The human C-C chemokine receptor type 7 (CCR7) has two endogenous ligands, C-C chemokine ligand 19 (CCL19) and CCL21, displaying biased agonism reflected by a pronounced difference in the level of β-arrestin recruitment. Detecting this preferential activation generally requires the use of separate, pathway-specific label-based assays. In this study, we evaluated an alternative methodology to study CCR7 signalling. Cellular electrical impedance (CEI) is a label-free technology which yields a readout that reflects an integrated cellular response to ligand stimulation. CCR7-expressing HEK293 cells were stimulated with CCL19 or CCL21, which induced distinct impedance profiles with an apparent bias during the desensitisation phase of the response. This discrepancy was mainly modulated by differential β-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. Pathway deconvolution revealed that Gαi-mediated signalling contributed most to the impedance profile, but Gαq- and Gα12/13-mediated pathways were also involved. To corroborate these results, label-based pathway-specific assays were performed. While CCL19 more potently induced β-arrestin2 recruitment and receptor internalisation than CCL21, both chemokines showed a similar level of Gαi protein activation. Altogether, these findings indicate that CEI is a powerful method to analyse receptor signalling and biased agonism. Chemokine receptors are members of the rhodopsin-like class A G protein-coupled receptors (GPCRs). They regulate immune cell activation and migration, and play a fundamental role in tissue development and organisation ,2,3. TheWithin the human chemokine signalling system, consisting of about 20 chemokine receptors and 50 chemokines, substantial promiscuity exists. Many chemokine receptors interact with more than one chemokine and often a given chemokine can stimulate multiple receptors. Although this was initially seen as signalling redundancy, it is now appreciated that, for several members of the chemokine receptor family, ligand bias occurs naturally, which contributes to the finetuning of chemokine signalling . Biased The human C-C chemokine receptor type 7 (CCR7) is a prime example of a chemokine receptor for which ligand bias was described. CCR7 is essential for homing various immune cells to secondary lymphoid organs and defining their position within the organ architecture ,12,13,14Studying chemokine receptor signalling is complex, especially when receptors are activated by multiple ligands. Determination of potential ligand bias generally requires the comparison of chemokines in multiple cellular assays, each looking into a separate signalling cascade or pathway. An alternative approach to determine ligand bias could be to employ assays with a readout that encapsulates the chemokine receptor response in its entirety, rather than comparing subsets of signalling pathways over multiple assays. Cellular electrical impedance (CEI) is a label-free, real-time cellular analysis methodology for which the readout reflects an integrated cellular response. CEI measures changes in the impedance of cells that adhere to a gold microelectrode surface. When these cells are subjected to a small alternating electrical current, they impede this current at the electrode–solution interface. The magnitude of the impedance depends on various cellular properties such as cell morphology, cell–cell contacts and cell adhesion. GPCR signalling induces morphological changes through G protein coupling-dependent rearrangement of the actin cytoskeleton . As suchIn this study, we investigated to what extent a CEI-based assay can capture the natural ligand bias previously described for human CCR7. For this, CCR7 was stably expressed in human embryonic kidney (HEK293) cells that were subsequently used for both CEI-based assays as well as more conventional label-based assays . Our data show that CEI is capable of detecting specific CCR7-mediated signalling events with clearly distinct response profiles for the two endogenous ligands (CCL19 and CCL21). Detailed analysis and modelling of the differential CEI responses for CCL19 and CCL21 and comparison with data obtained from the more conventional label-based assays led us to conclude that CEI could be a valuable alternative methodology to identify and study ligand bias at GPCRs.We investigated the capability of CEI to measure human CCR7 agonist-induced signalling. To this end, HEK293 cells stably expressing CCR7 were seeded in E-plates and an impedance profile was acquired following treatment with either CCL19 or CCL21 A. StimulHEK293 cells not expressing CCR7 did not respond to CCL19 addition, which validated that the observed response was indeed CCR7-specific. However, CCL21 did display a small increase in CI over time in these cells .[0–3] values were observed for both CCL19 and CCL21, they were more pronounced upon CCL19 stimulation . CCL21 reached higher AUC[20–120] values than CCL19, which rapidly reached a plateau  and larger (p < 0.01); however, this still resulted in an AUC[20–120] increase, although it was less significant than for CCL19  cell lines to map the effect of different transducers and their respective signalling pathway. To limit the effect of variable receptor expression levels, all KO variants were stably transfected with CCR7 and sorted for similar expression to the CCR7-expressing wild-type HEK293 cells . Due to emokines A–D. Nexton onset B,D. Furtd larger D, respecor CCL19 D. CMPD10[20–120] A,B,E–H. [20–120] C,D. We f[20–120] C,D. Inte[20–120] C,D.To validate the mechanisms that could explain the similarities and differences in the impedance profile induced by CCL19 and CCL21, we investigated CCR7-mediated signalling and function by employing various label-based assays. To minimize system bias, all these experiments were performed in the same cellular background used for the CEI experiments. First, as a proxy for Gαi activation, agonist-dependent inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) production was measured in HEK293 cells stably expressing CCR7 transiently transfected with a cAMP GloSensor plasmid. Both CCL19 and CCL21 induced robust Gαi activation with similar efficacy and potency . As expemax value similar to CCL19 could be predicted. When cells were treated with the specific GRK2/3 blocker CMPD101, there was minimal β-arrestin2 recruitment inhibition for both chemokines  to DNA ratio. The FuGENE® HD Transfection Reagent/DNA mixture contained 20 ng/µL DNA and was incubated for 10 min at ambient temperature before adding it to the cell suspension. When a co-transfection of two plasmids was carried out, half the DNA concentration was used per plasmid.Cellular transfections were performed in suspension per the manufacturer’s protocol. Briefly, 1.5 × 10The transfection setup for nanoBRET-based G protein activation assay differed slightly and is explained below.2. Cellular growth was monitored overnight every 20 min until a steady state was reached after 20–24 h. Following overnight incubation, E-plates were washed with 100 µL serum-free DMEM, 100 µL serum-free DMEM was added to each well and cell stabilisation was measured each minute for 4 h. Before ligand addition, a short normalisation measurement consisting of 5 total measurements, one every 5 s, was performed. Thereafter, 25 µL ligand at a 5× concentration was added and receptor stimulation was measured every 20 s for 4 h. When investigating the effect of compounds  on receptor stimulation, cells were washed and 80 µL serum-free DMEM was added. Forty minutes before the ligand addition, 20 µL of 5× concentrated compound was added. For PTX, which was incubated overnight, 25 µL of 5× concentrated compound was added two hours after seeding the cells.The xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument  was used to measure changes in cellular impedance following ligand stimulation. Briefly, RTCA E-plate VIEW 16 plates with embedded golden electrodes  were coated with 10 µg/mL fibronectin  for 30 min and air-dried for one hour. A mandatory reference measurement was performed with 50 µL of growth medium per well to establish background CI values for each well. Thereafter, HEK293 cells were seeded at a density of 30,000 cells/well in a final volume of 100 µL. E-plates were placed at room temperature for 15 min and then transferred to the xCELLigence RTCA instrument, located in an incubator at 37 °C and 5% CO4 cells/well in white, clear flat-bottom 96-well plates  coated with 100 µg/mL poly-D-lysin  and incubated for 48 h at 37 °C and 5% CO2. Next, cells were washed with CO2-independent medium  supplemented with 10% FBS and incubated in 100 µL CO2-independent medium/10%FBS further supplemented with 300 µM 3-isobutyl-1-methylxanthine  and 2% GloSensor cAMP reagent  for 2 h at 37 °C. The plate was transferred to the FLIPR Tetra  and baseline luminescence was measured for 30 s every 5 s. Thereafter, 25 µL of 5× ligand was added automatically to the cell plate by the FLIPR Tetra and changes in bioluminescence were monitored in real time for 10 min every 5 s. Next, 25 µL Forskolin  was added to a final concentration of 5 µM and changes in bioluminescence were monitored in real time for 40 min every 5 s. When required 25 µL, 5× concentrated PTX was added 24 h post-transfection.HEK293 cells stably expressing CCR7 were transfected in suspension with pGlosensor-22F. Transfected cells were seeded at a density of 1.5 × 10® HD Transfection Reagent to DNA ratio with a final acceptor concentration of 1 µg plasmid DNA per mL. The FuGENE® HD Transfection Reagent/DNA mixture contained 10 ng/µL DNA and was incubated for 10 min at ambient temperature before adding it to the cell suspension. Transfected cells were seeded at a density of 3.0 × 104 cells/well in white, clear flat-bottom 96-well plates coated with 100 µg/mL poly-D-lysin and incubated for 48 h at 37 °C and 5% CO2. Next, cells were washed with an assay buffer , 20 mM HEPES buffer , 0.5% FBS, pH 7.4) and incubated with 90 µL of a 1:100 Nano-Glo® Vivazine™ working solution  for 45 min at 37 °C and 5% CO2. The plate was transferred to the FLIPR Tetra and allowed to stabilise for 15 min. Baseline BRET was measured for 15 s every 2.5 s. Thereafter, 10 µL of 10× ligand was added automatically to the cell plate by the FLIPR Tetra and changes in BRET were monitored in real time for 25 min every 2.5 s. Measurements in the FLIPR tetra were carried out using a 440–480 nm donor emission filter and a custom 615 nm AT600lp acceptor emission filter .G protein activation was monitored using a modified NanoBRET protein:protein interaction system . NanoLuc cDNA was inserted into Gαi-1, Gαi-2 and Gαi-3 at position 91 in a pcDNA3.1(+) vector. Additionally, Gγ2 was N-terminally fused to LSS-mKATE2 and cloned into a pcDNA3.1(+) vector. HEK293 cells stably expressing CCR7 were transiently co-transfected in suspension. Briefly, pcDNA3.1(+) Gαi1-91-Nluc, pcDNA3.1(+) Gαi2-91-Nluc or pcDNA3.1(+) Gαi3-91-Nluc and pcDNA3.1(+) Gγ2-LSSmKATE2(N) were transiently co-transfected in suspension at a 1:10 donor-acceptor ratio using a 3:1 FuGENE4 cells/well in white, clear flat-bottom 96-well plates coated with 100 µg/mL poly-D-lysin and incubated for 48 h at 37 °C and 5% CO2. Next, cells were washed with an assay buffer , 0.5% FBS, pH 7.4) and incubated with 100 µL of a 1:100 Nano-Glo® Live Cell Substrate  working solution for 40 min at 37 °C and 5% CO2. The plate was transferred to the FLIPR Tetra and baseline luminescence was measured for 30 s every 5 s. Thereafter, 25 µL of 5× ligand was added automatically to the cell plate by the FLIPR Tetra and changes in bioluminescence were monitored in real time for 40 min every 5 s. When required, 20 µL of 5× compound was added to the working solution Nano-Glo Live Cell Substrate. In the case of PTX, 25 µL of 5× concentrated compound was added after 24 h of incubation.