O-glucoside (M3G) against the toxicity of Cd on female reproduction in KGN cells (human ovarian granulosa-like tumor cells) were investigated. After treating cells with 10 µmol/L cadmium chloride, the results showed that M3G lessened Cd-induced KGN cell cytotoxicity better than malvidin and malvidin-3,5-O-diglucoside. Additionally, M3G significantly decreased the Cd-induced generation of reactive oxygen species, inhibited the Cd-induced arrest of the G2/M phase of the cell cycle, and increased estradiol (E2) production. According to transcriptomic results, M3G reduced the abnormal expression of genes that responded to estrogen. Additionally, M3G promoted the endogenous synthesis and secretion of E2 by controlling the expression of CYP17A1 and HSD17B7. The current findings indicated that M3G is of great potential to prevent Cd-induced female reproductive impairment as a dietary supplement.Cadmium (Cd) is a frequent environmental pollutant associated with biological toxicity that can harm female reproduction. Anthocyanins have been reported to reduce the toxicity of Cd. In the present study, the protective effects and underlying mechanisms of malvidin-3- Cadmium (Cd) is a toxic heavy metal that is widely used in manufacturing processes, such as batteries, pigments, and solder . People Anthocyanins are present in a wide variety of fruits, vegetables, and flowers; they have been linked to significant health-promoting properties, such as anti-oxidant, anti-inflammation, anti-apoptosis, anti-cancer, and so on . ResearcO-glucoside (M3G), and malvidin-3,5-O-diglucoside  are the most typical anthocyanins in this family , malvidin-3-s family A. Herein2 humidified chamber. KGN cells, human ovarian granulosa-like tumor cells, were purchased from the ATCC ; they were cultured on Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12  containing 10% fetal bovine serum  and 1% penicillin/streptomycin/amphotericin  at 37 °C in 5% CO4 cells per well in 96-well plates. The cells were exposed to different concentrations of MV, M3G, and M3,5G  for 24 h after adhering for 12 h. The medium was discarded, and 100 µL of CCK8 solution  diluted 10 times with DMEM/F12 medium was added. The plates were then incubated at 37 °C for 2 h and measured using a microplate reader at 450 nm . Cell viability tests were also used to investigate the effect of cadmium chloride (CdCl2)  on KGN cells, as well as the effects of MV, M3G, and M3,5G  on 10 µmol/L CdCl2-treated KGN cells. Finally, the control group was used as the 100% cell viability; the value of other groups was calculated according to the measured absorption.The stock solution of 200 µmol/L MV, M3G, and M3,5G  was prepared by dissolving the powder in DMSO. KGN cells were seeded at a density of 1 × 104 cells per well for 12 h in 96-well plates. Then 10 µmol/L CdCl2 was added, along with various concentrations of MV, M3G, and M3,5G . Cell morphology was observed under an inverted phase microscope  after 24 h of incubation.KGN cells were seeded at a density of 1 × 105 cells per well in 6-well plates and were treated with 10 µmol/L CdCl2 and M3G  for 24 h. The culture medium was collected after treatment and centrifuged for 10 min at 4 °C at 1000 rpm. The levels of E2 in the collected supernatant were determined using an E2 enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.KGN cells were cultured at a density of 4 × 104 cells per well in 96-well plates, washed twice with phenol red-free and serum-free medium, and then incubated for 20 min at 37 °C with 30 µL of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) solution diluted by the medium. After that, any extra DCFH-DA solution was washed away twice with phenol red-free and serum-free medium. Regarding the cells treated with 10 µmol/L CdCl2 and M3G , the fluorescence of cells was assessed using a fluorescence plate reader  at 10, 20, 40, 80, and 160 min.The reactive oxygen species (ROS) levels in KGN cells were determined by a ROS assay kit . Cells were plated at a density of 1 × 105 cells per well for 12 h. After 24 h of treatment with 10 µmol/L CdCl2 and M3G , trypsinization was used to harvest the cells, which were then fixed in 2 mL of 70% ice-cold ethanol overnight at −20 °C. The ethanol was entirely removed by centrifugation, and the cells were then washed with PBS. The PBS was then removed, and the cell cycle was examined using propidium iodide (PI), a fluorescent dye probe. For each sample, 8 × 103 cells were collected and measured using a CytoFLEX flow cytometer , which were then analyzed with ModFit software version 5.0 .For cell cycle measurements, KGN cells were plated in a 6-well plate at a density of 4 × 105 per well in 6-well plates for 12 h; then, they were treated with 10 µmol/L CdCl2 and 200 µmol/L M3G separately or in combination for 24 h. After treatment, total RNA was extracted by the Trizol reagent  according to the manufacturer’s protocol. Then, RNA purity and integrity were assessed using the NanoDrop 2000 spectrophotometer  and the Agilent 2100 Bioanalyzer . Following the construction of the libraries with the TruSeq Stranded mRNA LT Sample Prep Kit , the library was sequenced on the Illumina HiSeq X Ten platform to generate a 150 bp paired-end read. Using Trimmomatic, raw reads in fastq format were processed to remove low-quality reads and obtain clean reads. Clean reads were mapped to the human genome (GRCh38) with HISAT2.Cells were seeded at 4 × 105 cells per well for 12 h and then treated with 10 µmol/L CdCl2 and 200 µmol/L M3G separately or in combination for 24 h. The extracted RNA was reverse-transcribed into cDNA with Evo M-MLV Reverse Transcription Kit , followed by RT-PCR using SYBR Green Premix Pro Taq HS qPCR Kit . Based on NCBI Primer-Blast, primers were designed for RT-qPCR and are listed in −ΔΔCt method.KGN cells were seeded in a 6-well plate at a density of 4 × 105 cells per well and exposed for 24 h to 10 µmol/L of CdCl2 and 200 µmol/L of M3G. Following this, the KGN cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing phenylmethanesulfonyl fluoride (PMSF) and protease inhibitors and were centrifuged for 10 min at 12,000 rpm. The BCA method  was used to determine the protein concentration of the samples. Then, using PBS and protein loading buffer, 2.5 μg/μL of protein samples for separation were prepared. For each sample, 20 µg of protein were subjected to 10% SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. Primary antibodies HSD3B , CYP17A1 , HSD17B7  at 1:1000 dilution, and GAPDH  at 1:5000 dilution were incubated overnight at 4 °C with blots, followed by incubation with the horseradish peroxidase (HRP)-conjugated secondary antibodies . The protein bands were detected using an imaging analysis system  and quantified using Image J software .The cells were cultured in 6-well plates at a density of 4 × 10p < 0.05 indicate statistical significance. Each experiment included three replications.The data are expressed as the mean ± standard deviation (SD). GraphPad Prism 8 software conducted the differences between groups through a one-way ANOVA. Results with a 2 concentrations on cell viability in KGN cells was investigated. The CCK8 assay revealed that CdCl2 concentrations of 1.25 and 2.5 µmol/L did not have a significant impact on cell viability. In the subsequent concentrations, CdCl2 decreased cell viability in a dose-dependent manner, with cell viability decreasing to 82.5% for 5 µmol/L and 76.9% for 10 µmol/L, respectively. At the 20 and 40 µmol/L doses, CdCl2 toxicity was observed at 27.4% and 7.8%  was observed. The cells in the control group grew more densely and with an irregular polygon morphology, as seen in 2 treatment and cell adhesion deteriorated, resulting in a gradual decrease of adherent cells and an increase of floating cells. The M3G treatment group (treatment with 10 µmol/L CdCl2 and M3G at various concentrations) improved cell morphology and cell adhesion compared with the CdCl2 treatment. Moreover, there was no observable difference in cell growth morphology and distribution between the MV, M3,5G treatment group  and CdCl2 treatment groups when considering the cell size, density, and shape.Using an inverted phase microscope, the cell morphology of KGN cells that had been exposed to 10 µmol/L CdCl2, and M3G as the efficient anthocyanin was considered to ameliorate the E2 production in vitro. As shown in 2 treatment and was significantly different. The E2 levels were noticeably higher, at 59.6 ± 3.3 pg/mL and 60.5 ± 2.6 pg/mL, when the M3G dose was 100 µmol/L and 200 µmol/L, respectively. This indicated that M3G protected E2 production in KGN cells exposed to CdCl2.Since Cd treatment is detrimental to the cell survival quality, we further examined the function of the reserved cells, regarding that E2 synthesis is the critical biofunctionability of KGN cells. Therefore, the cells were subjected to 10 µmol/L of CdCl2 and 50–200 µmol/L M3G. The intensity of the fluorescence rose with the number of ROS produced by cell generation. In the CdCl2-treated group, the ROS production of KGN cells was increased significantly, with a fluorescence intensity of 126.5% at 10 min, and peaked at 20 min with an intensity of 136.5%. ROS production by cells in the CdCl2 group was decreased after 40 min, and their fluorescence intensity was reduced to 127.3%, 120.0%, and 113.3%, at 40 min, 80 min, and 160 min, respectively , G1 (early DNA synthesis phase), S (DNA synthesis phase), G2 (late DNA synthesis phase), and M phase (division phase), is the stage sequence of cell proliferation. Evidence suggests that Cd damages cellular DNA, causing cells to halt their cell cycle at the G1-S or the G2-M transition point . The cel2 exposure (10 µmol/L) and the protective effect of M3G (200 µmol/L). As shown in the volcano map in 2 and M3G treatment group altered a few genes; 94 genes had a differential expression, with 66 upregulated and 28 downregulated. Analysis of gene ontology (GO) enrichment pathways revealed a variety of enrichment biochemical pathways linked to differentially expressed genes, in the CdCl2-only group and the CdCl2-M3G group, including pathways in response to hormones, apoptotic signaling pathways, response to endoplasmic reticulum stress, and positive regulation of mitochondrial organization  and RT-qPCR techniques were used to measure the expression levels of critical proteins and genes in E2 synthesis in KGN cells to learn more about the potential mechanism of abnormal E2 secretion caused by Cd. As shown in eased it D. In teror Gper1 E–I.Cd is a widely occurring metal and environmental contaminant that harms the female reproductive system in various ways. Particularly, serving as the factory of