Understanding the unique features of immune functions of HLA-E to inform future clinical exploitation requires detailed knowledge of HLA-E transport. He et al. characterize the basic trafficking patterns of HLA-E within cells and how this trafficking is regulated. + T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)–restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions.Interest in MHC-E–restricted CD8 MHC-E is a highly conserved non-classical major histocompatibility complex class I (MHC-I) molecule. Although HLA-E (human MHC-E) cell surface expression is universal, it is normally low compared with classical HLA-Ia molecules . The mai+ T cells, though reports are scarce compared with classical MHC-Ia molecules. HLA-E can activate CD8+ T cells through the presentation of Mycobacterium tuberculosis (Mtb) peptides  peptides to CD8+ T cells and inhibit S. typhi pathogenesis  or HLA-A*03:01 (HeLa.A3) tagged with EGFP. HLA-A*03:01, which is not known to share the binding of peptides with HLA-E, was selected as the control. To examine the proportion of HLA-E expressed on the cell surface, we compared the mean fluorescence intensity (MFI) of permeabilized samples, which indicated the total protein expression level, to the MFI of surface staining. While HLA-A3 was mainly found on the plasma membrane in HeLa.A3 cells, HLA-E was mostly found intracellularly in HeLa.E cells , which iThus, HLA-E differs from HLA-A3 by enrichment in the ER as well as in late and recycling endosomes, with only a tiny fraction expressed on the cell surface.As ER retention and low surface expression of HLA-E may result from a delay in antegrade transport, we compared the trafficking of HLA-E and HLA-A3 using the RUSH (retention using selective hooks) system. In the RUSH system, HLA molecules are trapped within the ER via attachment to an ER-retained “hook” protein from which subsequent release can be triggered by biotin addition to synchronize the ER exit . Given tWhen the hook and the reporter were cotransfected into HeLa cells, both HLA-E and HLA-A3 were localized in the ER . After sHLA-A3 could be clearly detected at the plasma membrane within 10–15 min after ER release, and the surface signal was significant at 30 min . As imagA proportion of HLA-A3 was detected in what appeared to be endosomal structures 1 h after biotin addition, which became more obvious after overnight biotin release . This isTaken together, although the proportion of HLA-E and HLA-A3 molecules exiting the ER differs, they are trafficked with similar dynamics upon ER export.To investigate the antegrade transport of endogenous HLA-E in THP1-derived macrophages, we exposed the cells briefly to citric acid buffer to strip off surface HLA-I proteins  and subsTo examine the contribution of the recycling pathway to the recovery of surface proteins, we also included control samples with brefeldin A (BFA), which was added to prevent newly synthesized HLA-I molecules from reaching the cell surface . BFA addTo summarize, newly synthesized HLA-E and HLA-Ia are trafficked similarly to the cell surface upon ER exit in THP1-derived macrophages, which is consistent with our results obtained in HeLa cells.In addition to ER retention, low surface stability could also account for the low surface expression of HLA-E. Therefore, we incubated HeLa.E and HeLa.A3 with BFA and measured the remaining surface signal of HLA-E and HLA-A3 over time. HLA-E disappeared rapidly from the cell surface, with a half-life of less than 1 h . On the We then examined how the surface expression level of HLA-E is influenced by HLA-E binding peptides and β2-microglobulin (β2m), which together with HLA-E form the functional heterotrimer complex. We cotransfected a fixed amount of HLA-E plasmid with an increasing amount of plasmids encoding either β2m or different peptide minigenes into HEK293T cells and examined how these factors influence HLA-E surface expression.VL9 (VMAPRTVLL), the canonical signal peptide that dominates HLA-E binding in normal cells , could eOverexpression of β2m also enhanced HLA-E surface expression , as illuTherefore, while both β2m and HLA-E binding peptides regulate HLA-E surface expression, peptide availability appears to play the dominant regulatory role.Given that HLA-E is enriched in the ER and is unstable on the cell surface, HLA-E binding peptides may increase its surface expression by promoting the ER export and/or surface stability of HLA-E.To investigate the role of HLA-E binding peptides, we cotransfected the RUSH system with different peptide minigenes into HEK293T cells. In the presence of the VL9 peptide minigene, we observed a rapid increase of surface HLA-E signal after biotin addition , confirmTo further characterize how HLA-E binding peptides affect its surface stability, a BFA decay assay was performed. We cotransfected HEK293T cells with HLA-E and different peptide minigenes and assessed the surface HLA-E level following various BFA incubation times . While tWe further validated the function of the endogenous VL9 peptide via confocal microscopy. Cotransfection of the VL9 peptide minigene alleviated HLA-E ER retention . When coIn conclusion, HLA-E binding peptides like VL9 increase HLA-E surface expression primarily by promoting ER exit but do not enhance its surface stability under physiological conditions.As the surface stability of HLA-E molecules driven out of ER via endogenous VL9 peptide expression was not improved, we hypothesized that they likely ended up in endosomes. Therefore, we cotransfected different peptide minigenes and HLA-E into HeLa cells and used confocal microscopy to investigate the endosomal distribution of HLA-E. The cotransfection of VL9 peptide minigene led to the enrichment of HLA-E in early endosomes  and lysoPrevious studies have shown that the cytoplasmic tail of MHC-I molecules influences endocytosis , degradaHLA-EA3 surface expression was ∼1.7-fold of that of HLA-E , and HLAAs most HLA-E was retained in the ER, we then examined if the HLA-A3 cytoplasmic tail could improve HLA-E ER egress. Compared with HLA-A3, HLA-A3E had a greater ER colocalization . In HLA-Taken together, these results indicate that the HLA-E cytoplasmic tail downregulates HLA-I surface expression and facilitates ER retention, although the effect appears minor compared with peptide supply.After determining the effect of the cytoplasmic tail on ER export and surface expression level, we explored how the cytoplasmic tail influences the surface turnover of HLA molecules. HLA-E cytoplasmic tail shortened the surface half-life of HLA-I molecules compared with their counterparts with the HLA-A3 cytoplasmic tail . The halTo further explore if the cytoplasmic tail regulates internalization, we employed an acid-stripping assay to measure the internalization rate of surface HLA molecules as previously described . After sMoreover, we found that the difference in internalization rate introduced by the cytoplasmic tail appears to dictate some of the difference in surface stability, at least at early time points. 1 h after BFA addition, 20% more HLA-E disappeared from the cell surface compared with HLA-EA3 . SimultaAs the HLA-E cytoplasmic tail regulates endocytosis, we then investigated whether it affects the distribution of HLA-I molecules in different endosomal compartments. While HLA-E was more enriched in late endosomes and recycling endosomes than HLA-A3 in HeLa cells , there wHowever, swapping the cytoplasmic tail did not completely reverse the endosomal distribution of HLA molecules. While HLA-EA3 was less enriched in recycling endosomes compared with HLA-E, the late endosomal enrichment was retained, implying that factors other than the cytoplasmic tail also contribute to the enrichment of HLA-E in late endosomes. The slight difference between HLA-EA3 and HLA-A3 in the enrichment of recycling endosomes also suggests the existence of other regulatory factors. No difference was observed in the distribution of these different constructs in early endosomes and lysosomes . Taken t+ T cells have been reported in different infections. HLA-E–restricted CD8+ T cells are a prominent component of the immune response against Mtb in humans  were purchased at a purity of 85% (GenScript), reconstituted to a final concentration of 200 mM in DMSO, aliquoted, and stored at −80°C.All plasmids were prepared using QIAprep Spin Miniprep kits (Qiagen). Primers  and GeneHLA-E, HLA-EA3, HLA-A3, and HLA-A3E without the EGFP tag were amplified from the corresponding EGFP-fusion constructs using EGFP-N1 F and corresponding reverse primers .The β2m expression plasmid was created by inserting a GeneArt string encoding β2m with a C-terminal Picornavirus 2A “slip” sequence between the HindIII and AgeI restriction sites of pEGFP-N1 so that β2m and EGFP would be expressed as separate proteins in transfected cells. Peptide expression constructs were generated by annealing complementary oligonucleotides encoding the desired peptide sequences with appropriate overhangs to allow insertion between the HindIII and AgeI sites of pEGFP-N1.The plasmid expressing the Sec61B RUSH hook was created by inserting a GeneArt String encoding an str-Sec61B fusion into pEGFP-N1 HindIII and NotI. The HLA-SBP-EGFP RUSH reporter plasmids were created by inserting HindIII-EcoRI fragments encoding HLA-E or HLA-A3  and an EcoRI-NotI fragment encoding an SBP-EGFP fusion  between the HindIII and NotI sites in pEGFP-N1.Lentiviral constructs were created by replacing the EGFP of pLenti CMV GFP Puro , while THP-1 cells and K562 cells were cultured in RPMI 1640 Medium (Life Technologies). All culture media were supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich) and penicillin/streptomycin , and cells were maintained at 5% CO-E*01:03  were genHEK293T cells and HeLa cells were seeded at 50–80% confluence in 6-well plates 24 h before transfection. HEK293T cells were transfected with 1 µg of plasmid DNA using GeneJuice (Merck), and HeLa cells were transfected with 2 μg of plasmid DNA using Lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer’s instructions and were harvested after 24 h. For immunofluorescence, HeLa cells were seeded in 24-well plates and transfected with 0.5 µg of total plasmid DNA. For the RUSH assay, hook and reporter plasmids were transfected at ratios of 5:1 and 10:1 for HLA-E and HLA-A3, respectively, to ensure optimal ER trapping. When plasmids expressing peptides were included in the RUSH assay, an equal amount of the peptide minigene plasmid and the RUSH plasmids were transfected. In HEK293T cells co-transfected with HLA-E and different amounts of peptide minigene plasmid or β2m plasmid, 0.2 µg HLA-E plasmid was transfected for each sample, and the total amount of plasmid is topped up to 1 µg by the addition of EGFP plasmid.g for 30 min at 32°C. 24 h after transduction, the virus was removed and 2 ml fresh media was added. 