β-arrestin recruitment was monitored using the NanoBiT PPI system (Promega). CCR7 and β-arrestin2 were C-terminally fused with LgBiT and N-terminally fused with SmBiT, respectively, and cloned into a pEF1α-IRES vector. HEK293 cells were transiently co-transfected in suspension with pEF1α-IRES CCR7-LgBiT(C) and pEF1α-IRES SmBiT-ARRβ2(N), as previously described. Transfected cells were seeded at a density of 1.5 × 105 cells per condition in a Falcon® 5 mL round bottom polystyrene tube . Cells were stimulated with 25 µL 5× agonist for 30 min at 37 °C and 5% CO2. Next, cells were quickly washed twice with and resuspended in 2 mL and 100 µL ice-cold DPBS/2%FBS, respectively. Cells were stained with 5 µL PE mouse anti-human CCR7 , PE mouse IgG2a κ isotype control  or DPBS/2%FBS and incubated for 1 h on ice. Cells were washed twice with 2 mL ice-cold DPBS and, finally, were resuspended in 200 µL ice-cold DPBS and kept on ice. Fluorescence was measured using the BD FACS Celesta (BD Bioscience) and data were processed with the BD FACSDiva Software (BD Bioscience) and FLOWJO™ version 10 (BD Bioscience).HEK293 cells stably expressing CCR7 were harvested with 0.25% trypsin, incubated for 2 h at ambient temperature, and washed twice with 2 mL DPBS  containing 2% FBS. Thereafter, cells were resuspended in 100 µL DPBS/2%FBS at 2 × 10stats (version 3.6.2) R package. Statistical analysis was performed as described in figure or table legends.Raw relative light units (RLU) (cAMP modulation assay and β-arrestin recruitment assay), raw CI values (CEI assay) or a BRET ratio (G protein activation assay) were used as a starting point for data manipulations. BRET ratios were first calculated by dividing acceptor RLU by donor RLU values. All data were normalised to the baseline before ligand addition to reduce inter-well variation, an approach commonly used for kinetic fluorescent and bioluminescent readouts. This baseline was defined as the mean of a 5-point run-in time before ligand addition. Normalisation was performed by dividing all timepoints following ligand addition by the initial baseline. The technical replicates of these normalised readouts were averaged and then background-corrected by subtracting the values of their respective vehicle controls at each timepoint yielding a normalised background-corrected measurement. Dose-response curves were fitted to three parameters-log(agonist) vs. response model in GraphPad V9.3.1  unless otherwise stated. Decay rates were fitted from the global maximum with a one-phase decay model in R version 4.0.5 using the SSasymp function from the


X-ray diffraction revealed a pure cubic spinel phase of all samples with space group Fd-3m. Fourier transform infrared spectrometry disclosed the vibrational modes of metal oxides in the spinel structure. Scanning electron microscopy and transmission electron microscopy disclosed a uniform distribution of cuboidal shape NPs with a decreased average NPs size from 22.72 ± 0.62 to 20.85 ± 0.47 nm as the Co content increased. X-ray absorption near edge spectroscopy results confirmed the presence of Zn2+, Co2+ and Fe2+/Fe3+ in Co-doped samples. The pore volume, pore size and specific surface area were determined using N2 gas adsorption/desorption isotherms by the Brunauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH) techniques. Electrochemical properties of supercapacitors, having active CoxZn1−xFe2O4 (x = 0.0–0.4) NPs as working electrodes, indicated pseudo-capacitor performance related to the Faradaic redox reaction. Interestingly, the highest specific capacitance (Csc), 855.33 F/g at 1 A/g, with a capacity retention of 90.41% after 1000 GCD cycle testing was achieved in the Co0.3Zn0.7Fe2O4 electrode.In this work, Co Generally, a SC cell consists of two separate electrodes immersed in an electrolyte. The chemical reactions between charges/ions in the electrolyte and electrodes materials are key functions that directly impact the performance of a SC cell. Practically, two important types of SCs are pseudo-capacitors (PCs) and electric double layer capacitors (EDLCs)5. Principally, each type of SC has similar cell structure, but a different charge storage mechanism and electrode material. For EDLCs, charges are stored in a very thin double layer on electrodes surface. Whereas in PCs, the reversible redox reactions are the major response for charge storage. In both types of SCs, the cell efficiency strongly depends on many parameters such as surface area, pore structure, and conductivity of active materials used for electrode fabrication4. Thus, a search for novel electrode materials with these good properties is an active area for researchers6. Usually, for PCs, various transition metal oxides (TMOs) and conducting polymers are generally employed for electrode fabrication because they can provide higher specific capacitance (Csc) and energy density (Ed). This due to their fast Faradaic redox reaction during charging/discharging compared with carbon materials7. Moreover, numerous nanosized (5–50 nm) TMOs have been extensively studied. Many of them were found to be suitable for SCs electrodes with high electrochemical performance. This is due to their increased electroactive sites which offer appropriate conductive pathways and extensive nanopaths to promote efficient transportation of charges/ionic species10. During the past decade, spinel TMOs of the AB2O4 structure  of CuFe2O42, MnFe2O44, NiFe2O46, CoFe2O49 and ZnFe2O415 were extensively studied as electrode materials for PCs. This is due to their advantage of a polymorphic structure of normal, inverse and mixed spinel forms in which metal ions with a + 2 oxidation state (M2+) and (Fe2+/Fe3+) can be distributed at A or/and B sites. In normal spinel, M2+ and (Fe2+/Fe3+) ions are at A and B sites, respectively. For inverse spinel, M2+ ions replace some of (Fe2+/Fe3+) ions at the B site, while (Fe2+/Fe3+) ions can occupy both the A and B sites. In the case of mixed spinel, M2+ and (Fe2+/Fe3+) ions can be distributed at both A and B sites. As reported by earlier researchers, the Csc values of various AB2O4 ferrites of different morphologies vary over a wide range from a hundred to more than a thousand. For example, Ni0.4Co0.6Fe2O4 nanoparticles (NPs) (237 F/g at 1 A/g)5, CoFe2O4/NiFe2O4 nanospheres (269 F/g at 1 A/g)6, electrospun carbon/CuFe2O4 nanofibers (191 F/g at 10 mV/s)2, NiFe2O4@CoFe2O4 core–shell nanofibers (480 F/g at 1 A/g)3, ZnFe2O4 NPs (712 F/g at 2 mV/s)15, CoFe2O4 NPs 1 and Ni1−xMnxFe2O4 NPs 4. Among numerous AB2O4 materials, spinel zinc ferrite (ZnFe2O4) is of great interest. It has been extensively investigated due to its fascinating properties, natural richness, low cost, and environmental friendliness15. However, the development of high-performance ZnFe2O4 electrode materials remains a greater challenge due to their poor stability in cycling tests, as reported by Vadiyar et al.12. Owing to this problem, different approaches such as using material composites13 and structure modifications by doping with various transition metals (TMs) in MxZn1−xFe2O4  have been attempted. However, doping with TMs was suggested as the effective method because it can significantly affect the ion distribution at A and B sites of spinel crystals of these materials and on the surface of ferrites. This makes them more active and sensitive to Faradaic redox reactions during charging/discharging process16. From literatures, Co was considered to be one of the effective TM that had been intensively used as dopant with successful improvement many properties of materials19.Currently, multi-functional nanomaterials with intriguing properties are very important and necessary for many emerging novel technologies. They are essential parts of many electronic devices, specifically as electrodes in the energy collectors of fuel cells, batteries and supercapacitors (SCs). Moreover, increasing need for them is seen due to the immense increase in the demand for clean and sustainable energy. Particularly, SCs are a unique type of electrochemical device that stores energy via a collection of charges/ions on the electrodes surface, which can offer many benefits over conventional batteries and capacitorsxZn1−xFe2O4 (x = 0.0–0.4) NPs using a hydrothermal method, because it is a facile process that can generally occur at low temperature, without toxicity due to the use of metallic salts that can be dissolved in water21. Moreover, a high yield NPs of narrow size distribution can be obtained as compared to other method22. The influence of Co doping on the physical properties of the materials such as their phase, crystal structure, morphology, functional groups, surface area and oxidation states of metal ions, were studied using various techniques. Furthermore, the electrochemical properties of the obtained products were studied for