estrogen production, the human ovarian granular cell has been well identified as the target of Cd toxicity . Anthocy2 was established and the protective effects of various molecular structural anthocyanins, including MV (C3-OH on a C-ring), M3G (C3-O-glc on a C-ring), and M3,5G (C3-O-glc on an A-ring and C5-O-glc on a C-ring) were evaluated. The structure of anthocyanins has a substantial impact on the suppression of aberrant proliferation of injured cells. Previous studies have shown that in a damaged cell model, anthocyanins with the monosaccharose substitution dissolved in DMSO had a significantly stronger protective effect than those with the disaccharide substitution against oxLDL-induced endothelial injury associated with increased cell viability, promoted NO release, and so on [An in vitro model of KGN cell injury induced by 10 µmol/L CdClnd so on . Analogond so on . ConsistCd exposure was found to significantly enhance oxidative stress in ovarian granulosa cells, playing a causal role in Cd-induced reproductive toxicity ,23. Ther2 treatment, the results revealed an increased number of cells in the G2/M phase. Given that Cd is recognized as a spindle poison, Cd can stop mitosis and is more sensitive to attack cells in the G2/M phase [Cell proliferation requires an orderly cell cycle progression. In the present study, we investigated the cell cycle changes induced by Cd. After 24 h of 10 µmol/L CdCl/M phase ,27. Howe/M phase . Additio/M phase ,30. Anth/M phase ,32. Simi2 + M3G group.Granulosa cells are responsible for estrogen (steroid hormones) synthesis and secretion in females. The three primary endogenous forms of estrogen are estrone (E1), estradiol (E2), and estriol (E3), which are in charge of the development and regulation of the female reproductive system and the maintenance of its function. Particularly E2 is the master and most biologically active estrogen . EarlierIn light of the improved E2 level after M3G intervention, the biogenesis of steroids involving a variety of steroidogenic enzymes was subsequently investigated. The first step in steroidogenesis is the transformation of cholesterol into pregnenolone, which is then changed into progesterone by HSD3B (3β-hydroxysteroid dehydrogenase). With the assistance of CYP17A (17α-hydroxysteroid dehydrogenase), progesterone is further transformed into androgens, such as androstenedione, testosterone, and dehydroepiandrosterone. In addition, androgens are converted to estrogen in granulosa cells, where androstenedione is converted to E1, which is then converted to E2 by the enzyme HSD17Bs (17β-hydroxysteroid dehydrogenase). Changes in the expression of steroidogenic enzymes influence steroid hormone production . HSD3B aThe conversion of E1 to E2 is catalyzed by the HSD17Bs’ enzymes family including HSD17B1, HSD17B7, and HSD17B12 ,39. HSD1Estrogen receptors are necessary for reproduction, and their absence not only disrupts hormone signaling but also affects follicular growth. The estrogen receptors include estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and G-protein coupled estrogen receptor 1 (GPER1) . GPER1, Together, the present result demonstrated the critical role of M3G in the protection of Cd-induced dysfunction of granule cells. Considering the application of anthocyanins in dietary supplementation, pure M3G is expensive and hardly accessible, and the anthocyanins extracts containing M3G, or other types of functional anthocyanins targeted to granule cells, could be considered after the in vitro and in vivo verification. More studies related to the improvement of M3G accessibility to ovary granule cells and the corresponding animal study should be conducted in the future.2-damaged KGN cells compared to MV and M3,5G. Additionally, M3G treatment in KGN cells that had been damaged by CdCl2 decreased ROS production, prevented the arrest of the G2/M phase, and increased estradiol synthesis by alleviating the aberrant expression of the key protein involved in estrogens including CYP17A1 and HSD17B7. As a result, this study provides a basis for M3G as a dietary supplement to protect against Cd-induced damage to female reproduction, which is critical to populations with a high risk of Cd exposure. However, the underlying mechanism by which anthocyanins protect KGN cells from Cd remains unclear.In conclusion, the present study indicated that M3G, as the typical anthocyanin, performed protection against Cd-induced dysfunction of human granule cells considering the estrogen secretion. Briefly, M3G has a better impact on cell proliferation and cell morphology of CdCl


O-linked glycans on the MCAM, induces MCAM dimerization and clustering on cell surface and subsequent activation of downstream AKT signalling. This leads to the increases of a number of important steps in melanoma progression of cell proliferation, adhesion, migration, and invasion. Thus, galectin-3 is a natural binding ligand of MCAM in melanoma, and their interaction activates MCAM and promotes MCAM-mediated melanoma progression. Targeting the galectin-3–MCAM interaction may potentially be a useful therapeutic strategy for melanoma treatment.Melanoma cell adhesion molecule  is a heavily glycosylated transmembrane protein and a marker of melanoma metastasis. It is expressed in advanced primary melanoma and metastasis but rarely in benign naevi or normal melanocytes. More and more evidence has shown that activation of the MCAM on cell surface plays a vital role in melanoma progression and metastasis. However, the natural MCAM binding ligand that initiates MCAM activation in melanoma so far remains elusive. This study revealed that galectin-3, a galactoside-binding protein that is commonly overexpressed in many cancers including melanoma, is naturally associated with MCAM on the surface of both skin and uveal melanoma cells. Binding of galectin-3 to MCAM, via  Melanoma is one of the most common cancers in the world, particularly in Western countries . AlthougOver the past years, increasing evidence has demonstrated that MCAM expression plays a vital role in melanoma progression and metastasis ,6,7. VacN-linked carbohydrate structures of cell membrane glycoproteins including MCAM, showed to substantially reduce melanoma metastasis in mice [Recently, while investigating the influence of galectin-3 on endothelial cell behaviours, we have revealed that galectin-3 can bind to the MCAM expressed on the surface of endothelial cells . This in in mice . Pre-inc in mice .These discoveries led us to speculate that galectin-3 may be the unidentified natural binding ligand of MCAM in MCAM activation and MCAM-mediated melanoma progression. A series of experiments were conducted in this study to test this possibility, and the results strongly support this notion.O-glycanase  was purchased from Agilent . ShRNA plasmid DNA for CD146/MCAM (TRCN0000151337), control shRNA plasmid (SHC002v), and N-glycanase PNGaseF (F8435) were from Sigma Aldrich . Matrigel matrix was obtained from Corning Life Sciences .Monoclonal antibodies against CD146/MCAM (mab 932) and galectin-3 (mab 1154), biotinylated goat anti-galectin-3 polyclonal antibody (BAF1154) were from R&D Systems . Antibodies against AKT (9272S) and Phospho-AKT (Phospho T308) were purchased from Cell Signalling . Antibody against CD146/MCAM rabbit polyclonal antibody was obtained from Proteintech . Protein A/G PLUS-Agarose beads were from Santa Cruz Biotechnology . Calcein AM cell labelling solution was obtained from Invitrogen. Human skin melanoma A375 cells  were cultured in DMEM containing 200 mM L-glutamine, 0.4% penicillin and streptomycin, and 10% foetal calf serum (FCS). Human uveal melanoma Mel270 and 92.1 cells  were cultured in RPMI containing 200 mM L-glutamine, 0.4% penicillin and streptomycin, and 10% foetal calf serum.E. coli as previously described [The full-length human galectin-3 and a C-terminal carbohydrate recognition domain of galectin-3  were produced in escribed .4 cells/well) and cultured at 37 °C. At a different time, 5 µL/well Calcein AM was added for 30 min before the cells were washed twice with PBS, and the fluorescence intensity was measured by a microplate reader .Sub-confluent cells were seeded into 96-well plates  or BSA  was then added, with or without 100 µg/mL lactose, for up to 72 h before the cells were washed and fluorescence intensity was measured.5 cells/mL) were cultured in 3-well silicone inserts  until monolayer formation. The medium was then replaced with a serum-free medium containing 0.25 mg/mL BSA for 16 h before the inserts were removed and 10 µg/mL galectin-3 or 10 µg/mL BSA was introduced. The gaps were imaged and measured every 12 h until closure.Cells (2 × 105 cells/mL) with 10 µg/mL galectin-3 or 10 µg/mL BSA. After incubation for 30 min at 37 °C, the plates were gently washed three times with PBS, and the adhesion cells between three and five randomly selected fields/well were counted under a microscope  with a 20× objective.Twenty-four-well plates were coated with 250 µL pre-cooled Matrigel matrix (20 µg/mL) for 1 h at 37 °C. The plates were washed three times with PBS before introduction of 250 µL cell suspension (1 × 105 cells/mL) with 10 µg/mL galectin-3 or 10 µg/mL BSA in DMEM containing 1% FCS was added to the transwells, and a 500 µL culture medium containing 10% FCS was added to the bottom wells of the plates. After 16 h incubation at 37 °C, the Matrigel and uninvaded cells in the inserts were gently removed with cotton swaps. The inserts were washed once with PBS and fixed in 2% formaldehyde/PBS for 20 min. After washing with PBS, the inserts were stained with 5% crystal violet dye for 5–10 min. The insert membrane was removed by a scalpel and mounted to slides, and the stained cells at the bottom side of the membrane between three and five randomly selected fields/well were counted under a microscope  with a 20× objective.Transwell inserts in 24-well plates were incubated with a 100 µL/insert Matrigel matrix (20 µg/mL) for 2 h at 37 °C. After gentle wash with PBS, 150 µL/well cell suspension (1 × 10Sub-confluent cells were seeded (100 cells/well) into a 6-well plate and cultured for two days at 37 °C. Recombinant galectin-3 (10 µg/mL) or BSA (10 µg/mL) was then added to the culture medium every 48 h. After 10 days of culture, the cells were fixed with 2% paraformaldehyde/PBS for 15 min before being stained with 0.5% crystal violet for 1 h. After two washes with PBS, cell colonies containing >50 cells were counted.MCAM shRNA or control shRNA plasmids (100 ng) were mixed with a Metafectin transfection reagent (1:4) in a serum-free, antibiotic-free DMEM for 30 min before introduction to 50% confluent A375 cells in an