After feeding, adipose tissue lipoprotein lipase (LPL) activity should be maximized, therefore the potent LPL-inhibitory activity of angiopoietin-like protein 4 (ANGPTL4) must be blocked by ANGPTL8 through formation of ANGPTL4/8 complexes. ANGPTL4/8 tightly binds and protects LPL but also partially inhibits its activity. Recently, we demonstrated ANGPTL4/8 also binds tissue plasminogen activator (tPA) and plasminogen to generate plasmin that cleaves ANGPTL4/8 to restore LPL activity. Although fully active LPL in the fat postprandially is desirable, ANGPTL4/8 removal could subject LPL to profound inhibition by ANGPTL3/8 (the most potent circulating LPL inhibitor), inhibition by other LPL inhibitors like ANGPTL4, ANGPTL3, and ApoC3 or interfere with ApoC2-mediated LPL activation. To understand better these potential paradoxes, we examined LPL inhibition by ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 and LPL stimulation by ApoC2 in the presence of ANGPTL4/8 + tPA + plasminogen. Remarkably, ANGPTL3/8-mediated LPL inhibition was almost completely blocked, with the mechanism being cleavage of fibrinogen-like domain–containing ANGPTL3 present in the ANGPTL3/8 complex. The LPL-inhibitory effects of ANGPTL4, ANGPTL3, and ApoC3 were also largely reduced in the presence of ANGPTL4/8 + tPA + plasminogen. In contrast, the ability of ApoC2 to stimulate LPL activity was unaffected by ANGPTL4/8-mediated plasmin generation. Together, these results explain how plasmin generated by increased postprandial ANGPTL4/8 levels in adipose tissue enables maximal LPL activity by preventing ANGPTL3/8, ANGPTL4, ANGPTL3, and ApoC3 from inhibiting LPL, while permitting ApoC2-mediated LPL activation to occur. After feeding, lipoprotein lipase (LPL) in adipose tissue should be maximally active on capillary luminal surfaces so that circulating triglycerides (TG) can be hydrolyzed to yield FAs that can then be taken up into adipocytes for re-esterification and subsequent storage as intracellular TG , 2. For Intact ANGPTL4 works mainly in an autocrine/paracrine manner, as its circulating levels are much lower than those of other ANGPTL proteins and complexes . ProtectThis observation suggested a possible conundrum, however. Although fully active LPL in the fat would be desirable after feeding, plasmin-mediated cleavage of ANGPTL4/8 on luminal capillary surfaces could also leave LPL in an unprotected state, susceptible to inhibition by ANGPTL3/8, the most potent circulating LPL inhibitor and whose levels increase dramatically after feeding , 12, 13.To understand better each of these potential paradoxes, we examined the respective abilities of ANGPTL3/8, ANGPTL3, ANGPTL4, and ApoC3 to inhibit LPL activity and the ability of ApoC2 to stimulate LPL activity after incubation of LPL with ANGPTL4/8 in the presence of tPA + plasminogen. In our current study, we show that ANGPTL4/8-mediated plasmin generation largely blocks the ability of ANGPTL3/8, ANGPTL3, ANGPTL4, and ApoC3 to inhibit LPL activity, while still allowing ApoC2 to be able to stimulate LPL activity. Together, these data provide a plausible explanation for how ANGPTL4/8-mediated plasmin generation in adipose tissue enables LPL to be maximally active in the fat in the postprandial state.Recombinant human tPA ab92637) was purchased from Abcam. Human plasminogen (1939-SE), anti-ApoC2 rabbit antibody (MAF4497), anti-ANGPTL3 rabbit polyclonal antibody (AF3829), and anti-LPL antibody (AF7197) were purchased from R&D systems. An anti-His antibody was generated internally using methods previously described 2637 was . C-termiTo study the effects of ApoC3 and ApoC2 on LPL enzymatic activity, human HEK293 LPL stable expressing cells were incubated with increasing amounts of each protein. Incubations were carried out at 37°C using assay medium (DMEM/F12 medium containing 0.1% FA-free BSA). After overnight incubation of the LPL stable expression cells in growth medium, the medium was replaced with 80 μl of assay medium containing increasing concentrations of ApoC3 or ApoC2 protein, and cells were incubated for 60 min. Afterward, 20 μl of 5× working solution, freshly prepared with 0.05% Zwittergent detergent 3--propanesulfonate (Sigma) containing EnzChek lipase substrate BODIPY dabcyl–labeled TG analog (Invitrogen), were added to achieve a final substrate concentration of 1 μM. At the end of each experiment, fluorescence was monitored with a Synergy Neo2 plate reader at 1 and 30 min with an excitation wavelength of 485 nm and an emission wavelength of 516 nm.70-90 for ANGPTL3/8, ANGPTL4, and ANGPTL3 -propanesulfonate (Sigma) containing EnzChek lipase substrate BODIPY dabcyl–labeled TG analog (Invitrogen), were added to achieve a final substrate concentration of 1 μM for a further incubation of 30 min. At the end of each experiment, one part of each sample had its fluorescence monitored with a Synergy Neo2 plate reader with an excitation wavelength of 485 nm and emission wavelength of 516 nm. In another part, Western blotting was performed as described below.At the end of the final incubation period for each LPL activity assay, the reaction was stopped in a portion of the sample by the addition of an equal volume of SDS-PAGE sample buffer, and samples were boiled for 5 min. Samples were separated on gels and transferred to separate PVDF membranes. The membranes from cells treated with ANGPTL3/8 and ANGPTL3 were probed with biotinylated anti-ANGPTL3 rabbit polyclonal antibody, and detection was performed with Alex-680–conjugated streptavidin. Membranes from cells treated with ApoC3 were probed with a biotinylated anti-His antibody, and detection was performed with Alex-680–conjugated streptavidin. Membranes from cells treated with ApoC2 were probed with rabbit anti-ApoC2 antibody, and detection was performed with Alex-680–conjugated anti-rabbit antibody. All images were recorded using a LI-COR Odyssey imaging system. For the membranes from the cells treated with ANGPTL3, quantification of the intensity of the ANGPTL3 bands was performed using LI-COR software.The ability of tPA to convert plasminogen to plasmin in the presence of ANGPTL4/8, ANGPTL4/8 + ANGPTL3/8, ANGPTL4/8 + ANGPTL3, ANGPTL4/8 + ANGPTL4, ANGPTL4/8 + ApoC3, and ANGPTL4/8 + ApoC2 was determined using a colorimetric plasmin activity assay as previously described  with minTo characterize the effects of ANGPTL4/8-mediated plasmin generation on LPL, HEK293 LPL stable–expressing cells were first incubated with 100 nM ANGPTL4/8 for 1 h at 37°C in assay medium. Afterward, cells were incubated in the absence or presence of tPA (0.1 nM) + plasminogen (30 nM) for 2 h at 37°C. Cells were washed, fresh media supplemented with 0.1 U/ml heparin was added to detach membrane-bound LPL, and the incubation was continued at 37°C for 1 h. The amount of membrane-bound LPL in each sample was analyzed via Western blotting as previously described . Cells rt test to compare the respective treatment group to the most relevant condition. A P-value of <0.05 was considered to indicate statistical significance.In all LPL activity assays, the fluorescent signal was recorded at 1 and 30 min, and the 1 min reading was subtracted from the 30 min reading to correct for background. In each experiment, the vehicle control fluorescent signal corresponded to 100% LPL activity, and the amount of LPL activity present in all other samples was expressed as a percent of the vehicle control. In each LPL activity assessment, significance for the effect of the combination of proteins of interest on LPL enzymatic activity was calculated using an unpaired 80 concentration. As expected, ANGPTL3/8 alone potently inhibited LPL, however this inhibition was almost completely reversed when ANGPTL4/8 + tPA + plasminogen were added  without interfering with LPL activation by ApoC2.A somewhat surprising finding from our study was that ANGPTL4/8-mediated plasmin generation resulted in suppression of ApoC3-mediated LPL inhibition, while having no effect on ApoC2-mediated stimulation of LPL. These data were consistent with the subsequent observation that the addition of ANGPTL4/8 + tPA + plasminogen resulted in cleavage of ApoC3 but not ApoC2. While plasmin-mediated cleavage of ANGPTL3 as part of the ANGPTL3/8 complex is consistent with the concept that ANGPTL3 contains a fibrinogen-like domain and that fibrin is a natural substrate for plasmin , 19, 20,Our findings build upon those of many other researchers who have studied the ANGPTL3/4/8 protein family, including Zhang and colleagues, who provided some of the earliest insights into ANGPTL8 biology, proposed an elegant model for how this family of proteins works to control TG metabolism in the fed versus fasted state and then further refined their model , 34, 35.Angptl8 knockout mice (Angptl8 knockout (which eliminates the liver-derived circulating ANGPTL3/8 complex) was beneficial and resulted in markedly reduced TG levels, adipose-specific Angptl8 knockout  caused significantly increased postprandial circulating TG concentrations  and the ANGPTL3/4/8 protein family. Clearly, much additional investigation will be required to understand in greater depth the exact mechanisms mediating each of these various interactions as well as their functional relevance to TG metabolism in vivo.All data are included in the main text and figures.The authors declare no conflicts of interest with the contents of this article.


