fractions were withdrawn for immunoblotting analysis. Beads were washed three times with IP buffer and the bound fractions were eluted by boiling for 5 min at 95 °C in X2 sample loading buffer.Brain/SC tissue extracts (100 µg), or recombinant SOD1g at 4 °C for 30 min and the supernatant (soluble fraction) was collected and stored at −80 °C until use. The pellet was resuspended with 1 mL ice-cold soluble buffer  and centrifuged at 17,000× g at 4 °C for 30 min. Next, the pellet (insoluble fraction) was resolved using 300 µL of 8 M urea and sonicated for 2 h at 4 °C. Protein concentration of soluble fractions was measured by the Bradford method using bovine serum albumin as standard, and protein concentration of insoluble fractions was measured by the protein determination (BCA) kit .Spinal cord tissues were dissected out, cut in half, homogenized on ice in 200 µL of ice-cold soluble buffer , 0.1 M PBS), and incubated at 4 °C for 2 h while rotating. Homogenates were then centrifuged at 17,000× Desired proteins were separated on a 10%, 12%, or 15% acrylamide SDS-PAGE gel in running buffer . Separated proteins were then blotted on a nitrocellulose membrane in transfer buffer . Blotted membranes were stained with ponceau-s  to verify protein presence and then washed several times with Tris-buffers saline with Tween20 (TBST) until color disappearance. The membranes were then incubated in 5% skimmed milk powder  in Tris-buffers saline (TBS) for 1 h while shaking to block exposed areas. The blocked membranes were incubated overnight at 4 °C while shaking with desired primary antibodies, including: mouse anti-SOD1 , mouse anti-GAPDH , rabbit anti-ChAT , mouse anti-GFAP , and rabbit anti-Iba1 . The next day, the membranes were washed for 3 min in TBST followed by 3 × 3 min in TBS while shaking. Horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies  were used and detected by the EZ-ECL reagent kit  containing the Luminol substrate for HRP. The membranes were photographed by Fusion Solo X . Reprobing of the membranes was performed in stripping buffer  for 15 min while shaking, followed by TBST and TBS washings.g 4 °C degrees. Samples were snap-frozen and stored at −70 °C until subsequent analysis. An amount of 200 µL of acetonitrile was added to 50 µL of serum, and after centrifugation, the supernatant was used for mass spectroscopy analysis. Brains were harvested and lysed in 500 µL PBS using 18 G needle, and then 1 mL of acetonitrile was added. After the addition of the acetonitrile, the samples were homogenized again with the same needle. Spinal cords (SCs) were harvested and lysed in 250 µL PBS using 18 G needle, and then 500 µL of acetonitrile was added. After the addition of the acetonitrile, the samples were homogenized again with the needle. All procedures were conducted on ice. LC/MS from Agilent Technologies  was used for PK analysis. Data were processed using mass L-ynX ver. 4.1 calculation and deconvolution software . Spiking and calibration curve were generated using serum of the nontreated mice. A total of 26 mice (8–9 weeks old) were used in this study. The animals were randomly assigned to the treatment groups before the pharmacokinetic study. Six sampling time points  were set for the experiment. Each of the time point treatment group included 3 animals. Test compounds: C4 (100 mg/kg) and C5 (25 mg/kg) were injected IP. Mice were sacrificed by cervical dislocation, and after that, the blood samples were collected by cardiac puncture, settled for 20 min, and then centrifuged for 10 min at 3000× TM mounting solution (Thermo), dried at room temperature overnight, and stored at 4 °C until imaging. Images were acquired on a NIKON C2Plus laser unit dock to a Nikon Eclipse Ti unit of the confocal microscope by using 10× and 20× objectives and 60× oil immersion objective. Scanning settings were reused across the samples.Mice were anesthetized via inhalation of 1 mL isoflurane, followed by perfusion with 50 mL 0.1 M PBS, and then switched to 4% paraformaldehyde (PFA) in 0.1 M PBS. The spinal cords were dissected out and postfixed in 4% formaldehyde in 0.1 M PBS at 4 °C overnight, cryoprotected in 20% sucrose in 0.1 M PBS 48 h at 4 °C, and afterwards embedded in Optimal Cutting Temperature (OCT) matrix compound . Sections were cut from the lumbar spinal cord (35 µm thickness), and the free-floating sections were stored in 0.1 M PBS with 0.02% sodium azide at 4 °C to conserve until staining. Sections were stained using standard protocol. First, sections were washed (3 × 15 min) in 0.1 M PBS with 0.03% Tween20 and then blocked for 1 h in blocking and permeabilization buffer  with gentle agitation at room temperature. Sections were washed (3 × 15 min) in 0.1 M PBS with 0.03% Tween20 and then incubated overnight at 4 °C with gentle agitation, with primary antibodies made in 0.1 M PBS, 1% donkey serum, 1% BSA, 0.15% triton, including mouse anti-misfolded SOD1 , mouse anti-glial fibrillary acidic protein , goat anti-ionized calcium-binding adaptor molecule 1 , and rabbit anti-neuronal nuclei antigen . The following day, the sections were washed (3 × 15 min) in 0.1 M PBS with 0.03% Tween20 and then incubated for 1 h at room temperature, gently shaking, with fluorescent conjugated secondary antibodies in 0.1 M PBS, 1% donkey serum, 1% BSA, 0.15% triton, including donkey anti-mouse , donkey anti-goat , donkey anti-mouse , and donkey anti-rabbit . Sections were then washed (3 × 15 min) in 0.1 M PBS and mounted on slides using Immu-MountQuantification of band intensity across experiments was done using Evolution-Capt Edge software . The data was transferred and statistically analyzed using OriginPro software . Values are reported throughout as mean ± SEM. After confirming a normal distribution by the Shapiro–Wilk normality test, a one-way ANOVA was performed to compare the database between the 3 experimental groups. For nonlinear data, a Kruskal–Wallis test was performed instead. Significance was set at a confidence level of 0.05. ThT aggregation assay analysis was done using GraphPad Prism .


