The as-fabricated ATP@Fe3O4-NH2Cs composite was characterized by Fourier Transform Infrared Spectroscopy (FTIR), Thermal Gravimetric Analyzer (TGA), Scanning Electron Microscope (SEM), Zeta potential (ZP), Vibrating Sample Magnetometer (VSM), Brunauer–Emmett–Teller method (BET) and X-ray photoelectron spectroscope (XPS). A significant improve in the adsorption profile was established at pH 2 in the order of ATP@Fe3O4-NH2Cs(1:3) > ATP@Fe3O4-NH2Cs(1:1) > ATP@Fe3O4-NH2Cs(3:1) > Fe3O4-NH2Cs > ATP. The maximum removal (%) of Cr(VI) exceeded 94% within a short equilibrium time of 60 min. The adsorption process obeyed the pseudo 2nd order and followed the Langmuir isotherm model with a maximum monolayer adsorption capacity of 294.12 mg/g. In addition, thermodynamics studies elucidated that the adsorption process was spontaneous, randomness and endothermic process. Interestingly, the developed adsorbent retained respectable adsorption properties with acceptable removal efficiency exceeded 58% after ten sequential cycles of reuse. Besides, the results hypothesize that the adsorption process occurs via electrostatic interactions, reduction of Cr(VI) to Cr(III) and ion-exchanging. These findings substantiate that the ATP@Fe3O4-NH2Cs composite could be effectively applied as a reusable adsorbent for removing of Cr(VI) ions from aqueous solutions.An efficient composite was constructed based on aminated chitosan (NH One of the most toxic pollutants that pose jeopardy to human health as well as the entire environment is hexavalent chromium (Cr(VI)). Therefore, Cr(VI) has been deemed to be carcinogenic, mutagenic and displayed higher toxicity due to its enormously mobile in the surroundings4. Subsequently, the World Health Organization has authorized that the maximum limit of Cr(VI) in potable water should not exceed 0.05 mgL−16. Despite these mentioned risks, there are number of significant industries mainly based on Cr(VI) including steel, textile, dyeing, cement, electroplating and leather tanneries8. Therefore, several researches have been focused on the removal of Cr(VI) ions from their aqueous solutions via diverse techniques including coagulation9, chemical precipitation10, adsorption12, membrane separation13, catalysis14, ion exchange15 and electrodialysis16. Principally, there are strict criteria to select the appropriate removal technique such as low energy consumption, process simplicity, renewability and the low operational cost17. Accordingly, adsorption can be considered as the most preferred technique to remove Cr (VI) from aqueous solution19. Consequently, a plethora of the adsorbents such as activated carbon, clay materials, polymers, natural products, and metal/mixed oxide nanoparticles have been used for the removal of Cr(VI) from wastewater21.Indeed, the recent prosperity of industries has obvious positive impacts on economic growth; however, disposal of the industrial effluents into the water bodies without reasonable treatment can undoubtedly eradicate mankind23. Owing to its unparalleled merits such as biocompatibility, polyelectrolyte properties, recyclability, hydrophilicity, biodegradability and adhesion properties, chitosan has a significant deal of interest as an efficient cationic adsorbent for removal of heavy metals, pharmaceutical pollutants and organic dyes from their aqueous solutions25. Besides, the presence of the chemically active groups in the chitosan structure such as amino and hydroxyl groups enable the formation of chitosan-derivatives with worthy properties by Schiff base formation, grafting, carboxymethylation, and amination28. For instance, aminated chitosan is a newly-established chitosan derivative with extra amine groups, which is expected to enrich the adsorption characteristics of the native chitosan30. Although all these features of chitosan, it possesses serious drawbacks including low adsorption kinetic, low surface area, high tendency to agglomerate, poor mechanical strength and low adsorption capacity32.Chitosan (Cs) is a cationic polysaccharide polymer that is easily obtained via N-deacetylation of chitin, the essential component of the exoskeleton of crustaceans like shrimp, fungi, crab and insects35. Amongst these clays, attapulgite is a subset of hydrous magnesium aluminum silicate with concrete features including hydrophilicity, non-toxicity, low price, abundant resources as well as its high surface area and high porosity37. Therefore, attapulgite has been vastly utilized in vital fields such as agriculture, catalysis, anticorrosion and wastewater treatment40.Incorporation of clays into chitosan matrices is a feasible solution to overcome its flaws since clays have great features; good adsorptive properties, low cost, high thermal stability, high surface area and special catalytic activity2Cs derivative and ATP clay as well as to allow their beneficial adsorption features to be combined. Herein, ATP@Fe3O4-NH2Cs magnetic composite was successfully synthetized and well-characterized using several analyses tools. Moreover, the aptitude adsorption of the developed adsorbent toward of Cr(VI) ions was achieved using a batch adsorption technique under several studied conditions. Furthermore, isotherms, kinetics and thermodynamics studies were thoroughly studied. Besides, the ability of the developed adsorbent composite to be reuse for ten consecutive adsorption cycles was also examined.Based on aforementioned interests, an attempt was made in this study to fabricate a new adsorbent composite for efficient adsorptive removal of Cr(VI) ions from their aqueous solutions with a highly adsorption performance and better recyclability. Taking advantages of NHPara-benzoquinone , Iron chloride hexahydrate (≥ 99%) and Ferrous chloride tetrahydrate (≥ 99%) were delivered from Sigma-Aldrich (Germany). Ethylenediamine , Glutaraldehyde (25%) and Sodium hydroxide (98%) were brought from Aladdin Industrial Corporation (China). Attapulgite (ATP) was supplied from the Huaiyuan Mining Industry Co., Ltd. (China). Hydrochloric acid (37%), Ammonium hydroxide (99%) and Acetic acid (98%) were acquired from Loba Chemie (India).Chitin (degree of acetylation = 0.94) was purchased from Daejung (Korea), Potassium dichromate (Assay ≥ 99%), 3O4 was made-up by co-precipitation technique41. Exactly, FeCl3·6H2O (0.092 mol) and FeCl2·4H2O (0.046 mol) were dissolved into 300 mL distilled H2O water under N2 atmosphere. After that, ammonia solution (25%) was slowly added to the reaction solution until pH reaches 9 and then the solution kept under magnetic stirring for 80 min at 70 °C. Ultimately, the formed particles were separated utilizing an external magnet, washed with ethanol and ultimately dried at 60 °C for 12 h.Magnetic Fe2Cs was synthesized according to the authors preceding work with a slight modification42.Transformation of chitin to NH2Cs was achieved via three main steps. The first step involves the activation of –OH− groups of chitin in which 8 g of chitin was soaked into PBQ solution  which acts as an activator agent. The reaction mixture was conducted under continuous stirring for 6 h at 60 °C. The resultant activated chitin was washed with distilled H2O to remove the excess of PBQ molecules. The second step includes the formation of amino-chitin, since, and followed by dispersion in EDA solution (6.9 mM) for 6 h under constant stirring at 60 °C. The obtained aminated chitin was separated and washed several times using distilled H2O to remove the unreacted EDA molecules. Finally; the third step involves deacetylation of aminated chitin which was achieved by immersing it in NaOH (50%) solution for 22 h under magnetic stirring at 140 °C. The gotten aminated chitosan (NH2Cs) was filtrated, washed with distilled H2O and dried at 60 °C.NH2Cs was dissolved into 20 mL of acetic acid  aqueous solution under ultrasonic stirring for 45 min. Next, 0.02 g of Fe3O4 was slowly added into NH2Cs solution, and then kept under vigorous stirring for 90 min until the reaction solution became totally homogenous. Then after, an appropriate amount of ATP clay was added to the reaction mixture, and followed by adding 4 mL of glutaraldehyde  solution. The composite mixture was left under continuous stirring at 60 °C for another 90 min. lastly, the resultant composite was separated, washed with ethanol and left overnight for drying at 45 °C. The ATP@Fe3O4-NH2Cs composite was prepared with different weight ratios of ATP and Fe3O4-NH2Cs composite namely; ATP@Fe3O4-NH2Cs (1:3), ATP@Fe3O4-NH2Cs (1:1) and ATP@Fe3O4-NH2Cs (3:1), respectively.A specific amount of NH3O4-NH2Cs magnetic composite was depicted in Fig. A schematic representation for the fabrication of ATP@Fe3O4-NH2Cs composite as well as NH2Cs and ATP clay a Scanning Electron Microscope  was employed under a voltage potential of 20 kV. The examined samples were placed on aluminum stumps and coated with a thin layer of gold via a sputter coating system. The thermal stability was examined under nitrogen atmosphere by Thermal Gravimetric Analyzer , while the temperature was raised from 10 to 700 °C at constant heating