SCs application. To the best of our knowledge, the electrochemical properties of hydrothermally obtained Co-doped ZnFe2O4 have never been reported.Herein, it is our aim to synthesize CoxZn1−xFe2O4 (x = 0.0–0.4) NPs were prepared by a hydrothermal method, as schematically shown in Fig. 3)3.9H2O, 5 mmol), zinc nitrate (Zn(NO3)2.6H2O, with 2.5-x mmol) and cobalt nitrate (Co(NO3)2.6H2O, with x = 0.0, 0.1, 0.2, 0.3 and 0.4 mmol) were dissolved in 25 mL deionized (DI) water, using a magnetic stirrer for 2 h at room temperature (RT). Subsequently, 10 ml ethylene glycol (EG) and 6 M KOH were added into the solution and magnetically stirred for 12 h. The obtained solution was hydrothermally treated in a tightly closed autoclave at 200 °C for 12 h in an oven . The obtained products were rinsed with DI water until they became neutral. Finally, the materials were dried in an oven at 80 °C for 6 h and ground to fine powders for further study.CoxZn1−xFe2O4 (x = 0.0–0.4) NPs were characterized using X-ray diffraction  and Barrett–Joyner–Halenda (BJH) techniques . A potentiostat/galvanostat station  was employed for electrochemical property studies via cyclic voltammetry (CV), galvanostatic charge–discharge (GCD) and electrochemical impedance spectroscopy (EIS) measurements in an aqueous 3 M KOH electrolyte using a three-electrode configuration.The phase and crystal structure of CoxZn1−xFe2O4 (x = 0.0–0.4) NPs as active materials with platinum wire and Ag/AgCl as counter and reference electrodes, respectively. CV testing was done at various scan rates (5–100 mV/s) in a potential range from −0.2 to 0.6 V. GCD measurements were performed at 1, 1.5, 2, 2.5, 3, 4, 5, 7.5 and 10 A/g. The stability of electrodes was evaluated after 1000 GCD cycle testing at 5 A/g. Specific capacitance (Csc) was determined using Eq. , Δt is the charging/discharging time (s), m and ΔV represent mass of active materials (g) and working potential window (V), respectively. The specific energy density  of electrode was directly related to the Csc value and evaluated using Eq.  can be derived from Ed and discharge time t as shown in Eq.  NPs in a 22O4 standard data (JCPDS Card No. 22–1012) within the Fd-3m space group, in agreement with the work of Perumal et al.10 and Mohamed et al.18. Moreover, all these XRD patterns correspond to a cubic spinel structure of crystalline Fe3O4 and CoFe2O4, as well, since these TMOs have the same crystalline structure shown in Fig. 23 These structures illustrate a typical spinel model of Fe3O4, a normal spinel of ZnFe2O4, an inverse spinel of CoFe2O4 and a mixed spinel of CoxZn1−xFe2O4 ferrites, respectively. According to our previous study on the magnetic properties of hydrothermally synthesized CoxZn1−xFe2O4 ferrites24 and similar work of Gözüak et al.25, Mohamed et al.18, Mathew and Juang26 and Feng et al.27, it can be concluded that our hydrothermally synthesized CoxZn1−xFe2O4 (x = 0.1–0.4) NPs are ferrites of a mixed spinel structure. Moreover, the absence of impurities and other secondary phases confirms the purity of all samples and implies that Co ions can be successfully substituted for Zn ions in the ferrite structure. Furthermore, the lattice parameter (a) and average (Av.) crystallite size (DXRD) of all samples were determined by Rietveld refinement using the dominant (220), (311), (400), (422), (511), (440) reflection planes and Scherrer’s formula presented as Eq. . Calculated a and DXRD values of CoxZn1−xFe2O4 NPs are tabulated in Table a and DXRD decrease with increasing Co content, in agreement with the work of Malik et al.28. Slight decreases in a and DXRD with Co loading are subjected to substitution of the smaller ionic radii Co2+ ions  on Zn2+ ions , including the displacement and redistribution of Fe2+ ions  and Fe3+ ions , as reported by Mohamed et al. (2019) in a study of CoxZn1−xFe2O4 NPs18. The oxidation states of Co, Zn and Fe ions will be further discussed with the XANES analysis below.In Fig. , (220), 11, (222)xZn1−xFe2O4 (x = 0.0–0.4) NPs in Fig. vs. Co content is also shown in Fig. 1−xZnxFe2O4 presented the lowest value for x = 0.2 and then increase with Co content (for x = 0.3 and 0.4). This might be dependent on different growth rates. Co cations may be substituted at A and B sites in different amounts during the preparation process. The homogeneous distribution of NPs was suggested to provide an enhanced active surface area with beneficial pathways for the electrolyte penetration and fast ion/electron transfer. This would result in enhanced electrochemical performance, as reported by Feng et al.6.SEM micrographs of CoxZn1−xFe2O4 (x = 0.0–0.4) NPs with insets displaying selected area electron diffraction (SAED) patterns and the histograms of estimated Av. particle size are shown in Fig. 1−xZnxFe2O4 to its lowest value for x = 0.2 and then its increase with Co content (for x = 0.3 and 0.4), as shown in Figs. 2O4 phase, indicating the polycrystalline nature of these materials. Moreover, these indexed diffraction rings have been verified to correspond with a Fd-3m space group, agreeing well with the XRD results and the study of Mohamed et al.18. From the XRD, SEM and TEM results, Co doping can significantly reduce the crystallite and particle sizes of CoxZn1−xFe2O4 (x = 0.0ؘ–0.4) NPs.TEM images of Co−1. The obtained spectra are displayed in Fig. −1 correspond to O–H stretching and bending modes, respectively. This may be due to the absorption of water molecules by KBr pellets during spectroscopic studies. In the case of Co0.3Zn0.7Fe2O4 and Co0.4Zn0.6Fe2O4 NPs, the peak at 1626 cm−1 shifts to a slightly lower frequency upon substitution of Co, implying that both samples are sensitive to moisture and water molecules29. Additionally, the peak at 1435 cm−1 corresponds to the bending modes of anionic carboxylate C-H by ethylene glycol (EG) coated on the surfaces of nanoparticles31. EG can function as a stabilizer that uses surfactant molecules to form coordination bonding or strong interaction. This can prevent aggregation of mixed spinel Co0.3Zn0.7Fe2O4 and Co0.4Zn0.6Fe2O4 NPs. Moreover, it can kinetically control the growth rates of crystals and the morphology of NPs, as reported by Gözüak et al.25. Additionally, it is established that small particle sizes and good dispersion are helpful for electrolyte ions to penetrate the porous surfaces of the electrodes and to provide for high specific capacitance. The spectra at 448 to 562 cm−1 were attributed to the characteristic vibrational modes of metals ion and oxygen atoms in the spinel structure34. Specifically, the wavenumber at 448 cm−1 was ascribed to the stretching mode of metal and oxygen atoms at the B site . However, the same vibration mode at 562 cm−1 was attributed to the stretching mode of metal and oxygen atoms at the A site . Moreover, as the Co content increases, the peak position of vibrational mode at A site slightly shifts to a higher frequency region due to displacement of a heavy atom (Zn) by a lighter atom (Co)35.FTIR measurements were performed over a wide range of wavenumbers from 400 to 4000 cmxZn1−xFe2O4 (x = 0.0–0.4) NPs, as well as Zn foil and ZnO standards. As seen in Fig. 38. For our samples, all the spectra display their maximal gradients in the range of 9660.20–9661.67 eV, implying that the oxidation state of the Zn cation is + 2, in agreement with the work of Wiriya et al.37. In Fig. 4), and Co3O4 standards reveal, respectively, the maximum gradient Co K-edge XANES spectra at energy positions of 7709, 7720.35, and 7726.68 eV. These energy positions correspond to the oxidation states of 0, + 2, and , respectively. Similarly, our samples (x = 0.1–0.4) reveal their maximum gradient Co K-edge XANES spectra at energy positions of 7721.75, 7722.56, 7722.84 and 7722.88 eV, respectively, which are close to that of Co(SO4). Therefore, it is suggested that the oxidation state of the Co cation is + 2 with redistribution of the ions at A- and B-sites in the spinel structure40. In Fig. 4), Fe3O4, and Fe2(SO4)3 appear, respectively, at 7112, 7122.74, 7125.07 and 7127.54 eV, corresponding to the oxidation states of 0, + 2,  and + 3, respectively. In the case of our samples with x = 0.0, 0.1, 0.2, 0.3 and 0.4, the maximum gradient Fe K-edge spectra appear at energy position of 7125.52, 7125.23, 7125.18, 7125.20 and 7125.16 eV, respectively, suggesting oxidation states + 2 and + 3 of Fe cations for these samples. Regarding our results and others related to the studies of Nilmoung et al.2, Wiriya et al.40 and Liang et al.41, Co2+ ions can replace Zn2+ ions at both the A and B sites, while Fe2+ and Fe3+ ions can be distributed at both sites of a mixed spinel CoxZn1−xFe2O4 (x = 0.1–0.4) NPs, as shown in Fig. [A] site [B] site2O4 [Zn2+] [Fe2+/Fe3+]O42-Normal spinel ZnFe2O4 [Fe2+/Fe3+] O42-Inverse spinel CoFexZn1−xFe2O4  O42−Mixed spinel CoFigure 2 adsorption–desorption isotherms of CoxZn1−xFe2O4 (x = 0.0–0.4) NPs. These results were found to be typical type IV isotherms with small hysteresis loops, representing a mesoporous nature1. Clearly, Co0.3Zn0.7Fe2O4 (x = 0.3) NPs exhibit the maximum BET surface area, 84.30 m2 g−1. Other electrodes, with x = 0.0, 0.1, 0.2 and 0.4, show lower surface areas, 37.05, 58.84, 64.38 and 75.31 m2 g−1, respectively. Based on the BJH model and the plot of pore volume vs. pore width shown in Fig. xZn1−xFe2O4 (x = 0.0–0.4) NPs were calculated and found to be 0.01833, 0.02491, 0.2306, 0.03127 and 0.0322 cm3/g, respectively. Moreover, the pore size of CoxZn1−xFe2O4 (x = 0.0–0.4) NPs were determined to be 16.93, 16.90, 14.33, 14.84 and 17.11 nm, respectively. It is clear that Co doping can significantly impact the surface area, pore volume and pore size of CoxZn1−xFe2O4 (x = 0.1–0.4) NPs, and gives evidence of increased active sites and shortened transport paths at electrodes surfaces7.Figure xZn1−xFe2O4 (x = 0.0–0.4) NPs electrodes recorded at a scan rate 5 mV/s over a potential window −0.2 to 0.6 V are shown in Fig. 42. For the ZnFe2O4 (x = 0) electrode, anodic and cathodic peaks appear at around 0.341 V and 0.184 V, respectively, which can be ascribed to stepwise oxidation and reduction of Zn2+ and Fe3+/Fe2+ in a KOH electrolyte, similar to earlier literature reports42. The charging process in ZnFe2O4 normal spinel is due to coupling between Zn2+ at its A site and Fe3+/Fe2+ at the B site. During discharge, Fe3+/Fe2+ and Zn2+ can become distributed among both sites, introducing a reversible redox reaction. However, in CoxZn1−xFe2O4 (x = 0.1–0.4) NPs electrodes, the anodic peaks were shifted from 0.372 to 0.422 V, while the cathodic peaks ranged from 0.186 to 0.139 V with increasing Co content. These anodic and cathodic peaks are related to the Co2+ and Zn2+ ion occupancy at the B site and Fe3+/Fe2+ at both A and B sites. This is a result of redox couples with the assistance of OH− in the KOH electrolyte43. Furthermore, it is observed that Co0.3Zn0.7Fe2O4 (x = 0.3) electrode shows the largest CV area, corresponding to the highest Csc value among these products. Figure xZn1−xFe2O4 NPs electrodes performed at various scan rates from 5–100 mV/s. With increasing Co content and scan rate, the anodic and cathodic peaks reveal increased current values and areas of redox peaks. Optimal values were observed in the x = 0.3 electrode, indicating that the charge storage process is dominated by a diffusion-controlled redox reaction43. Moreover, the anodic and cathodic peaks at all potential values of every electrode were slightly shifted towards higher and lower potentials, respectively. This might be caused by a polarization effect due to an increased Co content. As seen in Fig. 0.3Zn0.7Fe2O4 (x = 0.3) electrode is larger than those of other electrodes at different sweep rates, implying the best electrochemical performance.The CV results of CoxZn1−xFe2O4 (x = 0.0–0.4) NPs electrodes, as shown in Fig. 44 for all electrodes. The GCD results of the Co0.3Zn0.7Fe2O4 (x = 0.3) electrode show the highest specific capacitance among these products, agreeing well with the CV results. Moreover, the GCD curves of all CoxZn1−xFe2O4 NPs electrodes display a very low internal resistance, suggesting good conductivity of electrode materials21.The GCD results measured over a potential window 0–0.45 V at 1 A/g and various current densities of 1–10 A/g were used to determine the capacitive performance of CoCsc) of CoxZn1−xFe2O4 (x = 0.0–0.4) NPs electrodes were calculated with Eq.  electrode exhibits the maximum Csc value, 855.33 F/g, at a current density of 1 A/g, while other electrodes with x = 0.0, 0.1, 0.2 and 0.4 show lower Csc values of 391.51, 530.86, 662.75 and 733.53 F/g, respectively. Figure xZn1−xFe2O4 (x = 0.0–0.4) NPs electrodes over a potential window of 0.0–0.45 V and at various current densities of 1–10 A/g in a KOH electrolyte. It is clearly seen in Fig. 0.3Zn0.7Fe2O4 (x = 0.3) electrode delivers the highest energy density. This enhanced electrochemical performance is suggested to arise from improved electroactive sites with shortened conduction pathways, as well as numerous nanopaths that promote transport of various ionic species due to the presence of Co2+ ions in the structure21. Furthermore, Fig. xZn1−xFe2O4 (x = 0.0–0.4) NPs electrodes after 1000 GCD cycle testing at 5 A g−1. The capacity retention was found to be 54.83, 73.80, 81.63, 90.41 and 82.70% for CoxZn1−xFe2O4 electrodes with x = 0.0, 0.1, 0.2, 0.3 and 0.4, respectively. As can be seen, the Co0.3Zn0.7Fe2O4 (x = 0.3) electrode exhibits better cycling stability among the electrodes. It is suggested that this might be due to more effective contact of the Co0.3Zn0.7Fe2O4 electrode and KOH electrolyte. This promotes simultaneous faster ion/charge transport in the bulk of electrode and at the electrode/electrolyte interface to these achieve excellent properties38. Moreover, this electrode has a higher active area via a uniform distribution of nanosized particles that prevents particle agglomeration, as shown in the SEM, TEM, and BET results. This enhances the electrical conductivity of the electrode10. The highest Csc value obtained in the Co0.3Zn0.7Fe2O4 (x = 0.3) electrode is superior to values reported in the literature on spinel TMOs (AB2O4), as illustrated in Table Furthermore, the specific capacitances  electrode is superior to earlier related work reported in literature on spinel TMOs (AB2O4), as illustrated in Table 0.3Zn0.7Fe2O4 (x = 0.3) electrode prepared by a hydrothermal method, shows a higher Csc value than those obtained in many of TMOs (AB2O4) ferrites. The high specific capacitance of the Co0.3Zn0.7Fe2O4 (x = 0.3) electrode might be due to a homogeneous distribution of nanosized particles and the mesoporous nature of the material, which could promote electrolyte access. Moreover, with the high cycling stability retention of Co0.3Zn0.7Fe2O4 (x = 0.3) electrode, this material is a good candidate for supercapacitor applications. It is remarkable that Co ions in Co-doped ZnFe2O4 NPs can play a significant role in improving the electrochemical properties of these materials. Co ions can influence the interstitial sites of a ZnFe2O4 lattice, which can enhance the electrochemical activities of the electrode material. Moreover, the morphology of nanosized cuboidal shape-particles with good dispersion are supportive of electrolyte ion penetration of the porous electrode surface. This could result in a high specific capacitance. Additionally, Co doping could possibly increase cation transfer  between A and B sites, resulting in more redox couples with the assistance of OH− in KOH electrolyte.The highest xZn1−xFe2O4 (x = 0.0–0.4) NPs electrodes, where the plots of the real part (Z') vs. an imaginary part (Z'') were obtained of a frequency range of 0.01 Hz to 10 MHz using an applied AC voltage of 10 mV. The EIS analysis data for each electrode is given in Table 7. Initially, the Z' axis intercept in the high frequency range represents the solution resistance (Rs) of the 3 M KOH electrolyte and working electrode interface43. The Rs value for each CoxZn1−xFe2O4  NPs electrode was found to be 0.55, 0.67, 0.71, 0.68 and 0.82 Ω, respectively. However, the slight increase of Rs causes a reduction in the conductivity of the KOH electrolyte38. In the next frequency range, where semicircular loops are observed, the diameter of each semicircular loop is related to electron charge transfer resistance (Rct) at the electrode and electrolyte interface, resulting from the Faradaic redox process44. Accordingly, the Rct values were respectively found to be 4.56, 4.46, 3.83, 3.63 and 3.72 Ω for CoxZn1−xFe2O4  NPs electrodes. It is clear that the Rct value of the Co0.3Zn0.7Fe2O4 (x = 0.3) electrode is lower than that of other electrodes, indicating higher electrolytic ion diffusion. This might be due to the homogeneous surface of electrode that could provide area for effective active sites for the OH− ions of the KOH electrolyte, promoting their easy access. This is in good agreement with the lowest Rct value43. Last, in the low frequency range, the observed slope of the straight-line portion of the curve reflects the Warburg resistance (Rw). This represents the ion diffusion process of redox material in the KOH electrolyte38. The increased slope in this frequency range indicates that the materials have become more purely capacitive32. The slope values of CoxZn1−xFe2O4  NPs electrodes were determined to be 4.08, 5.31, 6.75, 7.32 and 5.60, respectively. As seen, the slope value of the Co0.3Zn0.7Fe2O4 (x = 0.3) electrode is highest among the electrodes, suggesting its faster ion transfer from the electrolyte and providing a greater Csc value44.Figure xZn1−xFe2O4 (x = 0.0–0.4) NPs samples were successfully synthesized using a hydrothermal method. All CoxZn1−xFe2O4 NPs have a cubic spinel structure as revealed by XRD. The a, DXRD and Av. particles sizes of CoxZn1−xFe2O4 NPs slightly decreased with increasing Co content . This was due to the substitution of Co2+ ions of smaller ionic radius at the A and B sites in a suggested mixed spinel structure of CoxZn1−xFe2O4 NPs. SEM and TEM images displayed the homogeneous distribution of NPs of a cuboidal shape, which could significantly increase the active surface area of electrodes (supported by the BET results) and beneficially provide optimal penetration paths for fast ion/electron transfer. The results in electrochemical enhancement of electrodes. The presence of Zn2+, Co2+ and Fe2+ and Fe3+ ions in all samples was confirmed by the XANES results. According to the suggested mixed spinel ferrites of CoxZn1−xFe2O4 NPs, Co2+ could replace Zn2+ ions at A and B sites, while Fe2+ and Fe3+ ions could be redistributed at both sites as well. The distribution of these ions could well provide for the Faradaic redox reactions of a pseudo-capacitive characteristic of the materials, as illustrated by the CV, GCD and EIS results. From these results, it is emphasized that Co ions could significantly influence the structure, morphology and electrochemical properties of CoxZn1−xFe2O4 NPs. The highest Csc value, 855.33 F/g at 1 A/g with a 90.41% capacity retention after 1000 GCD cycle testing, was achieved in a working Co0.3Zn0.7Fe2O4 (x = 0.3) NPs electrode. Based on these results, the Co0.3Zn0.7Fe2O4 NPs are suggested as a suitable candidate material for supercapacitor electrodes.Co