antibiotic-free, serum-containing DMEM in a 96-well plate. After 6 h culture at 37 °C, the media were replaced with a fresh DMEM containing 10% serum and 0.5 µg/mL puromycin for 72 h. The surviving cells were released from the plates by trypsinization, suspended in very low density, and seeded into 96-well plates. Wells containing a single cell were identified under a microscope and allowed to proliferate before they were analysed for MCAM expression by immunoblotting. The two selected subclones of MCAM-suppressed cells (55.6 and 55.3) were maintained in the same culture condition as the parental A375 cells.4 cells/well) for 24 h before introduction of galectin-3 (10 µg/mL) or BSA (10 µg/mL) for 30 min at 37 °C. The cells were washed in ice-cold Ca2+- and Mg2+-free PBS and incubated with 3 mM BS3 cross-linker on ice for 30 min. After excess BS3 was quenched with 250 mM glycine for 10 min at 4 °C, the cells were lysed in an SDS-sample buffer without β-mercaptoethanol and analysed by immunoblotting.A375 cells were cultured in 12-well plates  before incubation with mouse anti-galectin-3 monoclonal antibody and rabbit anti-MCAM polyclonal antibody (Proteintech)  for 2 h at room temperature. Control wells were incubated with 1% BSA in PBS. After three washes with PBS, the cells were incubated with Texas Red-conjugated anti-mouse antibody  at 1:300 dilution and FITC-conjugated anti-rabbit  at 1:300 dilution for 1 h at room temperature. The cells were washed five times with PBS before being mounted with DAPI-containing fluorescent mounting media  and analysed by a Zeiss LSM 800 Airyscan confocal microscope.In some experiments, the cells were treated with 10 µg/mL recombinant galectin-3 or 10 µg/mL BSA for 1 h before they were fixed and analysed for MCAM and galectin-3 localization by confocal microscopy.6) in T-75 flasks were washed twice with ice-cold PBS before being lysed with 1 ml of a RIPA buffer  for 10 min on ice. After centrifugation of the lysate at 100,000× g for 10 min at 4 °C, the supernatant was mixed with 20 µL protein A/G PLUS-agarose beads for 1 h on a rotary shaker at 4 °C. The beads were removed by centrifugation at 1000× g for 3 min at 4 °C. The cleaned supernatant was collected, and 0.5 mL supernatant was incubated with 5 µg anti-MCAM antibody or 5 µg mouse immunoglobulin on a rotary shaker for 1 h at 4 °C before introduction of Protein A/G agarose beads (20 µL) overnight at 4 °C. After centrifugation at 1000× g and removal of the supernatant, the pellets were washed twice with an ice-cold RIPA buffer and twice with ice-cold PBS before the addition of 50 µL PBS and 50 µL SDS-sample buffer and subsequent analysis by immunoblotting.Sub-confluent cells  at 2 µg/mL and one incubated with control mouse immunoglobulin (2 µg/mL) overnight at 4 °C. Eighty microlitres of protein A/G Plus-agarose beads was then added to each aliquot, incubated on a roller for 4 h at room temperature. After two washes with PBS, one MCAM antibody-treated aliquot was incubated with N-glycanase PNGaseF (5U) and one with O-glycanase (10 mU) overnight at 37 °C. The third aliquot and the immunoglobulin control aliquot were incubated with PBS. After three washes with PBS, the proteins were released from the beads using an SDS-sample buffer and analysed by immunoblotting. Galectin-3 immunoblotting was carried out using biotinylated, goat anti-galectin-3 polyclonal antibody at 1:4000 dilution, followed by Avidin-Peroxidase at 1:8000. MCAM immunoblotting was performed using mouse anti-MCAM monoclonal antibody at 1:5000 dilution and followed by peroxidase-conjugated anti-mouse antibody  at 1:8000. The blots were developed using a chemiluminescence Super Signal kit and visualized with the Molecular Imager® Gel Doc™ XR System .Sub-confluent A375 cells were washed twice with PBS and incubated with galectin-3 (5 µg/mL) in a serum-free medium for 2 h at 37 °C. The cells were washed with PBS and then lysed with a lysis buffer (1% Triton X-100 in PBS) for 15 min on ice. After centrifugation at 4500× p < 0.05.One-way analysis of variance (ANOVA) followed by Bonferroni correction was used for multiple comparisons. Differences were considered significant when To determine the possible association of galectin-3 and MCAM in melanoma, two types of human melanoma cells, skin A375 and uveal 92.1 cells, were selected and investigated in this study. MCAM immunoprecipitation revealed the presence of galectin-3 in the MCAM immunoprecipitates of both A375 A and 92.As MCAM dimerization is known to occur in the activation of MCAM in melanoma , we thenMany previous studies have shown that MCAM activation in melanoma cells is closely associated with the induction of AKT signalling . To see N-glycanase PNGaseF  or O-glycanase  and galectin-3–MCAM interaction then analysed by MCAM immunoprecipitation and immunoblotting. Treatment of the cells with N-glycanase, but not O-glycanase, led to MCAM shift in electrophoresis  and O-linked glycans [N-glycans on MCAM [O-linked carbohydrates on the MCAM. Such a difference of galectin-3 binding to the MCAM in melanoma and normal endothelial cells is very interesting. Given that changes of the glycosylation of cell membrane glycoproteins very commonly occur in cancer cells including melanoma [Galectin-3 is a multifunctional protein and is involved in various steps in cancer development, progression, and metastasis by binding to a number of galactose-terminated,  glycans ,36. For  glycans ,38 and clycanase . MCAM is glycans ,33 with  glycans . Our ear glycans , is medi on MCAM . In the melanoma , the binmelanoma , one invReceptor clustering is a common feature of galectins after their binding to cell surface receptors . For exaOverall, galectin-3 is identified in this study as a natural ligand of the MCAM in melanoma cells, and their interaction induces MCAM clustering and subsequent AKT activation. This leads to the increases in a number of steps in the melanoma progression of cell proliferation, adhesion, migration, and invasion. Targeting the galectin-3–MCAM interaction therefore may potentially be a useful therapeutic strategy for melanoma treatment.


The morphological and structural evolution of the electrodes was studied via FEG-SEM, HRTEM, FTIR, XRD, and Raman spectroscopy. The phase transition and enhanced degree of crystallinity were observed with increasing temperature. The orthorhombic structures of the hydrate WO3·H2O (W80), the mixed-phase with mesoporous structure (W200), and finally the monoclinic phase of WO3 structures (W400) were achieved at annealing temperatures of 80 °C, 200 °C, and 400 °C respectively. The electrochemical performance of electrode W200 showed the highest specific capacitance of 606 F g−1 as compared to electrode W80 (361 F g−1), and was two-fold greater than electrode W400 (302 F g−1) at a current density of 1 A g−1. Moreover, electrode W200 exhibited excellent cyclic stability of 89% at an ultrahigh scan rate of 100 mV s−1 after 4000 cycles. The results highlight that the mixed-phase WO3 nanoplates would make a suitable electrode material for supercapacitors with desired electrochemical features.The morphology and crystal structure of electrode materials have an enormous impact on their electrochemical properties for employment in supercapacitors for various applications. In this study, the transformations of the crystal structure of WO 3 nanoplates at various annealing temperatures confirmed by XRD and Raman spectra. The superior electrochemical performance is achieved by the mixed phase WO3 crystal structure.The schematic representation exhibits phase transformation of WO In the EDLC charge storage mechanism, the ions are electro-statistically adsorbing at the interface between the electrode and electrolyte. The pseudocapacitor charge storage mechanism can arise due to three major processes as follows. (1) Underpotential deposition, where metal ions or protons are adsorbed on a metal surface; (2) redox pseudocapacitance, where faradaic redox reactions arise on the surface or near the surface of electrode materials; (3) intercalated pseudocapacitance, where there is the fast faradaic insertion of ions into the tunnel or inter-layers of the electrode material.1,4 The supercapacitor performance is highly governed by the physical and chemical properties of the electrode materials, including porosity, chemical composition, chemical, and thermal stability, surface modification, electrical conductivity, crystal structure, and phase stability of the resulting structure.5–7 Transition metal oxides (TMO), such as MnO2, RuO2, Fe2O3, NiO, Co3O4, MoO3, are highly promising electrode materials due to their various active sites for redox reactions as compared to carbon-based materials, developing non-faradaic processes via the electric double layer.8–13 Nevertheless, the TMOs seriously suffer from low conductivity, high toxicity as well as high cost.14 To solve these issues, tungsten oxide (WO3) is preferred as a cheap and abundant material, which has also found superior applications in antibacterial, electrochromic, photocatalysis, gas sensor, and field emission applications.15–19 It has received significant attention as an electrode material due to its various oxidation states (W2+ to W6+), and high-density (>7 g cm−3) for electrochemical energy storage devices.20,21 Furthermore, its various crystal phases, including monoclinic, tetragonal, orthorhombic, cubic, and triclinic, may form hexagonal, pentagonal, trigonal tunnel, and cavities.22 Among these various structures, the hexagonal structure with a large tunnel size of 0.6 nm is favorable for electrochemical ionic intercalation.23 These polymorphic phases are highly sensitive to temperature changes and have attracted significant attention for their electrochemical charge storage performance.24The supercapacitor is a promising electrochemical energy storage device as compared to the battery, owing to its superior power density, rapid charge–discharge rate, and excellent life cycle.3·2H2O, orthorhombic WO3·H2O, and monoclinic γ-WO3 structure at room temperature, 120 °C and 350 °C, respectively.25 The γ-WO3 structure demonstrated the highest capacity of 420 C g−1 as compared to the monoclinic WO3·2H2O (360 C g−1) and orthorhombic WO3·H2O (386 C g−1). Augustyn et al. observed threefold increments in the capacitance of γ-WO3 (227 F g−1) as compared to monoclinic WO3·2H2O (75 F g−1).26 Hexing Li et al. observed a significant loss of capacitance after the annealing of h-WO3·H2O at 450 °C, which was attributed to the structural transformation from hexagonal to monoclinic WO3.27 Zongming Shao et al., reported the capacitance of 391 F g−1 for h-WO3·0.28H2O, however, after annealing