Ser329 phosphorylation in the presence of DYRK1B inhibitor. Therefore, these findings suggest that DYRK1B regulates CD4 T cell differentiation through FOXO1 phosphorylation and DYRK1B inhibitor has a potential as a novel agent for treatment of ACD.Allergic contact dermatitis (ACD) is a type IV hypersensitivity mainly mediated by Th1/Th17 immune response. Topical corticosteroid is currently the first-line treatment for allergic contact dermatitis (ACD) and systemic administration of immunosuppressive drugs are used in patients with severe disseminated cases. However, increased risk of adverse effects has limited their use. Thus, the development of a novel immunosuppressant for ACD with low toxicity is a challenging issue. In this study, we began our study by using a murine contact hypersensitivity (CHS) model of ACD to examine the immunosuppressive effects of DYRK1B inhibition. We found that mice treated with a selective DYRK1B inhibitor show reduced ear inflammation. In addition, a significant reduction of Th1 and Th17 cells in the regional lymph node upon DYRK1B inhibition was observed by FACS analysis. Studies in vitro further revealed that DYRK1B inhibitor does not only suppressed Th1 and Th17 differentiation, but also promotes regulatory T cells (Treg) differentiation. Mechanistically, FOXO1 signaling was enhanced due to the suppression of FOXO1 ACD is clinically important because it can occur in general population and be common occupational skin disorders having a socioeconomic impact. Currently, topical corticosteroid is the first-line medical treatment for ACD, but systemic administration combining with immunosuppressive drugs such as azathioprine, cyclosporin or tacrolimus are used in widespread ACD, with greater than 20% of the body involvement7. However, increased risk of various adverse effects of these systemic drugs limits their use, and a rebound flare-up may occur upon drug cessation. Therefore, novel compounds that are effectively and safely used for treatment of ACD are of high clinical need.Allergic contact dermatitis (ACD) is a form of inflammatory skin disease that is classified as a delayed or type IV hypersensitivity reaction and is mainly mediated by Th1/Th17 immune response10. Moreover, it also has been reported that elevated DYRK1B expression promotes the survival of colon carcinoma, pancreatic ductal adenocarcinoma, non-small cell lung cancer, and rhabdomyosarcoma14. However, in contrast to the roles of DYRK1B in the systems described above, its role in the immune system remains elusive.DYRK1B belongs to the DYRK family within the CMGC  superfamily of protein kinases. DYRK1B derives its kinase function via co-translational autophosphorylation on a conserved tyrosine that resides within its catalytic domain. DYRK1B is highly expressed in skeletal muscle and the testes, and it has been previously reported that DYRK1B plays roles in the differentiation of these cells50 of 7 nM, with low cytotoxicity15. Oral administration of AZ-DYRK1B-33 at 10 mg/kg to BKS db/db mice for three weeks was well-tolerated16. In this study, we used AZ-DYRK1B-33 to evaluate the therapeutic potential of DYRK1B inhibition for ACD using a contact hypersensitivity (CHS) murine model and investigate its potential effects on the differentiation of CD4 T cells.AZ-DYRK1B-33 is a small ATP-competitive inhibitor that potently inhibits the kinase activity of DYRK1B with an IC20 +IFN-γ+ and CD4+TNF-α+) and Th17 (CD4+IL-17A+ and CD4+IL22+) population  and Th17 (CD4+IL17A+) differentiation   differentiation in a concentration-dependent manner  analysis. We found 300 DEGs specific to samples treated with DYRK1B inhibitor and 331 DEGs specific control samples respectively , Th17 signature genes , Treg signature genes  and FOXO1 target genes  and found that in consistent with the above findings, DYRK1B inhibition suppressed the expression of Th1 and Th17 genes, while enhanced Treg and FOXO1 target genes , including Th1, Th2, and Th17, or Treg depending on the cytokine milieu program. Th cells serve as a key modulator in the activation of macrophages, cytotoxic T cells, and B cell maturation and function. In contrast, Treg plays a suppressive role in the maintenance of peripheral immune tolerance31. Consequently, an imbalance between Th1, Th2, or Th17 and Treg associates with various allergic inflammation and autoimmune diseases39. Interestingly, using in vitro T cell differentiation system, we found that inhibition of DYRK1B did not only suppress Th1 and Th17 differentiation, but also enhanced Treg differentiation. We therefore speculate that DYRK1B inhibitor may also have a potential to restore self-tolerance in immune dysregulation disorders that are mainly mediated through the impairment of Treg.After their activation by cognate antigens, naïve CD427. Unlike cancer cells, there is no evidence that suggests FOXO1 is downstream from DYRK1B in the immune system. This raises the question whether DYRK1B is an upstream kinase of FOXO1 in CD4 T cells. Based on our biochemical analysis, DYRK1B phosphorylates FOXO1 at Ser329 that subsequently inhibits FOXO1 transcriptional activity in CD4 T cells. FOXO1 is a transcription factor that plays an essential role in a variety of cellular processes, including metabolism, cell cycle progression, differentiation, and apoptosis43. T cell-specific deletion of Foxo1 led to autoimmunity because of decreasing FOXP3+ Treg cells while increasing T follicular helper in vivo46. The upregulation of FOXO1 could lessen inflammation in liver ischemia and reperfusion injury through the downregulation of NF-κB and NLRP3 inflammasome47. In CD4+ T cells, FOXO1 directly bound the promoter region of Ifng and suppressed its gene expression48. Furthermore, in the presence of TGF-β, T cell-specific deletion of Foxo1 results in impairment of Treg differentiation and misdirection toward Th144. In Th17 cells, FOXO1 bound the DNA binding domain of RORγt that led to the suppression of RORγt activity and hence decreasing Th17 differentiation22. In contrast, genetic deletion of Foxo1 in CD4+ T cells increased IL17A expression and Th17 differentiation49. Moreover, Foxo1 was directly repressed by miR-183C driving Th17 cell pathogenic function50. In Tregs, FOXO1 bound the promoter of Foxp3 and induced its gene expression, thus facilitated Treg cell differentiation24. Taken these reports together with our in vitro data suggested that the inhibition of DYRK1B promoted Treg differentiation while suppressed Th1 and Th17 differentiation through the regulation of FOXO1 inhibitory phosphorylation in vitro. However, the effect of DYRK1B on the phosphorylation of FOXO1 in vivo has not been confirmed and warrants further investigation.In breast and ovarian cancer cells, the phosphorylation of FOXO1 by DYRK1B resulted in decreasing transcriptional activity51. Moreover, it also plays important roles in cell-cycle progression and the survival of several cancer cell lines14. To activate its kinase function, DYRK1B auto-phosphorylates its tyrosine residue (Y273) in the activation loop during translation10. Furthermore, in human embryonic kidney (HEK 293) cells, a recent study suggests that DYRK1B is also activated via the phosphorylation of serine residue (S421) by ERK252. Notably, it has been reported that activation of the MAP/ERK2 pathway activates DYRK1B kinase function in cancer cells and further phosphorylates its downstream proteins, such as FOXO152. Interestingly, it was previously reported that activation of ERK2 is required for Th1 differentiation53. Moreover, it was also reported that inhibition of ERK2 suppressed Th17 differentiation, but induced FOXP3 expression and Treg differentiation54. Given that our results suggest similar suppression of effector Th1 and Th17 differentiation and enhancement of Treg differentiation upon DYRK1B inhibition, we speculate that ERK2 might be the upstream signaling molecule of DYRK1B in CD4 T cell differentiation. Further investigation should be pursued to confirm in the future.DYRK1B was previously reported to be a key regulator of skeletal muscle cell differentiation15, both indicate that inhibition of DYRK1B function by AZ-DYRK1B-33 had no clear cytotoxicity in vitro even at high concentration and the topical application of AZ-DYRK1B-33 in mice in vivo did not show any obvious side effects, we believe that this compound is a promising candidate drug for ACD. However, it should be noted that DYRK1B is highly expressed in skeletal muscle and testes. In skeleton muscle, DYRK1B was activated by Rho-family protein to promote myoblast differentiation51. In testes, DYRK1B interacted with Cold-inducible RNA-binding protein (Cirp) modulating p27 and cyclin D1 stability to fine-tune the proliferation of undifferentiated spermatogonia55. We therefore could not exclude the possibility that myopathy and impairment of spermatogenesis might occur upon long-term treatment. In addition, previous studies also suggested that DYRK1B may play roles in adipogenesis and glucose metabolism. It is therefore also possible that metabolic syndrome, such as central obesity, hypertension, and/or hyperglycemia, could develop upon the administration of DYRK1B inhibitor57. Further studies on the pharmacokinetics and long-term in vivo toxicity are warranted to unravel the therapeutic potential of DYRK1B inhibitor in human ACD.Given that our data . All the human experimental protocols and procedures were performed in accordance with the relevant guidelines and regulations, and were approved by the Human Research Protection Unit of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (assurance number COA no. 663/2564[IRB1]).Female 8-week-old C57BL/6NJc1 mice were purchased from Nomura Siam International . All mice were maintained in a specific pathogen-free condition with free access to standard rodent feed and water. All procedures were conducted in accordance with the guidelines of Mahidol University and the National Research Council of Thailand, and in complied with the ARRIVE guidelines. The number of animals used in each experiment was determined according to the number of animals used in similar experiments conducted in previously published studies. All experimental protocols were approved by the Siriraj Animal Care and Use Committee of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (assurance number COA no. 002/2565).20. All mice were sensitized by painting 25 μL of 0.5% DNFB diluted in acetone and olive oil  on the shaved abdomen (day 0) with an average coverage area of 2 cm2. On day 5, mice were elicited by painting 20 μL of 0.3% DNFB in the same vehicle on the dorsal and ventral pinna of both ears. For topical application of chemical and drug compounds, mouse pinnae were painted with either AZ-DYRK1B-33 , dexamethasone , or no treatment  starting on day 4 for 3 consecutive days. Mice sensitized and elicited with vehicle alone were used as a non-sensitized control. The ear thickness of each ear of each individual mouse was measured at pre-challenge and at 6-, 24-, and 48-h post-challenge using a Vernier caliper .DNFB was used as a hapten for inducing murine CHS as described by Honda et al. and Thitilertdecha et al.At 48-h post-challenge, all mice were sacrificed by intraperitoneal injection of thiopental sodium (50 mg/kg). The mouse ears were resected, fixed with 4% paraformaldehyde, embedded in paraffin. Sections (5 μm thick) were stained with hematoxylin and eosin (H & E) for histopathologic evaluation of CHS. Inflammatory cell infiltration in each section was quantified by counting in each visual field and divided by area of quantification using Image J.Human blood samples were obtained from healthy donors after obtaining written informed consent, and the procedures were conducted in accordance with the requirements of the Human Research Protection Unit of the Faculty of Medicine Siriraj Hospital. All experimental protocols were approved by the Human Research Protection Unit of the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand (assurance number COA no. 663/2564[IRB1]).