The PAN fibers can serve as a stable flexible support, while CuS flowers assembled from nanosheets can act as reactive materials, showing high performance in the degradation of dyes. Moreover, these heterostructures can be recovered easily without a decrease in their photocatalytic activity, thus showing favorable recycling capability.“Flowering branch”-like PAN-CuS hierarchical heterostructures were  in situ synthesized through a facile hydrothermal sulfuration growth process on PAN-based fibers prepared by electrospinning.“Flowering branch”-like PAN-CuS hierarchical heterostructures were  Biochemical methods have high selectivity, but their technology is complex. Through photocatalytic oxidation, the organic macromolecular pollutants can be oxidized directly or indirectly to non-polluting molecules, such as CO2 and H2O.10,11 This technology also has the advantages of mild operating conditions, significant degrading effects, thorough purification, and a lack of secondary pollution. Photocatalytic oxidation is expected to be one of the most effective treatments. Many previous studies have shown that metal oxide/chalcogenide semiconductors play an important part in the photocatalytic process.12–16 However, for now, there are still challenges regarding the large-scale construction of recyclable photocatalytic materials through mild methods.“Green hill and clear water” is not only a “China dream”, but a shared vision of the whole world. As issues related to the environment are drawing increasing attention, many researchers have focused on the degradation of pollutants in industrial wastewater.17–20 Recently, copper sulfides have attracted a great deal of attention owing to their widespread applications in solar cells, optical filters, photoelectric transformers, superconductors and sensors.21–23 Particularly, covellite (CuS) has a strong p-type metallic character with the highest concentration of free carriers among copper sulfide materials.24 Many previous researchers have demonstrated that CuS has important applications for photocatalysis. For instance, Saranya et al. prepared a CuS catalyst to treat organic pollutants, showing a degradation time of over 60 min.25 Thuy et al. reported CuS and CuS/ZnS core/shell nanocrystals for photocatalytic degradation of dyes under visible light, and showed that the pollutants almost bleached over a period of 2 h.26 Varieties of CuS micro- and nanostructures.27–35 have been synthesized using different methods including solid-state synthesis, solvothermal solution based methods, sacrificial templating and chemical vapor deposition techniques.28,30,36–40,44 Similar to most reports, CuS has mainly been synthesized in powder form, which is not convenient for recycling. For developing recyclable photocatalytic material, it is important to explore the large-sale preparation methods with low cost. It is known that electrospinning is a powerful method to fabricate one-dimensional functional materials with large specific surface-areas, high porosity, good flexibility and high stability.41–43Metal oxide/chalcogenide semiconductors have the outstanding attributes of unique optical, electronic, magnetic and thermal properties as well as potential applications in energy conversion and catalysis, particularly in the field of photocatalysis.2+ composite nanofibers from a simple electrospinning method. Then, the PAN-CuS composite nanostructures were prepared through a further in situ sulfidizing process. Then, the degradation activity for the dye pollutants and recyclability of the as-synthesized PAN-CuS nanostructures were studied. The superior photocatalytic activity can be attributed to the stable fibrilous structure and abundant active sites of the CuS nanoflowers assembled from nanosheets. Herein, we elaborately developed stringed “flowering branch” PAN-CuS with hierarchical architecture combining fibrous PAN skeleton and active CuS material. First, we obtained PAN-Cu2+, 1 mmol, 1.5 mmol, or 2 mmol Cu(CH3COO)2 was added to PAN/DMF to synthesize PAN-Cu-1, PAN-Cu-2, PAN-Cu-3 nanofilms, respectively. Fig. S1a and b2+ ions move to the surface to produce CuS nanoflowers, and the nanofiber structure is well-maintained to function as the primary structure. As shown in Fig. S1c and d,44 reported the formation process as follows:To explore the effects of different amounts of Cu2− ions are formed through the reduction of S by H atoms, as shown in 2+ ions move from the inside to the surface and then form the CuS crystal nucleus. When the ion concentration was moderate, the crystal nucleus grew into nanosheets and further self-assembled into nanoflowers. As shown in 2+ corresponds to more CuS flowers. However, as Cu2+ further increased, the CuS of the PAN-CuS-3 mainly showed the morphology of nanosheets. This phenomenon was proposed to be attributed to the increased CuS nucleation number with high Cu2+ content; this will result in a lower crystal growth process, thus limiting the formation of CuS nanoflowers.45 In addition, we prepared CuS on pure PAN fibers for comparison. The SEM image is shown in Fig. S2.in situ synthesis of CuS on PAN-Cu2+ fibers, Fig. S2in situ sulfuration method of PAN-Cu2+ films.The reaction temperature was chosen to be 150 °C to exceed the melting point of sulfur (120 °C). First, acetaldehyde can be generated by the dehydration of EG at high temperatures, where acetaldehyde can donate a hydrogen atom and act as a reducing agent, as shown in 46,47 Other diffraction peaks can be well assigned to CuS (JCPDS no. 6-464). No additional peaks for other phases were observed, indicating the high purity of CuS. The EDX spectrum  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O and C–C stretching vibration, while the vibration peaks at 1359.91 cm−1 and 1450.46 cm−1 are associated with the CH3 and CH2 stretching vibrations, respectively. The peak at 1730.24 cm−1 can be attributed to the CO stretching vibration. The peak at 2241.64 cm−1 is attributed to the stretching vibration of 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. The peaks at 2934.24 cm−1 can be attributed to the C–H stretching vibration in CH3. Furthermore, the presence of vibrational peaks at 612.71 cm−1 indicates the presence of Cu–S stretching modes. The surface chemistry of the as-developed PAN-CuS film was studied by XPS measurement. In Fig. S5a,O (289 eV), demonstrating the presence of the carboxyl carbon.The XRD pattern of PAN-CuS-2 films  shows a spectrum  further spectrum  inset in2+ film, and PAN-CuS film. All the tests were conducted in the presence of H2O2 under UV light.We explored the catalytic properties of different samples: pure PAN film, PAN-Cu2O2 was added, photoelectrons were consumed, thus preventing photonic electrons from recombining with holes, which improved quantum efficiency. −1, 0.08035 min−1, and 0.07099 min−1. The first order rate constant is described as follows:k is the apparent rate constant, C0 is the original concentration of MB, and Ct is the equilibrium concentration of MB at the relative reaction time. The calculated results show that the PAN-CuS-2 film has the highest k among the samples. As mentioned above, more CuS nanoflowers lead to better catalytic properties, indicating that CuS indeed plays a decisive role. 2O2 without PAN-CuS catalyst, the degradation of MB was only 17%, and the pure PAN film also had a mild effect on MB due to its adsorption capacity (the degradation degree was about 45%). However, the pure PAN film corroded after one cycle and could not be reused. For PAN-Cu2+ and PAN-CuS, the degradation of MB was 70% and 83%, respectively, which demonstrates the advantage of the in situ sulfuration method of PAN-Cu films. Ct/C0 as a function of time; the