rate of 20 °C/min and flow rate of 40 mL/min. In addition, the chemical composition of ATP@Fe3O4-NH2Cs composite was explored by Fourier Transform Infrared Spectroscopy , while the absorbance was scanned in the wavenumber range 500–4000 cm–1. Besides, a vibrating Sample Magnetometer  was utilized for evaluating the magnetic property, while Zeta potential  was utilized to determine the surface charge. X-ray photoelectron spectroscope  was employed for examining the elemental-surface composition of the developed adsorbent. Furthermore, the specific surface area was measure using Brunauer–Emmett–Teller method .To investigate the surface morphologies of developed ATP@Fe3O4-NH2Cs composite. An accurate 0.01 g of ATP@Fe3O4-NH2Cs composite was soaked into 20 mL of Cr (VI) solution with different concentration ranging from 50 to 200 mg/L at a constant stirring speed (200 rpm min−1). To optimize pH medium, pH of Cr (VI) solution was adjusted ranging from 1 to 8 by utilizing an aqueous solution of a strong acid and/or base. Moreover, the effect of ATP@Fe3O4-NH2Cs composite dosage onto adsorption of Cr (VI) was studied in the range 0.001–0.025 g, as well as the temperature effect, was studied in range 25–55 °C. After each experiment, the magnetic adsorbent was separated by an external magnet and the remaining concentration of Cr (VI) was detected via a spectrophotometer at λmax = 540 nm. The adsorption capacity (q) and the removal percent (R%) were calculated from Eqs. o and Ct, are the Cr(VI) initial concentration and its concentration at time t, respectively. While, V and W are the volume of Cr(VI) and the weight of ATP@Fe3O4-NH2Cs composite, respectively.Batch experiments were executed for evaluating the adsorption profile of ATP@Fe3O4-NH2Cs magnetic composite. In brief, the magnetic ATP@Fe3O4-NH2Cs composite was collected after completion the adsorption process by an exterior magnet, and followed by immersing in 25 mL of the desorption medium comprising of Methanol/NaCl solution mixture under stirring for 1 h. After complete the desorption process, ATP@Fe3O4-NH2Cs composite was separated magnetically for reuse for ten consecutive cycles.From the economical point of view, the selection of an efficient adsorbent strongly depends on its recycling characteristic quality. Therefore, recyclability test was executed to assess the reuse aptitude for the ATP@FeAll experiments were conducted in triplicate, and the results obtained were represented as the means corrected by standard deviation (± S.D.).3O4, NH2Cs, ATP and ATP@Fe3O4-NH2Cs composite. The spectrum of Fe3O4 reveals absorption broad at 3437 cm−1 which is ascribed to stretching vibration of –OH− group43. In addition, the detected bands at 1639 and 892 cm−1 are assigned to –OH− bending and vibrating modes, correspondingly44. Furthermore, the observed bands at 557 and 1405 cm-1 are related to Fe–O stretching45. FTIR spectrum of NH2Cs points out absorption bands at 2901, 2216 and 1619 cm−1 which correspond to CH2, COH stretching and N–H bending vibrations, respectively46. Besides, the broad bands at 3441 and 1062 cm−1 which are ascribed to stretching vibration of –OH− and C–N groups, respectively. Additionally, there are two bands at 2907 and 1402 cm−1 which attributed to C–H stretching vibration and in-plane bending vibration, respectively31. The spectrum of ATP shows a peak at 3563 cm-1 belongs to M–OH bonds stretching vibration, where M; Si, Mg and Al. Also, bands at 3404 and 1653 cm−1 are related to vibrations of OH bending and OH stretching, respectively36. Moreover, the band at 484 cm−1 is ascribed to Si–O–Si bond binding vibration39. FTIR spectrum of ATP@Fe3O4-NH2Cs composite elucidates the fundamental peaks of the pristine materials , inferring the successful fabrication of ATP@Fe3O4-NH2Cs composite.Figure 3O4-NH2Cs composite was scrutinized by XPS analysis. The wide-spectrum  and Fe3+ 48. Moreover, the high-resolution spectrum of O1s  significantly increased. Besides, the BET isotherm represents type IV with H4 hysteresis loop, indicating the existence of mesoporous. Moreover, the specific surface area and the total pore diameter were 164.24 m2/g and was 1.50 nm.Figure 3O4-NH2Cs composite is 6.9. Thence at pH < 6.9; ATP@Fe3O4-NH2Cs surface is positively charged due to the protonation of NH2 groups, providing columbic interactions between the positive charges on the ATP@Fe3O4-NH2Cs composite surface and the negatively charged Cr(VI) ions. Contrariwise, beyond pH 6.9 ATP@Fe3O4-NH2Cs composite displays negative charges, causing electrostatic repulsion forces with the anionic Cr(VI).Figure 3O4-NH2Cs(1:3) (96.68 mg/g) > ATP@Fe3O4-NH2Cs(1:1) (77.14 mg/g) > ATP@Fe3O4-NH2Cs(3:1) (69.16 mg/g) > Fe3O4-NH2Cs (65.88 mg/g) > ATP (42.1 mg/g). These results confirm the improvement in the adsorption characters of the three composites comparing with the pristine as a result of the generated synergetic effect between Fe3O4-NH2Cs and ATP clay. On the basis of these results, the synergetic effect of ATP was calculated and listed in Table 3O4-NH2Cs. Moreover, the increase in the adsorption capacity with increasing the Fe3O4-NH2Cs dosage in the adsorption medium is most likely due to increasing the surface positive charges resultant from the extra amine groups of aminated chitosan (NH2Cs). Accordingly, ATP@Fe3O4-NH2Cs was picked out for the subsequent studies.A comparative test was executed to compare the efficacy of the different synthesized composites to determine which ratio has the finest adsorption capacity towards the Cr(VI) ions under the same adsorption conditions. Moreover, the same test was conducted for the pristine materials to trace the improvement in their adsorption behaviors after combination. Figure 3O4-NH2Cs:3  which leads to a decrease in the columbic interactions between the cationic groups of ATP@Fe3O4-NH2Cs composite and the neutral H2CrO4 molecules7. However, at pH 2, there are resilient electrostatic interactions between the protonated NH3+ positive groups on the adsorbent surface and the negative charges of Cr(VI) species. One the other hand, beyond pH 2 there is an anticipated decrease in the number of protonated amine groups on the ATP@Fe3O4-NH2Cs surface. Consequently, the electrostatic interactions between ATP@Fe3O4-NH2Cs composite and Cr(VI) decreases, so the removal percentage and the adsorption capacity directly dwindle from 90.81% and 96.54 mg/g to 38.57% and 39.85 mg/g, respectively58. Based on these results, pH 2 was selected as an optimum pH value for the following adsorption studies.Indeed, pH dominates the form of Cr(VI) in the aqueous solution, where Cr(VI) presents as H3O4-NH2Cs composite dosage from 0.005 to 0.025 g leads directly to a dramatically reduction in the adsorbed quantity of Cr(VI) from 136.07 to 41.34 mg/g, respectively, which may be attributed to the aggregation of ATP@Fe3O4-NH2Cs particles. Contrariwise, the Cr(VI) removal % was gradually increased from 63.17 to 97.17% with increasing the composite dosage as a result of increasing the adsorption active sites on the composite surface59.Figure 3O4-NH2Cs surface with increasing the initial Cr(VI) concentration. On the contrary, Figure . The linearized isotherm equations are listed in Table S161.To deduce the interaction sort between Cr(VI) and ATP@Fe2 values  onto ATP@Fe3O4-NH2Cs composite is 294.12 mg/g, agreeing with the actual maximum adsorption capacity (270.68 mg/g). Moreover, Freundlich constant evinces the favorability of the Cr(VI) adsorption onto ATP@Fe3O4-NH2Cs composite at which n > 2. In addition, Temkin infers that the Cr(VI) ions adsorb onto ATP@Fe3O4-NH2Cs composite via physical adsorption, agreeing with D-R model result since the calculated bonding energy  adsorption onto ATP@Fe3O4-NH2Cs composite requires a low adsorption energy62.The computed Langmuir parameters clarify that q3O4-NH2Cs composite was identified utilizing Pseudo 1st order, Pseudo 2nd order and Elovich  adsorption mechanism of onto ATP@Feich Fig. A–C. The 3O4-NH2Cs best fits pseudo 2nd order based on the R2 values  initial concentration, suggesting the chemical adsorption process of Cr(VI) onto ATP@Fe3O4-NH2Cs composite59. Moreover, the computed Elovich coefficients elucidate that the rate of Cr(VI) adsorption is vaster than the desorption since the α values exceed the β values63.It was concluded that the Cr(VI) adsorption process onto ATP@Fe3O4-NH2Cs composite, the thermodynamics parameter such as, change in entropy (ΔSº), change in enthalpy (ΔHº) and change in free energy (ΔGº) were reckoned from Eqs. e and CAe are the Cr(VI) concentration in the solution and onto ATP@Fe3O4-NH2Cs surface at equilibrium, respectively. R and T are gas constant and adsorption temperature, respectively.To assess the impact of change the reaction temperature on the nature of Cr(VI) adsorption process onto ATP@Fe3O4-NH2Cs is randomness and endothermic process owing to the positive values of both ∆So and ∆Ho that have been reckoned from Van't Hoff Plot  adsorption onto ATP@Fet Figure . Also, ∆3O4-NH2Cs magnetic composite to reuse for several adsorption cycles, ten