+ T cells were isolated via magnetic cell sorting using a human naïve CD4+ T cell isolation kit . Complete RPMI supplemented with 10% FBS, 50 µM 2-mercaptoethanol, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10 mM HEPES was used as a culture medium. The isolated naïve CD4+ T cells were stimulated with immobilized anti-CD3 Ab  and 1 µg/mL soluble anti-CD28  for 96 h before analysis. The culture medium was supplemented with 15 ng/mL TGF-β1  and 100 U/mL IL-2  for Treg differentiation; with 10 ng/mL IL-12 , 5 µg/mL anti-IL-4 Ab , and 50 U/mL IL-2  for Th1 differentiation; with 30 ng/mL IL-4 , 5 µg/mL anti-IFN-γ Ab , and 50 U/mL IL-2  for Th2 differentiation, and with 5 ng/mL TGF-β1 , 25 ng/mL IL-6 , 50 ng/mL IL-23 , 5 µg/mL anti-IL-4 Ab , and 5 µg/mL anti-IFN-γ Ab  for Th17 differentiation. In each different culture condition, the DYRK1B inhibitor  was added on day 0 at the concentration of 0.1, 0.3, 1, or 3 µM.Human PBMCs were isolated from healthy donor blood using Ficoll-Hypaque  gradient centrifugation. Naïve CD4Surface staining  was performed in PBS supplemented with 2% FBS for 30 min at ambient temperature. For intracellular staining of iTreg, the cells were fixed and permeabilized using a BD Pharmingen Stain Buffer Set , followed by washing and staining with APC-conjugated anti-human FOXP3  for 30 min. For evaluation of Th1, Th2, and Th17, the cells were stimulated with 50 ng/mL PMA  and 1 μg/mL ionomycin  for 4 h at 37 °C in the presence of GolgiPlug protein transport inhibitor  before surface staining. BD Cytofix/Cytoperm  was used as a fixing and permeabilizing reagent for intracellular cytokine staining. The cells were then washed and stained with APC-conjugated anti-human IFN-γ , IL-4 , or IL-17A  and incubated on ice for 30 min. Flow cytometry was performed using a Cytoflex flow cytometer , and the data were analyzed using CytExpert software (Beckman Coulter Life Sciences).GAPDH. Primer used for PCR amplification are forward: 5ʹ TTCATCTGTGGCATCATCCG 3ʹ, reverse: 5ʹ TCGCATGTTGTGGAACTTGA 3ʹ for FOXP3, forward: 5ʹ TGTGGAGACCATCAAGGAAGACA, reverse: 5ʹ GGCGACAGTTCAGCCATCAC 3ʹ for IFNG, forward: 5ʹ AAAGCTCCAACCGGCAGCAA 3ʹ, reverse: 5ʹ CAAGATGACCAACAGAGCGAC 3ʹ for IL7RA, forward: 5ʹ GCGTGTCCCCCTATCCTTCC 3ʹ, reverse: 5ʹ GGGGGCCTTCTCAGTCCTTC 3ʹ for TBX21 and forward: 5ʹ AAATTCCATGGCACCGTCAAG 3ʹ, reverse: 5ʹ TGGTTCACACCCATGACG-AA 3ʹ for GAPDH.The cells were harvested and lysed in Trizol reagent . RNA was extracted using an RNAeasy Mini Kit , and then assessed for concentration and purity using a Nanodrop spectrophotometer . cDNA synthesis was performed using iScript Reverse Transcription Supermix . KAPA SYBR FAST qPCR Master Mix (Sigma-Aldrich) was used for quantitative PCR, which was run on QuantStudio 5 software (Thermo Fisher Scientific) and analyzed using QuantStudio design and analysis software version 1.3.1 (Thermo Fisher Scientific). Gene expression levels were normalized to + T cells were stimulated with anti-CD3 and anti-CD28 under Treg-polarizing conditions for 24 h in the absence or presence of 1 µM DYRK1B inhibitor. Total RNA were isolated using an RNAeasy Mini Kit (Qiagen), and the RNA concentration was assessed using a Qubit™ RNA Broad Range Assay Kit (Thermo Fisher Scientific). Bioanalyzer analysis using an Agilent 2100 analyzer  showed the RNA integrity (RIN) values for all samples to be ≥ 7.8. mRNA were enriched at the poly-A tail using oligo-dT attached beads. Libraries were prepared using an Ultra II Directional RNA Library Prep Kit for Illumina. Paired-end libraries were sequenced using an Illumina HiSeq™ system. The original raw data were transformed to sequenced reads via base-calling. Raw data were recorded in a FASTQ file that contained sequence information (reads) and corresponding sequencing quality information. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference human genome (hg38) using Hisat2 v2.0.5. Differential gene expression analysis was performed using edgeR software (v3.38.2). The resulting p-values were adjusted using the Benjamini–Hochberg method for controlling the FDR. Genes with an FDR < 0.05, and a Log2 fold change of <  − 1.2 or > 1.2 were defined as differentially expressed. The GO database, the KEGG database, and the Reactome Pathway database were used for gene set enrichment analysis using clusterProfiler R software. The model used, the company that is was purchased from, and the headquarters location of that company need to be sourced and entered for every product in the preceding paragraph.Human naïve CD4Ser-329  and mouse anti-human β-actin . The following secondary antibodies were used: goat anti-rabbit IgG H&L  and goat anti-mouse IgG H&L . The IRDye® infrared fluorescent dye signal on the cellulose membranes was detected and calculated using an Odyssey® CLX imaging system and Image Studio software, respectively.Cells were harvested, washed twice with cold PBS, lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific), and incubated on ice for 30 min with vortexing for 10 s at 15-min intervals. The protein concentration was determined using a Bradford protein assay (Bio-Rad Laboratories), after which equal amounts of total protein were run on poly-acrylamide gels that were prepared in-house. After electrophoresis, the proteins were transferred using an iBlot semi-dry transfer system (Thermo Fisher Scientific). The following primary antibodies was used: mouse anti-human FOXO1 , rabbit anti-human pFOXO1All data summarized in the bar/line graphs are presented as mean ± SEM. Statistical comparisons were performed using Student’s unpaired t-test (2-tailed) or 1- or 2-way ANOVA with subsequent Tukey’s test. These comparisons were performed using GraphPad Prism . A p-value < 0.05 was considered statistically significant.Supplementary Legends.Supplementary Figures.


The fabricated hybrid nanozymes displayed synergistic peroxidase (POD)-like activities. It showed a remarkable level of hydroxyl radicals (·OH) in the presence of a low dose of H2O2 (0.97 mM). Further, the hybrid FMA nanozymes exhibited excellent biocompatibility and favourable antibacterial effects against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The animal experiments indicated that the hybrid nanozymes promoted wound repair with adequate biosafety. Thus, the well-designed hybrid nanozymes represent a potential strategy for healing bacterial wound infections, without any toxic side effects, suggesting possible applications in antimicrobial therapy.Bacterial wound infections are a serious threat due to the emergence of antibiotic resistance. Herein, we report an innovative hybrid nanozyme independent of antibiotics for antimicrobial wound healing. The hybrid nanozymes are fabricated from ultra-small Au NPs via in-situ growth on metal-organic framework (MOF)-stabilised FeThe online version contains supplementary material available at 10.1186/s12951-023-02186-6. Bacterial infection is one of the most serious risk factors threating public health worldwide . AntibioThe emergence and rapid development of antibacterial nanomaterials such as silver nanoparticles (NPs), zinc NPs, and copper NPs have attracted tremendous attention due to their superior antibacterial performance –9. Howev3O4) NPs exhibit reaction kinetics and catalytic mechanisms similar to those of natural horseradish peroxidase [Nanozymes, which are nanomaterials with natural enzyme-mimetic catalytic performance, have been used extensively in biomarker detection , cancer roxidase , many naroxidase , 20. Nanroxidase –23.4O7 NPs, silica-loaded AuNPs, and Pd NPs, exhibit bactericidal effects. However, due to their weak catalytic activity, high concentrations are required to achieve optimal antimicrobial effect. Hence, polymetallic nanozymes maybe a promising strategy to achieve highly catalytic performance [2O2) is a common antibacterial agent using for sterilisation and disinfection, which can effectively destroy bacterial proteins and DNA leading to cell lysis and death [2O2, which is usually needed in high concentrations for bactericidal effects [2O2 can corrode healthy skin tissues and exacerbate pathological inflammation, and even damage the central nervous system in humans [2O2 for antibacterial treatment [Many monometallic nanozymes, such as Tbformance –26. Hydrnd death , 28. How effects . Neverthn humans , 31, perreatment –34. Ther3O4 NPs with intrinsic POD-mimetic performance have been utilised in biomedical applications. However, bare Fe3O4 NPs are highly unstable and easily aggregate due to the large surface energy, which greatly reduced their catalytic performance. A coating layer of mesoporous materials has been reported to prevent the agglomeration of Fe3O4 NPs and further improved their stability. Additionally, the synergetic interactions between Fe3O4 NPs and another catalyst can enhance the enzyme-mimetic activity. Therefore, the fabrication and stabilisation of a hybrid nanozyme based on Fe3O4 NPs has been shown to improve antibacterial treatment. Among the various coating layers available, metal–organic frameworks (MOFs) with high specific surface area, high porosity, tunable size, accessible active sites, and significant capacity to accommodate guest molecules by anchoring them in proper position, have attracted substantial interest [For more than a decade, Feinterest –37. A va3O4 NPs were initially prepared via chemical coprecipitation. Porous IRMOF-3 were then used for surface decoration of Fe3O4 NPs as linkers, resulting in functional modification. Ultrasmall Au NPs were successively integrated into IRMOF-3 to construct heterogenous bimetallic composite nanozymes, i.e., Fe3O4@MOF@Au NPs (FMA NPs). In principle, these FMA nanocatalysts induce an ROS-mediated antimicrobial mechanism in bacteria, thereby significantly minimising bacterial growth. This well-designed FMA nanozymes have prominent advantages as follows: (i) The synergistically enhanced POD-like activities via cascade reaction of Au and Fe3O4 NPs greatly accelerating catalytic reaction rate and further improving the antibacterial efficiency; ii) the improved dispersion stability, reduced oxidation and biosafety by surface encapsulation of Fe3O4 NPs using MOF nanoshell layers; (iii) the bimetallic nanozymes combined with the low concentration of H2O2 (0.97 µM) for achieving excellent therapeutic effects.Herein, we designed an innovative nanocatalytic antibacterial strategy based on MOF-modulated bimetallic hybrid nanozymes for infected wound healing without employing any antibiotic drugs Scheme . MagnetiTherefore, the FMA hybrid nanozymes were expected to exhibit improved enzymatic activity and effective antibiotic treatment.All experimental materials and methods of characterisation are described in detail in the Supporting Information.Approximately 5-week-old Kunming (KM) mice  were obtained from the Animal Experiment Center of Zhengzhou University  and fed under specific pathogen-free (SPF) conditions. Animal experiments were approved by the Animal Care and Use Committee of the School of Biological Engineering, Henan University of Technology .3O4 NPs were synthesised via chemical co-precipitation [3O4 NPs were synthesised using a high-pressure one-pot method [3O4 NPs were mixed with 5 mL of anhydrous ethanol and 5 mL of N-N dimethylformamide (DMF), followed by treatment with 200 mg of polyvinylpyrrolidone (PVP) as a dispersant under ultrasonication for 20 min. Simultaneously, 67 mg of zinc nitrate and 16 mg of 2-aminoterephthalic acid were dissolved in 4 mL of DMF to form a mixture. Subsequently, the two solutions were combined and sonicated for 10 min in an ultrasonic emulsion disperser. The mixture was transferred to an autoclave and heated for 4 h at 100 °C. Brown products were acquired via magnetic decantation under a magnetic field, and the residue was dispersed again into 20 mL of DMF. The products were washed with deionised water (DI) six times to obtain Fe3O4@ IRMOF-3 NPs.Superparamagnetic Fepitation , 39. Addt method . Briefly3O4@ IRMOF-3 NPs (80 mg), prepared as above, were dispersed into 40 mL of DI, followed by 20 min of sonication. Next, 4 mL of HAuCl4 solution (20 mM) was added and sonicated for 20 min, followed by 10 mL of freshly frozen prepared 0.1 M NaBH4. The suspension was stirred at 120 rpm for 1 h. The fabricated Fe3O4@MOF@Au NPs (FMA NPs) were washed three times using DI water for further use.Fe3O4 NPs, Fe3O4@MOF NPs, and FMA NPs was investigated using 3, 3’, 5, 5’-tetramethylbenzidine (TMB) as the chromogenic substrate. Next, 50 µL of various nanocomposites (1 mg/mL) were added to 500 µL of NaAc-HAc buffer solution consisting of 20 µL H2O2 (30%) and 10 µL TMB (10 mg/mL), respectively. The absorbance at 652 nm was measured with a microplate spectrophotometer (Bio Tek Instrument Inc. USA). Catalytic activities of nanozyme mimetics or natural enzymes were controlled by reaction pH, reaction temperature and reaction time [3O4, Fe3O4@MOF, and FMA NPs under different pH levels (2.8 ~ 8.4), temperature (20 ~ 60 °C), and reaction time (0 ~ 30 min) were analysed. The natural POD was used as a positive control.The POD-like catalytic performance of the as-prepared Feion time . TherefoThe analysis of steady-state kinetics is described in detail in the Supporting Information.2O2 (0.2 M) after 15 min. Quantitative analysis of ·OH production was performed using a microplate.The production of ·OH in the presence of the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) was first analysed qualitatively via electron spin resonance (ESR) spectroscopy using 400 µL of FMA NPs (400 µg/mL) and 20 µL of HFresh blood (5 mL) acquired from KM mice was dispersed in 0.9% saline solution, and centrifuged at 2500 × g for 5 min three times. The supernatant was then discarded. The acquired red blood cells (RBCs) were washed