at 500 °C, the capacitance drastically decreased to 42 F g−1 with the formation of the monoclinic WO3 structure.28 Shim Joo et al., obtained the as-prepared orthorhombic WO3·0.33H2O nanorods, which were further transformed into the hexagonal and monoclinic structures via post-annealing at 400 °C and 500 °C, respectively.29 These monoclinic WO3 nanorods demonstrated the highest photocurrent density of 2.26 mA cm−2 at 1.23 V. Thus, it is important to understand the fundamental characteristics of the phase transformations of the WO3 nanostructure, as its various applications demand annealing and/or higher temperature operations.Recently, Mitchell reported the monoclinic WO30–32 The template-assisted, chemical etching and pyrolysis at high temperatures are typical preparation methods for forming porous structures.33 The template-assisted method is highly suitable for creating pores in the bulk materials using numerous templates such as anodic aluminum oxide (AAO) and silicate-based periodic mesoporous templates, namely, MCM-41, MCM-48, HMS, SBA-15, and so on.34,35 However it is difficult to create the pores in metal oxide nanosheets, and significant efforts are required to remove the template, such as the etching method. Practically, a pyrolysis method would be an easier and simpler approach to converting metal hydroxide sheets into the metal oxide; it significantly collapses the morphology and improves the structure architecture.A porous network of electrode materials is highly remarkable for energy storage and conversion, which ensure abundant active sites for redox reactions and provide plentiful voids to accommodate ions during the charge–discharge process.3·H2O nanoplate annealed at 80 °C (W80). Further annealing at 200 °C (W200) and 400 °C (W400) resulted in the phase transition to the tri-phase of the orthorhombic/hexagonal/monoclinic (W200) and pure monoclinic WO3 nanostructures (W400), respectively. The FESEM and HRTEM images revealed the creation of the pore structure on the W200 and W400 samples. The electrochemical performances of all samples were investigated using cyclic voltammetry and a charging/discharging process. The fundamental charge storage kinematics was explored by scan-rate-dependent electrochemical analysis and electrochemical impedance spectroscopy. The importance of crystal structure engineering in enhancing the electrochemical performance of the hydrate WO3·H2O nanoplate was systematically examined for suitable electrode materials of supercapacitors.In this work, we report the preparation of the orthorhombic phase of the hydrate WO2.2.12WO4·2H2O), concentrated sulfuric acid (H2SO4), and ethanol were purchased from SD Fine, India. Nafion was purchased from Alfa Aesar, India. All the aqueous solutions were prepared using double distilled water (DDW).Sodium tungstate dihydrate . Afterward, the solution was heated at 110 °C for 1 h and then naturally cooled down to RT. The obtained precipitate was washed thoroughly with DDW and finally with ethanol. The preparation of the WO3·H2O nanoplates can be represented by reaction The WOThe powder was post-annealed at 80 °C, 200 °C and 400 °C for 4 h to obtain samples W80, W200 and W400, respectively.2.3λ = 1.54056 Å in the 2θ range of 10–80 was used to study the crystal structure. Raman Microscopy ; Raman spectral range of 100 cm−1 to 4000 cm−1) was used to examine the purity and for structural analysis. Fourier transform infrared spectroscopy (FTIR) (Varian 660-IR) was used to identify the functional groups present on the surface of the samples. A field emission scanning electron microscope (FESEM),  and a high-resolution transmission electron microscope (HR-TEM) with selected area electron diffraction (SAED), (TECNAI G2-20-TWIN TEM operating at 300 kV) were used for morphological and structural investigations. Surface area and pore size were investigated by nitrogen adsorption–desorption at 77 K using the Quantachrome Autosorb 1C.An X-ray diffractometer (Bruker D2 Phaser) with CuKα radiation and 2.4The electrochemical measurements were carried out in a three-electrode setup using the PGSTAT302N Autolab system. A glassy carbon electrode, Hg/HgCl, and platinum wire were used as the working electrode, a reference electrode, and counter electrode, respectively.−2 was maintained and aqueous 1 M H2SO4 solution was used as the electrolyte for all electrochemical measurements.The working electrode was prepared by the drop-casting method: 5 mg of active material was added to 2 ml ethanol–water (1 : 1 ratio) with 10 μl Nafion as a binder and sonicated for 10 min. The 10 μl solution was loaded on the working electrode and further dried for 2 h under the IR lamp (200 W). The mass loading of 0.36 mg cm3.3.13·H2O structure with lattice constants a = 5.249, b = 10.711, and c = 5.1333 (JCPDS: 43-0679).36 This structure is composed of a highly distorted WO5(OH)2 octahedron coordinated with five oxygen atoms and one water molecule 2 polyhedron is held together by strong covalent bonds via a bridging O–W–O network along the ab-plane, and remaining terminal oxygen atoms, and other axial water molecules along the c-axis. It was impossible to make a bond with the nearest sheets due to the hydrate molecules, thus leading to a layered structure. These hydrate molecules were ordered and confined between the layers and stabilized the structure. The XRD pattern of W200 represents the combination of three phases. Plane (111) at an angle of 18.38° corresponds to the orthorhombic phase (WO3·0.33H2O) (JCPDS: 35-0270); the highly intense plane (002) at 24.14° characterizes the hexagonal structure (h-WO3) (JCPDS: 85-2460), and the remaining peaks above 24.14° illustrate the existence of the monoclinic phase.29,37–39 On further annealing at 400 °C, W400 exhibited a pure monoclinic phase with lattice constants a = 7.306, b = 7.540, and c = 7.69 (JCPDS: 01-072-0677). The diffraction peak of W200 along the (111) plane completely vanished and the plane (002) corresponding to the hexagonal plane became a triplet, with the (002), (020) and (200) planes at angles of 23.14°, 23.68°, and 24.37° respectively. Thus, annealing at 400 °C resulted in the removal of the confined, ordered water molecules between the layers and formed a strong bridging covalent bond (O–W–O) along the c-axis, realizing a 3D monoclinic WO3 structure  of W80, W200 and W400 were estimated by the Scherrer λ is 1.54056 Å and 0.94 is a constant for the CuKα radiation, β is line broadening in the radian (FWHM). The obtained crystalline sizes of W80, W200 and W400 were 36.4 nm, 11.3 nm and 27.2 nm, respectively. Raman spectroscopy  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O) at 948.3 cm−1. This peak strongly indicates the presence of confined hydrated molecules in the layered structure. However, the band at 640.1 cm−1 represents the stretching mode of the bridging ν(O–W–O) bond along the ab-plane, as expected from the 2D character of the WO3·H2O structure. However, the W400 inhibited the Raman band at 948.3 cm−1 and two strong characteristic bands appeared at 812.13 cm−1 and 711.80 cm−1, indicating the stretching mode of the tungsten atom with a neighboring oxygen atom ν(O–W–O). The band at 276 cm−1 represents the bending vibration of the δ(O–W–O) mode. These three special vibration bands indicated the 3D network of the pure monoclinic WO3 structure. Interestingly, W200 also depicted the characteristics hydrate band at the same position of 948.3 cm−1, with a lower intensity as compared to W80. W200 showed a similar Raman band at 812 cm−1 due to the stretching mode of ν(O–W–O) as the monoclinic structure; the other band at 699 cm−1 shifted to the lower frequency region as compared to the band position of the pure monoclinic WO3 phase at 717 cm−1 for W400. The band at 699 cm−1 represents the characteristics peak of the hexagonal structure of WO3 (h-WO3).41 The intensity ratio of the WO/W–O–W Raman band showed the crystallinity of W80, W200 and W400. Thus, the higher intensity of the WO band and lower intensity of the W–O–W band illustrated the lower crystallinity in WO3.42 The estimated intensity ratios of WO/W–O–W for W80, W200, and W400 were 1.19, 0.345, and 0.048, which indicated the systematic increase in the crystallinity with annealing temperature. 3·H2O to the anhydrous 3D monoclinic WO3 structure as a function of the annealed temperature. The FTIR spectra elucidated the various functional groups present on the surface of W80, W200, and W400 as shown in ν(OH) and δ(OH) corresponding to the water molecules at 3397 cm−1 and 1604 cm−1, respectively. The peak at 947 cm−1 is attributed to a terminal oxygen atom (WO) of the layered WO3·H2O structure. The band at 624 cm−1 is due to characteristic stretching vibrations of bridging ν(O–W–O) bonds. However, the W400 inhibits all the characteristic bands such as the terminal oxygen atom (WO) at 947 cm−1, stretching ν(OH) at 3397 cm−1, and the bending vibration δ(OH) at 1604 cm−1, except the band of the bridging ν(O–W–O) network at 624 cm−1. Interestingly, at the intermediate annealing temperature of 200 °C, W200 exhibited the major characteristic band of the structural water of the terminal WO at 1001 cm−1, which shifted to higher frequency. Also, W200 noticeably showed the bending δ(OH) vibration and bridging ν(O–W–O) bond at the same position as W80. The FTIR spectra of W80, W200, and W400 are in good agreement with the XRD patterns and Raman spectra. TGA analysis as shown in 3·H2O layers. Therefore, it is important to investigate the electrochemical properties of WO3 nanoplates within the temperature range of major weight loss.43The XRD measurements  illustrae Fig. S1. The WO5e Fig. S2. The crytroscopy  is a pow30 The inset of d-spacing of the (111) plane. The SAED pattern in 2 g−1, 124.7 m2 g−1 and 57.4 m2 g−1, respectively. 