successive adsorption–desorption processes were executed. Figure 3O4-NH2Cs magnetic composite. This fascinating behavior to the as-fabricated ATP@Fe3O4-NH2Cs composite is may be due to its magnetic property that provides a perfect separation to the composite after the adsorption process without losing in its mass.To evaluate the ability of ATP@Fe3O4-NH2Cs composite and other reported adsorbents in previous studies  ions (294.12 mg/g) and a fast adsorption process as it reaches equilibrium at about one hour. This fascinating adsorption property of ATP@Fe3O4-NH2Cs composite is most likely due to the synergistic effect between ATP and Fe3O4-NH2Cs. Over and above, the extra- amine groups boost the cationic nature of the composite surface, and consequently causing more electrostatic interactions between the anionic Cr(VI) and the positively charged surface of ATP@Fe3O4-NH2Cs composite.To assess the developing behavior of our novel adsorbent, a comparison study was executed between the as-fabricated ATP@Fees Table . Notably3O4-NH2Cs adsorbent was deduced depending on the gained FTIR and XPS data before and after the adsorption process. FTIR spectrum of ATP@Fe3O4-NH2Cs composite after adsorption of Cr(VI)  ions66. Moreover, XPS spectrum of ATP@Fe3O4-NH2Cs composite after the adsorption process  2p3/2 emerged at BE of 586.75 and 577.01 eV, respectively66. While, the peaks at BE of 592.98 and 579.82 eV are related to Cr(VI) 2p1/2 and Cr(VI) 2p3/2, respectively. Furthermore, there is a decline in the intensity of Al2p, Si2p and Cl2p peaks which may be interpreted by the partial exchange of attapulgite metal ions with Cr(VI) and Cr(III), suggesting the ion exchange mechanism67. On the other hand, Fig. 2 and NH groups, respectively. Whereas, the spectrum of N1s after the adsorption of Cr(VI) infers the protonation of the amine group at low pH, since the distinctive peak of NH3+ appeared at BE of 402 eV, suggesting the possibility of electrostatic interaction mechanism between the anionic Cr(VI) ions and NH3+ on the surface of the composite. Furthermore, the wide-spectrum of O1s after the adsorption process   adsorption onto ATP@Fe3O4-NH2Cs composite involve the electrostatic interactions, reduction of Cr(VI) to Cr(III) and ion-exchanging  adsorption onto ATP@FeVI) Fig. A illustring Fig. .Figure 93O4-NH2Cs composite was formulated with different proportions for efficient adsorption for Cr(VI) ions from their aqueous solutions. The utilized characterization tools elucidated the good thermal and magnetic characteristics of the as-fabricated ATP@Fe3O4-NH2Cs composite in addition to its higher surface area. Furthermore, batch adsorption experiments clarified that the best adsorption capacity values were attained at pH 2 and achieved by ATP@Fe3O4-NH2Cs(1:3). Moreover, isotherms studies revealed an analogy between the calculated maximum adsorption capacity under Langmuir isotherm model (294.12 mg/g) and the experimental one (270.68 mg/g). In addition, kinetics studies validated that the adsorption process follows the pseudo 2nd order kinetic model, while the thermodynamic parameters recognized the process to be endothermic, spontaneous and randomness. Furthermore, the results assumed that the adsorption process of Cr(VI) ions occurred via the electrostatic interaction between opposite charges, reduction of Cr(VI) to Cr(III) and ion-exchanging mechanisms. Finally, reusability test proved also the excellent potential of ATP@Fe3O4-NH2Cs adsorbent composite to be reuse for several times, which is a beneficial for its application for removing of Cr(VI) ions from contaminated water. It can be concluded that the as-fabricated ATP@Fe3O4-NH2Cs composite could be applied as sustainable and reusable adsorbent for removing Cr(VI) ions from wastewater.In this study, ATP@FeSupplementary Information.


During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation. Despite their important scientific contributions, most studies have relied on over-expression of reporter constructs, potentially interfering with the processes they are visualizing. The recent advances in gene editing and gene replacement technologies have now made it readily feasible to study and visualize signaling processes of endogenous genes at the single cell level4.Excluding mitosis and cytokinesis, all other cell cycle phases, including the progression from early to late G1 phase, are morphologically difficult to distinguish. Hence, only a limited number of studies have successfully visualized cell cycle progression outside of mitosis in living cells7. p27 is actively degraded in a phosphorylation-dependent and cell cycle-specific manner8. The kinase responsible for initiating p27 degradation is Cdk2 itself9, which generates a positive feedback loop that renders cell cycle progression unidirectional10. Genetic ablation of p27 induces a gene dosage-dependent increase of animal size, due to an increased number of total cells in all organs13. However, gross morphological abnormalities are absent making it feasible to use p27 as a sensor for Cdk2 activation.The gene product of the human CDKN1B locus  binds with high affinity to cyclin A/E:Cdk2 complexes, rendering the complex kinase-inactive14. Moreover, Sanidas et al. both confirmed cyclin D:Cdk4/6 mono-phosphorylation, but also expanded these observations showing that mono-phosphorylation is a code for Rb to find cellular target proteins during early G1 phase15. At the end of early G1 phase, Rb becomes hyper-phosphorylated by activation of Cdk2 to trigger progression into late G1 phase, release of E2F transcription factors and subsequent entry into S phase16. However, if Cdk2 is artificially deleted, due to cellular plasticity, Cdk1 can substitute for Cdk216. Interestingly, although mono-phosphorylated Rb is required to help mediate a correct DNA damage response, mono-phosphorylation of Rb is neither necessary nor required for successful cell cycle entry14, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. In contrast, the continuous presence of cyclin D:Cdk4/6 kinase activity in early G1 phase is essential for cell cycle entry18, though the critical non-Rb substrate(s) remain unknown.We have previously demonstrated that cells entering the cell cycle select for active, mono-phosphorylated Rb by cyclin D:Cdk4/6 complexes that generate up to 14 different mono-phosphorylated isoforms that regulate Rb’s preferential binding to different E2F family members and induce a correct DNA damage responseHere we used CRISPR/Cas9 gene editing and rAAV-mediated gene replacement to genetically tag endogenous p27 (Kip1) with mCherry in hTERT immortalized human retinal-pigmented epithelial cells (RPE1). Using this reporter for cell cycle entry, we performed single cell time-lapse microscopy and identified an essential cyclin D:Cdk4/6 phosphorylation gradient that is integrated with progression into late G1 phase. To reveal the identity of Cdk4/6 substrates, we performed whole proteomic and phospho-proteomic mass spectrometry and identified new early G1 phase cyclin D:Cdk4/6 substrates. Together, our findings demonstrate that G1 cell cycle progression proceeds by cyclin D:Cdk4/6 progressive phosphorylation of non-Rb substrates.2. However, the use of overexpressed reporters can biologically interfere with the very processes that they are intended to visualize. In contrast, endogenous proteins escape this obstacle, but are difficult to study on the single cell level. In an attempt to visualize late G1 phase entry in the most unperturbed way possible, we targeted the genomic locus of p27 in hTERT-RPE1 cells, which are immortalized human retinal pigmented epithelial cells, with a combination of CRISPR/Cas9 and rAAV to induce homology directed repair (HDR). We introduced mCherry-HA into the C-terminus of p27  genomic locus with an enhanced green fluorescent protein (eGFP)  for 48 h. After release from Palbociclib by extensive washing, we recorded single cell eGFP and mCherry signals by time-lapse microscopy over the next 22 h. After normalizing and aligning all single cell traces of eGFP and mCherry, we noted that the initial p27-mCherry intensity rose slightly before the presence of a sharp inflection point at ~ 1.8 h after Palbociclib washout  along with two related proteins, p107 (RbL2) and p130 (RbL3). However, our previous data demonstrated that cyclin D:Cdk4/6 complexes exclusively mono-phosphorylates Rb throughout early G1 phase with no multi-phosphorylation observed until Cdk2 hyper-phosphorylationlib Fig. A. After nce Fig. . Based o14, and artificial phosphorylation of Rb in a test tube with cyclin D:Cdk4/6 results in phosphorylation at all sites, generating hyper-phosphorylated Rb. Furthermore, Rb depletion and/or artificial forced hyper-phosphorylation resulted in premature cell cycle entry, hence, a model that cyclin D:Cdk4/6 progressively multi-phosphorylates Rb, thereby negatively regulating its transcriptional repressor activity, appeared plausible. In line with this, genetic ablation or chemical interference of cyclin D:Cdk4/6 kinase activity results in early G1 cell cycle