with 0.9% saline solution, and diluted with an appropriate amount of 0.9% saline solution to prepare 2% RBC suspension. Next, 1 mL RBC suspension was added to solutions containing varying concentrations  of FMA NPs. Saline solution (0.9%) and DI water served as negative and positive controls. respectively. The resulting products were centrifuged at 3000 rpm for 5 min after 4 h of stabilisation at room temperature (RT). The absorbance of 100 µL of each supernatant was measured at 540 nm to calculate the hemolysis rate as follows.ODa is the absorbance of the positive control; ODb is the absorbance of the negative control; and ODc denotes the absorbance of the samples at 540 nm.where 2 and 37 °C.Normal liver L-02 cells and human umbilical vein endothelial cells (HUVECs), acquired from the Shanghai Cell Bank of the Chinese Academy of Sciences , were cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin (100 U/mL), and 1% streptomycin (100 µg/mL), in a cell incubator at 5% COS. aureus, and the Gram-negative E. coli bacteria, obtained from China General Microbiological Culture Collection Center were cultured in Luria-Bertani (LB) liquid medium. They were incubated at 37 °C.Gram-positive Normal liver L-02 cells and HUVECs were used to assess the cytotoxicity of FMA NPs. The L-02 cells and HUVECs were treated for 24 h with various concentrations of FMA NPs. Subsequently, 20 µL of CCK-8 was added to each micro well and cultured for another 4 h in the dark. The absorbance at 450 nm was measured using a multifunctional microplate reading instrument to evaluate the toxic effects of FMA NPs.The cell viabilities of treated liver L-02 cells and HUVECs were evaluated via PI and FDA double-staining assay, respectively. Cells were treated with different formulations for 24 h. The control groups were treated with serum-free medium. After washing twice with sterile PBS (pH 7.4), the treated cells were supplemented with a solution containing 100 µL PI (20 µg/mL) and 100 µL FDA (1 µg/mL). After incubation in the dark for 15 min at 37 °C, the samples were photographed under an inverted fluorescence microscope .S. aureus and E. coli using the plate count method [600nm value of the bacterial suspension was around 0.7. The bacterial suspension was then diluted with sodium acetate buffer (pH 4.5) to a concentration of 107 CFU/mL, followed by the addition of 50 µL of the bacterial suspension to various groups including control, H2O2 group (0.97µM), FMA NPs group, and the groups treated with (low dose) FMA NPs + H2O2 (200 µg/mL), and (high dose) FMA NPs + H2O2 (400 µg/mL). The minimum inhibitory concentrations (MICs) of FMA NPs were also determined under different concentrations of FMA NPs and under similar conditions to analyse the MICs of S. aureus and E. coli. Bacterial suspensions were cultured on LB agar plates and the number of CFU was calculated after 24 h of incubation at 37 °C. The bacterial inhibition rate was defined using the following equation:We investigated the antibacterial effects of various formulations against t method , 43. TheC represents is the number of colonies in different experimental groups, and CO denotes the number of colonies in the control group.In the equation above, S. aureus and E. coli treated with various formulations for 2 h were collected by centrifugation at 2000 rpm for 10 min, and the supernatant was discarded. The bacteria were then fixed with 2.5% glutaraldehyde at 4 °C for 12 h. Subsequently, the fixed bacteria were washed with PBS and then dehydrated for 20 min using a gradient of 30%, 50%, 70%, 80%, 90%, and 100% ethanol. After drying in a critical point drying apparatus for 90 min, the treated bacteria were sprayed with gold for 150 s and the morphological changes were observed via SEM .The morphology of the treated bacteria was analysed via scanning electron microscopy (SEM). Samples of E. coli (107 CFU/mL) and various formulations for 24 h at 37 °C. The biofilms were carefully washed twice with sterilised PBS to remove the supernatant. The remaining biofilms were stained for 30 min with 20 µL for 30 min with 20 µL of 2% crystalline violet solution at room temperature (RT), followed by the addition of 250 µL anhydrous ethanol solution to dissolve the crystalline violet for further semi-quantitative analysis of biofilm. The OD values of the samples were detected with a microplate spectrophotometer at 590 nm. The biofilm formation was evaluated and S. aureus infection was detected.The biofilm formation and detection was evaluated as described previously with minor modification . The bioE. coli (107 CFU/mL) was first added to 24 microplates. The biofilm formation was detected at the bottom of the microplates after incubation for 24 h at 37 ℃. Subsequently, various groups were treated with the formulations and incubated at 37 ℃ for another 24 h. The inhibition of biofilm was measured via crystalline violet staining as described above [To evaluate the inhibitory effects of the biofilm, a 250 µL suspension of ed above .S. aureus samples were stained in the dark for 15 min with the nucleic acid dye SYTO 9 and PI at RT. The stained bacteria were visually analysed with an inverted fluorescence microscope .We used SYTO 9/PI double-labelling to evaluate the vitality of the treated bacteria. Live bacteria were stained with SYTO 9, displaying green fluorescence. Dead bacteria were stained with PI, displaying red fluorescence. Following treatment for 60 min at RT with various formulations, the treated S aureus (107 CFU/mL) was added dropwise to the wound to create an S. aureus-infected wound. The infected mice were randomly divided into 5 groups and treated for 8 days using different formulations. The wounds were photographed every day. The treated mice were executed on day 9 after treatment. The skin tissues of the infected mice were obtained from each group separately, fixed using 4% formaldehyde, paraffin-embedded, and stained using hematoxylin-eosin (H&E).To assess the antibacterial activity of FMA nanozymes in vivo, KM mouse models were established. The mice were first dehorned. A circular skin measuring about 6–8 mm in diameter was cut off the backs of mice with scissors. A 20 µL solution of To further evaluate the biosafety of FMA nanozymes, the major organs including heart, liver, spleen, lung, and kidney of wound mice were dissected and fixed with 4% formaldehyde to prepare paraffin sections for H&E staining.The methods of statistical analyses are described in the Supporting Information.3O4 NPs displaying synergistic POD-mimicking activities for infected wound healing without using any antibiotics. Scheme 3O4 NPs via partial reduction chemical co-precipitation; (ii) modification of Fe3O4 NPs with highly porous IRMOF-3 shells to fabricate Fe3O4@MOF NPs using the hydrothermal method; and (iii) incorporating ultrasmall Au NPs into the pores of IRMOF-3 shells in situ, which enhanced their POD activities via a cascade reaction to form flexible multiphase catalysts FMA NPs.We developed an innovative nanocatalytic antibacterial nanosystem using IRMOF-3-modulated biomimetic hybrid FMA NPs. The FMA NPs were assembled with ultrasmall Au NPs and Fe3O4 NPs, Fe3O4@MOF NPs and FMA NPs were characterised by transmission electron microscopy (TEM). TEM image reveals a spherical or elliptic structure of Fe3O4 NPs with a diameter of around 10 nm . The characteristic Fe-O-Fe spectral band appeared at 576 cmNPs Fig. B. Follow3O4@MOF and FMA NPs, the crystal structures of the complexes were analysed via X-ray diffraction (XRD). The diffraction peaks at 2θ = 35.2°, 41.5°, 50.6°, 63.0°, 67.3°, and 74.2° correspond to the (220), (311), (400), (422), (511), and (440) crystal planes, respectively, of the Fe3O4 NPs (JCPDS 65-3107)  in DI water. As shown in Fig. 2O2 into•OH radicals. The combination of Fe3O4 NPs and ultrasmall Au NPs displayed a synergistic POD-like activity. The colourless TMB was oxidised in the presence of •OH, resulting in a blue color. Compared with the catalytic activity of single Fe3O4 NPs or ultrasmall Au NPs, the addition of Au NPs significantly increases the catalytic activity synergistically Additional file 2O2 showed a weak absorption peak, while other reaction systems with different catalysts exhibited a strong absorption peak at 652 nm. In addition, the simulated enzyme activity of Fe3O4@MOF NPs was slightly below that of the naked Fe3O4 NPs, which can be attributed to the adsorption of MOF on the surface of Fe3O4 NPs, leading to the reduction of catalytic centre. However, the catalytic ability of FMA NPs was significantly enhanced by the Au NPs incorporated in situ, which was attributed to the synergistic effects of the combination of Fe3O4 NPs and Au NPs. The simulated enzymatic activities of catalysts at different concentrations were also compared. The POD-mimicking activities of various catalysts increased with increasing concentration, and the absorbance of FMA NPs was the highest, indicating the strongest POD activities  were derived from the Lineweaver-Burk plot. The Km values were 0.34 and 0.24 mM for TMB and H2O2, respectively, based on Lineweaver-Burk double reciprocal plot  of FMA NPs was the largest, which indicates the highest catalytic efficiency per nanoparticle.The kinetic parameters of the hybrid FMA nanozymes were determined under varying TMB or Hdel Fig. F and G. Nano-biomaterials exhibit excellent biocompatibility for clinical application, and blood compatibility is an important indicator of biocompatibility . TherefoHUVECs and normal hepatocytes L-02 cells were used to determine the in vitro cytotoxicity of FMA NPs. As shown in Fig. We also used fluorescence FDA/PI double labelling to assess the viability of L-02 cells and HUVECs after treatment with FMA NPs. The living cells were stained by FDA, and showed green fluorescence, while the dead cells were stained by PI and displayed red fluorescence . As show2O2 compared with the control, indicating that FMA NPs or H2O2 did not have a significant effect on bacterial viability. In contrast, the treatment with FMA NPs and low-dose H2O2 had a remarkable inhibiting effect on bacterial growth. The antibacterial rates were 76% and 72% for E. coli and S. aureus at low concentrations (200 µg/mL) of FMA NPs and H2O2, respectively. Especially, the antibacterial activities of E. coli and S. aureus reached 97% and 98% following treatment with 400 µg/mL FMA NPs and H2O2, which was attributed to the production of abundant toxic·OH radicals. As shown in Additional file 2O2 resulted in increased inhibition against E. coli and S. aureus. The MIC was achieved by increasing the concentration of FMANPs to 400 µg/mL, and when the concentration reached 500 µg/mL, the inhibition of the two bacteria was greater than 99%.The antibacterial performance of FMA NPs was measured in vitro using the plate counting method. As shown in Fig. E. coli and S. aureus treated with different formulations were determined by SEM. The untreated E. coli and S. aureus showed rod-like and spherical morphology with smooth surface, respectively. When treated with H2O2, E. coli and S. aureus rarely displayed significant changes. Treatment with FMA NPs led to insignificant changes in the overall bacterial morphology, indicating a slight antibacterial activity against E. coli and S. aureus. Co-treatment with high levels of FMA NPs and H2O2 disrupted bacterial integrity and destroyed the bacterial morphology with unclear borders between bacteria, different degrees of surface depression and dents due to the generation of toxic ·OH  staining was performed for histological analysis of the wound tissues. As shown in Fig. ivo Fig. B.3O4 NPs. The well-designed FMA hybrid nanozymes displayed significantly enhanced POD-mimetic catalytic activities due to the synergistic effects between Au NPs and Fe3O4 NPs. The FMA nanozymes exposed to low doses of H2O2 generated a large amount of ·OH and accelerated the antibacterial efficiency in vitro and wound healing in vivo. Further, the FMA nanozymes showed negligible biotoxicity even at high concentrations. In summary, this study demonstrates the promise and potential of non-toxic nanozymes for clinical antibacterial applications.In summary, we report the fabrication of an innovative bimetallic FMA hybrid nanozyme for antibacterial wound healing. The nanozymes were constructed with ultrasmall Au NPs-anchored MOFs stabilising FeAdditional file 1: Fig. S1.A TEM images of Fe3O4 NPs, B TEM images of Fe3O4@MOF NPs. Fig. S2. Analysis of the POD mimicking activities of different formulations using UV–vis spectra . Table S1. Comparison of the kinetic parameters Km and Vmax of FMA NPs, HRP, and other reported materials. Fig. S3. The ESR spectra of FMA NPs (400μg/mL), H2O2 used as control. Fig. S4.A The determination of MIC of E. coli. B The determination of S. aureus. Fig. S5. A Inhibitory effects of FMA NPs on E. coli biofilm. B Inhibitory effects of FMA NPs on S. aureus).