samples were the same as those in where 46 (1) The ability of the catalyst to maintain its high activity over time is critical. It is known that the photocorrosion or photodissolution of photocatalysts may occur on the surface during the photocatalytic reaction. To test the stability of MB photodegradation on PAN-CuS films, we reused the catalyst three times. As shown in Recycling as well as maintaining high photocatalytic activity is a critical issue for the long-term use of catalysts in practical applications. Consequently, two factors need to be considered.48 can be ascribed to the formation of hydroxide radicals in the presence of CuS and H2O2. The hydroxyl radicals, in turn, react with and degrade organic substrates such as RhB. 2O2 under UV light.For studying the universality of the as-prepared PAN-CuS photocatalytic material, PAN-CuS-2 was also used to degrade the Rhodamine B (RhB) dye. The degradation result is exhibited in in situ sulfidation on electrospun fibers. This unique structure can provide abundant active sites, controllable morphology, and excellent charge separation, thus exhibiting enhanced photocatalytic activity in the decomposition of MB and RhB under UV light irradiation. Furthermore, these PAN-CuS hierarchical heterostructures with good flexibility can be easily recycled without a decrease in the photocatalytic activity. The results show that PAN-CuS-2 film has the strongest degradation effect, highlighting the role of CuS nanoflower assembly of nanosheets. It is expected that the development of such nanofibers by electrospinning with hierarchical heterostructures represents a very simple and cost-effective approach for the degradation of dye and will greatly promote their practical application to eliminate organic pollutants from wastewater.In summary, stringed “flowering branch” PAN-CuS hierarchical heterostructures were successfully fabricated through There are no conflicts to declare.RA-008-C8RA08293F-s001


W and TaC react to form the W2C phase at a TaC content of 50 vol%. The Vickers hardness of the composite increases from 3.06 GPa for WTA1 to 10.43 GPa for WTA5. The flexural strength reached 528 MPa in the W-40 vol% TaC composite. The thermal conductivity of W-20 vol% TaC composite was 51.2 ± 0.2 W·m−1·K−1 at 750 °C. The addition of TaC improved the ablation resistance of W-TaC composites. The mass ablation rate of W-30 vol% TaC composite was 0.0678 g·s−1. The ablation products were mainly W oxides and complex oxides of W-Ta-O.Transition metal carbide reinforcement can improve the performance of pure W. W-(10–50) vol% TaC composites were prepared by spark plasma sintering at 2100 °C. The effect of TaC content on the microstructure, mechanical properties, and thermal conductivity of the composites was studied. The ablation resistance of the W-TaC composites was evaluated under an air plasma torch. The addition of TaC into the W matrix enhanced the densification of W-TaC composites, the density of W-40 vol% TaC composite exceeded 93%. TaC particles inhibited the growth of W grains during sintering. Reactive diffusion occurred between W and TaC, forming the solid solutions of  Refractory metals and their alloys, carbon–carbon composites, and ultra-high temperature ceramics  are common candidates ,2,3,4,5.−1·K−1 ,7. Howev−1·K−1 ,10,11,12f pure W . Among tf pure W . However2C composites by in situ reactive sintering, with a micro Vickers hardness of 13.4 GPa, a flexural strength of more than 584 MPa, and a fracture toughness of 8.3 MPa·m1/2 [−1 and 0.0233 mm·s−1 [−1·K−1 [Teague et al. prepared W-49 vol%TaMPa·m1/2 ,16. Jian3 mm·s−1 , respect3 mm·s−1 . Miao et [−1·K−1 . The res [−1·K−1 ,21. HoweIn this research, W-TaC composites with increasing TaC content from 10 vol% to 50 vol% were prepared by SPS, and the effects of TaC content on the microstructure, mechanical properties, thermal conductivity, and ablation resistance were investigated.3N4 balls as the milling medium. The ball-to-powder ratio was 3:1. After milling for 24 h, the mixed powders were passed through a 200 mesh sieve. The mixed powders with designed compositions were then put into the graphite mold and densified into composite discs (30 mm in diameter and 3.5 mm in thickness) using discharge plasma sintering (SPS-10T-10-IV). The sintering was performed at 2100 °C for 10 min with a pressure of 40 MPa. The heating and cooling rates were 100 °C·min−1 and 200 °C·min−1, respectively. The compositions and codes of the as-prepared composites are listed in Commercial W and TaC powders were used as raw materials with average particle sizes of 1 μm (W) and 0.5 μm (TaC), respectively. The mixed powders of W and TaC were weighed and milled in polyethylene tanks using Si−1 and 0.02°, respectively. The XRD spectra of the samples were fitted with the ICCD data. The composites were polished to a mirror surface. The microstructure was characterized by scanning electron microscopy  equipped with energy-dispersive X-ray spectroscopy (EDS) for elemental analysis. The hardness of the composites was measured using a Vickers hardness tester , and the flexural strength of the composites was tested by the three-point bending method  on a Shimadzu-type universal testing machine (chamfered specimens with dimensions of 2 mm × 4 mm × 20 mm ). The thermal conductivity of the composites (12.5 mm in diameter and 3 mm in thickness) was tested under flowing argon gas using a laser thermal conductivity tester of type LFA457 with laser voltage of 1538 V and test temperatures of 750 °C, 850 °C, and 950 °C. The ablation resistance of the composites (12.5 mm in diameter and 3 mm in thickness) was estimated by the air plasma torch of an LGK-80YM plasma cutting machine. The distance between the gun tip and the surface of the specimens was 4 mm. The operating power of the air plasma was 8000 W with a current of 50 A and an air pressure of 0.5 MPa. The ablation time was set as 3 s. The mass ablation rate Rm was calculated by the formula: Rm = (m1 − m2) /t, where m1 and m2 represent the mass of the sample before and after ablation, and t is the ablation time.The density of the composites was measured by Archimedes’ method, and the porosity was calculated. The phase composition of the specimens was tested using an X-ray diffractometer  The scanning rate and step were 4°·minss and Css [2C appeared. Combined with the analysis of the W-Ta-C ternary phase diagram, there is a “W+TaCx+W2C” region in the tie-line between W and TaC when the TaC content is over 10 mol% [2C diffraction peak shifted to lower angles, probably because TaCx further interacted with W2C to form 2Css. The TaC and W2C diffraction peaks are shifted by 1°~2°. The SPS non-equilibrium sintering and the lack of C in the TaC and W2C cells are also important factors in the diffraction peak shift. It indicates that the W-TaC system has a similar phase compositions to that of the W-NbC system [Ta,W)Css ,12,22. T 10 mol% . Comparess phase.2C, the WTA5 composites have a density of 91.6 ± 0.2%, which is similar to those of the W-TiC and W-ZrC composites [mposites ,25. The ss and Css phases. The strong bonding of solid solution phases between the W and TaC grains positively affects the composite’s strength. Meanwhile, the strength of the W matrix is extensively enhanced by refining and solid solution strengthening. However, excessive TaC reacts with W to form W2C, and the TaC particles agglomerate to increase the porosity of the composites, and the poor interfacial bonding reduces the flexural strength of the composites [With the increased TaC content, the flexural strength of the composites increased from 292 ± 14 MPa for WTA1 to 528 ± 22 MPa for WTA4. The TaC second phase particles effectively increased the strength of the W matrix. The inter-diffusions at the W/TaC interfaces generated mposites .