72 h after transduction, media containing puromycin (Life Technologies) was added at a final concentration of 2 μg/ml for selection for a week.HeLa cells stably expressing EGFP-fusions of HLA-E, HLA-A3, or the EA3 and A3E hybrids were generated by lentiviral transduction. To produce lentivirus, HEK293T cells were seeded in 6-well plates at 50% confluence 24 h before transfection. Transfection was done by cotransfecting 1 μg transfer plasmid, 0.5 μg pMD2.G envelope plasmid , and 0.5 μg pCMV-dR8.91 packaging plasmid using GeneJuice (Merck) following the manufacturer’s instructions. Media was replaced with antibiotic-free media 8 h after transfection, and viral supernatant was collected 48 h after transfection and filtered through a 0.22 µm syringe filter (Merck). HeLa cells were seeded 24 h before transduction, and the media was replaced by 0.5 ml viral supernatant and 1.5 ml antibiotic-free media containing polybrene (Santa Cruz Biotechnology) at a final concentration of 10 μg/ml. Samples were then centrifuged at 800 ×For surface staining, cells were washed twice with Dulbecco’s PBS , stained for 20 min on ice in 100 μl DPBS, washed twice in DPBS, and fixed for 20 min on ice in 100 µl Cytofix (BD Biosciences). For intracellular staining, cells were washed twice with DPBS, fixed, and permeabilized in 100 μl Cytofix/Cytoperm Solution (BD Biosciences) for 20 min on ice, washed twice in Perm/Wash buffer (BD Biosciences), stained for 20 min on ice in 100 μl Perm/Wash buffer, washed twice with Perm/Wash buffer, once with DPBS, and resuspended in DPBS. Cells were acquired using a Cyan ADP Analyser (Beckman Coulter) or an Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed using FlowJo 10.4. Details of antibodies used are listed in 2O, with the addition of protease inhibitor cocktail [Roche], PhosSTOP [Roche], and PMSF [Stratech Scientific]). Lysates were centrifuged at 13,000 rpm (4°C) for 15 min to remove cell debris. Samples were then treated with 1,000 U of Endo Hf (New England Biolabs) for 2 h at 37°C following the manufacturer’s protocol. Equal volumes of lysates were mixed with LDS Loading buffer (Life Technologies), run on 4–12% Bis-Tris gels (Life Technologies) in 3-(N-morpholino)propanesulfonic acid buffer (Life Technologies), and then transferred to polyvinylidene difluoride membrane (Merck) in 2× NOVEX Transfer buffer (Life Technologies) on a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Membranes were blocked for 1 h at room temperature in blocking buffer , then incubated for 1 h at room temperature with primary antibodies diluted in blocking buffer. For the HeLa stable cell lines, mouse anti-EGFP was used to detect HLA-E_EGFP or HLA-A3_EGFP. For THP1-derived macrophages, MEM-E/02 and HC10 were used to detect endogenous HLA-E and HLA-I, respectively. Membranes were washed three times with DPBS+0.05% Tween-20 (10 min each), incubated with IRDye 800CW secondary antibody diluted in blocking buffer for 1 h at room temperature, washed as before, and imaged using a LICOR Odyssey X. Details of the antibodies used are listed in Cells were lysed by incubating for 20 min on ice in RIPA buffer  and fixed with 4% formaldehyde  for 15 min. For the RUSH assay, biotin diluted in media was added to reach a final biotin concentration of 50 μM, and cells were directly fixed with 4% formaldehyde at different time points after biotin addition without DPBS washing. After three washes with DPBS, samples were permeabilized and blocked by incubating for 1 h in blocking buffer , stained with primary antibody diluted in blocking buffer for 1.5 h, washed quickly three times with blocking buffer, stained with secondary antibody diluted in blocking buffer for 2 h, washed as before with the blocking buffer, washed twice with DPBS, and then mounted with DAPI fluoromount-G (Cambridge Bioscience). Images were obtained using a Zeiss LSM880 inverted confocal laser scanning microscope with a Plan-Apochromat 63×/1.4 oil objective. Images were processed and analyzed in Fiji/ImageJ. ROI was selected for individual cells excluding the nuclear region, and colocalization was calculated using the Coloc2 plug-in to determine PCC. Details of the antibodies used are listed in Live cell imaging was adapted from To analyze the dynamics of MHC-I transport in THP1-derived macrophages, cells were washed twice with cold DPBS and resuspended in citric acid buffer (pH 3.0) for 3 min on ice to strip off surface MHC-I proteins. Cells were then neutralized with 20 volumes of cold media and washed twice with cold DPBS. Cells were resuspended in prewarmed media and incubated at 37°C to allow for the recovery of surface MHC-I. BFA (Cambridge Bioscience) was added at 10 µg/ml to some samples to inhibit the antegrade transport of MHC-I. Cells were collected at appropriate times and fixed with 100 µl Cytofix (BD Biosciences). Surface expression of HLA-E or total MHC-I was further assessed by flow cytometry. The amount of protein recovered was calculated as the percentage of the initial surface signal before acid stripping.To analyze the internalization rate of different HLA-I molecules, HeLa cells or THP1-derived macrophages were stained on ice for 30 min with 3D12-APC (HLA-E), GAP.A3-APC (HLA-A3), or W6/32-APC (HLA-I). Cells were washed twice with cold DPBS to remove unbound antibodies and then incubated at 37°C to allow for internalization in the presence of 0.3 mM primaquine (Enzo Life Sciences) for different time lengths. Acid stripping was performed as described above, and samples were fixed with 100 µl Cytofix (BD Biosciences) and then assessed by flow cytometry. The amount of protein internalized was calculated as the percentage of the initial surface signal before acid stripping. Details of the antibodies used are listed in Cells were incubated with BFA (Cambridge Bioscience) for different time lengths at a final concentration of 10 µg/ml, with or without a 3-h preincubation of exogenous peptides (100 μM), and surface expression of HLA-E or total MHC-I protein was assessed by flow cytometry. Protein transport was stopped by keeping all samples strictly at 4°C during surface staining. Surface stability was calculated as the percentage of the initial surface signal without BFA incubation. Details of the antibodies used are listed in t test with Welch’s correction  or one-way ANOVA with Tukey’s post-hoc test (three or more groups) was performed for comparison between groups.Data were analyzed in GraphPad Prism 9 and presented as mean ± SD . Two-tailed Student’s Table S1lists primers for plasmid construction.Click here for additional data file.Table S2lists antibodies.Click here for additional data file.SourceData F1contains original blots for Fig. 1.Click here for additional data file.SourceData FS1contains original blots for Fig. S1.Click here for additional data file.


O-glycans and glycosaminoglycans [GAGs]) are critical nutrient sources and colonisation factors for Bacteroidetes of the human gut microbiota (HGM); a complex ecosystem comprising essential microorganisms that coevolved with humans to serve important roles in pathogen protection, immune signalling, and host nutrition. Carbohydrate sulfatases are essential enzymes to access sulfated host glycans and are capable of exquisite regio- and stereo-selective substrate recognition. In these enzymes, the common recognition features of each subfamily are correlated with their genomic and environmental context. The exo-acting carbohydrate sulfatases are attractive drug targets amenable to small-molecule screening and subsequent engineering, and their high specificity will help elucidate the role of glycan sulfation in health and disease. Inhibition of carbohydrate sulfatases provides potential routes to control Bacteroidetes growth and to explore the influence of host glycan metabolism by Bacteroidetes on the HGM ecosystem. The roles of carbohydrate sulfatases from the HGM organism Bacteroides thetaiotaomicron and the soil isolated Pedobacter heparinus (P. heparinus) in sulfated host glycan metabolism are examined and contrasted, and the structural features underpinning glycan recognition and specificity explored.Sulfated host glycans (mucin  O-glycans (cMOs)  or α-L-iduronic acid [IdoA]) and α-D-glucosamine (D-GlcN) A. The ursition 3 . It sharP. heparinus (formerly Flavobacterium heparinum) from soil [B. theta [P. heparinus uses three exo-acting S1 sulfatases from S1_8, S1_9, and S1_11. The S1_9 enzyme, Phep_28252S-Δ4,5UA, first acts on disaccharides produced by polysaccharide lyases (PL), to remove O2 sulfation [GH88 from glycoside hydrolase family 88 (GH88), to cleave the disaccharide into monosaccharides; an essential step that enables the S1_11 enzyme, Phep_28276S-GlcNAc, to desulfate 6S-GlcNS to GlcNS [2S-GlcN, which is inhibited by O6 sulfation, then acts on GlcNS to produce GlcN [O3 sulfatase has been identified in P. heparinus able to remove O3 sulfation from 3S,6S-GlcNAc or 3S-GlcNS.Two Bacteroidetes species, rom soil  and B. tB. theta ,44 have ulfation , allowinto GlcNS . The S1_uce GlcN   was grown on Hep, HS, and desulfated-Hep, showing a ∼50%, ∼25%, and no reduction in growth, respectively, correlating broadly with the proportion of GlcNS in each polysaccharide [bt4655 spent growth media on Hep showed that GlcNS remained in the media, whereas it was absent from wild-type spent growth media [In S-Δ4,5UA . Sulfateulfation ,44. De-N unknown B. BT4655ccharide . Analysith media . These dB. theta have been structurally characterised and their specificity drivers elucidated. BT15962S-ΔUA desulfates at position-2 of all disaccharides generated by the PLs of PULHep/HS, acting on IdoA and GlcA to produce the same product, a 4,5-unsaturated uronic acid . The 0 subsite of BT15962S-Δ4,5UA is specific for Δ4,5UA and employs the carboxylate of the 0 subsite uronate as a major specificity determinant, forming bidentate interactions via Arg230  and BT19183S,6S-GlcNAc. The S1_11 enzyme BT46566S-GlcNAc/GlcNS has specialist adaptations for HS/Hep sulfation, not observed in the cMO S1_11 BT31776S-GlcNAc, but conserved in HGM orthologues in HS/Hep PULs. B. theta, in contrast with P. heparinus, which uses an S1_8 enzyme to desulfate GlcNS, utilises a novel sulfatase family that is, again, conserved in homologous HGM Bacteroidetes HS/Hep PULs.In contrast with cMO catabolism, all sulfatases in HS/Hep degradation are O2 of GlcA and at positions 4 or 6 of GalNAc. In DS, sulfation is exclusive to O4 of GalNAc  and 1,3 linked to β-GalNAc. In CS, sulfation can occur on f GalNAc A.B. theta [4S-GalNAc, BT33336S-GalNAc, and BT15962S-Δ4,5UA from subfamilies S1_27, S1_15, and S1_9, respectively, perform CS and DS desulfation in B. theta. BT15962S-ΔUA, described above, can de-2-O-sulfate disaccharides produced from both CS/DS and HS/Hep [2S-Δ4,5UA and BT33336S-GalNAc are exo-acting, BT33494S-GalNAc is endo-acting and contains an SpII signal peptide, suggesting membrane association [4S-GalNAc acts on intact CS and DS prior to PL action but, its activity is significantly reduced by O2 sulfation and also, to a lesser degree, O6 sulfation [4S-GalNAc may therefore act on regions of CS and DS containing only O4 sulfation and act simultaneously with, or prior to, PLs. CS that contains O2, O4, and O6 sulfation, may hinder BT33494S-GalNAc activity to the extent that the PLs act first producing disaccharides for BT15962S-Δ4,5UA to de-2-O-sulfate, thereby allowing BT33494S-GalNAc to operate more efficiently. Only after tri- and disulfated disaccharides have had both O2 and O4 sulfation removed by BT15962S-Δ4,5UA and BT33494S-GalNAc, respectively, is the GH88 enzyme BT3448GH88 able to produce the monosaccharide product 6S-GalNAc. BT33336S-GalNAc, then desulfates 6S-GalNAc to produce GalNAc [The PUL responsible for CS and DS metabolism, as well as the accessory proteins, has been characterised in B. theta  (Figure d HS/Hep ,27. All ociation . BT33494ulfation  (Figure 4S-GalNAc reveals that the glycan chain runs across the surface of the protein enabling its endo activity, and exhibits two major glycan-binding sites, a 0 subsite and a –1 subsite. The former utilises a Trp for aromatic stacking and an Asp to co-ordinate O6 (O2 and O3, and Arg forming a