Xenopus laevis oocytes for proton current recording using a macro-patch voltage clamp. Our findings reveal that all three isoforms are mammalian mHv1 channels, with distinct differences in their activation properties. Specifically, the longest isoform, mHv1.2, displays a right-shifted conductance–voltage (GV) curve and slower opening kinetics, compared to the mid-length isoform, mHv1.3, and the shortest canonical isoform, mHv1.1. While mHv1.3 exhibits a V0.5 similar to that of mHv1.1, mHv1.3 demonstrates significantly slower activation kinetics than mHv1.1. These results suggest that isoform gating efficiency is inversely related to the length of the N-terminal end. To further explore this, we created the truncated mHv1.2 ΔN20 construct by removing the first 20 amino acids from the N-terminus of mHv1.2. This construct displayed intermediate activation properties, with a V0.5 value lying intermediate of mHv1.1 and mHv1.2, and activation kinetics that were faster than that of mHv1.2 but slower than that of mHv1.1. Overall, these findings indicate that alternative splicing of the N-terminal exon in mRNA transcripts encoding mHv1 isoforms is a regulatory mechanism for mHv1 function within MDSCs. While MDSCs have the capability to translate multiple Hv1 isoforms with varying gating properties, the Hvcn1 gene promotes the dominant expression of mHv1.1, which exhibits the most efficient gating among all mHv1 isoforms.Voltage-gated proton channels (Hv1) are important regulators of the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in mice and have been proposed as a potential therapeutic target to alleviate dysregulated immunosuppression in tumors. However, till date, there is a lack of evidence regarding the functioning of the Hvcn1 and reports on mHv1 isoform diversity in mice and MDSCs. A computational prediction has suggested that the Hvcn1 gene may express up to six transcript variants, three of which are translated into distinct N-terminal isoforms of mHv1: mHv1.1 (269 aa), mHv1.2 (269 + 42 aa), and mHv1.3 (269 + 4 aa). To validate this prediction, we used RT-PCR on total RNA extracted from MDSCs, and the presence of all six predicted mRNA variances was confirmed. Subsequently, the open-reading frames (ORFs) encoding for mHv1 isoforms were cloned and expressed in  The mHv1 proton channel mediates the movement of protons across cell membranes, supporting diverse vital cellular processes. These processes include intracellular pH regulation, apoptosis, proliferation, and migration and are well-documented in the immune system, such as in B and T lymphocytes . mHv1 chHvcn1 gene and also of empirical evidence of mHv1 isoform diversity in mice. These factors currently hinder the realization of mHv1 as a therapeutic target for MDSCs. However, it is intriguing to note that computational predictions regarding the Hvcn1 gene suggest the possibility of alternative splicing, potentially resulting in the expression of three isoforms with varying N-terminal lengths in mice.While genetic and pharmacological approaches hold promise for restoring homeostasis in MDSC immunosuppression through mHv1, our current understanding of the Hv1 gene in mice and its function remains limited. Additionally, there is a lack of comprehensive characterization of the Hvcn1 gene isoforms by conducting reverse transcription polymerase chain reaction (RT-PCR) on total RNA extracted from MDSCs. This validation confirmed the presence of all six predicted Hvcn1 transcripts. Then, we successfully cloned three open-reading frames (ORFs) associated with potential mHv1 isoforms. Furthermore, we observed a nonlinear voltage-dependent behavior of currents, characteristic of ion channels, in all cloned ORFs using a macro-patch voltage-clamp. Next, we biophysically characterized these channels using inside-out macro-patches and observed that all cloned ORFs functioned as bona fide, mammalian mHv1 channels. Since these mHv1 isoforms possess a different amino-terminus, we were intrigued by the potential differences in their functionality. When focusing on their activation process, we noticed significant differences among the isoforms. The longest isoform, mHv1.2, exhibited a V0.5 shift of nearly 1 kT (25 mV) toward depolarized potentials and slower opening kinetics compared to the mid-length isoform, mHv1.3. Interestingly, the mHv1.3 isoform displayed no differences in its V0.5 when compared to mHv1.1, the canonical and shortest isoform, but exhibited slower activation kinetics than mHv1.1. Based on these findings, we propose that the different isoforms display differences in their activation. Specifically, mHv1.1 exhibits the most “efficient” gating, followed by mHv1.3, and lastly, mHv1.2, which shows less efficient activation. In this regard, we will use the term gating “efficiency” to denote an increased probability of the channel being open at a given voltage range, which includes activating in a more negative voltage interval and/or opening more rapidly. Interestingly, the only reported Hv1 isoform across species is found in humans, known as HVCN1S, a shortened isoform of the canonical HVCN1L, which lacks the initial 20 amino acids and exhibits slightly slowed activation. Given that the isoforms reported here differ in their N-terminal length due to alternative splicing at the initial exons, we investigated whether the length of the amino-terminus regulates the gating of mHv1 isoforms. To explore this, we engineered a truncated form of the longest mHv1.2 isoform lacking the initial 20 amino acids, designated as mHv1.2 ΔN20. We predicted and confirmed a gating phenotype intermediate between the longest isoform, mHv1.2, and the shortest isoform, mHv1.1. The conductance–voltage (GV) curve was shifted to the left compared to mHv1.2 and to the right compared to mHv1.1. These differences were consistently observed at the single-channel level, and the opening kinetics was faster than that of mHv1.2 but slower than that of mHv1.1. In conclusion, our findings provide valuable insights into the genetics of Hvcn1 and the diversity of mHv1 isoforms in immune cells of mice.In our study, we first validated the predicted alternative expression of Hvcn1 gene alternatively splices at the initial exons of mRNAs encoding mHv1, thereby regulating the extension of their ORFs at the very beginning. Consequently, Hvcn1 can express three mHv1 isoforms with different activation properties, determined by their N-terminal length, which in turn regulates their gating. Redundant mRNA expression of Hvcn1 in MDSCs favors the expression of the shortest mHv1 isoform, which demonstrates a more efficient gating. It is possible for these longer isoforms to be still expressed, presumably depending on the genetic/phenotypic state of the cell.These results were discovered in mouse MDSCs, where the Mus musculus Hvcn1 gene, located on Chromosome 5–[NC_000071.6 (122206805-122242297)], predicts the presence of up to 13 exons , 2 (NM_028752.3), and 3 (NM_001359454.1). Notably, there is still a predicted isoform (variant X3: XM_006530472.3) encoding the same amino acid sequence, suggesting that the expression of the mHv1.1 isoform plays a significant role, possibly related to its translational redundancy among different mRNA transcripts.Computational analysis of nucleotide sequences encoding transcript variants of the 13 exons . These e13 exons , left. PHvcn1 gene, four  begin processing at exons 2, 4, 3, and 1, respectively. The untranslated regions (UTRs) are located in these exons. Although the initiation codon for protein translation is found in exon 6, all of these transcripts encode the same 810-base pair (bp) product up to exon 13 , transcription processing begins at exon 8, where the start codon for translation is also located. This results in an elongated ORF that codes for a 936-bp product . This prSimilarly, for Vx2 (XM_006530471.4), the transcription processing occurs at exon 7, where the transcription initiation site is located. This produces an mRNA with an ORF of 822 pb . This alAfter the N-terminal variable region, all of the ORFs include the same coding sequence from exons 9 to 13. This results in all of the amino acid differences among mHv1 isoforms being concentrated in the initial N-terminal domain .A comprehensive analysis of the primary sequences of the predicted longer isoforms revealed a high degree of conservation in the typical structural features for functional Hv1 proton channels , which iHvcn1 gene has the potential for alternative splicing, which could result in the production of up to six mRNA transcript variants. These variants, in turn, translate into three isoforms of the mHv1 channel protein with different N-terminal region lengths: mHv1.1, mHv1.2, and mHv1.3.In the earlier prediction, it was mentioned that the To further investigate this, we designed specific primers targeting the full length of the six predicted transcript variants, including the 5′ and 3′UTRs. We then screened a messenger library derived from mouse MDSCs using RT-PCR with these primers .The RT-PCR analysis results revealed the presence of four bands matching the expected sizes for variants 1–3 and variant X3 mRNAs, which encode the mHv1.1 isoform . The unitary conductance values obtained for mHv1.2 and mHv1.3 were 115.1 ± 34.8 fS (n = 3) and 164.4 ± 24.8 fS (n = 4), respectively. Additionally, the maximum open probabilities for mHv1.2 and mHv1.3 were 0.86 ± 0.03 (n = 3) and 0.80 ± 0.03 (n = 4), respectively.Using nonstationary noise analysis , we deten = 4) for mHv1.1, 63.1 fS for mHv1.2 (n = 1), and 165.4 ± 79 fS for mHv1.3 (n = 3), with a maximum open probability of 0.85 ± 0.04 (n = 4) for mHv1.1, 0.99 for mHv1.2, and 0.89 ± 0.08 (n = 3) for mHv1.3. All these data are consistent with previously reported unitary properties for Hv1 channels  coupled to the channel opening  to ensure gating equilibrium during measurements of mHv1.1, mHv1.2, and mHv1.3 currents, which were then transformed into macroscopic conductance and graphically represented in a log-linear plot against voltage . This anectively . These vHvcn1 gene expression in MDSCs are indeed three mammalian isoforms of mHv1 channels: mHv1.1 (the shortest), mHv1.2 (the longest), and mHv1.3 (of mid-length). Although these isoforms exhibit clear hallmarks of mammalian mHv1 channels, do they exhibit functional differences among them?All the previous gathered evidence suggests that the three cloned ORFs from Hvcn1 gene in mice expresses up to six mRNA transcripts, from which three ORFs are identified, expressing three different mHv1 isoforms. Upon inspecting their primary sequences, one can observe that nearly all sequences are conserved. However, at the very beginning of the N-terminal domain, there are 42 additional amino acids in mHv1.2 compared to mHv1.1  and at the kinetic level (activation kinetics).The sole reported isoform of Hv1, HVCN1terminus . HVCN1S 0.5 of mHv1.1 and the V0.5 of mHv1.3 in the GV plots . These kinetics findings suggest that a longer N-terminal end leads to slower activation of mHv1.Next, we investigated the activation kinetics of these isoforms. We determined voltage-dependent time constants, which were then plotted against voltage , with a maximum open probability of 0.92 ± 0.04 (n = 4).At the microscopic level, by nonstationary noise analysis of mHv1.2 ΔN20 current fluctuations at ΔpH = 2 and pHi = 6 , we estip < 0.05) and slower than mHv1.1 (*p < 0.05), suggesting that mHv1.2 ΔN20 activation kinetics was more accelerated than that of mHv1.2 and less