The NAP family contains well-characterized and dedicated histone chaperone domain called the NAP domain, and the NAP–histone interactions are key to deciphering chromatin assembly. Our comparative structural analysis of the three three-dimensional structures of NAPs from S. cerevisiae, C. elegans, and A. thaliana in complex with the histone H2A–H2B dimer reveals distinct and diverse binding of NAPs with histones. The three NAPs employ distinct surfaces for recognizing the H2A–H2B dimer and vice versa. Though histones are highly conserved across species they display diverse footprints on NAPs. Our analysis indicates that understanding of NAPs and their interaction with histone H2A–H2B remains sparse. Due to divergent knowledge from the current structures analyzed here, investigations into the dynamic nature of NAP–histone interactions are warranted.Nucleosome assembly proteins (NAPs) are histone chaperones that play a central role in facilitating chromatin assembly/disassembly which is of fundamental importance for DNA replication, gene expression regulation, and progression through the cell cycle. In vitroThe online version contains supplementary material available at 10.1186/s13072-022-00452-9. A nucleosome, the fundamental structural unit of nucleoprotein complexes, is made by the association of DNA with highly basic nuclear proteins called histones. For nucleosome formation, the genomic DNA wraps around a core histone octamer which consists of two molecules of H2A, H2B, H3, and H4 each , 2. ThisHistone chaperones are the extrinsic factors that promote proper interaction/mounting/unmounting of histone on DNA by shielding the basic histones and thus preventing promiscuous interactions that can lead to cell death. Histone chaperones also modulate the availability of histones and histone variants and orchestrate the mounting of histone to facilitate nucleosome assembly/disassembly –8. By meNucleosome assembly protein (NAP) family, which is highly conserved across species from unicellular to multicellular organisms, is well-characterized and is a widely studied histone chaperone . NAP is Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pneumocystis carinii and the malaria parasites Plasmodium falciparum and Plasmodium knowlesi are available  [Caenorhabditis elegans, Arabidopsis thaliana, and Homo sapiens are also known [S. cerevisiae , C. elegans , and A. thaliana  [Several three-dimensional apo structures of nucleosome assembly protein (NAPs) from unicellular eukaryotic fungi like csb.org) , 16–18. so known –22. ThreD: 7C7X) , 20, 23.D: 7C7X) , 24, 25,S. cerevisiae (ScNAP1) in 2006, is seen to be conserved in all NAP structures available  of ScNAP1 are disordered and are not part of the core structure [Plasmodium falciparum  and NAP from Plasmodium knowlesi [NAPs are highly conserved across species and have a dedicated domain known as Nucleosome assembly protein 1-like fold . The ovetructure , 26. Domknowlesi . After tPlasmodium falciparum PfNapL is localized to the cytoplasm while PfNapS is most likely a resident of the parasite nucleus [The NAP family proteins exhibit variation in their nuclear export and localization patterns , 17. The nucleus , 25.S. cerevisiae NAP1 (ScNAP1) in complex with H2A–H2B dimer (from Xenopus laevis) shows the involvement of helices α4–α6 of the first NAP monomer and the helix α8 of the second monomer . HBR1 spans helices α4–α6 (194–205 amino acids) that interact with α1 of H2A, which is the first DNA region out of three binding regions in histone H2A–H2B dimer [. Furthermore, the deletion of ScNAP1 resulted in reduced deposition of the second H2A–H2B dimer required for histone octamer formation [The low-resolution structure (6.7 Å) of 2B dimer . This bi2B dimer . The aci2B dimer . From th2B dimer , 23. Sim2B dimer , 23. How2B dimer , 23. The2B dimer . The bin2B dimer , 30. FurC. elegans NAP1 (CeNAP1) in complex with H2B 1–H2A fusion protein (PDB ID: 6K00 and 6K09) and CeNAP1–H2AZ–H2B complex (PDB ID: 6K0C) are available [The three-dimensional structures of vailable . CeNAP1 vailable .Fig. 3SeA. thaliana NAP1–Related Protein 1 (AtNRP1) in complex with two H2A–H2B dimers (PDB ID: 7C7X) shows a highly distinct mode of AtNRP1 binding to H2A–H2B dimer [A. thaliana and CTAD from human NAP1 showed a conversed binding mode to H2A–H2B dimer [The three-dimensional structure of 2B dimer  as the N2B dimer . Intrigu2B dimer . Additio2B dimer , 31.Fig.C-terminal acidic domain (CTAD) is reported to be involved in histone binding [Our analysis shows that ScNAP1, CeNAP1, and AtNRP1 share ~ 35% sequence similarity but have similar overall structures. Sequence alignment of these NAPs shows relatively conserved NES and NLS sequences Fig. , 3. In a binding , 20, 23, binding . In comp binding , 20. IntX. laevis, C. elegans, and A. thaliana, respectively) highlights high sequence similarity between the histones  are histone chaperones that play an integral part in the production and maintenance of eukaryotic chromatin by facilitating nucleosome assembly/ disassembly. This chromatin assembly is critical for DNA replication, gene expression control, and cell cycle progression. NAPs interact with histone nuclear proteins H2A, H2B, H3, H4, and H1 to mount them onto DNA inside the nucleus. Our analysis of three three-dimensional structures of the NAP–histone complexes reveals distinct modes of histone H2A–H2B recognition by NAPs. Histones also exhibit varied footprints on NAPs. Though NAPs interact with the DNA-binding region of histone H2A and H2B, however, this binding is not consistent in the three complexes. Due to divergence in current complexes in terms of binding modes between NAPs and histones, it is paramount to continue investigations on the overarching mechanism of binding and interaction between NAPs and histones.Additional file: Figure S1: The NAP-histone complexes of ScNAP1, CeNAP1 and AtNRP1. The NAP dimers are shown as surfaces in two orientations. The NAP monomers are colored pink and tan. A ScNAP1; histone H2A–H2B dimer is shown as purple ribbon, B CeNAP1; the histone H2B 1-H2A fusion protein is shown as orange ribbon, and C AtNRP1; the two H2A–H2B dimers bound are shown as blue and green ribbon.


The treatment of osteosarcoma (OS) is still mainly surgery combined with systematic chemotherapy, and gene therapy is expected to improve the survival rate of patients. This study aimed to explore the effect of DEP domain 1 protein (DEPDC1) and kinesin super-family protein 4A (KIF4A) in OS and understand its mechanism. Th expression of DEPDC1 and KIF4A in OS cells was detected by RT-PCR and western blot. The viability, proliferation, invasion and migration of OS cells and tube formation of human umbilical vein endothelial cells (HUVECs) after indicated treatment were in turn detected by CCK-8 assay, EdU staining, wound healing assay, transwell assay and tube formation assay. The interaction between DEPDC1 and KIF4A was predicted by STRING and confirmed by co-immunoprecipitation. The expression of epithelial-mesenchymal transition (EMT)-related proteins, tube formation-related proteins and Hippo signaling pathway proteins was detected by western blot. As a result, the expression of DEPDC1 and KIF4A was all increased in U2OS cells. Down-regulation of DEPDC1 suppressed the viability, proliferation, invasion and migration of U2OS cells and tube formation of HUVECs, accompanied by the increased expression of E-cadherin and decreased expression of N-cadherin, Vimentin and VEGF. DEPDC1 was confirmed to be interacted with KIF4A. Upregulation of KIF4A partially reversed the effect of DEPDC1 interference on the above biological behaviors of U2OS cells. Down-regulation of DEPDC1 promoted the expression of p-LATS1 and p-YAP in Hippo signaling pathway, which was reversed by upregulation of KIF4A. In conclusion, down-regulation of DEPDC1 inhibited the malignant biological behavior of OS cells through the activation of Hippo signaling pathway, which could be reversed by upregulation of KIF4A. Osteosarcoma (OS) is a kind of malignant tumor derived from mesenchymal stem cells and most common in children and adolescents . About 8The DEP domain 1 protein (DEPDC1) is an oncoprotein containing a DEP domain, which has not been detected in 24 normal human tissues, including normal lung tissue, except testicular surface –9 and waDysregulation of kinesin super-family protein 4A (KIF4A) expression can induce mitosis and aneuploid cell formation . The higTherefore, this study aimed to explore the effect of DEPDC1 and KIF4A in OS and understand its mechanism.2.hFOB1.19, U2OS, SaOS-2 and MG-63 cells were obtained from Procell . Human umbilical vein endothelial cells (HUVECs) were provided from American Type Culture Collection (ATCC). All cells were cultivated within the Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C in an incubator under 5% COTo knock down DEPDC1 expression and overexpress KIF4A expression in osteosarcoma cells, short hairpin (sh)RNA targeting ANGPT2 (sh-DEPDC1#1 and sh-DEPDC1#2), pcDNA3.1-KIF4A as well as corresponding negative control (sh-NC) and pcDNA3.1-NC were obtained from RIBIO . When OS cells were reached to about 80% confluence, OS cells were transfected with different vectors using Lipofectamine 3000 according to the manufacturer’s protocol. The transfection efficiency was confirmed by RT-qPCR and western blot 48 h later.−ΔΔCt method [After indicated treatment, total RNA from cells was extracted using Trizol reagent (Invitrogen), and the PrimeScript® RT reagent Kit (Takara) was used to make RNA transcribed into cDNA according to the manufacturer’s instructions. Quantitative real-time PCR (RT-qPCR) was performed with SYBR Green qPCR Master Mix using a 7500 Thermocycler (Applied Biosystems). The relative expression levels of DEPDC1 and KIF4A normalized to GAPDH were calculated by the 2t method .The treated cells were lysed with ice-cold RIPA buffer for 30 min (Beyotime) to obtain the proteins, concentration of which was determined by a bicinchoninic acid protein assay kit (Beyotime). Proteins (20 μg) were isolated with sodium dodecyl sulfate–polyacrylamide gels electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane . After blocked with 5% nonfat milk at room temperature, the membrane was incubated with the primary antibodies including DEPDC1, PCNA, E-cadherin, N-cadherin, Vimentin, VEGF, KIF4A, p-LATS1, LATS1, p-YAP, YAP, p-MST1, MST1 and GAPDH overnight at 4 °C and then incubated with specific horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Finally, the proteins were detected using enhanced chemiluminescence reagents and bands density was analyzed by Image J 1.8.0 software.5 cells/well) were seeded into the 96-well plates and cultured for 24 h. After respective treatment for 24, 48 and 72 h, cells were treated with CCK8 solution (10 μL) and incubated at room temperature for another 4 h. Finally, the absorbance was determined at 450 nm using a microplate reader.Cells (1 × 105 cells/well) for incubation about 24 h. Then, cells were incubated with EdU labeling agent for another 4 h. Next, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then incubated with 0.5% Triton X-100 in PBS for 15 min at room temperature. After PBS washing, cells were stained with anti-EdU working solution at room temperature for 30 min. Finally, cells were incubated with DAPI at room temperature for 30 min, which were then observed by a fluorescence microscope.Cells received respective treatment and seeded into the 96-well plate (1 × 104 cells) and cultured nearly 100% confluence. A sterile plastic micropipette tip was used to scratch a wound along the center line of the well, followed by the incubation for 24 h. The images of the cells were photographed at 0 and 24 h