bidentate interaction with the carboxylate, locking the GlcA residue in place (O2 of the –1 GlcA explains why O2 sulfation causes major reduction in activity as it would cause both steric and charge repulsion (O6 sulfation of the 0 subsite GalNAc also causes some loss of activity since it must fit between a hydrophobic Trp and a negatively charged Asp [The structure of BT3349inate O6 C, while in place C. The stepulsion C. Additirged Asp  (Figure 6S-GalNAc, an enzyme that can desulfate 6S-Gal but has a preference for 6S-GalNAc, reveals a deep pocket where the substrate sits, justifying an exo-mode of action. A galcto-recognition triad of His, Asp, and Arg co-ordinate O3 and O4, identical with BT16246S-Gal/GalNAc and similar to S1_11 enzymes described previously [N-acetyl group (6S-Gal/GalNAc, but is conserved in S1_15 sulfatases in CS PULs amongst numerous Bacteroides species [The structure of BT3333eviously  (Figure yl group D. Intere species .endo- and exo-modes of action. All enzymes are periplasmic, but the rationale for the endo-mode of BT33494S-GalNAc is not entirely clear, nor is its predicted membrane attachment. BT33494S-GalNAc can desulfate both CS and DS disaccharides and polymers, but its activity is decreased with high levels of O2 and O6 sulfation, permitting the production of di- and trisulfated disaccharides by periplasmic PLs. BT15962S-Δ4,5UA will then remove O2-sulfates, allowing BT33494S-GalNAc to act more efficiently. Both O2- and O4-sulfation must be removed before the GH88 can generate the monosaccharide substrate 6S-GalNAc for BT33336S-GalNAc for completion of the catabolic process.The sulfatases involved in CS/DS catabolism utilise both Bacteroides of the HGM and S1 carbohydrate sulfatases are essential enzymes [exo-acting carbohydrate sulfatases targeting internally located sulfate groups as they require the action of other CAZymes to expose their target substrate. In contrast with the metabolism GAGs, the catabolism of cMOs requires exo-acting carbohydrate sulfatases at the bacterial cell surface to process the glycans prior to import into the periplasm [Sulfated host glycans are critical nutrient sources for the  enzymes ,50. Most enzymes ,52. Thiseriplasm . The evoexo-mode of action, S1 carbohydrate sulfatases involved in cMO metabolism are attractive drug targets that are amenable to small-molecule screening and subsequent engineering for the binding pocket. Inhibition of carbohydrate sulfatases, essential for GAG metabolism by the HGM whose role is not well-understood, will provide new ways to control Bacteroidetes growth, as well as ways to explore the influence of Bacteroidetes in the HGM ecosystem. Owing to their high specificity, carbohydrate sulfatases will also enable examination of complex glycan sulfation in healthy and diseased states.S1 family members are CAZymes capable of exquisite substrate recognition involving both sulfate position and sugar stereochemistry. Each S1 subfamily possesses recognition features conserved throughout the subfamily, correlating with the genomic and environmental context of the enzyme ,28. TogeBacterial carbohydrate sulfatases targeting host glycans are almost exclusively found in the S1 family of sulfatases.S1 carbohydrate sulfatases are exquisitely specific enzymes, demonstrating tailored adaptations to their carbohydrate substrate that can be mapped at a subfamily level.P. heparinus and HGM resident B. theta largely share common catabolic steps but differ in how some specific sulfoester linkages are targeted.The metabolism of HS by soil-dwelling B. theta can deploy sulfatases to remove every sulfate ester linkage identified in cMOs to date.The HGM organism exo-acting S1 carbohydrate sulfatases, and their critical importance in cMOs metabolism, makes these enzymes attractive potential drug targets for the treatment of chronic bowel diseases, such as UC.The exquisite specificity of


These novel catalytic architectures exhibited notable efficiencies in facilitating both the UOR and HER in alkaline solutions. Specifically, within urea-containing electrolytes, the UOR manifested desirable operational potentials of 1.43 V and 1.65 V versus the reversible hydrogen electrode (vs. RHE) to reach the current densities of 10 and 100 mA cm−2, respectively. Concurrently, the catalyst displayed a meager overpotential of 60 mV for the HER at a current density of 10 mA cm−2. Remarkably, the two-electrode urea electrolysis system, exploiting the designed catalyst as both the cathode and anode, demonstrated an outstanding performance, attaining a low cell voltage of 1.79 V to achieve a current density of 100 mA cm−2. Importantly, this voltage is preferable to the conventional water electrolysis threshold in the absence of urea molecules. Moreover, our study shed light on the potential of innovative Cu-based materials for the scalable fabrication of electrocatalysts, energy-efficient hydrogen generation, and the treatment of urea-rich wastewater.Water electrolysis represented a promising avenue for the large-scale production of high-purity hydrogen. However, the high overpotential and sluggish reaction rates associated with the anodic oxygen evolution reaction (OER) posed significant obstacles to efficient water splitting. To tackle these challenges, the urea oxidation reaction (UOR) emerged as a more favorable thermodynamic alternative to OER, offering both the energy-efficient hydrogen evolution reaction (HER) and the potential for the treating of urea-rich wastewater. In this work, a two-step methodology comprising nanowire growth and phosphating treatment was employed to fabricate Cu The rapid proliferation of the global population, and the advancement of civilization have engendered an exponential escalation in energy demand ,2, while2 ,292-NW/C3P phase d 30]. H. H2-NW/CP-O peak ,32. Addi3P-NW/CF electrode was subjected to a comprehensive investigation in two distinct electrolytes for both the UOR and the OER: 1.0 M KOH + 0.33 M urea and 1.0 M KOH  compared to that of the UOR (1.43 V vs. RHE) to achieve a current density of 10 mA cm−2. This observation suggested a more favorable electrochemical landscape for the UOR process, indicating a promising pathway for efficient hydrogen production. Furthermore, the Cu3P-NW/CF electrode demonstrated a far superior UOR performance compared to the Cu3P-P/CF electrode, exemplifying the beneficial effects of the ordered in situ nanowire arrays generated on the conductive CF substrate. This enhanced UOR performance further underscores the role of the unique architecture in significantly boosting the electrocatalytic activity [3P-NW/CF were found to be 81.3 mV dec−1 for the OER and 33.1 mV dec−1 for the UOR, respectively. These values indicated accelerated UOR kinetics compared to OER kinetics. In contrast, the Cu3P-P/CF electrode exhibited similar Tafel slope trends for both the OER and the UOR, suggesting a distinct advantage of the self-supported well-ordered architecture over conventional powder materials [3P-NW/CF electrode not only provided an abundance of active sites [The electrochemical performance of the Cu.0 M KOH . The purformance . This nospecimen , impartiactivity . To delvaterials . The unive sites , but alsve sites .Cdl) test, these samples’ electrochemical active surface areas (ECSA) were also assessed since a positive correlation was found to exist between them [3P-NW/CF electrode had a Cdl value of 7.5 mF cm−2, which was approximately 1.4 times higher than the value of the Cu3P-P/CF electrode (5.3 mF cm−2). These results suggest that the construction of the self-supported nanowire arrays through an in situ method can effectively improve the catalyst’s active area [3P-NW/CF and Cu3P-P/CF electrodes. 3P-NW/CF electrode had a smaller solution resistance and ion diffusion resistance compared to the Cu3P-P/CF electrode, demonstrating its fast reaction kinetics and contributing to its excellent UOR activity. The chronoamperometry method provided compelling evidence of the excellent stability and durability of Cu3P-NW/CF in alkaline electrolytes comprising 1.0 M KOH + 0.33 M urea. As shown in −2 after 30 h continuous operation at 1.6 V vs. RHE. The SEM images captured before and after the stability tests indicated no discernible alterations in the structure and morphology of the nanowire arrays , but higher than that of Pt/C (39.7 mV dec−1), demonstrating a higher HER catalytic reaction kinetic rate for Cu3P-NW/CF. In comparison, the Cu3P-P/CF powder-based electrode exhibited comparatively sluggish kinetics due to its detrimental structural characteristics that alleviated the HER on the catalyst surface [3P-NW/CF followed a Volmer–Heyrovsky mechanism [3P-NW/CF at the current density of ~102 mA cm−2 in 1.0 M KOH aqueous electrolyte for 30 h.In  surface . The HERechanism ,46, withechanism ,48. To a3P-NW/CF catalyst prompted an investigation into its potential application in energy-efficient hydrogen generation systems through urea electrochemical oxidation. A two-electrode urea-assisted electrolyzer was assembled, using both the working electrode and the counter electrode consisting of Cu3P-NW/CF. 3P-P/CF electrode (1.56 V and 2.01 V), the Cu3P-NW/CF electrode required unusually lower operating potentials of 1.50 and 1.79 V to easily attain current densities of 10 and 100 mA cm−2, respectively. Furthermore, as presented in 3P-NW/CF electrocatalyst subsequently required 1.81 V to meet 10 mA cm−2, which was 0.31 V higher than that under the urea-assisted system, implying a 20.7% reduction in the driving voltage for hydrogen generation. This demonstrated the cost-effectiveness of the urea-assisted hydrogen production strategy [3P-NW/CF manifested a remarkable stability with a consistent cell current of approximately 100 mA cm−2 over a 30 h period (3P-NW/CF electrode (The exceptional UOR and HER achievement of the Custrategy ,50,51,52h period b. Furtheh period . Moreoveh period . This oblectrode . These f3P-NW/CF catalyst can be attributed to several factors elucidated by the aforementioned findings. First, the method utilized for growing ordered nanowire arrays in situ on CF does not require the addition of binders, thereby enhancing its stability as well as the electron transfer ability across the catalyst and the substrate in an electrochemical process. This binder-free configuration ensures robustness and mitigates the possibility of performance degradation over time. Furthermore, the unique structure of the Cu3P nanowire arrays provides a higher density of active sites, thereby enabling enhanced catalytic activity. At the same time, the nanowire architecture facilitates the efficient removal of generated gases through its outermost layer, preventing gas bubble accumulation and reducing mass transport limitations. This advantageous feature distinguishes the Cu3P-NW/CF catalyst from the conventional powder catalysts with flat surfaces, leading to improved catalytic performances and increased reaction efficiency. These combined attributes of the Cu3P-NW/CF catalyst, including its binder-free construction, high surface area, and efficient gas removal mechanism, contribute to its exceptional durability and activity in the urea electrochemical oxidation and hydrogen generation processes.The outstanding durability and activity of the Cu3P nanowire array was synthesized using a simple two-step methodology. The resulting catalyst demonstrated exceptional catalytic prowess for the UOR and the HER owing to the heightened conductivity and augmented active sites. Of particular significance, a urea-assisted electrolysis system employing dual Cu3P-NW/CF electrodes achieved a current density of 100 mA cm−2 with an impressively low applied voltage of 1.79 V, surpassing both the comparative sample and the conventional alkaline water electrolyzer. This configuration holds considerable potential for energy-efficient hydrogen production. This investigation unveils an innovative approach for the intelligent fabrication and modification of Cu-based electrocatalysts, highlighting a specific focus on hydrogen generation and other energy-related applications. Moreover, the cost-effective and highly catalytic Cu3P-NW/CF bifunctional electrocatalyst not only facilitates the ecologically sound treatment of urea-containing wastewater, but also enables the development of low-pressure, high-efficiency hybrid water electrolysis systems. Furthermore, it engenders numerous prospects for advancing Cu-based catalysts in the realm of electrochemistry, encompassing oxygen reduction, CO2 reduction, and oxidation reactions involving other diminutive molecules.In summary, a Cu