accelerated than that of mHv1.1 . mHv1.1 is the ortholog of HVCN1L in mice, where the longer mHv1 isoforms exhibit the addition of amino acids at the beginning of the N-terminal domain, contrary to HVCN1S, which lacks the first 20 amino acids, while exhibiting activation kinetics slightly slower than that of HVCN1L  would adopt a gating phenotype between mHv1.2 and mHv1.3 . We founS leads to a slowing of their activation kinetics when compared to HVCN1L or mHv1.1. Ab initio models of the N-terminal domains of mHv1 isoforms show consistency with the predicted secondary structure  corresponding to the Hv1 transcripts or their open-reading frame were performed to determine similitudes among sequences and define structural motifs. A putative alternative edition among transcripts was predicted using the Splign program (https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi). Query blast and identity percentage were calculated using BLAST-p (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Primers used to amplify the full transcript or the codifying of the sequence (® (version 2.0.6) software, and their selectivity was evaluated by the Primer-BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) using the RefSeq mRNA database for M. musculus as the target organism. Prediction of secondary structure was done by using PSIPRED v4.0  were maintained in the University of Valparaiso bioterium . All experiments followed the institutional guidelines of the University of Valparaiso’s Animal Care and Use Committee. Bone marrow-derived cells (BM cells) were isolated from the femurs and tibias of C57BL/6 mice and cultured to obtain MDSCs following the protocol previously described by Once MDSC differentiation was observed, cells were detached from the Petri dish using 2 mM PBS/EDTA and then centrifuged at 200 g for 5 min to re-suspend them in PBS on ice. Total RNA was extracted using the RNA-Solv kit (OMEGA Biotek) and lysed using a 20G needle and syringe. To degrade genomic DNA, samples were treated with dsDNAseI (ThermoFisher Scientific). cDNA was synthesized using the Maxima H Minus First-Strand cDNA Synthesis Kit (ThermoFisher Scientific) with 0.7–1 µg of total RNA. PCR and overhang PCR (oPCR) reactions were performed according to the instructions of the Platinum SuperFi DNA Polymerase Kit (ThermoFisher Scientific) in a final volume of 20 µL. The PCR products were run on a 1% agarose gel, stained with GelStar (Lonza) 2X, and visualized using a 3UV transilluminator (Epi Chemi Darkroom). The O’Gene Ruler 1 Kb DNA Ladder (ThermoFisher Scientific) was used as a molecular weight marker.in vitro transcription and obtain cRNA, following the instructions of the T7 Message Machine Kit (Thermo Scientific). Finally, the cRNA was quantified and visualized by electrophoresis using agarose. cRNA was stored at −80°C until use. For mHv1 isoforms, 50 nL of cRNA at concentrations ranging from 0.5–4 μg/μL were injected into X. laevis oocytes. The oocytes were incubated at 12–25°C in ND96 or SOS medium, supplemented with antibiotics and 2–5 mM pyruvate, for approximately 24–80 h post-injections before recording.First, the oPCR products were purified using the PCR purification kit (Qiagen), and then they were site-directly cloned into the BamHI/HindIII restriction sites from the pGEM-3 vector. After identifying Hv1 transcripts by Sanger nucleotide sequencing, selected clones were linearized with the BamHI restriction enzyme to perform 2), 1 mM ethylene glycol-bis (b-aminoethyl) N, N, N′, N″- tetra acetic acid (EGTA), and 160 mM N-methyl-D-glucamine (NMDG)—methane sulfonic acid (MeSO3) to reach an osmolarity of ∼300 mOsm for solutions at pH 5.5 and 100 mM HEPES buffer, 30 mM TEAOH, 2 mM MgCl2, 2 mM MgCl2, 1 mM EGTA, and 160 mM NMDG-MeSO3 (or solutions at pH 7.5). The solution used for pharmacological perfusion during proton current recordings contained 10 mM ZnCl2, 100 mM HEPES buffer, 30 mM TEAOH, 2 mM MgCl2, and 160 mM NMDG-MeSO3 at pH 7.5. Solutions used for proton current recordings in oocytes injected with cloned transcripts include 110 mM HEPES, 1 mM EGTA, 2 mM MgCl2, and 110 mM NMDG + MeSO3 at pH 5, 5, 7, and 5, respectively. All electrophysiology experiments were performed at room temperature (∼21°C).Borosilicate glass  used as recording micropipettes was pulled using a P-97 Flaming/Brown automatic pipette puller  and polished in an MF-830 Microforge . Micropipettes used for inside-out patch clamp recordings in oocytes had diameters ranging from 3 to 15 μm, with resistances between 1 and 8 MΩ, while micropipettes used for whole-cell patch clamp recording in MDSCs had a resistance of 7–12 MΩ. The Ag-AgCl reference electrode was connected to the bath solution through an agar bridge with 3M KCl solution and ground. The registered currents were obtained using an Axopatch 200B amplifier (Axon Instruments) by applying voltage protocols  acquired at 250 kHz, low-pass-filtered at 10 kHz (Frequency Devices), and analog-to-digital-converted using a Digidata 1550 (Axon instruments). Solutions used for proton current recordings in MDSCs for both bath and pipette were as follows: 100 mM MES buffer, 30 mM tetraethylammonium hydroxide (TEAOH), 2 mM magnesium chloride  from isoforms of Hv1, expressed in membranes of lsewhere  and wereFor the measurement of reversal potential, it is generally defined as follows:i represents the reversal potential of the ion i, and gi is the conductance of the ion i. Using solutions with no sodium and potassium chloride, Eq. In the previous equation, Ewhere leak can be very large or very small depending on the patch nature, but such values were corrected off-line by leak subtraction, leading The experimental determination of the reversal potential can be achieved by observing the intersection of fast ramp currents. The intersection of these currents can be represented by the expanded form of the total current as follows:k+CdVdt,where thWhen the voltage ramp matches Therefore, at Macroscopic measurements of mHv1 isoform currents were performed by using variable-duration pulse protocols, applied to diminish the proton depletion . All curFor the limiting slope method, slow and symmetrical voltage ramps (0.5 mV/s–1 mV/s) were used. Currents were leak-subtracted off-line and filtered to 10 Hz when external noise was contaminating the currents. The data points were decimated (average of five points) and mean-binned (100 bins).To estimate the effective charge coupled to the channel opening, a log-linear plot of the conductance vs. voltage was built, and the data were fitted to the equation at low open conductance values  as follows:0 represents a pre-exponential conductance factor. Subsequently, zδ was plotted against voltage using the following relationship:In Eq. 0 is the elementary charge.In Eq. For calculation of proton conductances, tail currents were transformed into conductance values measured from isochrones taken at the tail of the currents, whereAll the conductance values were plotted against the potential and fitted to a normalized Boltzmann function.max represents the maximum conductance. In a two-state channel model, the time constants for activation α (V) and deactivation β (V) can be represented as follows:act, was obtained by fitting the previous macroscopic ON currents to a standard exponential function:GIn the aforementioned equation, α (0) is the time constant at 0 mV, t represents the time, and C is a constant value. Assuming that MDSCs express the three mHv1 isoforms and that, at 0 mV, all these isoforms exhibit the same activation kinetic differences, then MDSC Hv1 currents at 0 mV were reproduced by the sum of three exponential functions:Each exponential returned three time constants, representing the presence of each mHv1 isoform. All the data were analyzed using Clampfit 10.7, GraphPad Prism 6.0, and custom-made Python 3.8 scripts. Statistics are presented as mean ± standard error of the mean (SEM), unless otherwise reported.


The prepared magnetite nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscope (SEM), Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric/differential thermogravimetric (TG/DTG) analysis. The water contact angles (θW) of Fe3O4@ODPA nanoparticles were more than 120°, indicating hydrophobic nature, and the diameter of the obtained spherical-shaped magnetite nanoparticles was 12–15 nm. The ODPA coating amount (AO) (coating weight per gram Fe3O4) and specific surface area (SO) of Fe3O4@ODPA were 0.124–0.144 g·g−1 and 78.65–91.01 m2·g−1, respectively. To evaluate the demulsification ability, stability, and reusability, the magnetite nanoparticles were used to demulsify an n-hexane-in-water nanoemulsion. The effects of the magnetite nanoparticle dosage (CS), pH value of nanoemulsion, and NaCl or CaCl2 electrolytes on the demulsification efficiency (RO) were investigated. The RO of Fe3O4@ODPA samples was found to be higher than that of bare Fe3O4 samples  under all CS values. With the increase in CS, the RO of Fe3O4@ODPA samples initially increased and then approached equilibrium value at Cs = 80.0 g·L−1. A maximum RO of ~93% was achieved at CS = 100.0 g·L−1 for the Fe3O4@ODPA sample S2. The pH and two electrolytes had a minor effect on RO. The Fe3O4@ODPA nanoparticles maintained high RO even after being reused for demulsification 11 times. This indicates that the hydrophobic Fe3O4@ODPA samples can be used as an effective magnetite demulsifer for oil-in-water nanoemulsions.To design more environmentally friendly, economical, and efficient demulsifiers for oily wastewater treatment, hydrophobic octadecylphosphonic acid (ODPA)-modified Fe At present, large amounts of oily wastewater are discharged from various industries, such as the petroleum ,2,3,4,5,3O4 nanoparticles, which are functionalized by organic and/or inorganic substrates, are used to demulsify oil-in-water or water-in-oil emulsions [3O4 nanoparticles [3O4 nanoparticles [3O4 nanospheres [Recently, the magnetite oil–water separation technique has received extensive attention, as it offers rapid and efficient separation, recyclability, simple operation, low cost, and no secondary pollution ,28,29,30mulsions ,34,35,36mulsions , which amulsions ,29. Sincmulsions ,29. Varimulsions , polyetharticles , cyclodearticles  or tertiarticles , and robospheres , have be2 and Cu(OH)2 facilitate the coating of ODPA on the metal surface [3O4 nanoparticles with ample hydroxyl groups under appropriate conditions. A possible schematic representing the interaction between ODPA and the Fe3O4 surface is shown in Notably, hydrophobic octadecylphosphonic acid (ODPA) coatings were prepared on oxidized copper mesh for self-cleaning and oil/water separation and on titanium dioxide for anti-fouling biomedical applications ,41. In a surface ,41. This3O4 nanoparticles (Fe3O4@ODPA) are synthesized using co-precipitation with or without cetyltrimethylammonium bromide (CTAB). The prepared nanoparticles are characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric/differential thermogravimetric (TG/DTG) analysis. The demulsification ability and recyclability of Fe3O4@ODPA nanoparticles are investigated by using them to demulsify an n-hexane-in-water (O/W) nanoemulsion under an applied magnetite field. Furthermore, the effects of demulsifier dosage, pH, and electrolytes such as NaCl or CaCl2 on the removal efficiency (RO) of oil from the nanoemulsion are examined. In this study, ODPA-modified Fe3 in water), n-hexane, ethanol, HCl, NaOH (granular), and oil-soluble Sudan III were