44–46The morphological analyses of W80, W200, and W400 were performed by FESEM and HRTEM as shown in 3.22SO4 as the electrolyte. −1 to 100 mV s−1 and similarly, Fig. S3(a) and (b)−1 to 100 mV s−1. The comparative CV curve  and galvanostatic charge–discharge (GCD) in a potential window between 0 V to −0.6 V using aqueous 1 M HCV curve  at a low−1 (−1) as compared to electrode W80 (386 F g−1) and W400 (336 F g−1) at a scan-rate of 2 mV s−1. −1. The GCD curves showed the distorted triangular curve representing the pseudocapacitive charge storage as indicated by the CV curves. −1. The specific capacitance at various current densities (−1 of the electrode W200 as compared to electrode W80 (361 F g−1) and W400 (302 F g−1) at a current density of 1 A g−1. Electrode W200 also revealed superior specific capacitance over other WO3 nanostructures reported earlier in the literature  and W400 (18 F g−1). Electrode W200 also demonstrated an excellent capability of 76%, which is three-fold higher than electrode W400 (26%) and better than W80 (64%) between the current densities of 3 and 10 A g−1.Electrode W200 demonstrated an irreversible faradaic reaction accomplished by the two-step intercalation of a proton into the porous network of the mixed-phase at a potential in the ranges of −0.1 V to −0.2 V, and −0.35 to −0.45 V. Electrode W400 showed the smallest area under the CV curve, with no indication of a redox reaction, and thus had the smallest capacitance as compared to electrodes W200 and W80. However, at a high scan-rate of 100 mV s−1 , electroensities  shows thterature . Howeverν)-dependent current contributions in the CV curve is given by I and ν are the current and scan rate, b is a constant value that can either be 0.5 or 1.48–50 The b-value of unity in eqn (2) reveals the direct relationship between current and scan rate, which highlight the dominant capacitive charge storage mechanism. However, a b-value equal to 0.5 indicates that the charge storage in the electrode materials is dominated by the ionic diffusion process. The b-value was obtained from the slope of the plot of the log(current) vs. log(ν) with the scan rate of 2–20 mV s−1. b-value of 0.98, 0.9 and 1.02 of electrodes W80, W200 and W400, respectively. The comparatively lower b-value of electrode W200 showed significant proton insertion into the electrode materials attributed to the mesoporous nature and high surface area. The voltage-dependent b-values  as a function of the scan-rate (mV s−1). At a high scan-rate, the insertion of a proton into the inner surface becomes more difficult. Hence, at the scan rate ν → ∞, all the inner surfaces of the electrode are excluded and q* tends toward the outer surface charge . However, as the scan-rate tends to zero (ν → 0), all inner surfaces are fully accessible and q* tends toward the total charge . The  of the electrode can be obtained by the plot of 1/q* vs. ν1/2  of 40 Ω across the interface of the electrode and electrolyte.52 Electrodes W200 and W400 did not exhibit the charge transfer resistance, revealing the higher conductivity. The x-axial intercept at the high-frequency region represents the equivalent series resistance (RESR) corresponding to the resistance of the current collector-electrode and the ohmic resistance of the electrolyte/electrode interface.53 The obtained RESR values were 3.11 Ω, 1.45 Ω and 1.51 Ω for electrodes W80, W200 and W400, respectively. Thus, the lower value of RESR suggests that the W200 structure enabled enhanced proton transport into the internal surface of the electrode. Furthermore, the obtained Warburg impedance (Zw) of electrodes W80, W200, and W400 were 83 Ω, 26 Ω, and 35 Ω, respectively. The lower Zw of electrode W200 revealed the fast proton diffusion and transport throughout the electrode.54,55 The inset of C, Zw, Rct and RESR. Thus, the W200 electrode facilitating the mixed crystal phase, porous network and agglomerated nanoplates morphology achieved by annealing at 200 °C provides highly suitable electrochemical features as an electrode material for future supercapacitor application.The cyclic stability test was performed at a high scan-rate of 100 mV s−1 , which d4.3 nanoplates were achieved by a post-annealing method for supercapacitor applications. Annealing at 80 °C, 200 °C, and 400 °C demonstrated the phase transition from the orthorhombic structure of the hydrate WO3·H2O (W80) to the mixed-phase (W200) and finally the pure monoclinic crystal structure of WO3 nanoplates (W400), respectively, owing to the escaping of the structural hydrate without disturbing the nanoplate morphology. The phase transitions and the improved degrees of crystallinity at 80, 200 and 400 °C were confirmed by XRD and Raman analysis. The W200 electrode demonstrated superior specific capacitance of 606 F g−1 as compared to the W80 electrode (346 F g−1) and it was two-fold higher than the W400 electrode (302 F g−1). The W200 electrode also exhibited an excellent rate capability of 76%, about three-fold higher than electrode W400 (26%). Moreover, the W200 electrode illustrated a better capacitance retention of 89% as compared to electrodes W80 (86%) and W400 (76%). Thus, the W200 electrode materials with mixed crystal phase, porous network, high surface area and agglomerated nanoplate architecture favor extremely suitable electrochemical qualities as potential electrode material for supercapacitors.The phase transition and the porous network in WOThere are no conflicts to declare.NA-002-D0NA00423E-s001


Simply mixed with carbon nanotubes (CNTs) during electrode preparation, this Cu0.33Co0.67S2 exhibits an overpotential of 284 mV vs. RHE at a current density of 10 mA cm−2 in 1.0 M KOH. The improved OER performance of the Cu0.33Co0.67S2 electrode can be attributed to the electrocatalytically active sites involved in octahedral coordination structures and further activated by Cu substitution. The encouraging results provide insight into further rational design of transition metal-based electrochemical catalysts towards OER applications.Developing cheap and efficient transition metal-based catalysts for the oxygen evolution reaction (OER) plays the key role in large-scale implementation of hydrogen production. However, there is still a lack of effective ways to tune the catalysts performance for the OER reaction from the aspect of structure design and element modulation simultaneously. Herein, a novel Cu Developing cheap and efficient transition metal-based catalysts for the oxygen evolution reaction (OER) plays the key role in large-scale implementation of hydrogen production. To gain efficient water splitting, great efforts have been devoted to exploring low-cost and high performance electrocatalysts for the hydrogen evolution reaction (HER) and oxygen evolution reaction (OER), in which OER is the bottleneck for the kinetically sluggish four-proton-coupled electron transfer in alkaline medium.3–8 Meanwhile, relatively high activation energy is needed to generate the O PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O double bond.9,10The depletion of conventional fossil fuels has sparked extensive research for renewable and clean energies, such as hydrogen energy, which has high gravimetric energy density.11–15 Moreover, the structure diversity of cobalt based sulfides offers more option for improving OER performance. With the aid of electrodeposition, Co–S with amorphous structure and nanosheet morphology was synthesized, exhibiting an overpotential of 361 mV at 10 mA cm−2 for the OER in 1.0 M KOH.16 Liu et al. synthesized the pure Co9S8 phase with the overpotential of 278 mV at 10 mA cm−2.17 Wang et al. employed Co3S4 as OER catalysts with an overpotential of 430 mV,18 while the CoS2 with overpotential of 278 mV is tested at the similar testing condition.19 Here comes a question that is there any relationship between structure and OER catalytical performance of cobalt based sulfides? Han et al. have pointed out that the precondition for this comparison is to synthesize various cobalt based sulfides with similar morphology.20 Given the multiple forms of cobalt sulfides, a big challenge still exists in the field of precisely controlling phase and morphology. For instance, Golberg et al. found Co9S8 is the product of incomplete sulfurization using Na2S as the sulfur source, while CoS2 is the product of over sulfurization using thioacetamide as the sulfur source.21 Nevertheless, it is rather difficult to obtain the single phase of cobalt based sulfides by using the same sulfur source. To solve this problem, Han et al. demonstrated that single phase of Co9S8, Co3S4 and CoS2 can be simply prepared through one-step facile hydrothermal method by just changing the amount of carbon disulfide (CS2) to 0.13 ml, 0.3 ml and 0.8 ml, respectively.20 However, CS2 is highly toxic, which even minor leak leads to danger. Convenient and safety method is still needed to controllable synthesize cobalt sulfides with desired phase and morphology.Among various OER catalysts, cobalt based sulfides have been widely investigated for the high catalytic activity and low cost.22–24 Amongst, the advantage of Cu element can be expressed as: low-cost, non-toxic and the similarity in atomic size and electro-negativity between Cu and Co.25 Peng et al. have proved that the addition of Cu can supply extra catalytic sites in Cu–CoS nanosheets, eventually leading to high performance in sensitized solar cells.26 It has been proved by theoretical calculation that intrinsic metallic nature and more band states near Fermi level could be obtained by introducing Cu to ensure the fast charge transfer, which will play an important role in charge transfer in OER performance.27,28 Moreover, Zhang et al. have demonstrated that the introduction of Cu element into CoS2 can enhance the activity of Co-sites and simultaneously activate the inert S-sites in CoS2. Introducing Cu into CoS2 is demonstrated to optimize the active species adsorption free energy of CoS2 catalyst.28 To the best of our knowledge, few investigations have been focused on the OER performance and the involved catalytic mechanism of Cu–Co–S system. The critical reason is that the controllable synthesis of single cobalt sulfides is still a big challenge, not to mention the bimetallic Cu–Co–S system which containing some complicated reaction between two metals. For instance, Bezverkhyy et al. indicated that the instead of Cu3Co6S8 pure phase which is previously desired, mixture of Cu2S and Co9S8 eventually formed upon sulfidation process of the Cu0.33Co0.67S2 precursor.29 Thus, it is rather desired but still a challenge to synthesize Cu–Co–S with a similar morphology and a controlled pure phase, which can be used to tune the catalysts performance for OER reaction from the aspect of structure design and element modulation.Recently, the research hotspot has been transferred from single cobalt-based sulfides to binary metal sulfide, as introducing another transition metal in cobalt-based sulfides is an effective approach to further enhance the catalytic activity for OER.0.33Co0.67S2, CuCo2S4 and Cu3Co6S8) have been successfully synthesized and their OER catalytical performance have been compared. Amongst, Cu0.33Co0.67S2 exhibits the lowest overpotential of 284 mV vs. RHE at a current density of 10 mA cm−2 in 1.0 M KOH, as compared with that of 310 and 320 mV in CuCo2S4 and Cu3Co6S8 phase, respectively. The improved OER performance of Cu0.33Co0.67S2 electrode can be attributed to the electrocatalytically active sites involved in octahedral coordination structure and further