using an optical microscope (Olympus Corporation).After indicated treatment, cells were seeded into the 6‐well plates (5 × 103 cells) was added to the upper chamber coated with Matrigel and the lower chamber was added with 600-μL DMEM containing 10% FBS. After incubation for 24 h at 37 °C, the invaded cells were fixed with 4% formaldehyde fixation and stained with 0.1% crystal violet for 30 min (Beyotime). Subsequently, the invading cells were observed by an optical microscope (Olympus Corporation).After respective treatment, cells were digested by pancreatin and suspended within the serum-free medium. The 200 μL of the cell suspension .g to collect the immunoprecipitates, which was analyzed by western blot.After respective treatment, total proteins in cells were extracted through immunoprecipitation buffer for 30 min on ice. Then, proteins were incubated with anti-DEPDC1 or anti-KIF4A overnight at 4 °C, followed by incubation with protein A/G magnetic beads (Thermo Scientific) at 4 °C for another 3 h. Subsequently, bead-antibody complexes were washed by immunoprecipitation buffer. The above mixture was then centrifuged at 3000P < 0.05 was considered as statistical significance.All data were presented as mean ± SD and GraphPad Prism 8.0.1 software was used to analyze the experimental data. A student’s t test was performed for comparisons between two groups and ANOVA followed by Tukey's post hoc test was carried out for comparisons among multiple groups. The expression of DEPDC1 was upregulated in osteosarcoma cells  compared with that in hFOB1.19 group, and the highest DEPDC1 expression was observed in U2OS cells (Fig. When U2OS cells were transfected with sh-DEPDC1, down-regulation of DEPDC1 inhibited the migration and invasion of U2OS cells (Fig. The images of tube formation of HUVEC are presented in Fig. STRING found a potential interaction between DEPDC1 and KIF4A (Fig. The expression of KIF4A was upregulated in U2OS cells transfected with Ov-KIF4A Fig. A and B. Upregulation of KIF4A promoted the migration and invasion of U2OS cells transfected with sh-DEPDC1 Fig. A and B bDown-regulation of DEPDC1 increased the expression of p-LATS1 and p-YAP, while decreased the YAP expression in U2OS cells, which was reversed by upregulation of KIF4A. The expression of LATS1, p-MST1 and MST1 was not obviously changed in U2OS cells whether or not receiving transfection Fig. .Fig. 7DEIn the current study, we found that DEPDC1 was upregulated in OS cell lines. The down-regulation of DEPDC1 inhibited the viability, migration, invasion, and proliferation of OS cells and tube formation of HUVECs. Our further study clarified that DEPDC1 was interacted with KIF4A. Upregulation of KIF4A could weaken the effect of DEPDC1 interference to improve the viability, migration, invasion, and proliferation of OS cells and tube formation of HUVECs.DEPDC1 has been shown to be highly expressed in most tumors. Amisaki et al. found that DEPDC1 expression was upregulated in hepatocellular carcinoma tissues compared with normal livers, and the high expression of DEPDC1 in tumor tissues was associated with tumor progression and poor prognosis . DEPDC1 Hippo signaling pathway is a highly conserved evolutionarily that regulates organ size and maintains tissue homeostasis by controlling cell proliferation and apoptosis . In receIn conclusion, the expression of DEPDC1 and KIF4A was increased in OS cells. Down-regulation of DEPDC1 inhibited the proliferation, invasion and migration of OS cells and HUVECs tube formation through the activation of Hippo signaling pathway, which could be reversed by upregulation of KIF4A. The expression of DEPDC1 and KIF4A in osteosarcoma tissues and the correlation of them will be investigated in future study.


The XRD data scans showed semicrystalline structures for all PVC/PVP/g-C3N4 polymer blend films. The FTIR and Raman measurements revealed intermolecular hydrogen bonding between the g-C3N4 surface and the OH− groups of the PVC/PVP network. ESEM morphology analysis for PVC/PVP/g-C3N4 nanocomposite films displayed homogeneous surface textures. The data of TGA showed improved thermal stability as the decomposition temperature increased from 262 to 276 °C with the content of g-C3N4 (0.0–1.0 wt%). The optical absorbance data for PVC/PVP films improved after the addition of g-C3N4. The optical energy gaps showed compositional dependence on the g-C3N4 content, which changed from 5.23 to 5.34 eV at indirect allowed transitions. The refractive index for these blend films enhanced (1.83–3.96) with the inclusion of g-C3N4. Moreover, the optical susceptibility for these nanocomposite films increased as the content of g-C3N4 changed from 0.0 to 1.0 wt%. Finally, the values of the nonlinear refractive index showed improvement with the increased percentage of g-C3N4. When g-C3N4 was added up to 1.0 wt%, the DC conductivity improved from 4.21 × 10−8 to 1.78 × 10−6 S/cm. The outcomes of this study prove the suitable application of PVC/PVP/g-C3N4 in optoelectronic fiber sensors.The present work considers the integration of g-C Polymers have been widely used in various applications due to outstanding properties such as their low cost, stability, easy fabrication, etc. The advantages of polymer matrix composites include their low cost and straightforward fabrication processes. Furthermore, polymer composites can be used as the primary material to create lightweight, flexible electronics, which is advantageous considering consumer demand. There are different types of polymers, for example, polyvinyl chloride (PVC) and polyvinylpyrrolidone (PVP). Moreover, polymers have different optical properties which are an essential factor to be investigated since these polymers have wide applications, i.e., energy, photonic, and optoelectronics ,2,3. In 3 for optoelectronics [A variety of procedures are performed on polymeric materials during polymer processing to maximize their utility. As the properties of polymers differ from one type to another, producing a blend of two polymers within a suitable ratio may lead to a polymer blend with combined properties which do not exist in a single polymer. Polymer blends have been used in various applications such as PVA/PVP/Ag film coating , PMMA/PVctronics , and PMMctronics . The prectronics . Doping ctronics , and thectronics . Hydrogectronics . Changinctronics ,6.3 [2O4 nanoparticles affected the physical properties of similar polymer blends [When nanosheets or other nanomaterials are added to the polymer matrix, the properties of the polymers can be changed, or frequently, new qualities are added. Nanosheets can either be produced inside the polymer matrix or synthesized previously and integrated to the polymer for homogeneous composite films. Doping the polymer blends with low percentages of nanoparticles is favorable due to excellent influences compared with conventional composites . In this3 . A previr blends .3C4) has a narrow band gap of 2.7 eV; therefore, it has good light absorption in the visible region [3N4 have comparable but distinct structural similarities. One by one, nitrogen and carbon atoms comprise the hexatomic ring. Every carbon atom forms a large planar network structure owing to covalent bonding with three nitrogen atoms. The g-C3N4 is a common kind of carbon nitride that has been extensively utilized in catalysis [3C4 has high potential to be used in optoelectronic applications, as reported in the literature [3C4 as a polymer dopant was reported. For example, the incorporation of g-N3C4 into the PVA polymer film enhanced its thermal conductivity and electrical insulation [Graphitic carbon nitride  were prepared via solution casting. The polymer blends’ nanocomposites were characterized by advanced techniques, i.e., XRD, Raman, FTIR, SEM, TGA, and optical absorption as well as dielectric measurements. We determined the linear and nonlinear optical parameters of this polymer blend’s nanocomposites when increasing the g-N3C4 proportions.Herein, PVC/PVP polymer blends doped with various g-N3N4 nanosheets were prepared from high-purity urea  by the polycondensation method at 500 °C at a heating rate of 3.0 °C per minute, and the process took 2.0 h [3N4 nanocomosite films, 0.9 g of PVC powder was dissolved in 30 mL of THF for 60 min at 300 K. In addition, 0.1 g of PVP powder was dissolved in 5.0 mL of THF and added to the PVC clear solution. The PVC/PVP mixture was stirred for another 60 min with the addition of 0.0, 0.1, 0.3, 0.6, and 1.0 wt% of g-C3N4 nanosheets. The solution mixture was subjected to ultrasonic waves for 30 min. Finally, the PVC/PVP/g-C3N4 nanocomposite solution was poured inside a polypropylene dish. The nanocomposite films were collected after drying inside an electric oven at 50 °C for 48 h.The g-Cok 2.0 h . The pro3N4 nanocomposite films. The Shimadzu FTIR–Tracer 100 spectrometer  recorded the transmittance vs. wavenumber for the PVC/PVP/g-C3N4 blend films. Quattro environmental scanning electron microscope (ESEM) produced surface morphology scans of the PVC/PVP/g-C3N4 nanocomposite films. A Shimadzu TGA-51 thermogravimetric  analyzer was used for TGA measurements at temperatures 30–600 °C, and the heating rate was 10 °C/min. The Cary 60 UV-Vis spectrophotometer recorded the optical absorption data for the PVC/PVP/g-C3N4 films. The DC conductivity data for the polymer films were completed on two-probe setup at 300 K on an MTZ-35 impedance analyzer.The Shimadzu XRD 7000  diffractometer produced the crystal structure data for the PVC/PVP/g-C3N4, as shown in 3N4 (002) is located at around 27.4°  was used to characterize the polymer PVC/PVP doped with nanosheet g-Cnd 27.4° a, which  PVC/PVP b reflectCI) for PVC/PVP/g-C3N4 nanocomposites, as described below [I24 represents the intensity of the peak at 2θ = 24°, and I18 at 2θ = 18°. Accordingly, the crystallinity index data for PVC/PVP films are 1.01, 1.20, 3.0, and 4.26 at 0.0, 0.3, 0.6, and 1.0 wt% of g-C3N4, respectively. From these data, a slight increase in CI was due to the small content of g-C3N4. The full width at half maximum (β) of a diffraction peak is inversely proportional to the crystallite size (D), as explained by the Scherer equation [λ = 1.54056 Å). As seen in 3N4 content. This, in accordance, led to a decrease in crystallite size.The Segal equation was used to calculate the crystallinity index (ed below :(1)CI=I2equation :(2)D=0.93N4 nanocomposites at different concentrations of nanosheet g-C3N4 are shown in 2 in PVC and the C–O in PVP. A broad absorptions peak at 3370–3420 cm−1 is ascribed to hydroxyl group O–H stretch vibrations, while a sharp absorptions peak at around 1655 cm−1 is assigned to O–H stretch bending vibrations, respectively [−1 are attributed to C-O and C=C vibrations, respectively [−1 correspond to C-H bending vibration out-of-plane [−1, while the C-Cl bonds are ascribed to the bands at 611 and 636 cm−1 [3N4 wt% increases, the position of the broad peak in the pure PVA/PVP spectrum shifts from 3370 to 3420 cm−1. Additionally, the characteristic peak at 1254 cm−1 related to C-N bending vibrations of the pure PVA/PVP shifts to 1290 cm−1. These variations in peak positions are attributed to the intermolecular hydrogen bonding between the g-C3N4 surface and the -OH groups of PVC/PVP, which indicates the success of the interaction between g-C3N4 nanosheets and PVC/PVP polymers. The distribution of potential energy throughout the host polymeric blend’s chains is significantly impacted by this interaction, which also modifies the backbone structure of the blend [3N4 filling-induced structural modification of the polymeric blend’s chains significantly affects the physical properties of the g-C3N4-filled polymeric nanocomposites [An efficient method for characterizing polymeric materials is Fourier transform infrared spectroscopy (FTIR). A unique fingerprint is recognized for a sample concerning the measured spectrum. The FTIR spectra of PVC/PVP/g-Cectively . The obsectively . The banof-plane ,32. The mposites .3N4 are shown in −1. Additionally, the intensity peak at 2915 cm−1 is the result of the stretching vibration of the C-H and C-H2 bonds [−1, while the defect structure of carbon material is related to the bands around 1330 cm−1 [The vibrational, rotational, and other low-frequency modes recorded by Raman spectroscopy allow us to assess the structural properties of materials. Thus, Raman scattering is used in Raman spectroscopy to identify molecular vibrations. The Raman spectra for polymer PVC/PVP doped with different concentrations of g-CH2 bonds . The str330 cm−1 .3N4 in the C-Cl region [Due to the asymmetric stretching vibration, this intensity of quenching validates the interaction between polymer PVC/PVP and nanosheet g-Cl region .3N4 nanosheets. The 2D sheet structure shown in 3N4 materials. Moreover, the image shows that g-C3N4 is stacked flakes. The ESEM surface morphology analyses for the PVC/PVP/g-C3N4 nanocomposite films are displayed in 3N4 and the PVC/PVP blend network.In comparison to an optical microscope, scanning electron microscopy is frequently employed to analyze the sample surface morphology. ESEM images were used to examine the morphology and structure of the synthesized g-C3N4 nanocomposites are displayed in 3N4 changed from 0.0 to 1.0 wt%. Therefore, the increase in g content resists the dehydrochlorination of the PVC/PVP polymer blend and improves the thermal stability.Thermogravimetric analysis (TGA) was used to characterize the thermophysical properties and examine the thermal stability of polymer films. Moreover, TGA is used to measure thermal stability of a sample, the amount of solvent still present, and the amount of water absorbed. This technique measures the mass changes in a sample as a function of time and/or temperature. The thermal stability of a copolymer is typically between that of two homopolymers and varies depending on the copolymer composition. Moreover, TGA analysis has also been used to investigate the effect of filler on a polymeric sample’s thermal stability. The TGA and DTG graphs for PVC/PVP/g-C3N4 increased from 0.0 to 1.0 wt%. Moreover, the residual mass % at 600 °C were 13.14, 17.50, 18.12, and 19.71% for the content of g-C3N4 . All these outcomes prove the enhanced thermal stability of PVC/PVP/g-C3N4 nanocomposites.A second degradative stage observed at temperatures higher than 400 °C belongs to the scission of covalent bonds in PVC/PVP chains . The dec3N4 concentrations of 0.0, 0.3, 0.6, and 1.0 wt%. The absorbance and optical transmittance curves of PVC/PVP/g-C3N4 nanocomposites have been investigated. 3N4 concentrations. 3N4 concentrations. The decrease in optical transmissions may be explained by an increase in the photon scattering due to the dense nanoparticles of the polymer blend [The absorption spectrum of a material is determined using ultraviolet–visible (UV-Vis) spectroscopy in the specified spectral band. This method is especially beneficial for polymers, because both the π-electrons and non-bonding electrons (n-electrons) enclosed in the molecules can absorb from the ultraviolet to visible ranges at the spectrum energy. In this section, we have studied the optical properties of the PVC/PVP polymer blend matrix doped with g-Cer blend .opt) from the variation in αR) vs. wavelength for the PVC/PVP/g-C3N4 blend matrix at different g-C3N4 concentrations. The values of R enhanced when we added 1.0 wt% of g-C3N4, while they reduced for 0.3 and 0.6 wt% of g-C3N4, respectively. We also estimated the refractive index (n) from the optical reflectance shown in n, we use the following Equation [kn increased when we doped different g-C3N4 concentrations  to the PVC/PVP polymer.Equation :(4)n=(1+3N4 nanosheets enhanced the refractive index (n), as shown in 3N4 in the visible light range [3N4 films have a refractive index 1.83–3.96, they are therefore suitable for optical sensors, LEDs, and cladding in optical frequency modulators.The integration of g-Cht range . Many apht range ,47, emisht range  require ht range . As the n, E0, dE, and hE0 and dE which are calculated from the slope and the intercept of the plots in E0 values decrease from 4.21 to 3.14 eV with an increase in the g-C3N4 concentrations (0.0–1.0 wt%), while the dE values enhance (9.88–46.08 eV) with the increase in g-C3N4.Equation (3) represents the refractive index dispersion as a function of photon energy :(5)(n2−1ectively . Table 1n0) is evaluated at From Equation (4), the static refractive index ( follows :(6)n02= is presented as follows [dE is the dispersion energy, and E0 is the single oscillator energy. f values improved when different g-C3N4 concentrations increased.The oscillator strength ( follows :(7)f=EdE3N4 nanocomposite films. The estimations of Nonlinear effects result from high-intensity light propagating across a material. The most basic of these is the Kerr effect, which is defined as a change in the refractive index in proportion to the optical intensity. Moreover, the nonlinear optical parameters such as linear optical susceptibility (elations :(8)χ(1)=3N4 films concerning the pure PVC/PVP. The enhanced optical susceptibility performance of these nanocomposite films after the addition of g-C3N4 nanosheet increase the applicability in optoelectronic devices.The data calculations are listed in 2) are estimated depending on the third-order nonlinear optical susceptibility n22 recorded in 3N4 nanosheet. These data of n2 for the present PVC/PVP/g-C3N4 are higher than those listed in the literature [The values of nterature ,55,56.DC) of the PVC/PVP/g-C3N4 films was measured at 300 K and is plotted in 3N4 was added up to 1.0 wt%, the DC conductivity improved from 4.21 × 10−8 to 1.78 × 10−6 S/cm. These findings indicate that the g-C3N4 nanosheet contributes more free ions and ion pairs to the PVC/PVP polymer blend. Higher charge carriers are observed in the polymer film containing 1.0 wt% of g-C3N4 due to the presence of more π-π∗ bonds. The reduced band gap in the g-C3N4 nanosheet accounts for the increased conductivity [Many factors influence the electrical conductivity of materials. The nature of the materials, their dimensions, the temperature, and frequency of electrical fields are the main factors affecting electrical conductivity. Moreover, the polymer–nanofiller interface contributes to the variation in electrical conductivity. The DC conductivity  after the addition of g-C3N4. Moreover, the optical band gaps for the PVC/PVP/g-C3N4 blend films changed with the integration of g-C3N4 nanosheets. Finally, the data of the nonlinear refractive index (n2) and the third-order nonlinear optical susceptibility (The current work investigated the preparation, structural, and optical properties of g-C


Time-course experiments revealed dKn2-7 was significantly more stable than Kn2-7 in 25% serum, with 78.5% of dKn2-7 and only 1.0% of Kn2-7 remaining after 24 h at 37°C.The optimized method significantly increased peptide recovery and allowed more accurate and consistent quantification of Kn2-7 and dKn2-7 serum stability. Antimicrobial peptides are a new class of molecules being studied for treatment of infections. These peptides can easily be broken down by enzymes present in the body. Removal of the peptides by the enzymes might limit the effect of the drugs against an infection. Our work discusses the importance of testing the stability of these peptides in human serum, a bodily fluid that contains a large amount of enzymes. We describe a method to decrease loss of two potential peptide drugs during sample processing. Further, we report results of testing the stability of these two peptide drugs in human serum. d-amino acid analogue antimicrobial peptides were more stable against host proteases.Peptide extraction was optimized for maximum recovery of antimicrobial peptides Kn2-7/dKn2-7 from serum for LC–MS analysis. Time course experiments revealed  This d-isomer is being tested as a strategy for improving stability of the peptide against proteolytic inactivation. Our recent in vitro studies have shown dKn2-7 has greater antifungal potency against Candida albicans biofilms than Kn2-7 [d-amino acid peptides are less prone to degradation by endogenous fungal proteases than their corresponding L forms. These data indicate that dKn2-7 may also be more stable against host proteases, such as those present in serum and wound exudate.Worldwide overuse of antibiotics has led to multidrug resistance in bacteria . Seriousisolates . Kn2-7 aan Kn2-7 . One posd-amino acid modifications to AMPs that could increase their stability against proteases, and hence their duration of activity in a wound environment, an optimized peptide extraction protocol was developed for LC–MS