purchased from Damao Chemical Reagent Factory, Tianjin, China. Analytical-grade reagents, including FeCl3∙6H2O, FeSO4∙7H2O, CTAB, NaCl, and CaCl2, and chemically pure Tween 60, were purchased from Sinopharm Chemical Reagent Co., Shanghai, China. Analytical-grade ODPA was purchased from Shanghai Bide Pharmatech, Shanghai, China. All of the reagents were directly used as received without further purification. The deionized water used in this study was obtained from a Hitech-Kflow water purification system from Hitech, Shenzhen, China.Analytical-grade ammonium hydroxide  was obtained, which was vigorously stirred for 30 min. Subsequently, nearly 0.50 g solid ODPA or alkaline ODPA solution  was added to the black magnetite suspension in the presence or absence of CTAB, and then the black suspension was continuously heated at 80 °C for 2 h. Next, the suspension was naturally cooled to room temperature, and the black magnetite nanoparticles were collected using a magnet, which were thoroughly washed with ethanol and deionized water in sequence. The obtained magnetite nanoparticles were dried in a vacuum oven at 60 °C to a constant weight, and Fe3O4@ODPA samples S1, S1T, and S2 were obtained. A previously reported ,43 modif3O4 nanoparticles  were also synthesized for comparison. S0 and ST were prepared in the absence and presence of CTAB without adding ODPA using the same process as that for S1 and S1T, respectively. A bare Fe3O4 nanoparticle sample (SN) was prepared using the same process as that for S2 without adding ODPA. The diameter and water contact angle (θW) of S0 were measured to be 11.3 nm and 29° at pH = 6.3 [Bare FeThe preparation conditions and characterization of the prepared magnetite nanoparticles are shown in −1 and a mean droplet size of 311 nm , and deionized water were mixed in a mass ratio of 1:1:8 and stirred using a DS-1 homogenizer  at 10,000 rpm for 10 min. The parent nanoemulsion was diluted with an oil content of 25.0 g·Lf 311 nm , measureRO), nanoemulsions were prepared using deionized water, whose pH value was previously adjusted by NaOH or HCl. To investigate the effects of NaCl or CaCl2 in the emulsion on RO, nanoemulsions were prepared using deionized water containing NaCl or CaCl2. The concentrations  of NaCl or CaCl2 in deionized water were both 0.30 g·L−1.To investigate the effects of the pH value of the emulsion on the demulsification efficiency  of the nanoemulsion (light red transparent liquid) was measured after settling it on a hand magnet. The designed amount of magnetite nanoparticles was thoroughly mixed with 20 mL freshly prepared nanoemulsion in a 50 mL glass vial, where the dosage (CS) of magnetite nanoparticles was 0.0–100.0 g·L−1. The vials with the prepared mixtures were shaken in a THZ-82 thermostatic water bath shaker  under a speed of 240 cycles·min−1 at 25 °C for 3 h. The demulsification kinetic tests indicated that shaking for 3 h was enough to achieve demulsification equilibrium (seen RO) could be calculated as follows:C0 (g·L−1) and Ce (g·L−1) are the initial and residual oil contents of the nanoemulsions, respectively.To evaluate the demulsification ability of Feium seen . Then, tRO values are the average values obtained from three parallel tests. For comparison, blank tests were conducted on nanoemulsions without the demulsifier. The relative error of the demulsification tests was <6.0%.The reported 3O4@ODPA, Fe3O4@ODPA sample S2 was collected by an additional magnet after the demulsification test. Subsequently, it was sequentially washed with dichloromethane, ethanol, and water and then dried in a vacuum oven at 60 °C. This recycling test has been used in our reports [3O4@ODPA nanoparticles were applied in the subsequent demulsification tests 11 times to monitor any reduction in demulsification efficiency (RO).To evaluate the reusability and stability of FeKα radiation (λ = 0.154184 nm) at 40 kV and 40 mA [3O4 surface was measured by TG/DTG analysis  [−1 in an N2 atmosphere. The conventional drop shape method was used to measure the water contact angles (θW) [θW value was the average value measured at three different locations on the disc surface. The morphology of magnetite nanoparticles was characterized using TEM  and SEM . The crystal structures of the samples were examined by powder XRD  with Cu nd 40 mA ,4. The cles (θW) . FirstlySa) of the magnetite nanoparticles was determined by N2 adsorption–desorption isotherm measurements , as shown in The specific surface area (D) of the prepared magnetite nanoparticles was measured to be 12.5–14.1 nm , (220), (311), (400), (422), (511), and (440) diffraction planes, respectively, of the cubic spinel structure of Fe3O4 [3O4@ODPA samples were similar to those of Fe3O4, indicating that the crystal structure of the magnetite particles were effectively retained after ODPA coating [The XRD patterns of the prepared magnetite nanoparticles are shown in of Fe3O4 ,34,45,4611), and 0 diffrac coating . −1 were attributed to the characteristic asymmetric and symmetric C-H stretching vibrations, respectively [−1 and 716 cm−1 were attributed to the ν(CH2) and ν(P-C) vibrations, respectively [−1 were assigned to νas(P=O) and νas(P-OH), and the bands at 1076 and 1005 cm−1 were assigned to the symmetric νs(PO3) and asymmetric νas(PO3) stretching vibrations, respectively [−1, which were attributed to O-H vibrations. This indicates that abundant hydroxyl groups were present on the surface of the Fe3O4 nanoparticle, proving the rationality of the scheme shown in −1 were assigned to Fe-O lattice vibrations [−1, which correspond to Fe3O4 [3O4. The FTIR spectra of solid ODPA and magnetite nanoparticles are shown in ectively ,45,47,48ectively . The absectively ,50,51. Ttric νsPO and asymbrations ,27,52,533O4@ODPA samples  exhibited the characteristic ODPA bands at 2922 and 2852 cm−1 and bare ST and SN bands at 3442, 1630, 633, and 591 cm−1. The weakened peak intensities at 3442 and 1630 cm−1 in the Fe3O4@ODPA samples were attributed to the fact that some hydroxyl groups covering the surface were replaced by ODPA. ODPA can modify the Fe3O4 surface by monodentate, bidentate, or tridentate bonding [−1 corresponding to νas (P=O) and νas (P-OH) vibrations indicates that ODPA was bound to the surface of Fe3O4 nanoparticles by tridentate bonding [The spectra of the Fe bonding ,50. The  bonding ,54. This3O4 samples (ST and SN) showed a weight loss of 2.39 wt% and 3.84 wt% in the range of 120 °C to 600 °C, which was attributed to the condensation of surface hydroxyl groups [3O4@ODPA samples  exhibited two weight loss stages in the range of 120 °C to 800 °C. The first weight loss stage occurred at 120–600 °C due to the loss of the ODPA coated on the surface of magnetite nanoparticles [32− and Fe. The other weight loss stage appeared above 650 °C with a derivative peak at 708–770 °C, which may have been caused by the reduction of Fe3O4 by the reducing substances produced during the ODPA degradation process. This result is consistent with the existing literature [The TG/DTG curves of magnetite nanoparticles are shown in l groups . The Fe3articles ,55,56. Iterature ,45,56,57AO) (g) on per gram Fe3O4 for the Fe3O4@ODPA samples was calculated using the net weight loss of ODPA from the TG data, and the results are shown in AO values of S1 and S2 were 0.127 and 0.124 g·g−1. Thus, the average AO value for S1 and S2 was approximately 0.126. However, the AO value of S1T was 0.144 g·g−1, indicating that a slightly higher amount of ODPA was coated on the Fe3O4 surface when CTAB participated in the synthesis process.The ODPA coating amount (CS) on the demulsification efficiency (RO) at PH = 6.9 and 25 °C. As CS increased, the RO of Fe3O4@ODPA nanoparticles  initially increased and tended to stabilize at CS ~80.0 g·L−1. The RO values of all Fe3O4@ODPA samples were found to be higher than those of the bare magnetite samples . The order of RO for the magnetite nanoparticles was S2 > S1T > S1 > bare Fe3O4 . The RO values of the three bare Fe3O4 nanoparticles were very close to each other, nearly 60%, indicating that the bare Fe3O4 nanoparticles had some interfacial activity. A maximum RO of ~93% was observed for the sample S2, verifying the potential of Fe3O4@ODPA nanoparticles in oil–water separation. The magnetite nanoparticles were used as a demulsifier for n-hexane-in-water nanoemulsion. Sa) of determination were determined to further analyze the demulsification performance. The Sa values of Fe3O4@ODPA samples  were 78.7, 87.0, and 87.0 m2·g−1, respectively, and less than those of the bare Fe3O4 samples (ST and SN) of 142.9 and 119.0 m2·g−1. The results do not seem to be related to demulsification performance. The relationship between specific surface area and demulsification ability needs to be studied further.The specific surface areas  were 15° and 19°, respectively, indicating a hydrophilic nature. This is consistent with the hydrophilic Fe3O4 surface containing abundant hydroxyl groups. After coating ODPA, the interfacial activity of the magnetite nanoparticles increased, causing an increase in their demulsification ability. The θW values of Fe3O4@ODPA samples  were 124°, 125°, and 133°, respectively, demonstrating that they were hydrophobic due to the ODPA coating. It has been reported that the amount of coating modifier on the Fefication ,59. TherθW of S2 may have been the reason for its better demulsification performance. According to the above results, the bare Fe3O4 nanoparticles had a similar size, similar shape, approximately similar θW (<90°), and approximately similar demulsifying ability. This indicates that the properties of the bare Fe3O4 nanoparticles were unaffected by the addition of CTAB or alkaline aqueous solution in the absence of ODPA during the preparation process. This suggests that samples S1, S1T, and S2 contained ODPA-coated structures, and the higher 2) on demulsification efficiency (RO) were investigated to assess the demulsifying property of Fe3O4@ODPA in different aqueous environments. RO of sample S2 under CS = 40.0 g·L−1 and 80.0 g·L−1 at 25 °C. It can be seen that the RO did not change significantly in the pH range of 4.8 to 10.5. This indicates that the pH value of nanoemulsion had a minor effect on the demulsification efficiency in the studied range. The result is consistent with an earlier report [RO of Fe3O4@PEI@β-CD and Fe3O4@OA was reduced from 96.8% to 95.3%, and there were no obvious differences as the pH of the emulsion increased from 4 to 10, respectively [2 with a concentration  of 0.30 g·L−1 on the RO of sample S2 in the nanoemulsion at pH = 6.9 and 25 °C is shown in RO changed slightly after adding a low concentration of NaCl or CaCl2 when CS increased from 20.0 to 80.0 g·L−1. At CS = 80.0 g·L−1, RO was 90.8%, 88.9%, and 91.1% for the bare nanoemulsion, nanoemulsion containing NaCl, and nanoemulsion containing CaCl2, respectively. This is consistent with an earlier report about the demulsification of Fe3O4@OA for cyclohexane-in-water nanoemulsion [The influences of pH and electrolytes  hardly affected the demulsification performance of Fe3O4@ODPA nanoparticles for n-hexane-in-water nanoemulsion.Consequently, the variation in the pH value or the addition of low-concentration NaCl or CaCl3O4@ODPA, the S2 sample was reused for demulsification 11 times. As shown in RO value after 10 cycles. Furthermore, the FTIR spectra and TG curves were obtained to observe the stability of the ODPA coating layer on the Fe3O4@ODPA surface (seen 3O4@ODPA nanoparticles.Compared with conventional chemical demulsifiers, magnetite demulsifiers can be recycled and reused to demulsify different nanoemulsions ,3,26. Toace seen , and no 3O4@ODPA nanoparticles were prepared using the modified co-precipitation method. The size, AO, and SO values of the Fe3O4@ODPA nanoparticles were approximately 12–15 nm, 0.124–0.144 g·g−1, and 78.65–91.01 m2·g−1, respectively. The θW of Fe3O4@ODPA nanoparticles was more than 120°, indicating hydrophobicity. The Fe3O4@ODPA nanoparticles exhibited efficient demulsification and excellent recyclability for n-hexane-in-water nanoemulsions under an external magnetite field. Further, the change in pH value of n-hexane-in-water nanoemulsion and the addition of a small amount of NaCl or CaCl2 in the nanoemulsion had a minor effect on the demulsification efficiency of Fe3O4@ODPA nanoparticles. Sample S2 exhibited a maximum RO of 93% and excellent recyclability, with the highest θW of 133°. Overall, the findings of the study verify that Fe3O4@ODPA nanoparticles are an excellent demulsifier and can be effectively used for oily wastewater treatment.Hydrophobic and quasi-spherical Fe