In addition, B-cell depletion therapies can lead to impaired CD8 T-cell responses. In this review, we attempt to answer 2 important questions: 1. the role of B cell antigen presentation and cytokine production in the regulation of CD8 T cell survival and cell fate determination, and 2. The role of B cells in the formation and maintenance of CD8 T cell memory.Activation of CD4 T cells by B cells has been extensively studied, but B cell-regulated priming, proliferation, and survival of CD8 T cells remains controversial. B cells express high levels of MHC class I molecules and can potentially act as antigen-presenting cells (APCs) for CD8 T cells. Several  T cell responses can be divided into primary immune response and immunological memory. The primary immune response is the activation of naive CD8 T cells in response to a specific antigen presented by antigen-presenting cells (APCs) and their subsequent proliferation and differentiation into effector T cells. Immunological memory is created after the primary immune response and provides protection against subsequent attacks by the same pathogen. Memory CD8 T cells also need to be activated by APCs with costimulatory activity to regenerate effector CD8 T cells. Effector CD8 T cells express various cytokines such as interferon (IFN)-γ and tumor-necrosis factor (TNF)-α and the effector molecules, perforin, granzyme B (GZMB) and Fas-ligand (FasL). T-bet and Eomesodermin (EOMES) are essential transcription factors expressed in activated CD8 T cells that control their cytotoxic capacity , 2. Duri22.1Autoimmune diseases are usually classified as organ specific , multiple sclerosis [MS], immune thrombocytopenia [ITP], bullous autoimmune dermatosis, primary membranous nephropathy) or multi-systemic -associated vasculitis).in vivo and/or in vitro. However, it soon became apparent that even in the presence of disease-specific autoantibodies, T cells were actively involved in cellular and/or tissue damage. With the progress of knowledge, notably through the development of mouse models, it became possible to classify these entities as either “humoral,” i.e., autoantibody-mediated and therefore B-cell-dependent, or “cellular,” i.e., T-cell-mediated. Twenty-five years ago, the immunotherapeutic arsenal for these patients was still limited to immunomodulatory and/or pleiotropic immunosuppressive drugs , and to plasmapheresis, an antibody depletion therapy. Over the past 20 years, the development of truly targeted therapies, i.e., biologics, has transformed the management of several autoimmune diseases. B-cell targeted therapies have been shown to be biologically and/or clinically effective in diseases associated with autoantibodies but thought to be T cell mediated, such as rheumatoid arthritis (RA), MS, and, to a lesser extent, T1D. This apparent paradox has led to the realization that a dichotomous view of T cell or B-cell mediated autoimmunity is outdated. It is increasingly recognized that the role of B cells goes beyond the production of pathogenic autoantibodies, as already suggested by earlier experimental data obtained in MRL/lpr mice that develop nephritis despite the absence of serum autoantibodies . One current explanation is that Rituximab refractoriness is characterized by the persistence of long-lived autoreactive plasma cells in the spleen and in other sites, the emergence of which may even be promoted by B-cell depletion itself , 13. HowMultiple sclerosis is another prototypical organ-specific T-cell-mediated autoimmune disease. As with other autoimmune diseases, most of the T-cell data have focused on CD4 T cells, particularly Th1, Th2, Th17, and Treg. Demonstration of the clinical benefit of B-cell depletion therapy has led to the consideration of B cells as a key architect of the immune response in MS Figure 1Finally, in T1D, CD8 T cells are considered key effectors of tissue damage. In non-obese diabetic (NOD) mice, B cells have been shown to promote the local CD8 T cell response, whereas B cell deficiency delays the development of diabetes  Figure.2.3+CCR7- T cells  (ANCA-associated vasculitis (AAV) and systemic lupus erythematosus (SLE) are multisystem immune-mediated diseases associated with pathogenic autoantibodies. In AAV, autoantibodies target neutrophil cytoplasmic proteins in a mutually exclusive manner: proteinase 3 or myeloperoxidase. In SLE, antibodies recognize numerous nuclear antigens such as dsDNA, histones, nucleosomes, ro/SSA52 TRIM21), Sm protein and others. Humoral and cellular immune responses have been implicated in the tissue damage of AAV and SLE. In 2010, McKinney et al. performed transcriptional analysis of CD4 T cells, CD8 T cells, B cells, monocytes, and purified neutrophils , Sm protFigure 1; TEMRA) .In SLE, several lines of evidence indicate that CD8 T cells are the effectors of tissue damage. In lupus nephritis, previous studies have reported that CD8 T cells are the predominant lymphocyte population in renal biopsy  and in u2.4Pneumocystis Jirovecii pneumonia compared with those receiving a TNF-α inhibitor  . RTX-trenhibitor . In addinhibitor   showed increased depletion of virus-specific CD8 T cell memory with impaired long-term viral control (-/-) mouse model infected with Listeria monocytogenesis showed impaired memory CD8 T cell responses. The authors demonstrated a normal phase of activation and expansion of Ag-specific CD8 and CD4 T cell responses in the absence of B cells, but there was a deeper contraction phase leading to a lower level of Ag-specific CD8 T cell memory. However, B cells played only a minimal role once memory was established  and interleukin-17A (IL-17A) producing cells when anti-CD20 was administered. Treatment of mice with recombinant IL-17A partially reversed the observed effect of B-cell depletion on CD8 T cells, indicating that IL-17A secretion by CD4 T cells may be important in the maintenance phase of the CD8 immune response  vaccine  is a primary immune deficiency resulting from the absence of Bruton’s tyrosine kinase (BTK), an intracellular signaling molecule essential for B cell differentiation and survival. XLA is characterized by recurrent bacterial infections due to the near-total absence of serum immunoglobulins and peripheral B cells. Liu et al. reported that CD8 cell responses to a trivalent inactivated influenza vaccine (TIV) were similar in XLA patients and healthy controls, suggesting that B cells are not involved in this CD8 vaccine response . Howeverbacteria   that enhance the function of cytotoxic T cells and adoptive T cell transfer with genetically engineered T cells. These treatment strategies have improved survival in patients with advanced cancer. However, the role of B cells in cancer immunity is increasingly recognized and there is evidence that tumor-infiltrating B lymphocytes (TIL-Bs) have an important impact in the elimination of malignant cells . This ha culture . Further culture . In addi culture , 55. How culture –59. This culture .via ADCC, or they contribute to tumor growth by promoting inflammation, angiogenesis or immunosuppressive mechanisms.Studies in humans demonstrate that the presence of a B-cell signature was associated with a better survival in sarcoma . In orop44.1in vitro and in vivo models. B-cells possess antigen-presenting capacities and can present exogenous antigens via the class I pathway when taken up by receptor-mediated endocytosis  models. In the same study, they also demonstrated that the antigen can be effectively acquired in vitro or in vivo by immune stimulating complexes (ISCOMS) that sustained the primary expansion of CD8 T cells in OVA-HEL ISCOMS-immunized mice , a receptor expressed by subsets of CD8 T cells and mediate antibody-dependent cellular cytotoxicity. Indeed, we have identified in kidney transplant recipients that effector memory expressing CD45RA CD8 T cells (TEMRA) express FcγRIIIA  Figure, Epstein4.3via supporting the accumulation of CD8 and CD4 T cells (IL-27 is a heterodimeric cytokine belonging to the IL-6/IL-12 family which binds to distinct IL-27R subunits (IL-27Rα and gp130) and induces downstream signaling through STAT1 and STAT3 pathways. IL-27 has a dual and opposite function with both pro- and anti-inflammatory properties. Upon combined stimulation of the TCR with IL-27, human naive CD8 T cells are rapidly activated into efficient and potent cytotoxic CD8 T cells . In cont T cells .4.4in situ memory CD8 T cells.IL-15, a member of the IL-2 family, is an important homeostatic and inflammatory cytokine contributing to the activation, proliferation and maintenance of memory CD8 T cells . In a br4.5257-264 coated B cells , has been shown to suppress immune response and support immune tolerance. More than two decades ago, Bennett et al. demonstrated that B cells can directly tolerize CD8 T cells after syngeneic injection of OVA B cells . To date B cells . The lit B cells –93. The antibody . Naturalantibody . Recentlantibody . Stimula5in vivo. B cells are thought to be key players in the amplification of self-reactive CD8 T cells because B depletion induces proliferation and memory development defects in CD8 T cells. However, B cells do not seem to play a role in the initial activation of cytotoxic lymphocytes as shown in different infectious disorders or autoimmune diabetes (via MHC class I and of secretion of different cytokines may play a central role. Cross presentation of exogenous antigens, under resting conditions induces CTL tolerance, but under inflammatory conditions can lead to CTL activation via the CD40 pathway. Within the cytokines of importance IL-2, IL-4, IL-15, IL-27, IL-17 and the IFN type I pathway seem to orchestrate the B-T cell interaction. Some evidence also points to the central role of CD4 T cells supporting B and CD8 T cell interaction.Evidence is accumulating to support the role of a cross-talk between B and CD8 T cells in controlling infectious disease, tumoral pathologies and the development of autoimmune disorders 2. Studidiabetes , 38, 64.The identification of the different pathways responsible for an effective B-CD8 T cell interaction is of utmost importance to understand disease pathology and is crucial for the development of effective vaccine and antitumoral strategies. The way is paved to unravel the complex interplay between B and CD8 T cells and between B cells and the different subtypes of CD8 T cells.TV, AN, SB and ND wrote the review, contributed to manuscript revision, read, and approved the submitted version.