−1·K−1 at 750 °C. The thermal conductivities of W and TaC are ~175 W·m−1·K−1 and 15–35 W·m−1·K−1, respectively [−1·K−1. This might result from the higher TaC content, distributed in the composite matrix as a second phase, and hinders the thermal conduction between tungsten crystals. Besides, the effect of residual pores on the thermal conductivity of the composite and the existence W2C phase with low thermal conductivity (~30 W·m−1·K−1) is non-negligible [ectively ,25. Consectively . For WTAgligible .2C have opposite trends, the different content of TaC makes the thermal conductivity of W-TaC composites change in different trends at the test temperatures. The increase in temperature increases the chance of collision between free electrons and metal ions, and the thermal conductivity shows a decreasing trend. The higher the W content in WTA1 and WTA2, the stronger decreasing trend of the thermal conductivity of the composites as the test temperature decreases. For WTA3, WTA4, and WTA5 composites with higher TaC contents, the thermal conductivity turns to an increasing trend with temperature. In WTA5, W reacts with TaC to form W2C, and the thermal conductivity is increased more significantly than those of WTA3 and WTA4.The thermal conductivities of WTA1 and WTA2 decrease with increasing temperature, while the values of WTA3, WTA4, and WTA5 increase. Since the thermal conductivity of W decreases with increasing temperature and those of TaC and W2) [2C phase enhance the composite’s thermal expansion coefficient. It is difficult to get rid of the more considerable thermal stresses during quick heating at the beginning of the ablation process [2) . As a re2) ,17. Comp2) :Rst=  of the reactive sintered W-30 vol%TaC composite ablated under an oxyacetylene flame [The mass ablation rates of W-TaC composites were collected in ne flame . The dif3, Ta2WO8, etc. [2O5 is 400 °C higher than that of WO3) [O8, etc.  There is of WO3) . Oxides Composites were prepared by SPS sintering with TaC as the second reinforcing phase. The results show that TaC improves the mechanical and thermal properties of W-based materials, and the main conclusions are as follows:ss + W2C + 2Css due to solid solution reaction, TaC and W2C cell C deficiency, and non-equilibrium sintering when the TaC content was 50 vol%.1. The densities of the composites exceeded 93% when the TaC content was 40 vol%, and W reacts with TaC to form C2. The hardness of the composites increased with the increase in the TaC volume fraction, and when the TaC content was 50 vol%, the hardness of the composites reached 10.43 ± 0.73 GPa. The flexural strength of the W-40 vol%TaC composite reached a maximum of 528 ± 22 MPa.−1·K−1, which decreased with increasing temperature.3. The thermal conductivity of the composites increased and then decreased with the increase in the TaC volume fraction. The thermal conductivity of W-20 vol% TaC at 750 °C was 51.2 ± 0.2 W·m−1 to 0.0678 g·s−1 with increasing TaC content from 10 vol% to 30 vol%. The ablation products were mainly W oxides and mixed oxides of W and Ta.4. The addition of TaC enhanced the ablation resistance of W-TaC composites. The mass ablation rate reduced from 0.0918 g·s


In this study, the effect and mechanism of PM2.5 exposure in HaCaT keratinocytes were investigated. PM2.5 stimulated the aryl hydrocarbon receptor (AhR) to produce reactive oxygen species (ROS) in HaCaT cells, leading to mitochondrial dysfunction and intrinsic mitochondrial apoptosis. We observed that the culture medium derived from a particular skin microbe, Staphylococcus epidermidis WF2R11, remarkably reduced oxidative stress in HaCaT cells caused by PM2.5-mediated activation of the AhR pathway. Staphylococcus epidermidis WF2R11 also exhibited inhibition of ROS-induced inflammatory cytokine secretion. Herein, we demonstrated that S. epidermidis WF2R11 could act as a suppressor of AhRs, affect cell proliferation, and inhibit apoptosis. Our results highlight the importance of the clinical application of skin microbiome interventions in the treatment of inflammatory skin diseases.The skin supports a diverse microbiome whose imbalance is related to skin inflammation and diseases. Exposure to fine particulate matter (PMThe online version contains supplementary material available at 10.1007/s12602-022-09922-8. The adhesion of contaminants, oxidizing gases, organic compounds, or transition metals to PM0.1 and PM2.5 introduces toxins to the body [Air pollution from particulate matter (PM) has detrimental effects on humans and animals . Industr≤ 10 µm) . PM0.1 athe body , 5.Propionibacterium species dominate, whereas, in moist areas, Staphylococcus and Corynebacterium species dominate. An imbalance in the composition of bacterial species—i.e., dysbiosis—destroys the diversity of the microbial community, causing deterioration and inflammation of the skin and a plethora of skin disorders [2.5 can cause severe skin diseases, including eczema, upon permeating the human skin cells [2.5-induced dermatitis have not been evaluated.The skin, the largest organ of the human body, is a multi-layered structure comprising the epidermis, dermis, and subcutaneous tissues , 7. It iisorders , 14–16. in cells . However2.5 mediate many biological effects, including carcinogenicity and developmental defects [CYP1 and Cox-2 are deemed important functional biomarkers involved in AhR-mediated signaling. In addition to the PAHs constituting PM2.5, PM2.5 comprises various transition metals and organic compounds, which can induce excessive ROS production in mitochondria and cell cytoplasm [PAHs contained in PM defects –20. Seve defects , 21. Mos defects –24. Thes defects . Therefoytoplasm . It induytoplasm .Table 1CStaphylococcus epidermidis WF2R11, a member of the human skin microbiome, alleviates the oxidative stress generated due to PM2.5-mediated ROS accumulation, and whether S. epidermidis WF2R11 inhibits PM2.5-induced apoptosis in human skin keratinocytes (HaCaT) in vitro.Simultaneously, increased free radical production in skin tissue stimulates the release of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukins (ILs). These cytokines induce activated neutrophil infiltration and phagocytic production of radicals , 29. ExcAll processed gene expression data used in this study were procured from the Gene Expression Omnibus repository with accession number GSE107871 . This RNThe study for the isolation of permanent bacteria on the skin was approved by the Institutional Review Board  of Korea National Institute for Bioethics Policy (KONIBP). All study protocols adhered to relevant ethical guidelines. In addition, all participants provided written informed consent before enrolment. In addition, this study protocols adhered to relevant ethical guidelines and regulations. Skin samples were collected from donors who had no history of skin disease. Before collecting the skin samples, the participants were asked not to wash their faces for longer than 12 h. Samples were obtained by rubbing the donor’s face for 1 min or 20 times vigorously with a sterilized cotton swab soaked in distilled water and were then placed in a 10-mL trophic soy broth (TSB) solution.