activated by Cu substitution. These encouraging results shed light on further rational design of transition metal-based electrochemical catalysts towards OER application.Herein, Cu-substituted cobalt sulfide hexagonal nanosheets with three different structures crystallographic (Cu0.33Co0.67(OH)2 precursor was prepared by a facile coprecipitation process according to the previous report.30 Specifically, 5 mmol CoCl2·6H2O , 2.5 mmol CuCl2·2H2O , and 45 mmol hexamethylenetetramine (HMT)  were dissolved in the oxygen-removal deionized water. This prepared solution was refluxed with nitrogen at 120 °C for 5 h under continuous magnetic stirring in a three-necked flask, and then cooled down to room temperature naturally. After filtrating, the coprecipitation product was centrifugally washed with deionized water and alcohol for several times and then dried at 60 °C overnight to obtain the Cu0.33Co0.67(OH)2 precursor powder.Firstly, the Cu0.33Co0.67(OH)2 precursor powders with sulfur powders . For the preparation of Cu0.33Co67S2, 50 mg Cu0.33Co0.67(OH)2 precursor powders and 100 mg sulfur powders were separately put on opposite ends of a quartz boat in CVD furnace, which was keeping at 250 °C for 3 h with the heating rate of 2 °C min−1 under a H2/Ar atmosphere, and then cooled down naturally to obtain the black power of Cu0.33Co67S2. Similarly, CuCo2S4 and Cu3Co6S8 can be synthetized at the temperature of 300 °C, 350 °C, 400 °C and 500 °C in the same way, respectively.The series of copper cobalt sulfides were synthesized by thermal annealing of Cuλ = 1.5406 Å, 0.02° per step) and further refined by a Rietveld program RIETAN-2000.31 To examine their morphologies and microstructures, scanning electron microscopy  and transmission electron microscopy  were carried out, respectively. And the elemental composition and chemical states of the samples were measured by X-ray photoelectron spectroscopy  with a C 1s peak at 284.6 eV as the standard signal.The phase structures of the obtained samples were characterized by X-ray diffraction  with Cu Kα radiation   were added before preparing the catalyst slurry, as reported previously.2 and dried at room temperature, and the working electrode was totally loading about 0.03 mg active materials.The catalyst slurry was prepared by a 30 minutes ultrasonic process of the mixture of 4 mg catalyst, 970 μL anhydrous ethanol and 30 μL Nafion solution Nafion  under 40 °C. Subsequently, 8 μL as-prepared catalyst slurry was coated uniformly on the glassy carbon electrode with the area of 7 mm−1. Subsequently, the obtained OER LSV data were treated with iR-compensation according to the equation: Ec = Em − Im × Rs, where Ec, Em, Im and Rs stand for the compensated voltage, measured voltage, measured current and electrolyte resistance, respectively. Unless specified otherwise, all the reported potentials were calibrated with reference to the reversible hydrogen electrode (RHE) based on the following equation: ERHE = ESCE + 0.059 × pH + Em. And the turnover frequencies (TOF) values were calculated according to the following equation: TOF (s−1) = (j × A)/(4 × F × n), where j, A, F and n represent the current density at a given overpotential, the geometric surface area of the working electrode, F is the Faraday constant, mole number of transition metal(s) loaded on the electrode, respectively. Moreover, cyclic voltammograms (CVs) of samples were carried out at different scanning rates , and the apparent electrochemical double layer capacitance (Cdl) was estimated at non-faradaic region. Half of the difference between charging current density and discharging current density (|jc − jd|/2) was plotted linearly versus scan rates, and Cdl value was reflected from the fitted slope. The electrochemical impedance spectra (EIS) were obtained in a frequency range from 10 mHz to 100 kHz at the amplitude of the sinusoidal voltage of 5 mV. The durability of Cu0.33Co0.67S2 was performed in current over 8 h at a fixed potential at 310 mV in 1 M KOH.The electrochemical performances of as-obtained catalysts were conducted on an electrochemical workstation (CHI760e) with a three-electrode cell configuration in oxygen-saturated 1 M KOH  solution at 25 °C, in which the slurry-coated glassy carbon electrode, a graphite rod and a saturated calomel electrode (SCE) were respectively used as working electrode, counter electrode and reference electrode. Linear sweep voltammetry (LSV) measurement was performed at a scan rate of 10 mV s0.33Co0.67(OH)2 structure, consistent with previous reported works.33 Moreover, the morphology of the Cu0.33Co0.67(OH)2 exhibits two-dimensional hexagonal sheet structure. Then, the Cu0.33Co0.67(OH)2 hexagonal sheets, mingled with sulfur at fixed ratio of 1 : 2, are subjected to thermal annealing, at the temperature ranged from 250 °C to 400 °C. The morphology evolution upon thermal annealing at different temperature is evaluated in 0.33Co0.67(OH)2 precursor. The crystal structure upon thermal annealing was characterized by XRD, as shown in Pa3̄ space group. It should be noted here that this phase is identical to CoS2 phase, how to ensure the introduction of Cu? The uniform distribution of Co, Cu and S elements were further confirmed by energy-dispersive X-ray (EDX) spectra, as illustrated in Fig. S3.0.33Co0.67S2 (250 °C) is described in 34 Lifted the annealing temperature to 300 °C, all diffraction peaks can be indexed to the standard JCPDS no. 42-1450 (CuCo2S4), indicating a normal spinel phase with the Fd3m space group. The schematic crystal structure is that Cu atoms occupy the tetrahedral sites while the Co atoms still locate at the octahedral sites.34 Further increasing the annealing temperature to 400 °C, all diffraction peaks can be assigned to the standard JCPDS no. 02-1459, which crystallized in the cubic Fm3̄m structure. The Cu3Co6S8 consists of tetrahedron and octahedron, in which every 24 tetrahedrons share the 6 corners of each octahedron.34,35 Based on above phase evolution information, the compositional design map adopted in Cu–Co–S can be depicted in 0.33Co0.67S2 to Cu3Co6S8, with CuCo2S4 as intermediated phase can be realized in our vapor transformation method. It is observed that under such lower sulfide annealing temperature (lower than 400 °C), the Cu is expected to substitute the Co position, as confirmed by the combination of previous results of XRD pattern and EDX mapping. Increasing the annealing temperature to 500 °C, the phase segregation occurs with the appearance of new Cu1.96S phase, as shown in Fig. S4.†The copper cobalt sulfides with different crystal structures were prepared by simple coprecipitation reaction followed by subsequent thermal annealing with sulfur powders, as illustrated in 0.33Co0.67S2 shows regular hexagonal morphology, as shown in 0.33Co0.67S2 with conspicuous lattice spacing, which shows the crossed lattice fringes of 0.260 nm and 0.331 nm, assigned to the (−210) and (111) interplanar d-spacings, as illustrated in d-spacings is slightly larger than that of the standard CoS2 lattice (JCPDS no. 89-1492). Moreover, the CuCo2S4 still exhibits hexagonal morphology, as shown in 0.33Co0.67S2 showing the crossed lattice fringes of 0.346 nm and 0.551 nm, indexed to the (220) and (111) interplanar d-spacings, as illustrated in 3Co6S8 with maintained hexagonal morphology is observed in 3Co6S8 showing the crossed lattice fringes of 0.178 nm and 0.304 nm, indexed to the (044) and (311) interplanar d-spacings, as illustrated in 0.33Co0.67S2, CuCo2S4 and Cu3Co6S8, as shown in Fig. S5,To further detect the morphology and structure evolution upon vapor transformation, the high-resolution transmission electron microscopy is employed, as shown in 0.33Co0.67S2 exhibits two peaks located at 779.8 eV and 794.9 eV, which belong to the Co2+ 2p3/2 and Co2+ 2p1/2 states.36 On the other hand, the fitting results Co 2p spectra reveal that the Co3+ and Co2+ cations coexist both in CuCo2S4 and Cu3Co6S8, respectively.37 Regarding the Cu 2p spectra in 2+ exists in Cu0.33Co0.67S2, CuCo2S4 and Cu3Co6S8.38 And in 0.33Co0.67S2 reveals the existence of S22− species, while the S 2p spectra of CuCo2S4 and Cu3Co6S8 confirms the existence of S2− species instead of S22− species.37 In addition, the peak at about 168.5 eV belongs to the peak of S–O bonding, which might owing to the exposure to air.39To detect the elemental compositions and the chemical states of Cu–Co–S sheets, X-ray photoelectron spectroscopy (XPS) is carry out. As obtained in 0.33Co0.67S2, CuCo2S4 and Cu3Co6S8 samples were evaluated in a standard three-electrode system with 1 M KOH electrolyte, respectively. 0.33Co0.67S2 manifests the optimal catalytic activity, with an overpotential of 284 mV at 10 mA cm−2. This overpotential value is lower than 310 mV and 320 mV of CuCo2S4 and Cu3Co6S8 samples, respectively. Moreover, the Tafel slope, which is an indicative value of catalytic kinetics of OER,40 of Cu0.33Co0.67S2 CuCo2S4 and Cu3Co6S8 phases are compared in 0.33Co0.67S2 (86 mV dec−1) indicates that the kinetics is more favorable as compared to that of CuCo2S4 (90 mV dec−1) and Cu3Co6S8 (91 mV dec−1), respectively. This enhanced OER rate performance of Cu0.33Co0.67S2 is attributed to the smaller charge transfer between the active sites, as evidenced in smaller loop in EIS observation (Rs) and a charge transfer resistance (Rct), as shown in Table S1.Rct value obtained from the diameter of the semicircles in the high frequency zone is Cu0.33Co0.67S2 (47 Ω) < CuCo2S4 (54 Ω) < Cu3Co6S8 (72 Ω), demonstrating faster electron transfer process upon OER for Cu0.33Co0.67S2.To investigate the relationship between crystal structure and oxygen evolution reaction (OER) activity, the as-prepared Cuervation . To furt41,42 Thus, to further unravel the possible causation of the differences in OER catalytic performance in term of the crystal structure, the electrochemical surface area (ECSA) and turnover frequencies (TOF) have been evaluated, respectively. To estimate the ECSA, the cyclic voltammograms measurements of these products are carried out by sweeping the potential across the non-faradaic region in static solution. 