Due to the heterogeneity of PCa, the clinical indicators used for PCa can't satisfy risk prognostication and personalized treatment. It is imperative to develop novel biomarkers for prognosis prediction and therapy response in PCa. Accumulating evidence shows that non-mutational epigenetic reprogramming, independent from genomic instability and mutation, serves as a newly added hallmark in cancer progression.In this study, we integrated multi-center cohorts (N > 1300) to develop a RNA 5-methylcytosine regulator-based signature, the m5C score. We performed unsupervised clustering and LASSO regression to identify novel m5C-related subtypes and calculate the m5C score. Then we assessed the role of m5C cluster and m5C score in several clinical aspects such as prognosis in various molecular subtypes, responses to chemotherapy, androgen receptor signaling inhibitor (ARSI) therapy and immunotherapy in PCa. Finally, we validated the cancer-promoting performance of ALYREF through clinical data analysis and experiments in vivo and in vitro.ALYREF, yielding the highest weighed coefficient, promoted PCa progression through in silico analysis and experimental validations (in vivo and in vitro).The investigation revealed that the m5C score could accurately predict the biochemical recurrence (BCR) in different subtypes (the PAM50 subtypes and immunophenotypes) and the responses to chemotherapy, ARSI therapy, and immunotherapy (PD1/PD-L1). A high m5C score indicated a poor BCR prognosis in every subtype of PCa, unfavorable responses in ARSI therapy and immunotherapy (PD1/PD-L1). Moreover, the m5C reader gene termed The m5C signature can function in many aspects of PCa, such as the development and prognosis of the disease, and multiple therapy responses. Further, the m5C reader, ALYREF, was identified as a prognostic biomarker and a potential therapeutic target for PCa. The m5C signature could act as a brand-new tool for predicting the prognosis of patients in different molecular subtypes and patients’ therapy responses and promoting customized treatments.The online version contains supplementary material available at 10.1007/s12672-023-00671-w. Currently, prostate cancer (PCa) is the second most common malignant cancer diagnosed in men worldwide. It is the 5th leading cause of cancer-specific death in male patients, with deaths expected to reach 740,000 by 2040 . The the6-methyladenosine (m6A) has the highest abundance and is well-studied. However, the role of 5-methylcytosine (m5C), another common modification of RNA, remains obscure in many tumors.m5C is a common modification of RNA in all creatures. In eukaryotes, this modification is installed by methyltransferases (writers) such as NSUN/DNMT proteins on diverse RNA species and can be dynamically regulated by demethylases (erasers) such as TET families, and binding proteins (readers) such as YBX1. In humans, m5C methylation is catalyzed by the NSUN protein family. These regulators have different roles; for example, NOP2 (NSUN1) and NSUN5 methylate very conserved residues in 28 s sRNA, whereas NSUN4 and DNA methyltransferase member 2 (DNMT2) target rRNAs and tRNAs [Chromatin and epigenetic disorders mediate tumor progression by generating transcriptional abnormalities and by promoting cancer cell proliferation, and these features have been defined as overall hallmarks of the cancer . Epigenend tRNAs , 10. Mecnd tRNAs , 12. In nd tRNAs . HoweverIn our study, we integrated over 1300 clinical samples with sequencing information, and we studied the profile of m5C regulators from a global multi-omics perspective to assess their overall relevance to PCa clinicopathological characteristics, molecular subtypes, prognosis, androgen receptor signaling inhibitor (ARSI) therapy benefits, and immune phenotypes to determine potential immunotherapy opportunities for PCa.The Cancer Genome Atlas  project and processed by a series of functions in the R package GDCRNATools [Gene expression sequencing files, DNA mutation, and copy number variation (CNV) data of prostate adenocarcinoma tissues were downloaded free from RNATools . A singlRNATools .https://www.cbioportal.org/).Five independent external PCa validation cohorts with detailed biochemical recurrence (BCR) status and survival data in the same sequencing platform, namely, GSE54460, DKFZ, Stockholm, CPC, and SU2C-PCF-2019 were downloaded from the Gene Expression Omnibus (GEO) and cBioPortal , GSE135222 , and Checkmate cohort . RNA-seq and RIP-seq of ALYREF were uploaded to GEO (GSE195701).Detailed information about these cohorts was in Additional file An unsupervised clustering algorithm was applied to cluster analysis of m5C RNA-modified “writers” consisting of 7 molecules , 4 m5C erasers , 2 readers including YBX1 and ALYREF from previous studies , 16. UnsWe identified the differentially expressed genes (DEGs) between m5C clusters and the m5C score groups by using the limma R package. The significance for determining DEGs was set as adjusted P < 0.05 and |logFC|> 1.5. To figure out the differences of RNA m5C modification regulators in biological processes, we used the “GSVA” R package to conduct Gene set variation (GSVA) analysis . The genm5C regulators with FPKM > 1 was analyzed with Cox regression; P < 0.05 was used as the significance cut-off to identify candidates associated with BCR, meanwhile, the median of gene expressions was used to confirm whether the sample belonged to the high-expression or low-expression group. Thirteen m5C regulators were selected to construct a tenfold cross-validation and penalty LASSO regression prognostic prediction model by R package glmnet. GSE54460 was used as a validation cohort. The m5C score was calculated by expression profile data and coefficient of the corresponding m5C regulators with the formula as follows:The n is the number of m5C regulators in the prognostic prediction model, Coef(i) represents the coefficient, and X(i) means the relative expression level of m5C regulators identified by LASSO regression.Kaplan–Meier method was employed to draw the survival curves and assess the BCR of high and low-risk score groups. Nomogram Construction by “rms” R package.https://www.cancerrxgene.org/) database. Furthermore, the T-cell dysfunction and exclusion score was used predict the clinical response in treatment of potential immune checkpoint blockade.To further investigate the drug sensitivity correlation of m5C score in PCa, the effects of the m5C score on drug response from the Genomics of Drug Sensitivity in Cancer  in the tumor samples . Normali2.BPH-1 and 22Rv1 cell lines were recovered from a Liquid nitrogen refrigerated storage tank in the Central Laboratory of Guangzhou First People's Hospital. LNCaP, C4-2, PC-3, and DU145 cell lines were purchased from BeNa Culture Collection. BPH-1, LNCaP, C4-2, and 22Rv1 cells were cultured in RPMI 1640 medium  and the others (PC-3 and DU145) were cultivated in DMEM . All media were supplemented with 10% fetal bovine serum  and 1% Penicillin and Streptomycin . All cell lines were cultured in at 37 °C with 5% COThe small interfering RNAs (siRNA) targeting ALYREF transcript were synthesized by GenePharm company. LNCaP and C4-2 cells were transfected with siRNAs using siRNA-Mate . The oligo sequences of all the siRNAs are listed in Additional file Cell viability was evaluated by CCK-8 kit  at a sequential time point including 0 h, 24 h, 48 h, 72 h, and 96 h. The plate colony formation assay was used to evaluate the clonogenic ability of the cells. Transwell assay was performed to detect the invasive ability of cells. Each assay was performed in triplicate and the detailed protocols can be referred to in our previous study [6 C4-2 cells mixed with Matrigel  was injected subcutaneously into the backside of the nude mice. Then the mice were randomized into two groups, followed by treated with si-NC or si-ALYREF#615 mixed with RNAi-Mate  in situ injection for three weeks. Tumor volume was inspected and calculated every 4 days. Finally, the mice were sacrificed and the tumor was weighed.All animal experiments were approved by the Animal Care Committee of Guangzhou First People's Hospital. Ten four-week male BALB/c nude mice were purchased from Guangdong Medical Laboratory Animal Center. 3 × 10Immunohistochemistry (IHC) was conducted to investigate the expression level of ALYREF in Prostate cancer Tissue microarrays  according to the protocol described previously, and the procedures were approved by the Ethics Committee of Guangzhou First People's Hospital .RNA immunoprecipitation (RIP) was performed on the C4-2 cell line. The cell was treated with cell lysis buffer. The 10% lysis sample was stored and named “Input”, and 90% was used in immunoprecipitation reactions with anti-ALYREF antibody) and named “IP”, and 10% was incubated with rabbit IgG  as a negative control and named “IgG”, respectively. More details were shown in our previous study .®, NO. DR08402, Wuhan Seqhealth Co., Ltd. China) following the instruction. PCR products corresponding to 200–500 bps were enriched, quantified, and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.Total RNAs were extracted from the C4-2 cell line using TRIzol Reagent. DNA digestion was carried out after RNA extraction by DNaseI. RNA quality was determined by with Nanodrop™ One/Onec Spectrophotometer (Thermo Fisher Scientific Inc). Qualified RNAs were finally quantified by Qubit3.0  2 μg total RNAs were used for stranded RNA sequencing library preparation (KCTM Stranded mRNA Library Prep Kit for IlluminaR (version 4.1.2) and related packages were applied to all statistical analyses. We used the t-test to compare continuous variables between two groups if they fit the normal distribution, and the Wilcoxon test was used to compare two independent non-parametric samples. The Pearson coefficients were used to analyze the correlation between continuous variables. For the m5C score, we applied the “survcutpoint” function to determine the cutoffs for low- and high- m5C score groups, except in the TCGA-PRAD cohort, in which we defined the median as the cutoff. And the Kaplan-Meier survival curves were verified with the log-rank test. We used chi-square to analyze the difference between categorical variables and mean ± standard deviation was used to describe the continuous variables in normal distribution while the median (range) was applied to testify the continuous variables in abnormal distribution. P < 0.05 was regarded as statistically significant.A total of 13 m5C regulators, including 7 writers, 4 readers, and 2 erasers from published articles were included in our study  sequencing of prostate cells with the uniform manifold approximation and projection (UMAP) and t-distributed stochastic neighbor embedding (t-SNE) algorithms and classified the cells into nine different cell types, including neuroendocrine epithelial cells, luminal epithelial cells, and fibroblasts , we used single-sample gene set enrichment analysis (ssGSEA) and the CIBERSORT algorithm to compare immune cell infiltration between the low and high m5C score groups  score was utilized to predict the response to immune checkpoint blockade (ICB) in the different m5C score groups of patients with PCa . NotablyIn previous results, we utilized several in silico methods to corroborate the role of m5C in prostate cancer. To further enhance the reliability of our results, we endeavored to validate the hub gene that was identified through our analysis. As presented in Fig. In Fig. vitro and in vivo, we first detected its protein expression level in five prostate cell lines, and the results suggested that ALYREF is more overexpressed in C4-2 and LNCaP cells than in BPH-1 cells . Next, we scrutinized the areas of the RNA sequences that bound to ALYREF in RIP-seq analysis: the PR_intron area accounted for 52% of binding, ranking first, and the NP_exon area accounted for 2%, ranking last  analysis and an ALYREF RNA immunoprecipitation sequencing (RIP-seq) analysis of C4-2 cells. For both analyses, we identified those binding molecules with fold