arrest, emphasizing the importance of cyclin D:Cdk4/6 kinase activity for early G1 phase progression.Rb is a cyclin D:Cdk4/6 substrate with 14 phospho-acceptor sites15. At the early to late G1 transition, Cdk2 performs the inactivating Rb hyper-phosphorylation to release E2F transcription factors, whereas if Cdk2 is artificially deleted, due to cellular plasticity, Cdk1 will substitute for Cdk216. Importantly, while Rb mono-phosphorylation is crucial for a DNA damage response, it is not essential for early G1 phase progression, suggesting additional, yet unknown functions and targets of cyclin D:Cdk4/6 during early G1 phase. We note, however, that entire cell's compliment of Rb is strictly mono-phosphorylated during early G1 phase in all normal and tumor cell types assayed .In direct contrast to this model, we and others have previously demonstrated that cyclin D:Cdk4/6 does not multi-phosphorylate Rb in cells, but strictly mono-phosphorylate Rb on 14 phosphor-acceptor sites, thereby regulating Rb’s binding to the E2F family of transcription factors and a multitude of cellular targetsHere we used CRISPR/Cas9 gene editing and rAAV gene replacement to generate a single cell reporter for the early to late G1 phase transition. Our p27-mCherry reporter leads us to several important conclusions: (1) single cell p27 levels are heterogeneous and independent of cell cycle entry, (2) single cells build up a cyclin D:Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry, and (3) the phosphorylation gradient is time-sensitive.p27 is a bona fide cyclin A/E:Cdk2 inhibitor, whose Cdk2 phosphorylation- and Skp2-mediated degradation induces a positive feedback loop to ensure the irreversibility of cell cycle entry. Over-expression of wild-type p27 blocks cells in early G1 phase with active cyclin D:Cdk4/6 and mono-phosphorylated Rb, demonstrating that an excess of p27 is not tolerated for G1 progression. Our data find the single cell level of p27 to be very heterogeneous with cellular differences of up to sixfold. Nevertheless and irrespectively of the p27 level, all cells entered the cell cycle with similar kinetics, suggesting the presence of a modulating mechanism that orchestrates p27 levels with cell cycle entry timing. Due to the fact that exogenous p27 over-expression blocks cells from S phase entry, we favor the presence of a single cell p27 level threshold, that when reached by intrinsic or external factors prevents cells from cell cycle entry and may lead to permanent cell cycle exit.24. Interestingly, FoxM1 has been characterized to regulate critical G1/S phase genes with multi- or hyper-phosphorylated FOXM1 being the most active24. Despite its proposed function, we failed to identify any FOXM1 phosphor-peptides in our proteomic screen, probably due to low FOXM1 abundance. However, FOXM1 is most likely not the only critical Cdk4/6 substrate. If a FOXM1 mutant, containing phosphor-mimetic residues at all 15 predicted phosphor-acceptor sites, contributes to Palbociclib resistance remains to be seen. However, we wish to emphasize that the textbook model that a cyclin D:Cdk4/6 substrate is progressively phosphorylated until a cell cycle-entering threshold is reached is factually incorrect. In fact, we believe that our previous and present data support this model; however, we conclude that Rb is not the critical progressive phosphorylation substrate of cyclin D:Cdk4/6 during early G1 phase.Rb is exclusively mono-phosphorylated during all of early G1 phase and only hyper-phosphorylated when Cdk2 activity becomes detectable. Hence, we argue that the cyclin D:Cdk4/6 phospho-gradient is Rb-independent. Other cyclin D:Cdk4/6 substrates have been described, among them the FOXM1 transcription factor26. With respect to the identified Cdk4/6-dependent phosphorylation-gradient, we propose its mechanistic linkage may be relevant to one or more of our identified phosphor-proteins. However, at this point in time, we do not yet know the identify of the critical cyclin D:Cdk4/6 targets.While Rb is clearly the most well studied cyclin D:Cdk4/6 substrate, there has been a paucity of cyclin D:Cdk4/6 substrate. However, up until recently, a comprehensive mass spectrometry approach has not been possible due to the lack of specific reagents. The development of Palbociclib has closed this gap. Hence, we performed whole proteome and phosphor-proteome mass spectrometry and compared early G1 phase cells treated either with DMSO or Palbociclib for 3 h. Our approach identified new cyclin D:Cdk4/6 substrates, as well as proteins that experience abundance changes in response to Palbociclib. Among them, we identified 11 canonical Cdk phosphor-acceptor sites that displayed significant down regulation. Gene ontology (GO) analysis revealed a significant increase of genes involved in metabolism. This goes well with the fact that Cdk4 has been linked to several aspects of metabolism, including glucose metabolism and mitochondrial biogenesisHuman hTERT-RPE1 cells were cultured at 37 °C, in 5% CO2 atmosphere and DME/F12 media (Life Technologies), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin (100 μg/ml). For synchronizations, cells were treated with 2 mM thymidine (Sigma-Aldrich), 4 mM hydroxy urea (Sigma-Aldrich), 9 μM RO3306 (Sigma-Aldrich), 15 μM Roscovitine (Sigma-Aldrich) for 24 h prior to release, or with 1 μM Palbociclib  for 48 h if not indicated otherwise. Cells were released by washing four times with full media and subsequently analyzed or treated as indicated.27. For cloning of human p27 left and right homology arms  the following DNA oligonucleotides were used: p27-LHA-f: 5′-GGCGGTCGTGCAGACCCGGGAGAAAGATGTC-3′, p27-LHA-r: 5′-CGTTTGACGT CTTCTGAGGCCAGGCTTCTTGG-3′, p27-RHA-f: 5′-GATCACTAAAGGAGCACGCACT GGAACCCGGGGCC-3′, p27-RHA-r: 5′-CGGCACAATTAGCATACACTGGCTGACGTGTTCCT GGATTGC-3′. Homology arms were amplified from genomic DNA of hTERT-RPE1 cells and cloned into pAAV-SEPT, flanking a C-terminal mCherry-2A-Neomycin tagging and selection cassette19. Infectious rAAV particles were generated as previously described3.A p27-specific gRNA (5′-GTCAAACGTAAA CAGCTCGGT-3′) was designed and cloned into pX330 using previously published online algorithms and protocols, respectively28.For cell cycle analysis, hTERT-RPE1 cells were seeded into either 6-well or 96-well chambers, treated as indicated and imaged using a Nikon ECLIPSE Ti microscope equipped with a CoolLED pE-1 excitation system and a × 20/0.75 air Plan Apo objective (Nikon). For single cell tracing, hTERT-RPE1 cells were seeded into 96-well chambers, treated as indicated and imaged using the high-throughput imaging systems Yokogawa CQ1 or Yokogawa CV7000S (Yokogawa Electric Corporation). Images were acquired by simultaneously recording of 2 × 3 or 3 × 3 positions every 10 or 20 min and automatically stitched and processed by Fiji (version 2.0.0-rc-43/1.50e)29 was used for single cell tracing with the following settings: DoG detector, 20 μm blob diameter with a threshold of 0.5, 15 μm of linking distance, 15 μm of gap-closing maximal distance and 2 frames for maximal gap-closing. Single cell traces were exported as csv-files for further analysis.Images were imported to Fiji and stack converted to binary files with the ‘Li’ method and ‘Dark’ background and through calculation of thresholds for each image. Subsequently, the TrackMate plugin (v2.8.1)30. Lysates were passed through a 21 gauge needle 20 times and sonicated for 5 min to ensure complete lysis. Cellular debris was pelleted via centrifugation at 14,000 RPM for 5 min, and resultant supernatants were used for processing.Cells were lysed in a buffer composed of 3% SDS, 75 mM NaCl, 1 mM NaF, 1 mM beta-glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM PMSF and 1X Roche Complete mini EDTA free protease inhibitors in 50 mM HEPES, pH 8.531. Reduced/alkylated proteins were precipitated by methanol-chloroform precipitation32. Precipitated proteins were re-suspended in 1 M urea, 50 mM HEPES, pH 8.5 for proteolytic digestion. Digestion was performed in a two-step process; proteins were first digested with LysC overnight at room temperature, then with trypsin for 6 h at 37 °C. Digestion was quenched by adding TFA, and peptides were desalted with C18 solid-phase extraction columns as previously described33. Eluate peptide concentrations were determined by BCA assay, and peptides aliquoted into 2 mg portions, which were lyophilized and stored at − 80 °C until phosphor-peptide enrichment.Protein disulfides were reduced with DTT and alkylated with iodoacetamide as previously described35. Briefly, 8 mg of titanium dioxide beads was used per 2 mg of peptide starting material. Enriched peptides were desalted with solid-phase extraction columns as described above, then lyophilized and stored at − 80 °C until they could be labeled for quantitation.Phosphor-peptides were enriched with an immobilized metal affinity chromatography (IMAC) approach as previously described37 as previously described38. TMT reagents, at a concentration of 20 μg/μL in dry acetonitrile were used for labeling. Lyophilized