analysis of Kn2-7 and dKn2-7. This improved protocol was then used to evaluate of the stability of these AMPs in the presence of human serum (wound fluid mimic).Peptide stability in human serum can be studied using liquid chromatography (LC) and mass spectroscopy (MS) techniques. However, the existence of high-abundance proteins and the large dynamic range of protein concentrations make any quantitative analysis of peptides in serum challenging . Furtherd-type amino acid isomer dKn2-7 were purchased from GenScript . Kn2-7 and dKn2-7 stock solutions were prepared in deionized water at 100,000 μg/ml and then stored at -80°C. Kn2-7 and dKn2-7 working solutions were prepared by dilution in deionized water to 1000 μg/ml. Acetonitrile (ACN), methanol and formic acid  were of MS grade. Human serum and Roswell Park Memorial Institute (RPMI) culture medium containing 165 mM MOPS buffer (pH 7.0) were purchased from Sigma-Aldrich, and RapiGest SF Surfactant was purchased from Waters . High performance LC grade water was used throughout the study.Internal standard AMP 1018 (VRLIVAVRIWRR) was purchased from Genemed Synthesis . Kn2-7 (FIKRIARLLRKIF) and its Pilot experiments were first conducted to optimize recovery of peptides from samples. For these pilot experiments, dKn2-7  was mixed with 25% human serum in RPMI culture media. One set of pilot samples was used to determine if incubation of the dKn2-7/serum mixture with a mass spectrometry-compatible surfactant, RapiGest SF surfactant, prior to processing helps reduce losses due to binding of peptides to serum proteins or other factors during precipitation. We also compared ethanol and ACN as precipitants in these experiments. The samples were either incubated with RapiGest SF Surfactant  or vehicA separate set of pilot samples (no RapiGest SF treatment) was used to compare the amount of dKn2-7 loss for two different solvents utilized for serum protein precipitation prior to liquid chromatography-mass spectrometry (LC–MS) analysis, in other words, ethanol  or 1% foAfter parameters were optimized to reduce peptide loss during sample processing, the extent of AMP degradation by proteases in human serum was determined via a time-course experiment. Kn2-7 or dKn2-7  was mixed with RPMI media alone or RPMI containing 25% human serum and incubated at 37°C in a water bath for up to 24 h. At predetermined time points , a 200 μl aliquot of each sample was collected and mixed with 400 μl of 1% formic acid in ethanol. Samples were incubated on ice for 15 min and centrifuged at 18,000 g for 10 min. Supernatants were transferred to fresh 500 μl tubes and stored at -20°C.Samples were diluted eight-times with 3% ACN and 0.1% formic acid in water and spiked with internal standard AMP 1018 ,16 at 1 A two-component LC system was used and consisted of mobile phase A (0.1% formic acid and 10% ACN in water) and mobile phase B (0.1% formic acid in 100% ACN). The flow rate was set at 0.4 ml/min. The ZORBAX Eclipse XDB 80Å C18 column at 40°C was allowed to equilibrate with 100% mobile phase A. Peptides were loaded to the column and washed for 0.5 min with 100% mobile Phase A. Analytes were separated using a linear gradient of 0–90% mobile Phase B for 2.5 min. Between 3.0 and 4.5 min, the mobile phase was set at 90% B. From 4.51 to 7.0 min, the mobile phase was set at 100% A. Retention times for Kn2-7/dKn2-7 and AMP 1018 were 2.44 and 2.40 min, respectively.2) least-squares linear regression analysis of peptide-to-internal standard peak-area ratios versus concentration. AMPs Kn2-7/dKn2-7 and 1018 were quantified with the most abundant multiple reaction monitoring transitions m/z 558.7/120.2 and 385.18/239.1, respectively. The MS/MS settings were: declustering potential (DP) 75 V, collision energy (CE) 39.0 V and collision cell exit potential (CXP) 14.0 V for m/z 385.2/239.1; DP 75 V, CE 45.0 V and CXP 20.0 V for m/z 385.2/115.1; DP 88 V, CE 65.72 V and CXP14.07 V for m/z 558.7/120.0; DP 88 V, CE 108.1 V and CXP 17.0 V for m/z 558.7/103.0. Source and gas parameters were set as: CAD (collision gas) 10 psi (nitrogen); CUR (curtain gas) 20 psi (nitrogen); GS1 (ion source gas 1) 35 psi (nitrogen); GS2 (ion source gas 2) 30 psi (nitrogen); IS (ionspray voltage) 5000 V; TEM (source temperature) 500°C.MS was conducted in multiple reaction monitoring  and posipost hoc Tukey tests with p ≤ 0.05 considered significant. GraphPad Prism software  was used for statistical analyses.For graphs showing LC–MS chromatograms, the two highest peaks for each peptide are included for identification, and Y-axis maximum values were matched to the highest peak intensity. The peak area of the highest peak for each peptide was used for quantification. Data for Kn2-7 and dKn2-7 stability in media or media with human serum over time are from three independent experiments (n = 3) and are presented as the mean ± SD. Statistical significance was determined using two-way analysis of variance (ANOVA) and Pilot experiments were conducted to optimize recovery of peptides from human serum because initial testing using 67% ethanol for protein precipitation as described in a previously published method  indicateIn our pilot experiments, dKn2-7 was mixed with 25% human serum in RPMI culture media. Addition of RapiGest SF Surfactant only modestly improved recovery of dKn2-7 from serum when ethanol was used as the precipitant and did not improve recovery when ACN was used to precipitate peptides. The amount of recovered dKn2-7 doubled from 0.24 μg/mL for ethanol alone A to 0.50The extent of AMP degradation by proteases in human serum was determined by conducting a time-course experiment. Kn2-7 or dKn2-7 at a final concentration of 50 μg/ml was mixed with RPMI media alone or RPMI containing 25% human serum and incubated at 37°C in a water bath for up to 24 h. Results revealed dKn2-7 was significantly more stable than Kn2-7 in human serum , 4 and 5Quantitative LC–MS analysis of AMPs in biological samples is challenging because of the greater reproducibility and sensitivity needed for accurate determination of low levels of small molecules and complexity of the sample components. AMP loss due to NSB in the sample matrix and at container surfaces is a major problem in quantification due to the highly amphiphilic topologies of this class of peptides . PeptideResistance to antibiotics is a continual threat to human health with no adequate solutions on the horizon. Every year multidrug resistant microbes infect millions of people worldwide, with many dying of the infection . As promA reliable and efficient protocol for peptide extraction was successfully developed for LC–MS quantification of antimicrobial peptides Kn2-7/dKn2-7.This optimized method significantly increased the peptide recovery and, therefore, allowed more accurate and consistent quantification of the serum stability of antimicrobial peptides Kn2-7 and dKn2-7.The stability results support our previous hypothesis that the increased antifungal potency of dKn2-7 is related to its higher resistance to proteolytic degradation compared with Kn2-7.Further, the greater stability of dKn2-7 will potentially increase its duration of activity and thus efficacy against biofilms in a wound environment.


The impact of different experimental parameters on supersaturated dissolution–recrystallization and preparation processes of α-CaSO4·0.5H2O was elaborated. At 3.5 mol/L HCl concentration, the dissolution temperature and time were 90 °C and 20 min, respectively. After eight cycles and 5–8 times cycles, total crystallization amount of CaSO4·2H2O was 21.75 and 9.97 g/100 mL, respectively, from supersaturated HCl solution. The number of cycles affected the shape and amount of the crystal. Higher HCl concentration facilitated CaSO4·2H2O dissolution and created a much higher supersaturation, which acted as a larger driving force for phase transformation of CaSO4·2H2O to α-CaSO4·0.5H2O. The HCl solution system’s optimum experimental conditions for HH whiskers preparation involved acid leaching of CaSO4·2H2O sample, with HCl concentration 6.0 mol/L, reaction temperature 80 °C, and reaction time 30 min–60 min. Under the third cycle conditions, α-CaSO4·0.5H2O whiskers were uniform in size, clear, and distinct in edges and angles. The length range of α-CaSO4·0.5H2O whiskers was from 106 μm to 231 μm and diameter range from 0.43 μm to 1.35 μm, while the longest diameter ratio was 231. Purity of α-CaSO4·0.5H2O whiskers was approximately 100%, where whiteness reached 98.6%. The reuse of the solution enables the process to discharge no waste liquid. It provides a new reference direction for green production technology of phosphogypsum.A simple and efficient approach for the high-purity CaSO Therefore, PG utilization may have potential application prospect and, for the preparation of high-purity α-HH whiskers, offers a new reference. There was no solid or water waste discharge during the whole preparation process. This acid-leaching strategy could have interesting potential in producing α-HH whiskers and DH whiskers comprehensively utilizing PG. According to the prediction of 1000 YUAN /1 ton hydrochloric acid, 290.88 kg HH whiskers can be produced. The price of hemihydrate calcium sulfate on the market is CNY 6–10 per kilogram. It is estimated that the profit of treating one ton of phosphogypsum is about CNY 1900. It shows that the scheme has certain feasibility in industry. It provides new thinking for the sustainable development of the wet phosphoric acid (WPA) process industry.We successfully produced high-purity α-HH from PG at atmospheric pressure and low temperature by recycling reaction filtrate. Optimized reaction conditions were as follows: 6.0 mol/L HCl concentrations, 80 °C reaction temperature, and 30 min reaction time. The particle size of α-HH whiskers were uniform, and the surface was bright and smooth, leveled off, clear, and with distinct edges and corners; the maximum length to diameter ratio was 231. The cycle number was three, the length range of α-HH whiskers was from 106 mm to 231 mm, and the diameter range was from 0.43 mm to 1.35 mm, reaching the nanometer scale. It can be used as nanometer calcium sulfate hemihydrate whisker material. Analysis of whiteness was 98.6%. α-HH content reached 100% according to XRD semi-quantitative analysis, and the determined chemical formula was α-CaSO