changes exceeding 2 and P < 0.05 as differentially expressed genes (DEGs)  . LumA tuIn many malignancies, cancer immunotherapy has been shown to be a successful and crucial treatment option. In this manuscript, we showed that the DEGs between the m5C score groups were significantly enriched in PD-L1 expression and the PD-1 checkpoint pathway. The m5C score was found to be positively related to immunomodulators, such as TGFB1, CXCL10, GZMA, PD-1, CTLA4, IDO1, HAVCR2, and LAG3, which are critical for the clinical benefit of immunotherapy. Similarly, the high m5C score group was correlated with poor clinical benefit of PD1/PDL1 inhibitor therapy in three different cancers. Overall, the m5C score may predict the response to immunotherapy in PCa. However, it should be noted that the relationship between the m5C score and the immune TME is complicated, and our study lacked samples from PCa patients who received immunotherapy. These factors could be discussed in further studies.The implementation of the m5C scoring system based on the expression of seven molecules in clinical practice may face significant challenges, particularly in terms of economics. To address this, ALYREF, the hub gene of the m5C score, could be used as a cost-effective alternative. Our study also demonstrated that ALYREF promotes PCa progression through in silico and experimental validations. Therefore, in exploring the potential clinical applications of AYREF in the future, detection of RNA expression in samples or immunohistochemical detection of protein levels are both viable options. Furthermore, our findings indicate that ALYREF is expressed in luminal epithelial and neuroendocrine epithelial cells. The heterogeneity of cancer refers to the presence of variable populations of cells with distinct genomic signatures within tumors or at different sites within the same patient. Approximately 25% of tumors in men who die of PCa are neuroendocrine tumors, which have been shown to be associated with poor prognosis.In conclusion, this study offers a new m5C-related predictor for determining prognosis and the response to multiple therapies in PCa. Our study provides both a prognostic biomarker and a potential therapeutic target for identifying and improving the prognoses of patients with different tumor molecular subtypes, predicting patient therapy response, and enhancing personalized therapy.Additional file 1: Table S1. Public datasets in this study. Table S2. Oligo sequence of siRNAs targeting ALYREF transcript. Table S3. Single cell sequencing expression profile. Table S4. COX & network of m5C regulators. Table S5. Cluster KEGG, and GO. Table S6. Coefficient. Table S7. m5C scores KEGG & GO. Table S8. GDSC. Table S9. ssGSEA & cibersort. Table S10. Tissue Microarray. Table S11. siALYREF RNA-seq. Table S12. ALYREF RIP-seq. Table S13. ALYREF Functional EnrichmentAdditional file 2: Figure S1. The overall study design and the workflow of the research. Figure S2. The m5C regulators’ expression landscape and their prognosis value. (A) The differential gene expression of the 13 m5C regulators in the MSI and non-MSI subgroups in the TCGA-PRAD cohort. *P < 0.05; **P < 0.01; ***P < 0.001. (B) The differential gene expression of the 13 m5C regulators in the TP53_wt and TP53_mut subgroups in the TCGA-PRAD cohort. (C) The differential gene expression of the 13 m5C regulators in the ERG-fusion and non-ERG-fusion subgroups in the TCGA-PRAD cohort. (D) The Kaplan–Meier survival curves show five m5C genes to be favorable factors. Log-rank test . (E) The Kaplan–Meier curves demonstrate another five m5C genes to be risk factors. Log-rank test . Figure S3. Depicting the 13 m5C regulator genes in prostatic cells with t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP). Figure S4. The unsupervised clustering of m5C regulators in the TCGA-PRAD cohort and prognosis differences under several scenarios of m5C cluster grouping. (A) BIC plot for models fitted. (B- F) The Kaplan–Meier curves of different cluster numbers of grouping. Log-rank test . Figure S5. The clinical relevance of the binary m5C clustering and the functional annotations for differentially expressed genes between the two m5C clusters. (A) The proportion of patients’ clinical factors in the two m5C clusters in the TCGA-PRAD cohort. (B & C) The GO and KEGG functional annotations for DEGs between the two m5C clusters. Figure S6. Construction of the m5C score system and the nomogram. (A) Selection of the optimal parameter (lambda) in the LASSO model. (B) LASSO coefficient profiles of the 13 m5C regulator genes. A coefficient profile plot was generated against the log (lambda) sequence. (C) The nomogram to predict BCR-free survival of PCa patients in the TCGA-PRAD cohort and the calibration plot for the 1-year (blue line) and 5-year (red line) BCR free survival predictions. Figure S7. The correlation of m5C clusters and m5C score, and GSVA enrichment of m5C scores. (A) The gene expression differences of m5C regulators in PCa patients within low- and high- m5C score subgroups using the TCGA-PRAD database . (B) The Sankey diagram shows correlations between m5C clusters and m5C score and BCR status. (C) The m5C score difference between 2 m5C clusters. (D) The heatmap shows the GSVA score distribution of representative pathways curated from the MSigDB database in two m5C score subgroups. Figure S8. The function annotations and mutation landscape of the m5C score subgroups in the TCGA-PRAD cohort. (A & B) GO and KEGG function annotations of DEGs between the two m5C score subgroups. (C & D) Mutation landscapes of significantly mutated genes in TCGA-PRAD in low- and high- m5C score subgroups, respectively. Each column represents one patient with gene mutations. The top bar plot shows tumor mutation load, and the right horizontal bar plot shows the mutation signatures of each gene in mutated samples of each m5C score subgroup. Figure S9. The molecular signatures relevance of the m5C score in the ARSI cohort. (A-D) Figures 1-4 show the correlations between m5C score and CCP score, m5C score and RB-loss score, m5C score and NEPC score, and m5C score and AR score, respectively, in the ARSI cohort. R represents Pearson’s correlation coefficient, and P stands for P-value . The m5C score positively correlated with both CCP score (A) and RB-loss score (B). Figure S10. The immune landscape of the m5C score groups in TCGA-PRAD cohort. (A & B) The fraction variation of tumor-infiltrating immune cells in two m5C score groups using the ssGSEA (A) and CIBERSORT (B) algorithms, respectively. Within each group, the scattered dot represents the TME cell expression value. The thick line represents the median value . (C & D) The heatmap shows the correlations between the m5C score grouping and the clinicopathological characters and between the m5C score grouping and the score of tumor-infiltrating immune cells, using the ssGSEA (C) algorithm and CIBERSORT (D) algorithm, respectively. Figure S11. The correlation between TME subtypes and the m5C score, cytokine and chemokine milieu among m5C score groups. (A) The differences in the m5C score between four PCa TME subtypes in TCGA-PRAD cohort. (B) The expression levels of macrophage polarization-related genes . (C) The expression levels of immune-checkpoint-relevant genes  in the m5C score groups. (D) The expression levels of immune activation-relevant genes  in three Gene-cluster groups. Within each group, the scattered dots represent gene values. The thick line represents the median value. The bottom and top of the boxes are the 25th and 75th percentiles (interquartile range). The whiskers encompass 1.5 times the interquartile range. The range of P-value is labeled above each boxplot with asterisks. . Figure S12. ALYREF promotes prostate cancer progression. (A) Western blotting validated the knockdown efficacy of siRNAs  targeting ALYREF in the LNCaP cell line. β-actin was the loading control. (B) Transwell assay confirmed silencing ALYREF diminished LNCaP cell invasion ability. (C) 4-day CCK-8 assay reflected the knockdown of ALYREF attenuated LNCaP cell proliferation. (D) Plate colony formation assay showed silencing ALYREF downregulated C4-2 cell colony formation ability. (E) ALYREF’s pan-cancer prognosis analysis , including 10 types of cancers ; OV, ovarian serous cystadenocarcinoma) Error bar indicates mean ± SD. *P <0.05, **P < 0.01, ****P < 0.0001. Figure S13. RNA-seq and RIP-seq co-analysis of ALYREF. (A)Venn diagram showed 376 overlapped genes between si-ALYREF RNA-seq data and ALYREF RIP-seq data. (B)The top 15 GO terms in the functional enrichment analysis of RNA-seq and RIP-seq co-analysis. (C)The top 15 KEGG pathways in the functional enrichment analysis RNA-seq and RIP-seq co-analysis


The P(VDF-HFP)@SiO2-GE presents high electrolyte uptake (~1000 wt%), thermal stability (up to ~350 °C), ionic conductivity (~2.6 mS cm−1 at room temperature), and excellent compatibility with an active Li metal anode. Meanwhile, F-doping carbon/silica composite nanofibers (F-C@SiO2) were also produced by carbonizing the P(VDF-HFP)@SiO2 film under Ar and used to make an electrode. The assembled F-C@SiO2|P(VDF-HFP)@SiO2-GE|Li half-cell showed long-cycle stability and a higher discharge specific capacity (340 mAh g−1) than F-C@SiO2|Celgard 2325|Li half-cell (175 mAh g−1) at a current density of 0.2 A g−1 after 300 cycles, indicating a new way for designing and fabricating safer high-performance LIBs.The aim of this work is to effectively combine the advantages of polymer and ceramic nanoparticles and improve the comprehensive performance of lithium-ion batteries (LIBs) diaphragm. A flexible film composed of electro-spun P(VDF-HFP) nanofibers covered by a layer of mesoporous silica (P(VDF-HFP)@SiO The organic/inorganic hybrid composite diaphragm showed good thermal stability, a high absorption rate of liquid electrolyte, and low interfacial resistance between the electrode and the separator. Wang et al. [Another way to enhance the comprehensive performance of secondary batteries is to improve the interfacial contact of the separator and solid electrode ,22,23,242) was fabricated through electrospinning followed by sol–gel preparation. It was then applied as a separator as well as scaffold to absorb organic electrolyte to make quasi-solid-state electrolyte  for LIBs. Meanwhile, F-doping carbon/silica composite nanofibers (F-C@SiO2) were also gained by carbonizing P(VDF-HFP)@SiO2 composite film. The electrochemical tests showed that the P(VDF-HFP)@SiO2 separator presented high electrolyte uptake, ionic conductivity, and excellent compatibility with active Li metal, and the F-C@SiO2 was a promising LIBs anode material. The assembled F-C@SiO2|P(VDF-HFP)@SiO2-GE|Li half-cell exhibited superior electrochemical performance as expected.In this work, we provided a facile way to fabricate a high-performance separator as well as improve interfacial contact between the solid separator and solid electrodes. A composite film composed of P(VDF-HFP) nanofibers covered by a layer of mesoporous silica (denoted as P(VDF-HFP)@SiO2 composite nanofibers are showed by their FE-SEM and TEM images @SiO2 membrane was tested and compared with Celgard 2325 and P(VDF-HFP) membranes, as is displayed in 2 membranes changed only a little. At the temperature of 170 °C, Celgard 2325 shrank further, P(VDF-HFP) showed serious shrinkage, while P(VDF-HFP)@SiO2 changed only slightly. This result shows that the coating of the SiO2 layer can effectively reduce thermal shrinkage of the polymer diaphragm, thereby inhibiting the battery short circuit problem and improving the battery’s safety.The morphology and structure of the obtained electro-spun P(VDF-HFP) nanofibers and P(VDF-HFP)@SiOM images a–d. The M images  disclose2 film was heated in nitrogen at 700 °C, F-C@SiO2 composite nanofibers were obtained and displayed in 2 composite comprises 63.3 wt% of F-doping carbon, as calculated from 2 composite nanofibers  crystal plane of graphite, indicating that the degree of crystallinity for carbon in the nanofibers is