peptides were re-suspended in 30% acetonitrile in 200 mM HEPES, pH 8.5 and 5 μL of the appropriate TMT reagent will be added to the sample. The labeling was conducted for 1 h at room temperature, and then quenched by the addition of 6 μL of 5% hydroxylamine, which was allowed to react for 15 min at room temperature. Labeled samples were then acidified by adding 50 μL of 1% trifluoroacetic acid. Differentially labeled samples were pooled into one multiplex experiment and then desalted via solid-phase extraction as described above, then lyophilized and stored at − 80 °C until fractionation.Phosphor-peptides were labeled with 10-plex tandem mass tag (TMT) reagents39. Lyophilized samples were re-suspended in 5% formic acid/5% acetonitrile and separated on a 4.6 mm × 250 mm C18 column on an Ultimate 3000 HPLC with a fraction collector, degasser and variable wavelength detector. Separation was performed over a linear gradient of 8 to 28% acetonitrile in 10 mM ammonium bicarbonate in 60 min with a flow rate of 0.5 mL/min. In total, 96 fractions were collected and combined in a concatenated manner as previously described33. Resultant fractions were lyophilized and re-suspended in 5% formic acid/5% acetonitrile for identification and quantification by LC-MS2/MS3.Samples were fractionated by basic pH reverse-phase liquid chromatography40. All LC-MS2/MS3 experiments were performed on an Orbitrap Fusion mass spectrometer with an in-line Easy-nLC 1000 with chilled autosampler. Peptides were separated on a home pulled, home packed column . The column was packed first with approximately 0.5 cm of C4 resin  followed by approximately 0.5 cm of C18 resin  and then to a final length of 30 cm with C18 . Peptides were eluted with a linear gradient from 11 to 30% acetonitrile in 0.125% formic acid over 165 min at a flow rate of 300 nL/minute and heating the column to 60 °C. Electrospray ionization was achieved by applying 2000 V at the inlet of the column.All aspects of mass spectrometry experiments performed here were used the same text as previously published40. The mass spectrometer was operated in data-dependent mode, with a survey scan performed over a mass to charge (m/z) range of 500–1200 at a resolution of 120,000 in the Orbitrap. For the MS1 survey scan, automatic gain control (AGC) was set to 5 × 105 with a maximum injection time of 100 ms and the s-lens set to an RF of 60. The most abundant ions observed in the survey scan were subjected to MS2 and MS3 analysis for identification and quantitation respectively. For this analysis, Top Speed mode was utilized, enabling the instrument to acquire a maximum number of spectra in a 5 s experimental cycle. Data collected at MS1, MS2 and MS3 levels were centroided.All aspects of mass spectrometry experiments performed here were used the same text as previously published40. For MS2 analysis, ions above an intensity threshold of 5 × 104 were selected for fragmentation. They were isolated in the quadrupole portion of the mass spectrometer with an isolation window of 0.5 m/z, and then fragmented with collision-induced dissociation with a normalized energy of 30%. Fragment ions are detected with rapid scan rate setting enabled in the ion trap, with an AGC setting of 1 × 104 and maximum injection time of 35 ms.All aspects of mass spectrometry experiments performed here were used the same text as previously published40. MS3 analysis was performed using the synchronous precursor selection setting to maximize sensitivity for quantification of TMT reporter ions36. A maximum of 3 MS2 ions are simultaneously isolated and fragmented for MS3 analysis. An isolation window of 2 m/z was utilized to isolate MS2 fragments, and these were fragmented further with high energy collision dissociation at a normalized energy of 50%. Fragment ions were detected in the Orbitrap at a resolution of 60,000 with a low mass of 110 m/z for MS3 analysis. The MS3 AGC was set to 5 × 104 with a maximum ion injection time of 150 ms. MS2 ions in the range of 40 m/z below and 15 m/z above the precursor (MS1) were excluded from selection for MS3.All aspects of mass spectrometry experiments performed here were used the same text as previously published41. The database was appended to include a decoy database comprised of all protein sequences in reversed order for downstream false discovery estimation43. Search parameters included a 50 ppm MS1 mass tolerance, static modifications of 10-plex TMT tags on lysines and peptide n-termini and carbamidomethylation of cysteines. Variable modifications included oxidation of methionines and phosphorylation of serine, threonine and tyrosine residues. Data were filtered to a peptide and protein false discovery rate of less than 1% using the target-decoy search strategy44. Peptides matching to multiple proteins were assigned to the protein containing the largest number of matched redundant peptides following the law of parsimony34.Data was processed using the ProteomeDiscoverer 2.1 software package. Sequest was used to assign identities to MS2 spectra searching against the Uniprot database of Human entries38. Data were normalized in a multi-step process, whereby they are first normalized to the average for each peptide, and then to the median of all averages. To account for slight differences in amounts of protein labeled, these values were then normalized to the median of the entire dataset and reported as final normalized signal to noise ratios per peptide per sample.TMT reporter ion intensities were extracted from MS3 spectra for quantitative analysis. Spectra used for quantitation had to meet the requirements of greater than 10 average signal to noise per label and isolation interference of less than 25%Movies from microscope were processed and exported as xls files separately for p27 and H2B measures. Files were merged by using a 1100 pixel radius around every H2B data point for every cell. For all H2B data points without a close p27 data point, p27 was set to zero. p27 data points without H2B data points in close vicinity were ignored. All cells without time point zero were filtered out, to avoid complicated alignment algorithms. To normalize for measurement inaccuracy and to fill missing data points each data point for H2B and p27 was smoothed by the median within a +/− 5 time point windows. Next, all cells with a measured time of less than 10 h (60 time points) and all cells with no p27 signal were filtered out. Finally the distribution of H2B and p27 over time per cell was scaled down to values between 0 and 1. All plots show the data smoothed with the median and normalized to 1. To show graphs about trends in the population, all p27 and H2B values for all cells at every specific time point were averaged, using the mean. For defining a cell as cycling an increase of 60% between the lowest H2B and the highest H2B value was required. Cells with no H2B signal increase or less than 60% were defined as not cycling. To calculate the inflection point, the maximum peak in the p27 data was found for every cell. To ensure the global maxima, the 10 time points before the restriction point were required to increase, whereas the 10 time points after the restriction point were required to decrease in signal. To calculate the turning point for the H2B signal the global minima was calculated, requiring the last 10 time points to decrease and the next 10 time points to decrease in signal.For siRNA transfections, 1 μL of a 100 μM siRNA stock solution was mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific Inc.) and Optimem (Gibco) in a ratio of 1:1:100 (100 nM:1 μL:100 μL). siCdk2 and siRB and control obtained from Thermo Fisher Scientific Inc. For plasmid DNA transfections, 2 μg of plasmid DNA was mixed with Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific Inc.) and Optimem (Gibco) in a ratio of 1:3:100 (1 μg:3 μL:100 μL). Both mixtures were incubated at room temperature for 30 min and subsequently added drop-wise to cells. While siRNA transfections were performed in the presence of full media, plasmid DNA transfections were done in the presence of serum-free media for 4 h after which full media was added back.45 using Tris lysis buffer  containing 100 mM NaF, 100 mM β-glycerophosphate, 20 μg/ml RNase A, 20 μg/ml DNase and 1/300 protease inhibitor cocktail  and 1/100 phosphatase inhibitor cocktail #2 . Antibodies used in this study were purchased from the following sources: rabbit anti-Cdk2 , mouse anti-tubulin , mouse anti-Cyclin A , mouse anti-cyclin E , rabbit anti-CDK4 , mouse anti-Rb , rabbit anti-p27  and anti-HA High affinity . Secondary antibodies used for western blot analysis were goat anti-mouse  and, goat anti-rabbit . Mouse anti-tubulin hybridoma cell line (clone #12G10) was developed by J. Frankel and E.M. Nelson under the auspices of the NICHD and maintained by the Developmental Studies Hybridoma Bank.Preparation of lysates and immunoblot analyses were performed as described previouslySupplementary Information 1.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.Supplementary Video 4.Supplementary Information 2.