–1–10–threefold using phosphate-buffered saline (PBS). Then, 100 μL of each diluted solution was spread onto R2A, TSB, Luria–Bertani, MRS , and blood (Columbia agar with 5% sheep blood) agar plates . The inoculated plates were incubated at 37 °C for up to 72 h; whereafter, the single colonies on the plates were picked up, and their 16S rRNA genes were amplified using colony PCR. The applied parameters for the PCR were as follows: initial denaturation at 95 °C for 15 min, then 32 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min and 45 s, and then a final extension step at 72 °C for 5 min. The primers for PCR were 27F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 1492R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ). Purification of the amplified DNA was carried out using the EZ-pure PCR Purification Kit , and the nucleotide sequences of the genes were determined using the ABI 3730xl system . For phylogenetic analysis, the 16S rRNA gene sequences were analyzed using the nucleotide BLAST program available at the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome).The skin samples collected from ten donors were diluted to 10For hemolysis testing of the isolated bacteria, 73 single colonies were obtained by streaking bacterial samples onto TSB plates using an inoculation loop. The colonies were re-streaked onto blood agar plates (trypticase soy agar with 5% sheep blood) and incubated at 37 °C. The color of the colonies, indicating the hemolysis pattern, was noted at 48 h. From the isolation and subsequent analysis of 16S rRNA gene sequences, 50 species and 135 strains, including a variety of skin bacteria, were identified (data not shown). Hemolysis analysis of the isolated bacteria showed that most of the microbes had a β (35 strains) or γ pattern (37 strains), and the α pattern (1 strain) was not frequently observed (data not shown). For further analyses, six strains that showed γ (gamma) hemolysis patterns were selected.2.5) was purchased from Sigma-Aldrich, St. Louis, MO, USA. The PM2.5 dose used in this study was established based on US EPA air quality standards for particulate pollution and AQI  revisions. The composition and AQI category of Urban PM NIST 1648a are indicated in Table 2.5 stock solutions  were prepared in DMEM and sonicated for 10 min to avoid agglomeration of the suspended PM2.5 particles. All experiments were performed within 1 h of stock preparation to avoid variability in PM2.5 compositions in the solution.Urban PM NIST 1648a (PM5 HaCaT cells were maintained at 37 °C in an incubator with a humidified atmosphere of 5% CO2 and are tested four times per year for mycoplasma using PCR. All studies were conducted within 6 months of the latest test date (11–2020). HaCaT cells were cultured in DMEM with 10% heat-inactivated FBS and an antibiotic–antimycotic   for no longer than 4 weeks before use. For PM2.5 treatment, HaCaT cells were inoculated in 6-well plates, incubated in an atmosphere of 5% CO2 at 37 °C, and grown till they achieved 80% confluence. After 12 h, the cells were washed once with PBS, and 2 mL of each concentration of PM2.5 stock solution was added to the cells, depending on the purpose of the in vitro assays. Then, 10% of conditioned S. epidermidis WF2R11 medium  was added to the cells together with a supplement-free medium simultaneously as PM2.5 treatment.HaCaT cells were purchased from PromoCell . A total of 3 × 10AhR and CYP1A1 mRNA was commercially synthesized by Bioneer, Daejeon, South Korea: siAHR ; siCYP1A1 . To transfect the siRNA oligo, we used Lipofectamine™ RNAiMAX  according to the manufacturer’s instructions. Briefly, 24 h before transfection, 3 × 105 HaCaT cells were seeded onto 6-well plates with 2.5 mL of growth medium without antibiotics to reach 50–60% confluence at the time of transfection. AhR and CYP1A1 siRNA were incubated with HaCaT cells for 24 h. The efficiency of gene silencing by siRNA was evaluated using real-time PCR (qPCR).siRNA against human 5) were seeded into 48-well plates and incubated for 24 h in 2 mL of the complete medium. Then, 1, 5, 10, 20, or 30% of S. epidermidis WF2R11 culture supernatant was added to the cells, respectively, and incubated for another 48 h. After washing the cells once with PBS, 3--2,5-diphenyltetrazolium bromide (MTT) solution was added to each well, and the plates were incubated for 4 h. Then, the medium was discarded, and dimethyl sulfoxide was added to dissolve the formazan crystals. Optical density was measured at 570 nm using a microplate reader and was normalized relative to the untreated control.HaCaT cells  according to the manufacturer’s instructions. cDNA was synthesized from 1 μg total RNA using the Reverse Transcription Premix  under the following reaction conditions: 45 °C for 45 min and 95 °C for 5 min. Gene expression was quantified using qPCR, and data were analyzed using the StepOne Plus™ software . qPCR amplification reactions were performed using SYBR Green PCR Master Mix with premixed ROX . The following primer pairs  were used in the ABI 7300 Cycler internal reaction according to the manufacturer’s protocol (Online Resource 5), seeded in 6-well culture plates, were pretreated with PM2.5  for 12 h and then treated with the supernatant of the S. epidermidis WF2R11 for 12 h. After the treatment period, aliquots of samples (100 μL/well) were collected from the experimental medium, and the production of cytokines  was measured using a Human ILs Quantikine ELISA Kit  according to the manufacturer’s instructions.The proinflammatory cytokines  produced were measured using an ELISA assay. HaCaT cells  supplemented with protease/phosphatase inhibitor cocktail on ice for 30 min. BCA kit was used to measure the concentration of the proteins and for protein quantification. Proteins (20 µg) were mixed with loading buffer, separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes . The PVDF membranes were blocked with 5% skim milk in Tris-buffered saline with Tween (TBST) for 2 h at 37 °C and incubated with primary antibodies against β-actin (1:2000), Bax (1:1000 dilution), Bcl-2 (1:500 dilution), p38 (1:1000 dilution), p-p38 (1:1000 dilution), JNK (1:1000 dilution), p-JNK (1:1000 dilution), ERK (1:2000 dilution), and p-ERK (1:2000 dilution) at 4 °C overnight. After washing 3 times with TBST, PVDF membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. The immunoblots were visualized with a chemiluminescence detection system. The optical density (OD) of western blot bands was measured using ImageJ software . The data was normalized to the level of reference proteins and then averaged and presented as a relative fold change of control, from at least three independent experiments.For Annexin V-Pi assays, HaCaT cells were stained with Annexin V-FITC and PI and assessed for mitochondrial apoptosis using flow cytometry according to the manufacturer’s protocol . Early apoptotic cells , necrotic cells , and viable cells  were classified using flow cytometry and fluorescence detection of annexin V bound to HaCaT cells. Apoptotic cells were filtered and determined using flow cytometry . The data processing was performed using the FlowJo 7.6.1 software.2.5 were detected with fluorescence microscopy using MitoSOX™ Red Mitochondrial Superoxide indicator  as a specific fluorescence probe. HaCaT cells treated with PM2.5 were incubated with 5 μM of the probe for 30 min at 37 °C in the dark. Then, cells were thoroughly washed with warm Hank’s balanced salt solution (HBSS) buffer and mounted for imaging. MitoSOX™ Red-stained cells were visualized at an excitation wavelength of 510 nm and an emission wavelength of 580 nm.For the measurement of mitochondrial peroxide, HaCaT cells treated with different concentrations of PMThe mitochondrial membrane potential and early stage of apoptosis were analyzed via fluorescence microscopy after staining with 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide , a lipophilic cationic fluorescence dye. JC-1 dye is characterized by green fluorescence emission at ~ 529 nm in the monomeric form of the probe, which changes to red (~ 590 nm) with the concentration-dependent formation of red J-aggregates.5 normal