low. In the Raman spectrum @SiO2 and F-C@SiO2 composite nanofibers are showed in 2 g−1/0.277 cm3 g−1 and 285.1 m2 g−1/0.453 cm3 g−1, respectively. The coarse fiber surface and plenty of micro- and mesopores within the composite nanofibers provide enough space for the holding of electrolyte and ensure the rapid transfer of lithium ions.When the P(VDF-HFP)@SiOnofibers . The fulnofibers  confirms pattern , a broadspectrum , D and G2 film was cut into a 16 mm diameter wafer and used to soak organic electrolyte (1M LiPF6 in 1:1 EC/DMC) till saturation, the P(VDF-HFP)@SiO2-GE membrane was gained. The electrolyte uptake of the P(VDF-HFP)@SiO2 membrane was calculated based on the mass difference in the membranes before and after sufficient immersion in the liquid electrolyte according to the following formula,dW and wW are the mass of the membrane before and after immersion in the liquid electrolyte, respectively. The electrolyte uptake is 1000 wt%, much higher than the commercial celgard 2325 membrane (~90 wt%) and many reported electro-spun P(VDF-HFP)-based scaffolds (2-GE were tested and are displayed in σ) of P(VDF-HFP)@SiO2-GE was investigated using AC impedance analysis on a stainless steel symmetrical cell (SS|P(VDF-HFP)@SiO2-GE|SS), which can be further calculated by the formula,l is the electrolyte membrane thickness, bR stands for the electrolyte bulk resistance measured by EIS, and S is the cross-sectional area. The value of σ increases with temperature due to a decrease in the ohmic resistance of the electrolyte, as is presented in + conduction can be calculated from A is the pre-exponential factor, aE stands for the activation energy, T is the absolute temperature, and R stands for the Boltzmann constant. The activation energy of ion conduction is estimated to be 7.9 kJ mol−1. The ionic conductivity of P(VDF-HFP)@SiO2-GE at 25 °C is 2.6 mS cm−1, which meets the minimum operating limit of LIBs at room temperature (1 mS cm−1) [2-GE at room temperature is 4.6 V, larger than commercial LIBs (4.5 V vs. Li/Li+) [2-GE|Li cell was tested in the voltage range of 2.5–4.2V. A periodic constant current (0.1 and 0.5 mA cm−2) was applied to the cell, and the stripping/plating behavior of lithium ions on the electrode surface was investigated by recording voltage changes, as shown in 2-GE membrane. The LiFePO4|P(VDF-HFP)@SiO2-GE|Li cell can work well at different rates (0.1–2 C), as is shown in 2-GE membrane exhibited superior electrochemical characteristics such as higher ionic conductivity and a better capacity retention ratio after long cycles at a high rate than many reported electro-spun P(VDF-HFP)-based gel electrolytes (When the P(VDF-HFP)@SiOcaffolds  [25,26,2mS cm−1) ,30. As F Li/Li+) , indicattrolytes , indicat2 to make the working electrode, metallic Li foil as the counter electrode, and P(VDF-HFP)@SiO2-GE as the separator as well as quasi-solid-state electrolyte, a CR2016 coin-type half-cell (F-C@SiO2|P(VDF-HFP)@SiO2-GE|Li) was integrated. Its electrochemical behavior was tested and is shown in −1, the cathodic peak at around 0.5 V is because of the decomposition of electrolyte and the formation of SEI film on the fiber surface [2 electrode at 0.2 A g−1 is shown in −1, respectively. The irreversible capacity loss is related to the formation of the SEI film and other lithiation reactions. After 300 cycles, a stable discharge capacity of 340 mAh g−1 is gained. It is interesting to find that the capacity of the cell using P(VDF-HFP)@SiO2-GE is nearly twice of the cell using the commercial celgard 2325 separator, which is probably related to the larger electrolyte absorption of the P(VDF-HFP)@SiO2 membrane and better interfacial contact between the P(VDF-HFP)@SiO2-GE and F-C@SiO2 electrode [2|P(VDF-HFP)@SiO2-GE|Li cell at various current densities is displayed in −1 at current densities of 0.1, 0.2, 0.5, and 1 A g−1 were achieved, respectively. When the current density returns to 0.2 and 0.1 A g−1, the recovery rate of the capacity is higher than 90%. In the Nyquist plot (2|P(VDF-HFP)@SiO2-GE|Li cell reduces from 255 Ω to 70 Ω, while that of the F-C@SiO2|Celgard 2325|Li cell reduces from 365 Ω to 224 Ω. P(VDF-HFP)@SiO2-GE shows better interfacial compatibility with the F-C@SiO2 electrode, which is due to their similar structure and composition. In addition, the sharp reduction in impedance suggests good electrical conductivity and a rapid charge transfer reaction, benefitting lithium-ion insertion/extraction.Using F-C@SiO surface . The almlectrode ,34. The ist plot d,e, afte2|P(VDF-HFP)@SiO2-GE|Li cell exhibited superior electrochemical performance, which was reasonably ascribed to the deliberately designed composition and structure as well as the enhanced interfacial contact between the electrodes and gel electrolyte. As is known, the more similar the composition and structure of the two phases, the better the compatibility between them is. The mesoporous SiO2-coated nanofibers which exist both in the gel electrolyte and F-C@SiO2 electrode undoubtedly improve the interfacial compatibility between them. Meanwhile, plenty of the mesopores on the nanofibers greatly increase the electrolyte uptake, benefitting the interfacial contact of the gel electrolyte (separator) and the metallic Li electrode. The hard SiO2 layer also enhances the strength of the nanofiber membrane, lessening the risk of piercing through by Li dendrite. As far as we know, this is the first report on assembling an electro-spun nanofiber separator and its derivate carbonaceous electrode in one battery.Herein, the integrated F-C@SiO4 was purchased from Shanghai D&B Biotechnology Co., Ltd. . Vinyl carbonate (EC) and dimethyl carbonate (DMC) were produced by Guotai Huarong Chemical New Materials Co., Ltd. . Cetyl trimethyl ammonium bromide  was acquired from Bede Pharmaceuticals Ltd. . Tetraethyl orthosilicate  and acetone derive from Jiangsu Qiangsheng Functional Chemical Co., Ltd. . Anhydrous ethanol (>99.7%) and ammonia  were provided by Sinopharm Chemical Reagent Co., Ltd. . Celgard 2325 was bought from Suzhou Sukrain Instrument Co., Ltd. . All materials were used as received without further purification.Polyvinylidene-hexafluoropropylene (P(VDF-HFP), Mw = 455,000), and N,N-dimethy-l-formamide  derive from Aladdin. LiFePOv/v = 7:3) and stirred at 50 °C for 3 h to obtain uniform spinning solution. P(VDF-HFP) nanofibers were produced by electrospinning. Regarding the experimental conditions, we applied a positive voltage of 10 kV, negative voltage of −5 kV, collection distance of 15 cm, injection speed of 0.08 mm s−1, and moderated at 40%. The collected nanofibers were dried overnight in a 60 °C oven.Electro-spun P(VDF-HFP) nanofiber film was synthesized according to previous work : P at 40 °C. Then, 50 mg of above-prepared P(VDF-HFP) electrospinning film was immersed in the solution and stood for 2 min. After that, 1 mL of TEOS followed by 0.1 mL of concentrated ammonia solution was added dropwise to the solution and it was left to stand at 40 °C for 8 h. The as-prepared composite film was washed with deionized water to remove the surface impurities, heated in ethanol with reflux for 12 h to remove CTAB, and dried at 70 °C for 24 h. Finally, P(VDF-HFP)@SiO2 composite nanofiber film was obtained.A total of 200 mg of CTAB was completely dissolved in 200 mL of EtOH/H2 composite nanofiber film was heated in nitrogen at 700 °C at a rate of 10.0 °C/min for 6 h. After naturally cooling to room temperature, F-doped carbon/silica composite nanofibers (designated as F-C@SiO2) were obtained. The fabrication process for P(VDF-HFP)@SiO2 composite nanofiber membrane and F-C@SiO2 composite nanofibers was illustrated in The prepared P(VDF-HFP)@SiO4 electrode, LiFePO4 (80 wt%) powder, acetylene black (10 wt%), and PVDF (10 wt%)  were evenly dispersed in NMP and stirred at room temperature for 5 h. The mixture was scraped onto aluminum foil, dried overnight in a 70 °C oven, and cut into 14 mm diameter rounds. Using the LiFePO4 electrode as the positive electrode, lithium metal as the counter electrode, and P(VDF-HFP)@SiO2-GE as the separator, the LiFePO4/Li battery was assembled in a glovebox filled with argon gas.For the preparation of the LiFePO2 electrode, F-C@SiO2 (80 wt%) powder, acetylene black (10 wt%), and PVDF (10 wt%)  were evenly dispersed in NMP and stirred at room temperature for 5 h. The mixture paste was scraped onto copper foil, dried overnight in a 70 °C oven, and cut into a 14 mm diameter wafer. Using the F-C@SiO2 electrode as the working electrode, lithium metal as the counter electrode, and P(VDF-HFP)@SiO2-GE and Celgard 2325 as the separators, the F-C@SiO2|P(VDF-HFP)@SiO2-GE|Li and F-C@SiO2|Celgard 2325|Li batteries were assembled in a glovebox filled with argon gas.For the preparation of the F-C@SiO+) with a scan rate of 5.0 mV s−1. The interfacial compatibility between the P(VDF-HFP)@SiO2-GE membrane and Li metal electrode was tested based on the charge/discharge cycling performance of the symmetrical Li|P(VDF-HFP)@SiO2-GE|Li battery with a Land CT2001A cell tester at room temperature.Electrochemical tests were conducted with an electrochemical workstation , and galvanostatic charge/discharge tests were carried out with LAND systems. The electrochemical impedance was acquired in a frequency range from 0.01 Hz to 100 kHz at different temperatures with an AC amplitude of 5 mV. The electrochemical stability window was measured using linear sweep voltammetry (LSV). The potential was swept between open circuit voltage and 7.0 V  was performed on a Thermal Analysis TG/DTA 6300 instrument with a heating rate of 10 °C min−1 under air from 25 °C to 800 °C. X-ray photoelectron spectroscopy (XPS) was performed on a Pass Energy 100.0 eV, Al K Alpha.Field emission scanning electron microscopy (FE-SEM) was performed using a Hitachi 4800 instrument at 3.0 kV. Transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) images, as well as selective area electron diffractions (SAED), were obtained using an FEI TecnaiG220 at 200 kV. Wide angle X-ray diffraction (WAXRD) patterns were collected with an X’Pert-Pro MPD X-ray diffractometer using Cu Kα radiation (λ = 0.154 nm). Raman spectra were recorded using a Jobin Yvon Horiba HR 800 LabRAM confocal microprobe Raman system with Ar laser excitation (514.5 nm) and a power of 10 mW. Specific surface area and pore-size distribution were determined based on the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halon (BJH) methods using N2 composite nanofiber film was synthesized via electrospinning followed by a sol–gel reaction. After carbonization, F-C@SiO2 composite nanofibers were gained. Electrochemical characterization disclosed that P(VDF-HFP)@SiO2-based gel electrolyte exhibited high ionic conductivity and thermal stability, while the F-C@SiO2-based anode showed good Li-storage capacity. Because of the enhanced interfacial contact between the electrodes and the gel electrolyte, the assembled F-C@SiO2|P(VDF-HFP)@SiO2-GE|Li cell exhibited superior electrochemical performance. On account of the economic raw materials, facile fabrication process, and good comprehensive properties, this work has demonstrated a new design for safe next-generation LIBs.In conclusion, P(VDF-HFP)@SiO