Bacillus subtilis BsDyP belongs to class I of the dye-decolorizing peroxidase (DyP) family of enzymes and is an interesting biocatalyst due to its high redox potential, broad substrate spectrum and thermostability. This work reports the optimization of BsDyP using directed evolution for improved oxidation of 2,6-dimethoxyphenol, a model lignin-derived phenolic. After three rounds of evolution, one variant was identified displaying 7-fold higher catalytic rates and higher production yields as compared to the wild-type enzyme. The analysis of X-ray structures of the wild type and the evolved variant showed that the heme pocket is delimited by three long conserved loop regions and a small α helix where, incidentally, the mutations were inserted in the course of evolution. One loop in the proximal side of the heme pocket becomes more flexible in the evolved variant and the size of the active site cavity is increased, as well as the width of its mouth, resulting in an enhanced exposure of the heme to solvent. These conformational changes have a positive functional role in facilitating electron transfer from the substrate to the enzyme. However, they concomitantly resulted in decreasing the enzyme’s overall stability by 2 kcal mol−1, indicating a trade-off between functionality and stability. Furthermore, the evolved variant exhibited slightly reduced thermal stability compared to the wild type. The obtained data indicate that understanding the role of loops close to the heme pocket in the catalysis and stability of DyPs is critical for the development of new and more powerful biocatalysts: loops can be modulated for tuning important DyP properties such as activity, specificity and stability. Pseudomonas putida PpDyP with improved catalytic efficacy for lignin-related phenolics, an upshift in the optimal pH to the alkaline range and enhanced resistance to H2O2 inactivation [2O2 biosensors [Pseudomonas fluorescens Dyp1B resulted in variants with increased kcat for 2,6-dichlorophenol and the oxidation of alkali kraft lignin [Biocatalysis is both a green and sustainable technology and redox biocatalysts offer eco-friendly advantages in comparison with conventional chemical reactions due to the selectivity, controllability and economy of their reactions. Lignin is the largest reserve of aromatics on Earth and is a key renewable source of chemicals and materials . Recent tivation , represeosensors . Moreovet lignin .BsDyP from the soil bacteria Bacillus subtilis was identified and characterized [BsDyP enzymatic mechanism was the first among DyPs to be elucidated, revealing that the reduction of Compound II is the rate-limiting step of the catalytic cycle [kcat/Km ≈ 102 M−1 s−1) is far from that observed for synthetic dyes (104 M−1 s−1) and from that required for biotechnological applications. In the present work, BsDyP was tailored to improve its catalytic efficiency for 2,6-dimethoxyphenol (DMP), a model lignin-derived phenolic, using directed evolution (DE) approaches. DE is a powerful engineering tool that mimics the principles of natural selection through iterative rounds of mutagenesis, recombination and screening [In a previous work, cterized . This iscterized ,23,24. Tic cycle . In spitcreening . The procreening ,27,28. HBsDyP has a 45-residue N-terminal twin-arginine signal peptide sequence (MSDEQKKPEQIHRRDILKWGAMAGAAVAIGASGLGGLAPLVQTA) with an Arg-Arg motif, recognized by the twin-arginine translocation (Tat) pathway, involved in the translocation of folded proteins from the cytoplasm and secretion to the extracellular milieu [BsDyP in Escherichia coli resulted in two forms with different molecular masses as assessed by SDS-PAGE (Thermobifida fusca [E. coli YcdB [BsDyP to achieve homogeneous preparations of recombinant enzyme. The heterologous production of the truncated (mature) form of BsDyP resulted in one single band in the electrophoresis gel (BsDyP) was used throughout this work.r milieu ,30. The da fusca  and E. coli YcdB , DyP memesis gel . The truThree rounds of evolution were performed and approximately 6000 clones were screened to identify a variant with improved catalytic efficiency for the lignin-related phenolic DMP . In the BsDyP towards DMP, the wild type and variants 1F9, 3G5, 54D6 and 5G5 were overproduced, purified and characterized. The Reinheitszahl values of purified enzymes vary between 1.6 and 2.5 and the UV–visible absorption spectra of the purified enzymes revealed the characteristic Soret band at 406–407 nm  the catalytic efficiency (kcatapp/Kmapp) remained analogous in the variants as compared to the wild type  increased 10-fold as compared to the wild type, which is reflected in a slightly lower catalytic efficiency (kcatapp/Km) for DMP as compared to the wild type. The lower Km indicates that mutation K317E introduced steric changes in the substrate-binding site(s) that negatively affected DMP binding to the enzyme. Note, however, that the turnover number (kcatapp) is considered the most important parameter for biotechnological applications since bioprocesses usually take place at high concentrations of substrate, i.e., the enzyme’s activity is not limited by substrate concentration but by the turnover number. In this respect, the hit variant 5G5 has a kcatapp for DMP 107-, 56- and 29-fold higher when compared, for example, with class I bacterial Thermobifida fusca TfuDyP (kcatapp = 0.026 s−1), class P P. putida PpDyP (kcatapp = 0.05 s−1) and class V Streptomyces avermitilis, SaDyP2 (kcatapp = 0.097 s−1), respectively [BsDyP evolution to increase the kcatapp parameter to values close to those of fungal counterparts, such as Auricularia auricula-judaeAauDyP, which shows a paramount kcatapp of 89 s−1 for DMP [An optimal pH around 4 was observed for DMP and for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) for all tested enzymes . The actild type . The varild type . Noteworectively ,31,33. 5 for DMP .BsDyP crystal structure  belongs2 and 38–48 Å2, respectively . A water molecule is located approximately at 3.5 Å apart from the iron atom . The open wide cavity gives access to the solvent-exposed heme propionate p6 group and likely represents an electron transfer route from substrates to the porphyrin radical [BsDyP structure PDB 6KMM unveiled the presence of two HEPES molecules: one is bound at 6 Å away from the propionate group in the heme cavity and another at 16 Å near the surface-exposed Y388 residue  with BsDyP PDB 7PKX (this work) shows that a hydrogen bond network comprising solvent molecules and amino acids from loop 1, loop 2 and the small α6-helix . The comparative analysis of available DyPs structures reveals that the loops and α-helix regions delimiting the heme pocket are structurally conserved in all DyPs (AauDyP [K317 (and D314 from loop 2) establishes salt bridges with D250 (and K248) from symmetry-related molecules in the wild-type crystal structure a. E312 fall DyPs . Members  of 1.4 M, and the native state is more stable than the unfolded state by 7 kcal mol−1. The overall structure of the 5G5 variant is 2.0 kcal mol−1 less stable than the wild type and the GdnHCl mid-point decreases to 1.1 M. Variants 3G5 and 54D6 also displayed a lower overall stability similar to 5G5, while variant 1F9 was found to be comparable to the wild type (The thermodynamic stability of the wild type and variants was assessed by probing the unfolding of the tertiary structure using the fluorescence emission of the tryptophan residues (with selective excitation at 296 nm) in the presence of the chemical denaturant GdnHCl . The wilild type .BsDyP and variants was also assessed. It was found that the insertion of mutations decreased the melting temperature  of all variants by 4–5 °C  and E. cloni 10G  were used for routine propagation and amplification of plasmid constructs. E. coli Tuner  and BL21 star  were used to heterologously express the bsdyp gene without signal peptide cloned in pET-21a (+) plasmid  (pVB4) and its variant genes. In E. coli BL21 star and E. coli Tuner, the target genes are under the control of the T7 promotor, induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). Luria-Bertani medium (LB) was used as a routine liquid medium to grow the different E. coli strains, supplemented with appropriate antibiotics when required.bsdyp-A45M-NdeI-F (5′-GACTCATATGAAGCCATCGAAAAAG-3′) and bsdyp-BamHI-R (5′-CGCTGAAAGGATCCTGTAAAGGCTTCTTTTATGATTCC-3′), resulting in plasmid pVB4. The A45M mutation was introduced to create a new starting codon. PCR was carried out in a 50 μL containing 3 ng DNA template, 1 μM of primers, 200 μM of dNTPs, NZYProof polymerase buffer and 2.5 U of NZYProof polymerase . After an initial denaturation period of 5 min at 94 °C, the following steps were repeated for 28 cycles in a thermal cycler : 1 min at 94 °C, 1 min at 57 °C, 2 min at 72 °C followed by a final 10 min period at 72 °C. The amplified products were purified using GFX PCR DNA and Gel Band Purification kit . The final PCR product was digested with NdeI/BamHI , cloned into pET-21a (+)  and transformed into electrocompetent E. coli DH5α cells.The signal peptide present in the N-terminus of BsDyP was identified using Signal P  and remobsdyp wild-type gene was amplified using the forward primer BsDyP-A45M-NdeI (5′-GACTCATATGAAGCCATCGAAAAAG-3′) and the reverse primer DyPBsRv (5′CGCTGAAAGGATCCTGTAAAGGCTTCTTTTATGATTCC3′). The epPCR was performed in a total volume of 50 µL reaction containing 3 ng of DNA template (pVB4), 1 µM of each primer, 200 µM of dNTPs, 4 mM MgCl2, Taq polymerase buffer and 2.5 U of Taq polymerase . The concentration of 0.01 mM MnCl2 was chosen for the construction of libraries of mutants. The PCR program was carried out in a thermal cycler  at the following conditions: 5 min initial denaturation at 94 °C, 28 cycles of 1 min at 94 °C, 1 min at 56 °C and 2 min at 72 °C and a final step of 10 min at 72 °C. The PCR product was purified using Illustra GFX PCR DNA kit , digested with NdeI/BamHI and cloned into pET21a (+) (Novagen). Electrocompetent E. coli BL21 star cells were transformed with BsDyP mutant libraries. The transformed cells were spread into LB solid medium supplemented with 100 µg mL−1 of ampicillin and 0.01 mM IPTG.In the first round of evolution, the bsdyp 1F9 gene: BsDyPFwdGA (5′ CCACCAGTCATGCTAGCCATTTATGATTCCAGCAAACGC 3′) and BsDyPRevGA (5′ TTTAAGAAGGAGATATACATATGAAGCCATCGAAAAAGG 3′). The underlined sequence is the region that will pair with the bsdyp gene template. The epPCR was performed in the presence of 0.1 and 0.15 mM MnCl2, and the annealing temperature was 59 °C. The amplification by PCR of the vector pET21a(+) was performed in high fidelity conditions with the forward primer pET21GA2F (5′ ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTC 3′) and the reverse primer pET21GA240R (5′ ATGGCTAGCATGACTGGTG 3′). The PCR was performed in a final volume of 50 µL reaction containing 3 ng of DNA template (pET21a(+)), 0.5 µM of each primer, 200 µM of dNTPs, Q5 reaction buffer and 1 U of Q5 High-fidelity DNA polymerase (New England Biolabs). The PCR program was carried out in a thermal cycler  at the following conditions: 30 s initial denaturation at 98 °C, 30 cycles of 10 s at 98 °C, 20 min at 63 °C and 3 min at 72 °C and a final step of 10 min at 72 °C. The PCR products were purified using Illustra GFX PCR DNA kit . To avoid the presence of the DNA template, both PCR products were digested with DpnI . The assembly of bsdyp variant genes to the expression vector pET21a(+) was performed using NEBuilder HiFi DNA Assembly Master Mix (NEB) and the mixture was used to transform E. cloni 10G electrocompetent cells (Lucigen). The colonies were washed with LB and the cells recovered by centrifugation at 4 °C, 3220× g, 20 min. The plasmids were extracted using GeneJET Plasmid Miniprep Kit . Electrocompetent E. coli BL21 star cells were transformed with bsdyp mutant libraries and were spread into LB solid medium supplemented with 100 µg mL−1 of ampicillin and 0.01 mM IPTG and plates were incubated overnight at 37 °C.In the second round of evolution and in order to perform ligation through Gibson Assembly , the fol2 for 20 min at 15 °C in a thermocycler. Digestion was stopped by adding 6 µL of 0.5 M EDTA. The PCR reassembly was carried out in a 20 µL reaction volume containing 5 µL of DNA fragments, 200 µM of dNTPs, NZYProof polymerase buffer and 2.5 U of NZYProof polymerase . After an initial denaturation period of 3 min at 96 °C, the subsequent steps were repeated for 45 cycles in a thermal cycler: 1 min at 94 °C, 90 s at 59 °C, 90 s at 56 °C, 90 s at 53 °C, 90 s at 50 °C, 90 s at 47 °C, 90 s at 44 °C, 90 s at 41 °C and 1 min + 5 s/cycle at 72 °C followed by a final 10 min period at 72 °C. The PCR reassembly products were amplified by PCR using the primers BsDyPFwdGA (5′ CCACCAGTCATGCTAGCCATTTATGATTCCAGCAAACGC 3′) and BsDyPRevGA (5′ TTTAAGAAGGAGATATACATATGAAGCCATCGAAAAAGC 3′). The amplified products were purified using GFX PCR DNA and Gel Band Purification kit . Ligation was performed through Gibson Assembly and the constructed library was firstly introduced into E. cloni 10 G electrocompetent cells, the plasmids were extracted and then introduced in E. coli BL21 star. The transformed cells were spread into LB solid medium supplemented with 100 µg mL−1 of ampicillin and 0.01 mM IPTG and plates were incubated overnight at 37 °C.In the third round of evolution, DNA shuffling was performed after amplification of genes coding for variants 3G5, 50F7, 51E9, 54D6 and 56C8 using primers pET21GA2F (5′ ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTC 3′) and pET21GA240R (5′ ATGGCTAGCATGACTGGTG 3′). A mixture containing 280 ng of each parental gene was digested with 0.15 U of DNase I in 200 mM Tris-HCl, pH 7, with 0.2 M MnCl−1 of lysozyme  in 20 mM Tris-HCl, pH 7.6, and the papers were placed at 37 °C for 2 h. After the incubation, the chromatographic papers were soaked in the reaction mixture containing 100 mM sodium acetate buffer, pH 3.8, 1 mM DMP and 0.2 mM H2O2. After this step, the appearance of an orange color at room temperature indicated the presence of enzymatic activity in colonies.Colonies were replica-plated onto chromatography paper  and the original culture-plate was re-incubated at 37 °C until colonies re-appeared (~6 h). The colonies on the filter papers were lysed by soaking the chromatography paper in a solution of 2 mg mL−1 of ampicillin. Four wells in each plate were used to inoculate the parental strain of each generation as a control. Plates were incubated for 6 h at 37 °C, 750 rpm in Titramax 1000-plate shaker , after which 0.01 mM IPTG and 15 µM Hemin were added. Cultures were cultivated overnight and cells were harvested by centrifugation  and disrupted using 3 cycles of freeze and thaw, and pellets were suspended in 100 µL of 20 mM Tris-HCl buffer, pH 7.6, and lysozyme (2 mg mL−1). Plates were centrifuged  and the supernatants were collected (cell crude extracts) and used for enzymatic activity.Colonies that screened positive using the “activity-on-plate” assay were picked from the original culture-plate and transferred to 96-well plates containing 200 µL of LB supplemented with 100 µg mL2O2). The progress of reactions was monitored at 468 nm  using a Synergy2 microplate reader . One unit (U) of enzymatic activity was defined as the amount of enzyme required to reduce 1 µmol of the substrate per minute.To test the enzymatic activity of variant libraries, 20 µL of cell crude extracts was transferred to 96-well plates containing 180 µL of reaction mixture , which were cultivated in 2.5 L of LB medium. Crude extracts were loaded onto a SP-Sepharose column , previously equilibrated with 20 mM Tris-HCl buffer, pH 7.6. Elution was performed by applying a 0–50% gradient with 1 M NaCl in 5 column volumes, followed by a second gradient of 50–100% in 2 column volumes. The active fractions were collected, pooled and concentrated, before applying onto a Superdex 75 HR 16/60 , pre-equilibrated with 20 mM Tris-HCl buffer with 0.2 M NaCl, pH 7.6 [280 = 36,900 M−1cm−1. The heme content was determined by the pyridine ferrohemochrome method using an extinction coefficient of ƐR-O 556 (28.32 mM−1cm−1) [The plasmid pVB4, containing the wild-type , pH 7.6 . The proM−1cm−1) . UV–visi2O2 in Britton–Robinson buffer  and ABTS  were performed in the presence of 0.2 (wild type) and 0.6 mM H2O2 . The rates varying the concentration of H2O2 (0.01–6 mM) were measured in the presence of 4 mM ABTS for the wild type and variants 3G5, 54D6 and 5G5, and 1 mM ABTS for variant 1F9. The enzymatic rates were calculated considering the active fraction of enzyme preparations . The apparent steady-state kinetic parameters were determined by fitting data directly into the Michaelis–Menten equation . All assays were performed at least in triplicate.The pH profiles of the wild type and variants were determined using 1 mM substrate (ABTS or DMP) in the presence of 0.2 mM of HBsDyP crystallization screenings, Structure Screen I and II and JCSG-plus , were performed at 20 °C using the vapor diffusion technique and a Mosquito nano-drops dispenser robot  to simultaneously test 1:1, 1:2 and 2:1 relative volumes of protein and crystallization solution droplets. Needle-shaped orange crystals appeared within three days in 0.05 M potassium phosphate monobasic and 20% (w/v) PEG 8K in a 2:1 protein:crystallization volume ratio. Crystals were optimized using manual handling at micro-liter scale. The best native crystal was obtained in 0.05 M potassium phosphate monobasic and 15% (w/v) PEG 8K. Crystals of BsDyP 5G5 variant were produced in 24 h upon mixing 1 µL reservoir solution with 0.1 M Tris-HCl, pH 8.5, 25% (w/v) PEG 4K, 0.2 M magnesium chloride and 2 µL of protein solution. Native and 5G5 variant crystals were soaked in cryo-protectant solutions, each composed of the corresponding crystallization solution supplemented with 20% (v/v) glycerol prior to flash-cooling to 100 K.Wild-type Wild-type X-ray diffraction data were collected at 100 K at ALBA synchrotron  on the BL13-XALOC beamline equipped with a PILATUS 6M detector and an MD2M diffractometer . 