cells onto LAB-TEK® chamber slides , treating the cells with 100 μg/mL PM2.5, and incubating the slides for 24 h at 37 °C. A thin smear of each HaCaT was stained using the diff-quik staining by manufacturer’s instructions. Briefly, the air-dried smears were sequentially dipped five times in a methanol fixative solution I, five more times in stain solution II (color reagent red), then dipped five times in stain solution III (color reagent blue), and gently rinsed with distilled water. The overall staining process took ~ 40 s. The slides were then cleared in xylol and mounted in a non-aqueous mounting medium. A diff-quik-stained smear of each HaCaT slide was analyzed by a board-certified pathologist (MGN).Diff-quik  staining preparations of HaCaT cells were obtained by spreading 3 × 105 normal cells onto LAB-TEK® chamber slides , followed by treatment with 100 μg/mL PM2.5, and incubation for 24 h at 37 °C. After removing the chamber wall, all the slides were fixed in neutral-buffered formalin. Endogenous peroxidase activity was blocked by incubating with 3% hydrogen peroxidase in methanol for 5 min. Epitope retrieval of Ki-67 was carried out by boiling in a pressure cooker in Tris–EDTA solution buffer at pH 9.0 for 15 min at 99 °C. After washing with PBS, the sections were incubated with the anti-human Ki-67 polyclonal antibody  at a dilution of 1:600 using an antibody diluent  for 60 min at 23–25.5 °C. The primary antibody against Ki-67 was detected with Polink-2 Plus HRP Rabbit with DAB Kit  for 30 min at approximately 23–25.5 °C. Diaminobenzidine  was used as the chromogen and incubated for 3 min at 25 °C. The slides were counterstained with hematoxylin. For the positive control, a normal tonsil tissue was used; for the negative control, the primary antibody was omitted, and PBS was used in each experiment. The Ki-67 index was quantified by estimating the number of positive HaCaT cells expressing nuclear Ki-67 (brown colored) among the total number of HaCaT cells. Both weakly and strongly labeled nuclei were included in the estimates of proliferating cells. All immunostained slides were evaluated twice by a blinded board-certified pathologist (MGN). A semi-quantitative assessment of the average percentage of Ki-67 positive cells in several fields comprising more than 1000 cells was performed.HaCaT cells were prepared by spreading 3 × 10p < 0.05.All data were tested for normality, and the dataset was analyzed using one-way ANOVA. The post hoc analysis was then carried out using the Bonferroni test for comparison between pairs. All results are presented as the mean ± SEM. Correlations were determined using Pearson’s correlation analysis. All statistical analyses were performed using GraphPad Prism 5.02  and R-4.2.0 for Windows. The statistical significance was set at 2.5 and ROS production [2.5, AhR stimulation, and ROS production, we performed RNA-seq analysis of whole skin using open-access data from a study by Swindell [2.5. AhR expression was positively correlated with ARNT expression  , unhealthy , hazardous . Similar to the correlation between the two genes presented in Fig. 2.5 concentration increased, suggesting the possibility that PM2.5 may act as a ligand for AhR and induce the AhR/ARNT complex formation   in the transcriptomic analysis  category, apoptosis  was compared and analyzed. Control HaCaT cells showed high intercellular density and evidence of persistent mitosis, whereas, in the PM2.5-treated cells, the intercellular density was low, and indicators of nuclear degeneration as well as apoptosis were evident -treated groups  and inoculated with Se solution. The treatment with Se solution reduced the mRNA level of AhR by more than 30% at each PM2.5 concentration group  in HaCaT cells using siRNA.To determine whether the Se solution downregulates AhR signaling and reduces ROS production, HaCaT cells were treated with different concentrations of PMoup Fig. a. Regardion Fig. b. As exp2.5 Fig. c. In addced Fig. d. Furtheoup Fig. e. Based AhR, ARNT, and CYP1A1 genes to less than half in all other groups compared to the si-control group respectively. However, the Cox-2 showed a lower mRNA expression level compared to other genes when treated with siAhR. In addition, the expression level of Cox-2 in the group treated with siAhR and PM2.5 was almost similar to that of the si-control group. The mRNA expression was significantly reduced in the Se solution treatment group compared to the PM2.5 treatment group , extracellular signal-regulated kinase 1/2 (ERK), and p38 mitogen-activated protein kinase (p38 kinase). After treatment with either PM2.5 (100μg/ml) or PM2.5 (100 μg /ml) and Se solution for 12 h, the phosphorylation level of protein kinase was determined , JNK, ERK, and p38 protein and calculated the Bax/Bcl-2 ratio for correlation with mitochondrial apoptosis mechanisms  showed a significant decrease in the Bax/Bcl-2 ratio compared to the si-control group following PM2.5 treatment, and a more significant decrease compared to the PM2.5 alone treatment group when Se solution was added. Furthermore, when AhR signaling was blocked by treatment with both siAhR and siCYP1A1, the Bax/Bcl-2 ratio was significantly reduced regardless of PM2.5 and Se solution treatment. Our results suggest that CYP1A1 is an important gene that mediates mitochondrial apoptosis signaling induced by PM2.5-induced intracellular ROS accumulation. In addition, S. epidermidis WF2R11 directly inhibits AhR to regulate the sub-mechanism of CYP1A1-induced sub-mechanisms. To further show that ROS accumulated in HaCaT cells via AhR signaling pathway induced mitochondrial apoptosis, we analyzed apoptosis by treating HaCaT cells with PM2.5 and PM2.5 and Se solution in siAhR-treated and untreated groups, respectively. We labeled cells with Annexin V-PI and observed the percentage of apoptotic cells using flow cytometry  gene  gene showed a significant positive correlation with CYP1A1  and heme oxygenase-1 (HO-1) [Nrf-2 was positively correlated with the hallmark genes of the AhR signaling pathway  with or without PM2.5 treatment by partially silencing AhR signaling pathway. The expression level of Nrf2 was correlated with the activation of AhR signaling; meanwhile, when both AhR and CYP1A1 were silenced, the expression level was significantly reduced , Se solution, and PM2.5 (100 μg/ml) and Se solution. The expression of Nrf2 was significantly decreased in the AhR silenced group (gray color) regardless of Se solution treatment with PM2.5 compared to the control. However, in the group (pattern) in which CYP1A1 was silenced, the expression of Nrf2 was increased more than threefold compared to the control when PM2.5 was treated. Meanwhile, when comparing the PM2.5-treated and the Se solution-treated groups, the expression of Nrf2 was significantly reduced in the Se solution-treated group, although the group in which both AhR and CYP1A1 were silenced (black color) showed almost no expression of Nrf2 regardless of PM2.5 and Se solution treatment  –50. Ther2.5 exposure on the AhR pathway and mitochondrial dysfunction-dependent apoptosis. To the best of our knowledge, this is the first study to analyze how a member of skin microbiota can downregulate the AhR pathway, which leads to the inhibition of skin inflammation and ROS production. We specifically investigated the effect of S. epidermidis WF2R11 on the progression of the AhR pathway. The main mechanism by which PM2.5 induces cellular oxidative stress is through the generation of ROS in skin keratinocytes [2.5 directly increases the level of ROS production and free radicals on the surface of skin keratinocytes, indicating potent redox activity in PAHs [2.5-binding AhR complexes to ARNT [2.5 acted