Genome damage has been related to the induction of autoimmune processes, chronic inflammation, and apoptosis. Recent studies suggest that some rheumatological diseases are associated with overall genomic instability in the T cell compartment. However, no data regarding leucocyte abnormalities in synovial fluid (SF) and their relationship with inflammation are available. The aim of this study was to investigate cellular phenotypes in SF collected from patients with different inflammatory arthropathies, including rhematoid arthritis (RA), psoriatic arthritis (PsA), crystal-induced arthritis (CIA), and non-inflammatory arthropathies, such as osteoarthritis (OA). We found high percentage of micronuclei in SF from CIA compared to the other groups and a high frequency of pyknotic cell in RA and CIA patients. A correlation between pyknosis and immature polymorphonuclear cells with local inflammatory indices was observed. The study of the apoptosis process revealed an increased BAX expression in CIA and RA compared to OA and PsA, while Bcl-2 was higher in CIA. Caspase-3 activity was increased in SF from RA patients and correlates with inflammatory and anti-inflammatory cytokines. In conclusion, our results showed that inflammatory SF is associated with genomic instability and abnormal cell subsets. Rheumatological joint diseases, including rheumatoid arthritis (RA), psoriatic arthritis (PsA), crystal-induced arthritis (CIA), and osteoarthritis (OA), are chronic conditions affecting a large proportion of the population, and they represent a leading cause of disability and comorbidities worldwide . AlthougSF leucocytes increase during an acute phase of disease, reaching thousands of different cells per mmc . The majIt is known that leucocytes can present abnormalities depending on cell state, aging, or upon mutations . These aIt has been hypothesized that genome damage might be related to the induction of autoimmune and chronic inflammatory diseases . Self-DNGenomic instability can lead to altered gene expression and induce cell apoptosis. Apoptosis can affect nuclear integrity disturbing, both the nuclear lamina and envelope, and resulting in cell death .Recent studies suggest that some rheumatological diseases, including rheumatoid arthritis (RA), are associated with overall genomic instability in the T cell compartment  and incr3. The most inflammatory degree, in terms of both WBC count and PMN percentage, was found in SF collected from CIA, followed by RA and PsA. SF from OA patients showed typical non-inflammatory features. Patients with PsA were the youngest among the groups.SF characteristics are shown in All the preparations were examined for MN, NA , and karyolitic, karyorrhectic, and pyknotic cells. The morphologies of the different cell types are illustrated in The hypersegmented neutrophils band, hyposegmented (immature PMN), and hypersegmented PMN were higher in SF from patients with RA, PsA, and CIA compared to those with OA. The percentage of MN was significantly higher in CIA patients than in the OA patients, but there were no statistically significant differences in the MN frequency between the other groups of patients. Binucleated monocytes were higher in OA patients with respect to those with CIA. No significant differences were found in the frequency of M and PMN with vacuoles between the patient groups analyzed.p < 0.05). Data are summarized in MN and other nuclear abnormalities are considered biomarkers of genotoxic events and chromosomal instability. MGG staining was used to detect MN in PMN and M from OA, RA, PsA, and CIA patients. p < 0.05) and CIA (p < 0.01) patients than in OA patients. The percentage of karyorrhectic and karyolitic cells were higher in RA patients with respect to OA and PsA patients (p < 0.05), although significance was reached only for karyorrhexis (p < 0.01)  D (Table  < 0.01) .SF cytokine, chemokine, and growth factor levels are presented in A strong positive correlation between local inflammatory cellular indices including WBC and PMN and the cytokines IL-1β, IL-6, IL-8, and IL-10 was found .Immature PMN percentage was positively correlated with WBC count and PMN percentage and with the levels of IL-1β, IL-6, IL-8, and IL-10; it was negatively correlated with M and L percentage . The frep = 0.02, Bcl-2 p = 0.05). More specifically, BAX expression was increased in CIA and RA compared to OA and PsA    (Table S < 0.05) . As rega < 0.05) . By cont < 0.05) .The aim of the study was to investigate the relationship between the presence of genomic instability and the degree of inflammation in the SF obtained from RA, PsA, CIA, and OA patients. Although genomic instability has been associated with both inflammatory and autoimmune diseases, there are no data in the literature describing abnormalities in SF leucocytes from chronic rheumatological conditions.The SF collected from patients with the inflammatory diseases RA, PsA, and CIA had classical inflammatory features, with CIA displaying a higher number of WBCs and PMN. OA, which is considered a low-grade inflammatory disease, showed non inflammatory cellular aspects . In the To investigate signs of genotoxicity/cytotoxicity, we searched for the following cytological abnormalities: (1) nuclear abnormalities , (2) cell death biomarkers (necrotic and apoptotic cells), and (3) biomarkers indicative of cell division rate or cell division failure (binucleated cells) . MicronuNeutrophils are the main actors in acute inflammatory responses. In different inflammatory conditions, immature neutrophils (with non-segmented nuclei) can be released from the bone marrow, causing a so-called left shift . Banded Cell death biomarkers evaluated in this study were pyknosis, karyolysis, and karyorrhexis. Pyknosis has been widely considered as a marker of cell death, and it is characterized by an irreversible condensation of chromatin. It commonly occurs in both apoptotic and necrotic cell death. Karyolysis is the disintegration and dissolution of the nucleus of a necrotic cell, usually associated with karyorrhexis, while the nucleus of an apoptotic cell usually dissolves after karyorrhexis . We founApoptosis is a complex and tightly regulated process divided into three phases: initiation, commitment, and execution. The Bcl-2 protein family regulates the commitment phase, while caspases regulate the execution of apoptosis. Caspase-3 is classified as effector caspase and, among all executioner caspases, is the most important ,24. AntiIn conclusion, inflammatory SF samples from RA, PsA, and CIA patients are associated with abnormal cell subsets and apoptosis that could be involved in the resolving phase of the inflammatory process. A deeper knowledge of different abnormal cell subsets and their function could lead to a better understanding of the underlying mechanisms for the resolution of inflammation in these diseases.® Lo-ROX Kit (BIO-94050) were purchased from Bioline . Caspase-3 activity was determined using a colorimetric assay kit from Abcam .May–Grunwald (63590) and Giemsa staining (1.09204.0500), Bradford (B6916), PBS (P4417), and methanol (179337) were from Sigma-Aldrich . ELISA kits for interleukin (IL)-1β (88–7261), IL-6 (88–7066), IL-8 (88–726), IL-10 (88–7106), and TGFβ (88–8350) were from Thermo Fisher Scientific , and TNFα was from BioLegend . The Total RNA purification kit was from Norgen Biotek . The SensiFAST™ cDNA Synthesis Kit (BIO-65054) and SensiFAST™ SYBRSynovial fluids (SFs) were collected by arthrocentesis from swollen knees of untreated adult patients in the acute stage with RA (n = 6), PsA (n = 12), CIA (n = 10), and OA (n = 7) who attended the outpatients’ clinic of the Rheumatology Unit at Padua University Hospital for acute swelling and were diagnosed according to ACR (R), CASPAR (r) and EULAR criteria ,30,31,32Synovial fluids samples were collected in EDTA and plain tubes and analyzed by ordinary and polarized light microscopy. Total white blood cell (WBC) count was performed using a Bürker counting chamber. Differential leucocyte count was performed by microscopic examination of 300 cells on MG-stained preparations. The percentage of polymorphonuclear neutrophils (PMN), monocytes (M), and lymphocytes (L) in the SF was recorded. Crystal search was performed using compensated polarized light microscopy. Monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals were distinguished by their shape, degree, and sign of birefringence . After eMay–Grunwald–Giemsa (MGG) staining was used for studying cellular morphology, including cytoplasm, granules, vacuoles, and the basement membrane, and to perform a cytogenic evaluation of leucocytes. Air-dried SF smears were fixed with methanol and stained using a stepwise procedure with MGG. The smear was transferred to May–Grunwald solution diluted 1:1 in water for 3 min. Excess stain was drained off on filter paper (without blotting), and the smear was then transferred to dilute 1:9 Giemsa solution for 7 min. The smear was transferred to water, rinsed for 30 s, and then dried. Oil immersion microscopy with 1000× magnification was applied for analysis; for each smear we examined at least 200 leucocytes. Leukocytes were identified on the basis of morphology; in each field the number of cell profiles corresponding to neutrophils, monocytes, and lymphocytes were recorded. All slides were examined for micronuclei (MN) (a micronucleus is defined as a round cytoplasmic inclusion having a diameter one-tenth to one-third that of the primary nucleus), and types of nuclear abnormality (NA) included binucleated cells (contains two nuclei that are not attached and similar in size), and karyolitic, karyorrhectic, and pyknotic cells . ApoptotThe following cytokines, chemokines, and growth factors were measured in SF after appropriate PBS dilutions using commercially available enzyme-linked immunosorbent assay (ELISA) kits: interleukin (IL)-1β , IL-6 , IL-8 , IL-10 , TGFβ  , and TNFα .® Lo-ROX Kit (Bioline) via Quant Studio 5 real-time PCR system (Thermo Fisher Scientific). Target genes were normalized to GAPDH and analyzed using the 2−ΔCt method. The primer sequences are given as follows:GeneForward PrimerReverse PrimerBCL-2ATGTGTGTGGAGAGCGTCAAACAGTTCCACAAAGGCATCCBAKTCATCGGGGACGACATCAACCAAACAGGCTGGTGGCAATCBIDCTTGCTCCGTGATGTCTTTCTCCGTTCAGTCCATCCCATTTBADCGGAGGATGAGTGACGAGTTGATGTGGAGCGAAGGTCACTBAXTCTGACGGCAACTTCAACTGTTGAGGAGTCTCACCCAACCGAPDHTGCACCACCAACTGCTTAGCGGCATGGACTGTGGTCATGAGSelected SF samples (n = 5) from PsA, CIA, RA, and OA patients were centrifuged at 1500 rpm for 10 min to collect cells and stored at −20 °C. RNA was isolated using a Total RNA purification kit (Norgen Biotek) according to the manufacturer’s recommendations and quantified with a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using the SensiFAST™ cDNA Synthesis Kit (Bioline). The expression levels of BCL-2, BAK, BID, BAD, and BAX were measured by real-time quantitative PCR (qPCR) using SensiFAST™ SYBRLeukocytes from synovial fluids were lysed with 50 μL lysis buffer  for 10 min at 4 °C. Supernatants were than collected for SDS-PAGE western blotting and centrifuged at 10,000 rpm for 15 min. Protein quantification was performed using a BCA assay kit . Proteins (60 μg) were separated on SDS-PAGE. Membranes were then blocked and probed using the anti-Bax and anti-Bcl-2 mouse primary monoclonal antibodies (Biolegend) and anti-actin mouse primary monoclonal antibodies . After washing, membranes were incubated with mouse secondary horseradish peroxidase-conjugated antibodies . Bands were detected through chemiluminescence using Western brightTM 143 Quantum . Images were acquired by the Alliance mini HD9 Imaging System . Densitometric analysis of bands was performed using Image J version 1.47 software .The same SF samples used for RNA extraction were tested for caspase-3 activity (n = 5). Caspase-3 activity was determined using a colorimetric assay kit based on the formation of the chromophore p-nitroaniline (p-NA) by cleavage from the labeled substrate DEVD-pNA (Abcam) according to the manufacturers’ instructions. Briefly, cells were lysed in the supplied lysis buffer for 10 min at 4 °C. Supernatants were collected (200 μg of protein in 50 µL) and incubated with the supplied reaction buffer containing dithiothreitol and DEAD-pNA as substrates at 37 °C for 2h. The reactions were measured by changes in absorbance at 405 nm using the Byonoy microplate reader .p value < 0.05 was considered significant.Data are reported as median and IQR. The Shapiro–Wilk test was used to analyze the distribution of continuous variables. For non-normal distributed data, the Kruskal–Wallis followed by Dunnet post hoc tests were used for multiple comparisons. Categorical variables were compared using the Chi-square test. Spearman correlation analysis was used to determine correlations. Statistical analysis was performed using GraphPad Prism version 8 . A