0.33Co0.67S2 and Fig. S62S4 and Cu3Co6S8, respectively, which revealed that the Cu0.33Co0.67S2 has a faster increase in current density at different scan rates. On the other hand, the capacitive behavior and OER performance of the as-synthesized three Cu–Co–S samples are compared according to CVs of Cu–Co–S product in Fig. S7.2S4 and Cu3Co6S8, the Cu0.33Co0.67S2 exhibits the well-defined rectangular shape, implying that a series of active sites with different levels of energies are formed at lower potentials.43 And the Cdl was calculated from cyclic voltammetry (CV) curves versus different scan rates, which can be used to estimate the value of ECSA.44Cdl which obtained from the value of the slop of linear plots, with a tendency of as Cu3Co6S8 (15.56 mF cm−2) < CuCo2S4 (31.26 mF cm−2) < Cu0.33Co0.67S2 (76.32 mF cm−2), indicating the more active catalytic sites were obtained in Cu0.33Co0.67S2 for OER.The effective electrochemical active site is the key for the OER performance.0.33Co0.67S2 is higher than CuCo2S4 and Cu3Co6S8 samples at different testing overpotential. Combining ECSA and TOF value of three samples, it is demonstrated that remarkably improved active catalytic sites are located in Cu0.33Co0.67S2, responsible for the enhanced OER performance with respect to lower overpotential and Tafel slope. Obtained from above results, some information can be provided: firstly, Cu0.33Co0.67S2, CuCo2S4 and Cu3Co6S8 samples all show acceptable OER catalytical performance with overpotential ranged from 284 mV to 320 mV at 10 mA cm−2, which are ahead of the most of as-reported transition metal compounds, as listed in Table S2.45 Secondly, given the same 2D morphology of the three samples, the performance difference of the three samples is originated from the crystal structural differentiation. In Cu0.33Co0.67S2 structure cell, the metal ions are all located in the MS6 (M = Cu/Co) octahedra, while for CuCo2S4, two-third metal ions are located at the centre of the MS6 (M = Cu/Co) octahedra. The lowest ratio of MS6 (M = Cu/Co) octahedra of one-ninth is obtained in Cu3Co6S8. Thus, relationship between crystal structure and oxygen evolution reaction (OER) activity can be established as: the octahedrally coordinated metal sites show higher activity when compared with that of tetrahedrally coordinated metal sites. Specifically, the more octahedral coordination structure MS6 is, the better the catalytic effect can be obtained.460.33Co0.67S2. The XRD pattern  octahedra increasing from 11% to 100%, the overpotentials at 10 mA cm−2 decreases from 320 to 284 mV.To further verify the tendency mentioned above, a new sample is synthesized at the temperature of 350 °C for 3 h in the same way as synthetizing Cu Fig. S8a and furt Fig. S8a indicate6 (M = Cu/Co) octahedra provide more active sites, there is still an open question that what is the role played by the Cu introduction in OER performance. To further elucidate the indispensable effect of Cu elements, the referential pyrite CoS2 has been synthesized through the same procedure without the addition of Cu element. Cu0.33Co0.67S2 and CoS2 were prepared at the same sulfidation temperature of 250 °C to minimize the effect of geometric aspects. As compared in 0.33Co0.67S2 displays an overpotential of 284 mV at the current density of 10 mA cm−2, outperforming 343 mV of pure CoS2. The Tafel slopes are fitted to be 86 mV dec−1 and 98 mV dec−1 with and without Cu addition, as described in 47 Moreover, Zhang et al. have demonstrated that the introduction of Cu element into CoS2 can enhance the activity of Co-sites and simultaneously activate the inert S-sites in CoS2.28In spite of the MS0.33Co0.67S2 with all the metal ions located in the MS6 (M = Cu/Co) octahedra, shows the most fascinating OER performance. With the ratio of MS6 (M = Cu/Co) octahedra increasing from 11% to 100%, the overpotential at 10 mA cm−2 decreases from 320 to 284 mV. Thus, the octahedrally coordinated metal sites show higher activity as compared with that of tetrahedrally coordinated metal sites, which are evidenced in ESCA and TOF results. Subsequently, Cu0.33Co0.67S2 and CoS2, with the same structure that all the metal ions located in the MS6 (M = Cu/Co) octahedra, still show the performance difference. This difference is attributed to the effect of Cu substitution, activating more catalytical sites which are previously inert. The number of catalytical sites is basically determined by MS6 (M = Cu/Co) octahedra. This can be observed in the ESCA value which represents the practical and potential catalytical sites show slightly increased after Cu substitution, however, dramatical increment is observed in TOF value implying potential catalytical sites are triggered by Cu.Based on above results, it can be concluded that structure design and element modulation both are effective ways to tune the catalytical performance for OER. Amongst, structure plays the prerequisite role in determining the number of catalytical sites, including practical and potential catalytical sites. In our Cu–Co–S system, the Cu0.33Co0.67S2 and CoS2. The cyclic voltammetry (CV) curves versus different scan rates for pure CoS2 are shown in Fig. S9.†Cdl values of 76.32 mF cm−2 for Cu0.33Co0.67S2, which is slightly higher than that of 67.03 mF cm−2 for CoS2. The addition of Cu indeed leads to more active catalytic sites in some degree, however, the increment is not as high as that of structure modulation, as shown in 0.33Co0.67S2 are much higher than CoS2 at different testing overpotentials. Thus, it is demonstrated that as compared with structure modulation, the addition of Cu actually boosts intrinsic activities of catalytical sites, as evidenced in TOF analysis. The durability of Cu0.33Co0.67S2 is shown in 0.33Co0.67S2 can retained for 90% of the initial value over 8 h, exhibiting long-term cycling stability for OER. There still exists a question why the addition of Cu increases the intrinsic activity of CoS2 as shown in TOF analysis. To elucidate the underlying mechanism of Cu addition, the XRD patterns of Cu0.33Co0.67S2 and CoS2 are compared in 2 without changing the pristine cubic phase, however, obvious peak shift to lower angles can be observed after Cu addition, indicating that the interlayer distances were increased. Further Rietveld refinement is employed to provide the detail crystal expansion after Cu substitution, as shown in 2 is a cubic phase with lattice parameter of a = b = c = 5.610 Å, while Cu atoms substitute for Co atoms in CoS2 matrix to form Cu0.33Co0.67S2, with slightly expanded lattice parameters of a = b = c = 5.625 Å. This increment in lattice parameter is due to that the ionic radius of Cu is larger than that of Co, thus the lattice is expanded when the Cu atoms substitute for Co atoms in CoS2 matrix.48 The estimated lattice expansion ratio reaching 1% is obtained in Cu0.33Co0.67S2 as compared with that of CoS2, in which lattice distortion and lattice tensile strain are involved.49 More recently, Fan et al. have demonstrated that massive lattice distortion lead to activate more catalytical sites which were previously inert, eventually boosts electrochemical performance.50 Moreover, with respect to lattice tensile strain, it can facilitate active intermediates adsorption, such as O*, OH* and OOH*, by enhancing binding strength between functional catalyst to active intermediates.51The electrochemical surface area (ECSA) and turnover frequencies (TOF) were compared for Cu0.33Co0.67S2, CuCo2S4 to Cu3Co6S8 with the similar hexagonal sheet shape. Benefited from the similar morphology, the relationship between structure and OER catalytical performance has been established. The Cu0.33Co0.67S2 with all the metal ions located in the MS6 (M = Cu/Co) octahedra manifestes the optimal OER performance, with lowest overpotential of 284 mV at a current density of 10 mA cm−2, indicating the MS6 (M = Cu/Co) octahedra exhibits higher catalytic activity. Moreover, the effect of Cu substitution is elucidated to trigger previously inert sites in MS6 (M = Cu/Co) octahedra by introducing lattice distortion and lattice tensile strain. The comprehensive investigation on OER performance of Cu–Co–S provide insight into further rational design of transition metal-based electrochemical catalysts towards OER application.In conclusion, we have successfully synthesized series of copper-substituted cobalt sulfide compounds, from CuThere are no conflicts to declare.RA-009-C9RA00640K-s001


NK cells were distinguished as CD3-CD16+CD8α+/dim cells and further subdivided using NKp46 (CD335) expression into NKp46-, NKp46+, and NKp46high NK cells. T-cell receptor (TCR) γδ T cells were defined by the expression of TCR-γδ and different subsets were determined by their CD2 and perforin expression. TCR-αβ T cells were classified by their CD8β+ or CD4 expression. For monitoring their differentiation, expression of CD27 and perforin was investigated for CD8β++ T cells and CD8α together with CD27 for CD4+ T cells. We clearly detected a postnatal development of immune cell composition and identified phenotypes indicative of differentiation within the respective leukocyte subsets. Examination of the development of the antigen-specific immune system after transfer to different distinct housing conditions and after vaccination against common porcine pathogens such as porcine circovirus 2 (PCV2) revealed a markedly increased presence of more differentiated CD8+ and CD4+ T cells with central and effector memory T-cell phenotypes. To complement the findings, a PCV2 vaccine-specific antigen was used for in vitro restimulation experiments. We demonstrated antigen-specific proliferation of CD4+CD8α+CD27+ central and CD4+CD8α+CD27- effector memory T cells as well as antigen-specific production of TNF-α and IFN-γ. This study of postnatal immune development defines basic cellular immune parameters of EGMs and represents an important milestone for the use of EGMs for immunological questions in experimental medicine.Interest in Ellegaard Göttingen Minipigs (EGMs) as a model in experimental medicine is continuously growing. The aim of this project is to increase the knowledge of the immune system of EGMs as information is still scarce. Therefore, we studied the postnatal maturation of their immune system from birth until 126 weeks of age. For the first 26 weeks of the study, animals were kept under pathogen-reduced conditions (SPF) and afterwards under conventional housing conditions. The development of the immune system was analyzed by monitoring changes in total numbers of leukocytes and lymphocytes of ten individuals and the composition of leukocyte populations by multi-color flow cytometry (FCM). We followed the presence of monocytes using monoclonal antibodies (mAbs) against CD172a The main objective of our study was to define reference values and to increase the knowledge about the development of the immune system of EGMs. We determined immunological characteristics of EGMs and discussed possible differences to other species, in order to strengthen the usability of EGMs in preclinical studies.The importance of swine and especially Ellegaard Göttingen Minipigs (EGMs) as a model in translational research and experimental medicine is steadily growing –9. In paTen EGMs  were used in this study. This breed was established in the 1960s in Göttingen, Germany. The background of these pigs are Minnesota Minipigs from Hormel Institute, Austin, TX, USA and Vietnamese potbellied pigs from Wilhelma Zoo, Stuttgart, Germany. In 1965 Vietnamese potbellied pigs from Friedrichsfelde Zoo, East Berlin, Germany were crossed in. Between 1965 and 1969, the German Landrace was crossed in, and the final breed was established  twice wiDuring the first six months of the study, blood was collected at two-week intervals starting 2 – 4 days after birth. After transfer to Vienna, pigs were bled starting at week 44 in intervals between two to ten weeks. For blood sampling, animals were fixed with a V-trough or sling (Denmark), or a nose snare (Austria). Blood was collected by puncture of the Vena jugularis externa or the Vena cava cranialis. In Denmark, a BD Vacutainer™ system (Na-Heparin) was used while in Austria blood was collected in heparin tubes .Isolation of peripheral blood mononuclear cells (PBMCs) was carried out using lymphocyte separation medium  or 1.077 g/mL (weeks 8 – 126), PAN-Biotech, Aidenbach, Germany). Centrifugation was carried out for 30 min at 920 × g at room temperature (RT). The isolation protocol was previously described , 21, 22.Whole blood for determination of leukocytes and lymphocytes and isolated PBMCs were counted using Türk`s solution  and a Neubauer counting chamber  by microscopy  for weeks 0, 2 and 6. For the first week of the study cell counts of only three animals were available. Therefore, a mean of the three samples was used to calculate the total cell numbers for all animals. For weeks 2 and 6, blood of all animals was counted and the percentage of lymphocytes within total PBMCs was determined by flow cytometry (FCM). For all other study days, samples of all animals were counted on a Sysmex XP300 . Isolated PBMCs were stored at -150°C in a freezing medium containing 50% (v/v) RPMI 1640 with stable glutamine (PAN-Biotech), 100 IU/mL penicillin and 0.1 mg/mL streptomycin (PAN-Biotech), 40% (v/v) fetal calf serum , and 10% (v/v) DMSO (Sigma-Aldrich).For FCM staining for phenotyping 1.5 – 2.0 × 106 freshly isolated PBMCs per sample were plated into 96-well round-bottom plates . Information about the monoclonal antibodies (mAbs) and second-step reagents are presented in For cultivated PBMCs as described in section 2.5 a staining buffer consisting of D-PBS (PAN-Biotech) with 3% (v/v) FCS (Gibco™) was used. For intracellular cytokine staining (ICS), PBMCs were fixed and permeabilized with BD Cytofix/Cytoperm™ Kit  according to manufacturer's protocol.7 cells/mL and 1 mL of a 5 µM CellTrace™ Violet solution  was added for each mL of cell suspension, followed by immediate vortexing. The suspension was incubated for 10 min at 37°C in a water bath and briefly vortexed several times. Thereafter, 2 mL FCS (Gibco™) were added to every 2 mL of the cell-dye suspension and cells were incubated for an additional 15 min at RT in the dark. 10 mL of cell culture medium were added and PBMCs were centrifuged as described above. Washing was repeated twice before PBMCs were counted. Validation of the successful cell labeling was performed on a CytoFLEX LX . Labeled cells were plated into 96-well round-bottom plates with 2 × 105 cells per well in a total of 200 µL volume. Cells were stimulated with recombinant PCV2-ORF2 protein or GP64 as a baculovirus-expressed control protein  and cultured for four days . Concanavalin A  stimulated PBMCs and PBMCs cultivated in medium alone served as positive and negative controls, respectively. After four days, microcultures were harvested, cells of the same stimulation group of the same animal were pooled and stained as described above in 96-well round-bottom plates.Following defrosting, PBMCs were resuspended in D-PBS (PAN-Biotech) and filtered through a Falcon Cell Strainer 70 μm Nylon  before counting. Cells were adjusted to 2 × 105 cells per well in a total volume of 200 µL. Cells were stimulated with a recombinant PCV2-ORF2 protein or GP64 as control protein . PMA/Ionomycin treated PBMCs  served as positive control and PBMCs cultivated in medium alone served as additional negative control. Cells were cultivated for 18 hours . Four hours prior to harvesting, 1 µg/mL Brefeldin A (BD Biosciences) was added to all microcultures to inhibit cytokine release. For the following FCM analyses, at least 12 wells of the same stimulation group were pooled, and cells were washed twice with D-PBS (PAN-Biotech) before staining (described in section 2.4) in 96-well round-bottom plates.After defrosting, PBMCs were plated in 96-well round-bottom plates with 5 × 10Samples were analyzed using a CytoFLEX LX (Beckman Coulter) equipped with six lasers (U-V-B-Y-R-I). At least 100,000 cells were analyzed per sample. For compensation, single stains were used to set up a compensation library. FCS files were analyzed with FlowJo software version 10.8. (Becton Dickinson). The gating strategy used in all experiments is summarized in +CD163+ expression (-CD79a+ lymphocytes (-CD8α+CD16+ lymphocytes and further subdivided using NKp46 (CD335) expression into NKp46-, NKp46+, and NKp46high NK cells  and CD8α+CD27- effector memory (TEM) CD4+ T cells  and week eight . An increase around week 60 was followed by a decrease in total cell counts within both groups (+ cell frequencies was present. This was independent of sex and the distinct increases in the median CD8+ T cells counts (e.g. week 80) likely reflects booster immunizations. As previously described, CD8β+ T cells show different phenotypes depending on their CD27 and perforin expression  can be characterized by their co-expression of CD3 and CD8βpression , 34, 35 ectively . In the + T cells, CD8α expression was studied in combination with the expression of CD27.  and a first increase was observed until week ten in males and week eight in females . At the end of our study, the three CD4/CD8α/CD27-defined phenotypes contained nearly equal levels of cells in females, whereas in males' naive CD4+ T cells were still slightly higher.The expression of CD4 was used as a marker to identify T helper cells. For a further identification of distinct differentiation stages of CD4of CD27. 7A. As s+ T cells and their proliferative capacity after in vitro restimulation with the vaccine antigen the baculovirus-expressed PCV2-ORF2 protein. In addition, we analyzed the production of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) as we expected a Th1 response within CD4 cells when using a vaccine against a viral pathogen . This confirms T cell memory function of CD8α+CD27+/- CD4+ T cell subpopulations in EGMs. The gating strategy is shown in To investigate the development of an antigen-specific T-cell response after vaccination we focused on CD4pathogen , 47. Thr+CD8α+ T cell subpopulations could be further characterized by intracellular cytokine staining for TNF-α and IFN-γ. Here, PBMCs of six randomly chosen pigs of our herd were restimulated with PCV2-ORF2 overnight. A control staining without TNF-α and IFN-γ can be found in + T cells cultivated in medium showed hardly any cytokine production of TNF-α and IFN-γ with less than 0.01% for all CD8α/CD27-defined T cell subsets  (6/mL ± 2.8 × 106/mL) within the EGM population at the end of our study. Nevertheless, the percentage of lymphocytes was higher in EGMs (59.9% ± 5.9% lymphocytes within leukocytes) versus 44.7% ± 10.2% in Large White breed  and 14.6 × 106/mL ± 2.7 × 106/mL  at 16 weeks of age. At one year of age, the average for males was 13.3 × 106/mL and 16.4 × 106/mL in females in Duroc - Jersey Swine, while EGMs had 15.6 × 106/mL ± 5.4 × 106/mL  and 17.0 × 106/mL ± 4.6× 106/mL  leukocytes at 60 weeks of age. A similar picture within the two breeds was found looking at the percentage of lymphocytes around 4 months of age: Duroc - Jersey Swine had an average of 63% of lymphocytes while EGMs had an average of 60.6% ± 10.7%. EGMs showed slightly higher levels when we compared ≥ 1 year old pigs. Duroc - Jersey Swine had 55%  and 54%  whereas EGMs had 65% ± 8%  and 68% ± 6%  of lymphocytes on average. Apart from this, our findings generally confirm the findings cited by Weiss and Wardrop that age related changes in EGMs are similar to domestic swine  and 7.9 × 106/mL ± 2.7 × 106/mL  were found and lymphocytes with an average of 36.7% ± 17.5%  and 39.6% ± 13.1%  in Macaca  and 8.8 × 106/mL ± 2.4 × 106/mL  of leukocytes which fits human reference values well, whereas we found a higher number of lymphocytes in EGMs: 58.9% ± 2.1%  and 60.7% ± 7.9% . When comparing EGMs to other species one has to consider age as our pigs at the age of week 126 relate more to young adult humans. Investigating total monocyte numbers and reference values in humans  , we founte breed . We havete breed , we findte breed , an averic swine . As EGMsn Macaca . In adulel, 2020 . Linkingn humans  3 – 7% wn humans  and to >n humans .In general, EGMs showed comparable numbers of B cells to domestic swine  over thedim/+NKp46high phenotype are in an activated state  cells and CD4+CD8α+CD27- are defined as effector memory (TEM) T cells. As expected, the CD4+CD8α-CD27+ phenotype was the most frequent during the early postnatal development in EGMs. Over time, an increase in the CD4+CD8α+CD27+ and CD4+CD8α+CD27- subsets occurred after transfer to Vienna and vaccination. This underlines the assumption that naive, central and effector memory phenotypes are also true for EGMs. Looking at the frequency of CD4+ T cells in different species we find comparable numbers that differ more in humans and pigs within one species compared to NHPs. Within humans, 32 – 50% CD4+ T cells are the relative numbers  and in Austria by the institutional ethics committee, the Advisory Committee for Animal Experiments , and Austrian Federal Ministry of Education, of Science and Research (reference BMBWF-68.205/0198-V/3b/2019).Concept development: CPSP, SJ, MWS, KHM and AS. Experimental work: CPSP, MS. Caretaking of the pigs and bleeding: AL, CK, SD, HK and VM. Data analysis: CPSP, AS. Data Interpretation: CPSP, KM, EL and AS. Figure Preparation: CPSP. Writing: CPSP, AS and SS. All authors read and approved the final manuscript.The authors declare that this study received funding from Merck HealthCare KGaA. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.We thank Wilhelm Gerner for helping in developing the panels for FCM analysis. We also thank the team of Ellegaard Göttingen Minipigs A/S for taking care of and bleeding the pigs until six months of age.Authors SJ and MS were employed by company Merck KGaA.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.