as a ligand for AhR, both the AhR and ARNT mRNA levels increased. Our results demonstrated that although S. epidermidis WF2R11 did not directly affect AhR, it reduced AhR and ARNT mRNA levels. It was initially speculated that the reduced expression of AhR and ARNT is caused by the metabolites secreted by S. epidermidis WF2R11, thereby affecting the formation of AhR/ARNT complexes. However, when AhR was knocked down in HaCaT cells, ARNT and CYP1A1 expression was inhibited regardless of treatment with PM2.5 or the S. epidermidis WF2R11 supernatant. Although previous hypotheses have speculated that the S. epidermidis WF2R11 metabolites inhibit the formation of the AhR/ARNT complex, this finding demonstrated that AhR activation to PAH is a prerequisite for ARNT and CYP1A1 expression. We therefore suggest that the S. epidermidis WF2R11 metabolites may operate by suppressing AhR activity on PAHs or act as a competitive ligand inhibitor, rather than directly inhibiting the formation of the AhR/ARNT complexes. AhR/ARNT heterodimers bind to xenobiotic responsive elements and activate the transcription of sub-target genes such as CYP1 [CYP1A1 was significantly increased after PM2.5 treatment, while S. epidermidis WF2R11 was involved in the inhibition of AhR activity, thereby reducing the expression of CYP1A1. Thus, the AhR/ARNT complex affects the activity of the CYP1 enzyme, thereby increasing the expression of CYP1A1 [Cox-2 overexpression in HaCaT cells critically exerts oxidative stress on various organelles, including mitochondria [CYP1A1 and in Cox-2 expression [Cox-2 was significantly reduced, which was restored following PM2.5 treatment to the same level as that of si-control. However, despite silencing CYP1A1, signal transduction of AhR caused an increase in the expression of Cox-2, which suggests that the expression of Cox-2 is determined somewhat independently of the sub-mechanism of CYP1A1, and its expression may be regulated by AhR activity. Moreover, PAH and oxy-PAH result in differential ROS generation and Ca2+ perturbation and promote electrophysiological instability, which increases mitochondrial inner membrane permeability. Furthermore, an increase in ROS not only produce a cytotoxic effect, but also activate MAPK pathways, including JNK, ERK, and p38 MAPK, which ultimately lead to mitochondrial stress and excessive free radical accumulation, resulting in mitochondrial dysfunction and apoptosis  [2.5 and Se solution treatment via fluorescent staining , suggesting that the metabolite of S. epidermidis WF2R11 is effective in suppressing PM2.5-induced oxidative stress. Furthermore, we demonstrated that the metabolites of S. epidermidis WF2R11 can significantly reduce phosphorylation of p38 kinase and JNK among ROS-activated MAPK subgroups. Bax is phosphorylated by stress-activated p38 kinase and/or JNK and phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis. The mitochondrial translocation of pro-apoptotic Bax can act as apopotosis stimulators or conditions that induce mitochondrial apoptosis [2.5-induced apoptosis upregulates Bax and downregulates Bcl-2 [Bax/Bcl-2 ratio suggests a decrease in mitochondrial dysfunction-dependent apoptosis in the S. epidermidis WF2R11-treated group. Although the mechanism by which increased ROS can activate ERK, JNK and p38 MAPK remain unclear [S. epidermidis WF2R11 did not show any significant difference in the regulation of Bcl-2 but exhibited downregulated Bax expression. In addition, since the PM2.5-derived ROS-induced mitochondrial apoptosis mechanism is increased through the AhR signaling pathway, the change in the Bax/Bcl-2 ratio was confirmed after suppressing the AhR or CYPA1 gene, or both. We confirmed that the key gene for PM2.5-derived ROS increase was the expression of CYP1A1, while S. epidermidis WF2R11 suppressed AhR, the upper gene of CYP1A1, to reduce PM2.5-derived ROS. Transcriptome analysis showed that the AhR signaling pathway not only increased the expression of CYP1A1 but also contributed to the activation of TNF-α signaling to induce mitochondrial apoptosis, or upregulate Cox-2, thereby contributing to the activity of p38 and JNK MAPKs to enable translocation of mitochondrial Bax. Taken together, excessive oxidative stress due to the AhR pathway specifically affects the expression level of Bax via phosphorylation of MAPKs and may be a major contributor to mitochondrial dysfunction [Bax, which suggests AhR as an important receptor that leads to apoptosis by causing mitochondrial dysfunction. Furthermore, the metabolites of S. epidermidis WF2R11 can inhibit the progression of ROS-mediated damage by reducing AhR activity. Endogenous ROS can also cause structural damage and cell degradation, leading to apoptosis in HaCaT cells [2.5 triggers an inflammatory cascade [S. epidermidis WF2R11 effectively reduced apoptosis and inflammatory responses from PM2.5-induced oxidative stress via AhR-mediated Nrf2 signaling. Previous studies have suggested two different mechanisms for the activation of Nrf2 by AhR: direct transcriptional activation of Nrf2 following AhR signaling activation or ROS generation by CYP1A1 induction [S. epidermidis WF2R11 influence initiation of Nrf2 signaling. AhR silencing significantly reduced the expression level of Nrf2 and sub-genes such as NQO1 and HO-1 regardless of PM2.5 or S. epidermidis WF2R11 treatment. However, when CYP1A1 was silenced, while treating with PM2.5, the expression of Nrf2 gene was significantly increased, whereas when PM2.5 and S. epidermidis WF2R11 were co-treated, the expression of Nrf2 was significantly decreased.This study demonstrated the effect of PMinocytes . PM2.5 d in PAHs , 53. In  in PAHs , 55. Wat in PAHs , and oxy in PAHs . Initiat to ARNT , 57. It  as CYP1 . CYP1 is as CYP1 , 60. In  as CYP1 , 37, 61.f CYP1A1 , 62. In chondria , 63. Sigpression . In partoptosis) –69. We vpoptosis , 71. PM2es Bcl-2 , 72–74;  unclear , we founfunction –78. The aT cells . Previou cascade . Activat cascade . The sec cascade , 80, 81.nduction , 83. AmoCYP1A1, has direct transcriptional activation function for Nrf2 signal and that two other pathways of AhR may be activated simultaneously, one to increase cellular ROS and the other to trigger an antioxidant response. Furthermore, the decrease in expression Nrf2 following inhibition of AhR may possibly reduce the activation signal for other ROS generation mechanisms involved in the AhR signaling pathway. Conversely, we consider that the reason for the increase in Nrf2 during PM2.5 treatment despite the silencing of CYP1A1 is probably attributed to ROS generation associated with the increased expression of Cox-2 based on the activity of AhR. Thus, the direction of pathway progression of AhR will depend on either the ligand, intracellular ROS accumulation, or additional factors, which will determine the agonist or compensatory antioxidant AhR pathway.These results indicate that AhR, not S. epidermidis WF2R11 can inhibit the activation of AhR. Therefore, further research is required to study the effects of metabolites secreted by the skin microbiome on the AhR signaling pathway. We will also investigate the association of AhR-Trp metabolism and explore the functional significance of microbial Trp metabolites in skin inflammation. Future studies should harbor the prospect of overcoming these limitations and treating skin diseases caused by PM2.5 exposure.Taken together, these findings provide new insights into the potential application of skin microbiome interventions in clinical practice. However, this study could not comprehensively explore the mechanism by which the metabolites of Supplementary file1 (DOCX 3184 KB)Below is the link to the electronic supplementary material.