S-acylation is a reversible lipid post-translational modification that allows dynamic regulation of processes such as protein stability, membrane association, and localization. Palmitoyltransferase ZDHHC9 (DHHC9) is one of the 23 human DHHC acyltransferases that catalyze protein S-acylation. Dysregulation of DHHC9 is associated with X-linked intellectual disability and increased epilepsy risk. Interestingly, activation of DHHC9 requires an accessory protein—GCP16. However, the exact role of GCP16 and the prevalence of a requirement for accessory proteins among other DHHC proteins remain unclear. Here, we report that one role of GCP16 is to stabilize DHHC9 by preventing its aggregation through formation of a protein complex. Using a combination of size-exclusion chromatography and palmitoyl acyltransferase assays, we demonstrate that only properly folded DHHC9-GCP16 complex is enzymatically active in vitro. Additionally, the ZDHHC9 mutations linked to X-linked intellectual disability result in reduced protein stability and DHHC9-GCP16 complex formation. Notably, we discovered that the C-terminal cysteine motif (CCM) that is conserved among the DHHC9 subfamily  is required for DHHC9 and GCP16 complex formation and activity in vitro. Co-expression of GCP16 with DHHCs containing the CCM improves DHHC protein stability. Like DHHC9, DHHC14 and DHHC18 require GCP16 for their enzymatic activity. Furthermore, GOLGA7B, an accessory protein with 75% sequence identity to GCP16, improves protein stability of DHHC5 and DHHC8, but not the other members of the DHHC9 subfamily, suggesting selectivity in accessory protein interactions. Our study supports a broader role for GCP16 and GOLGA7B in the function of human DHHCs.Protein  S-acylation is the process in which long-chain fatty acids  are added to proteins at cysteine residues via a labile thioester linkage. While the fundamental chemical effect of S-acylation is a local increase in protein hydrophobicity, the reversible nature of S-acylation can result in a range of consequences such as dynamic changes in protein stability, membrane affinity and binding, and protein trafficking and localization  are the enzymes responsible for catalyzing the addition of long-chain fatty acids to substrate proteins . MembersIn humans, DHHC9 was the first DHHC protein discovered to require an accessory protein—GCP16 —for its enzymatic function, based on sequence homology with the yeast Ras PAT Erf2-Erf4 . DHHC9 cvia standard molecular biology techniques into their respective expression vectors. Any mutations were generated via quick change PCR.Amino acid sequences were obtained from the UniProt database for the following human proteins: DHHC9 (Q9Y397), DHHC14 (Q8IZN3), DHHC18 (Q9NUE0), DHHC5 (Q8VDZ4), DHHC8 (Q9ULC8), DHHC3 (Q9NYG2), DHHC20 (Q5W0Z9), and GCP16 (Q7F5G4). Genes were synthesized based on protein sequences . The plasmid encoding GOLGA7B was obtained from the Harvard Medical School Plasmid repository. All DHHC genes used were PCR amplified to exclude the start methionine and to include flanking BamHI/XhoI sites. Similarly, GOLGA7 and GOLGA7B were amplified to exclude the start methionine and to include AgeI/NotI sites. All genes were subcloned For FSEC, we used a dual mammalian expression vector modified from the pIRES-EGFP RK6 vector provided by M. Mayer, National Institutes of Health, Bethesda, MD. The vector encoded a start methionine followed by mNeonGreen (A0A1S4NYF2), a BamHI/XhoI insertion site, an EMCV internal ribosomal entry site (IRES), an AgeI/NotI insertion site, and a stop codon. DHHC genes were incorporated using BamHI/XhoI, and GOLGA7 or GOLGA7B genes were incorporated using AgeI/NotI.For protein purification, genes were subcloned into a modified pFastBac baculovirus expression vector . DHHC9, DHHC9 TM , and GCP16 were encoded to express a C-terminal strep-tag. DHHC14 and DHHC18 were encoded to express an N-terminal strep-tag.Human embryonic kidney (HEK) cells were maintained in DMEM medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum , and 10 μg/ml gentamicin (Thermo Fisher Scientific) per manufacturer’s instructions. For FSEC experiments, cells were transfected at 80–95% confluency with 2.5 µg of expression plasmid using jetPRIME transfection reagent per the manufacturer’s instructions. Cells were grown at 37°C for 48 h. The media was aspirated, cells were suspended and washed in 2 ml of cold PBS, and samples were lysed in 150 µL of lysis buffer , 1x Roche protease inhibitor) for 30 min at 4 °C with rotation. Whole-cell lysates were cleared by centrifugation in an Eppendorf FA-45–24-11 rotor at 21,000 x g for 10 min at 4°C. The supernatant was further cleared by ultracentrifugation in a TLA 100.3 rotor at 265,000 x g for 20 min. An aliquot (50 µL) was applied onto a Superdex 200 Increase 10/300 GL column pre-equilibrated with running buffer . The eluate from the SEC column was passed through a fluorometer set to excitation, 480nm, and emission, 508 nm.For quantitation, peak intensities in arbitrary fluorescence units were obtained at the expected DHHC peak for every profile. For DHHC9 constructs, values were normalized to either DHHC9 or DHHC9 N2C1, depending on the set of constructs tested for that given day. Data sets across different days were then normalized to DHHC9 globally. For experiments that included other DHHCs, i.e., DHHC14, 18, 5, 8, 3, and 20, raw intensity values were used without normalization. For statistical analysis, we used two-tailed t-tests assuming unequal variances against the null-hypothesis that GCP16/GOLGA7B co-expression has no significant effect on DHHC protein signal.® baculovirus-insect cell expression system. Sf9 cells (1–2L) were infected at 2.5—4.0 × 106 cells/mL with P2 virus . Cells were incubated at 27°C for 24 h and moved to 18 °C for an additional 48 h. All purification steps were done on ice or with 4°C buffer unless stated otherwise. Cells were harvested by centrifugation at 2,040 x g and washed with 200 ml of PBS. Cells were solubilized in buffer A  containing 1% DDM w/v at a ratio of 250 mg DDM/g of cells. Solubilization was performed at 4 °C with stirring for 1 hour. Large debris were removed by centrifugation at 12,000 x g. The supernatant was further clarified by ultracentrifugation at 185,000 x g for 45 min to remove remaining insolubilized material. To the supernatant, 2 ml of equilibrated StrepTactin Sepharose High Performance resin  was incubated with stirring for 1-h. The resin was collected by centrifugation and transferred to a gravity column (Bio-Rad). The resin was washed with 10 resin bed volumes of wash buffer (buffer A with 0.5 mM DDM) and eluted with elution buffer . Protein was concentrated using Amicon ultra concentrators. Size exclusion chromatography was done using a Superdex 200  in SEC buffer .All constructs were expressed using the Invitrogen Bac-to-Bac3H]-palm-CoA. Each reaction was incubated at room temperature for 10 min. Reactions were quenched in SDS loading dye containing 25 mM TCEP. Samples were split and run on two SDS-PAGE gels in parallel. For fluorography, one gel was exposed to a solution of 1M sodium salicylate, 15% methanol, dried under vacuum for 2 h, and exposed to film for 4–15 days. For liquid scintillation spectroscopy, the gel was stained with Coomassie, H-Ras bands were extracted, and a section of gel around the expected DHHC protein size was extracted. Extracted gel pieces were dissolved in 500 µL Soluene overnight at 37°C. Ultima Gold scintillation fluid (5 ml) was added, and disintegrations per minute (DPM) was measured using a scintillation counter after allowing for stabilization for 8–24 h. For each experiment, we used DHHC20 as a positive control and H-Ras without enzyme to account for non-enzymatic acylation. Background DPM was subtracted from each sample before converting counts to picomoles based on the specific activity of palmitoyl-CoA.PAT assays were performed immediately following purification and SEC as described previously . ProteinOur initial attempts to express and purify DHHC9 from Sf9 cells were met with low protein yield, reduced purity, and extensive protein aggregation. This was consistent with a previous report that DHHC9 purified without GCP16 resulted in an increased fraction of DHHC9 protein being proteolyzed . Thus, wNext, we asked which regions and amino acid residues of DHHC9 are required for the GCP16-mediated stabilizing effect. We used evolutionary conservation to generate combinatorial N- and C-terminal deletions of DHHC9  and asseWe next sought to determine the exact residues between DHHC9 C1 and C2 that are required for GCP16-mediated stabilization. We made finer truncations between regions C1 and C2 in the N2C1 background and assayed for the effect of GCP16 co-expression using FSEC . Much liWhile FSEC is an efficient way to probe whether GCP16 expression influences DHHC9 stability, it cannot address whether the two proteins are interacting. To determine if DHHC9 and GCP16 form a complex, we co-expressed these two proteins in Sf9 insect cells, affinity purified, and assessed whether they co-elute in SEC . Across Next, we tested whether the CCM of DHHC9 is required for complex formation with GCP16. We co-purified DHHC9 TM with GCP16 and analyzed the SEC profile. We confirmed by SDS-PAGE that both DHHC9 TM and GCP16 were present in the elution from affinity purification  lane 1. S-acylation by DHHC proteins occurs via a two-step mechanism  sequence among DHHC proteins where cysteines centered around the “GCxxN” motif were palmitoylated, and these cysteines in DHHC5 and DHHC8 were palmitoylated in vivo . Subsequin vivo . The catin vivo . DHHC5 hin vivo . Howie ein vivo . Interesin vivo . Nonethein vivo . The CCMWhile this manuscript was in revision, a preprint deposited in bioRxiv reported the cryo-EM structures of human DHHC9-GCP16 and yeast Ef2-Erf4 . Severalin vitro. Whereas GCP16 potentiated all subfamily members, GOLGA7B potentiated only DHHC5 and DHHC8. Using purified components, we establish that both DHHC14 and DHHC18 when complexed with GCP16 can function as Ras palmitoyltransferases. Our research supports a broader role for GCP16 and GOLGA7B in the function of human DHHC proteins.Our study demonstrates that a subgroup of the larger DHHC protein family that includes DHHC9, 14, 18, 5, and 8 all require accessory proteins for their stability