GsMYB7 gene in soybean under aluminum acid stress, and its regulatory mechanism remains unclear.MYB transcription factor (TF) is one of the largest families of TFs in plants and play essential roles in plant growth and development, and is involved in responses to biological and abiotic stress. However, there are few reports on GsMYB7 held a constitutive expression pattern rich in roots. When AlCl3 concentration was 25 µM, the total root surface area (SA) of GsMYB7 transgenic lines were 34.97% higher than that of wild-type Huachun 6 (HC6). While the accumulation of Al3+ in root tip of transgenic plants after aluminum treatment was 17.39% lower than that of wild-type. RNA-sequencing analysis indicated that over 1181 genes were regulated by GsMYB7 and aluminum stress. Among all the regulated genes, the expression levels of glutathione peroxidase, protein kinase, cytochrome and other genes in the transgenic lines were significantly higher than those in wild type by acidic aluminum stress. The bioinformatics and qRT-PCR results showed that 9 candidate genes were induced under the treatments of acidic aluminum stress which were indirectly and/or directly regulated by GsMYB7. After AlCl3 treatments, the transcripts of these genes in GsMYB7 transgenic seedlings were significantly higher than those of wide-type HC6.The GsMYB7 protein is localized in the nucleus and has transcriptional activation ability. Quantitative real-time PCR (qRT-PCR) results showed that GsMYB7 may enhance soybean tolerance to acidic aluminum stress by regulating the downstream genes.The results suggested that The online version contains supplementary material available at 10.1186/s12864-022-08744-w. Aluminum (Al) is the third richest element in the earth's crust next to oxygen and silicon . AluminuGmMYB92, GmMYB76 and GmMYB177 in Arabidopsis can enhance the tolerance of transgenic plants to salt and cold injury   .Trizol solution . The complementary DNA (cDNA) templates were synthesized using the HiScriptII first strand cDNA synthesis kit  [TMII , the RNA transcripts of the relative genes were measured using a real-time CFX96TM system  for qRT-PCR [Actin-3 in soybean. Finally, the relative expression values were calculated using the comparative cycle threshold method 2−△△ct. The experiments were carried out with three independent organisms [The total RNA of samples was extracted from plant seedlings using , China) . Using S qRT-PCR . The folrganisms .http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/). The recombinant fragment containing the CDS sequence of GsMYB7 gene (without termination codon) was amplified and purified with the restriction sites of NcoI and SpeI at both ends  for culture and then mixed in the same volume. After sitting at room temperature for 3 h, the bacterial fluid was injected into the lower epidermis cells of 4-week-old leaf in Nicotiana tabacum, and the transformed leaves after being cultured for 48 h were observed by laser confocal microscopy .The subcellular localization of GsMYB7 protein was analyzed using the method described by Tamara et al.and and Yan et al., 60. TheGsMYB7 was analyzed using the method described by Ma et al. [GsMYB7 was inserted into the sites of BamHI and EcoRI restriction enzymes of pGBKT7 vector to form the fusion expression vector pGBKT7-GsMYB7. The constructed vector pGBKT7-GsMYB7 and the empty vector pGBKT7 were then transformed into yeast Y2H strain, respectively. The positive transformants were cultured on SD medium which was lacking Trp (SD/-Trp) at 30 ℃ for 2 or 3 days. The positive colonies were transferred from SD/-Trp medium to chromogenic SD/-Trp medium containing 200 μg X-α-gal. After cultured for 3 days, the status of colony growth could be detected on selective medium.Transcriptional activation assay of a et al. . To veriGsMYB7 amplified by PCR was inserted between the restriction sites of BamHI and KpnI from the modified intermediate vector pUC18 to construct the intermediate vector pUC18-GsMYB7. The pUC18-GsMYB7 was digested with a HindIII enzyme to recover the target fragment of 35S-GsMYB7-NOS. The DNA fragment containing the target gene was inserted into the HindIII site of pZY101 vector to construct GsMYB7-pZY101 vector which was then transformed into the competent cells of Escherichia coli DH5α. The positive clones of pZY101-GsMYB7 were identified by bacterial liquid PCR, enzyme digestion and DNA sequencing. The expression plasmid of pZY101-GsMYB7 was transformed into Agrobacterium tumefaciens EHA101 and then used for soybean genetic transformation. The soybean HC6 was used as receptor for GsMYB7 genetic transformation using the cotyledonary node method described by Zeng et al. [3 transgenic positive lines of GsMYB7 were further used to analyze the expression pattern response to aluminum stress, the phenotypes tolerant to aluminum stress, and molecular identification, etc. [The CDS sequence of g et al. . The obton, etc. . The priGsMYB7 transgenic lines and wild type (WT) for the phenotype analysis to aluminum stress were sterilized with 1.0% (v/v) NaClO for 5 min and germinated in sterilized moist vermiculite sand which was placed under a condition of photoperiod of 14-h day/10-h night and 28 /25℃ (day/night) temperature circulations for 3 or 4 days in light incubator. The soybean seedlings cultured in 0.5 mM CaCl2 (pH4.5) solution for 24 h were transferred to nutrition barrels separately which contained different concentrations of AlCl3 (0 and 25 μM) for 48 h. The data were analyzed by using the software Image J and Root Scanner [The full seeds of  Scanner .GsMYB7 transgenic lines and WT were germinated in darkness for 3 days and then transferred to Hoagland solution under light incubator 16 h (26◦C)/8 h (24◦C) day/night. After cultured for 3 days, the seedlings with relatively consistent length of taproots were chosen and immediately transferred to Hoagland solution with 25 µM AlCl3 (pH4.5) for 24 h [The seeds of for 24 h . The 0–2for 24 h .GsMYB7 transgenic soybean and HC6. The root samples were taken from the seedlings of GsMYB7 transgenic plants and HC6 treated with or without 25 µM AlCl3 . After the treatment for 8 h, the samples of 2 cm root tip were taken for transcriptome sequencing analysis [To determine the downstream genes and regulatory pathways regulated by TF GsMYB7 under acidic aluminum stress, the RNA-seq technology was used to find the differentially expressed transcriptome of analysis . Each bianalysis .*P < 0.05; **P < 0.01) [All experiments were repeated at least three times independently, with tissue samples from 20 plants in each experiment. The data were the mean ± SD. The SPSS Statistics (v.22) and GraphPad Prism 8 softwares were used to analyze and process the data. Differences were compared using Student’s t-test. The statistical significance of the difference for the data was indicated by an asterisk ( < 0.01) .Additional file 1:Table S1. Sequence information of GsMYB7. Table S2. Primers used in this study. Table S3. A cis-acting element in the upstream nucleotide sequence of the initiation codon of GsMYB7. Table S4. The information of MYB genes from soybean. Table S5.GsMYB7 downstream candidate gene information. Additional file 2:Table S6. G7L1VSG7L1_A_Gene_differential_expression. Table S7. WT_AVSG7L1_A_Gene_differential_expression. Table S8. WTVSG7L1_Gene_differential_expression. Table S9. WTVSWT_A_Gene_differential_expression.Additional file 3:Table S10. Bar primers were used to identify the transgenic lines by PCR.