5G5 varBsDyP structure was solved using MORDA [Streptomyces coelicolor DyP (PDB 4GRC) as a search model, while the 5G5 variant structure was solved using PHASER [The solvent contents of each crystal were estimated using the corresponding Matthews coefficients ,63. The ng MORDA  and the g PHASER , within g PHASER ,67, withg PHASER .http://skuld.bmsc.washington.edu/~tlsmd, accessed on 6 January 2020) [a.d.p.s), TLS refinement and automatic water solvent completion with hydrogen bonding distances within 2.45–3.40 Å. Although the refinement included standard stereochemistry libraries [Both structures were refined with PHENIX.REFINE  ,67,69,70ry 2020)  was usedibraries , inter-ao|−|Fc| and |Fo|−|Fc| electron density maps for manual model improvement with COOT [BsDyP and 5G5 variant were deposited in the Protein Data Bank  [Structure refinement was performed in iteratively repeated cycles of protein and solvent updating and refinement, alternated with inspection of σA-weighted 2|Fith COOT . MOLPROBith COOT  was usedith COOT . The anaith COOT  and PyMOith COOT ,78. Caviith COOT . ASA wasith COOT ,81. Figuith COOT ,78. Refist 2021)  with PDB−1 protein in 20 mM sodium acetate, pH 5, were placed onto a thermostatically controlled thermal block and heated at a rate of 1 °C/min up to 90 °C. For the equilibrium unfolding studies, guanidinium hydrochloride (GdnHCl) concentrations in the range of 0–2.5 M in 20 mM Tris–HCl containing 0.2 M NaCl buffer, pH 7.6, were used to induce protein unfolding. The excitation wavelength was 296 nm and the fluorescence emission was recorded between 310 and 450 nm. The stability of enzymes was analyzed based on a two-state process [kin were obtained by linear regression of (ln activity) versus t. Thermal inactivation appears to obey first-order kinetics, and the half-life value (t1/2) was calculated using t1/2 = ln2/kin. All assays were performed at least in triplicate.Thermal denaturation was assessed by steady-state fluorescence measured with a Cary Eclipse Spectrofluorometer  at excitation wavelength of 296 nm and emission wavelength of 330 nm . Prepara process . Thermal process . BrieflyBsDyP was evolved through rounds of epPCR and DNA shuffling followed by high-throughput screening. This led to the identification of the variant 5G5, showing almost 10-fold higher activity for a lignin-related phenolic and improved enzyme production yields. Furthermore, the biochemical and kinetic analysis of intermediate variants and the comparative examination of the X-ray crystal structure of the wild type and variant 5G5 revealed novel insights into the structure–function relationship contributing to advancing the knowledge of enzyme function and evolution in DyPs. It was evidenced that loops around the heme pocket in DyPs account for a local flexibility, which is critical for modulating important enzyme properties such as activity, specificity and stability. The obtained results suggest that it is possible to tune the dynamics of catalytically relevant loops, which might be essential for the improvement or emergence of new catalytic properties in these enzymes. This work opens perspectives to further evolve BsDyP towards the conversion of phenolic compounds in green chemistry and biorefinery fields and to advance fundamental biochemical insights within the DyP-type peroxidase family of enzymes.DyPs are potentially important biocatalysts for industrial oxidations and redox conversions of lignin-related phenolics. In this study,


The final formulation was made to 10 g with water, stirred overnight at 2–8 °C and then adjusted to pH 5.5. (3) Results: Formulations F3 (3% PS20) and F4 (3% PS80) were selected for further evaluation, as their gelation temperatures were near 28 °C. The hydrodynamic particle diameter of clozapine was 18.7 ± 0.2 nm in F3 and 20.0 ± 0.4 nm in F4. The results show a crystallinity change in clozapine to amorphous. Drug release studies showed a 59.1 ± 3.0% (F3) and 53.1 ± 2.7% (F4) clozapine release after 72 h. Clozapine permeated after 8 h was 20.8 ± 3.0% (F3) and 17.8 ± 3.1% (F4). The drug deposition was higher with F4 (144.8 ± 1.4 µg/g) than F3 (110.7 ± 2.7 µg/g). Both sol–gels showed no phase separation after 3 months. (4) Conclusions: Binary PS80-P407 mixed micelles were more thermodynamically stable and rigid due to the higher synergism of both surfactants. However, binary mixed PS20-P407 micelles showed better drug permeation across the nasal mucosa tissue and may be a preferable carrier system for the intranasal administration of clozapine.(1) Background: Clozapine is the most effective antipsychotic. It is, however, associated with many adverse drug reactions. Nose-to-brain (N2B) delivery offers a promising approach. This study aims to develop clozapine-encapsulated thermosensitive sol–gels for N2B delivery. (2) Methods: Poloxamer 407 and hydroxypropyl methylcellulose were mixed and hydrated with water. Glycerin and carbopol solutions were added to the mixture and stirred overnight at 2–8 °C. Clozapine 0.1%  Clozapine is the most effective antipsychotic for treatment-resistant schizophrenia . It is, b-poly(propylene oxide)-b-poly. The high hydrophobic PPO fraction of P407, along with the polymer molecular weight compared to other types of poloxamers, has the capacity to increase the drug loading of water-insoluble drugs through direct solubilization [Well-reported methods to enhance drug solubility include the use of surfactants and solubilizers ,8,9. Pollization . Designing the most suitable sol–gel formulation for the intranasal delivery of clozapine involves balancing a variety of parameters, including the micellarized drug, particle size, lipophilicity, formulation pH and molecular interactions between the drug and other excipients. Herein, we report on the development of clozapine-encapsulated binary polysorbate/poloxamer mixed micelles formulated into a thermosensitive sol–gel, intended for N2B delivery to reduce peripheral ADRs, increase CNS drug bioavailability and improve patient compliance. Accordingly, the aim of the present study was to investigate the feasibility of the optimized formulations to transport clozapine across nasal mucosa tissues in a controlled manner for a sustained therapeutic effect. w/w P407, without clozapine or additives, were prepared using Milli-Q water and evaluated for Tsol/gel. As seen in sol/gel of the formulations increases as the concentration of P407 decreases. Sol–gels that demonstrated Tsol/gel of 28 °C  were selected so that the gelation phase is completed around the nasal temperature of 32–34 °C [w/w P407 were selected for further investigations. Preliminary sol–gel formulations consisting of 13.5–16.5% 32–34 °C ,13. Therw/w P407 solution was 0.4 ± 0.04 mg/mL. Therefore, PS20 or PS80 was used to increase the concentration of clozapine to a final concentration of 1 mg/mL (0.1% w/w). As shown in w/w P407 and 3% w/w PS20, and 0.3 ± 0.2 mV for 15.5% w/w P407 and 3% w/w PS80. The zeta potential of native clozapine particles (as received) was measured to be −1.0 ± 0.5 mV, and may have reduced the overall zeta potential of the drug-encapsulated binary mixed micelles. The saturation solubility of clozapine in 15.5% sol/gel (p = 0.41) in Tsol/gel to those with PS80 at the same concentration. However, the turbidity was significantly lower for formulations with PS20 (p = 0.04). Sol–gels with 1% w/w PS20 (F1) or 1% w/w PS80 (F2) had similar Tsol/gel to those without polysorbate (Control 1 and Control 2). However, the turbidity of Control 2 decreased with the addition of 1% w/w PS20 (F1) and 1% w/w PS80 (F2). The addition of 5% w/w PS20 (F5) or 5% w/w PS80 (F6) had the lowest turbidity. However, both formulations displayed a gelation above the targeted Tsol/gel of 28 °C. F3 and F4 had turbidities ≤25 NTU with Tsol/gels near 28 °C, and were therefore chosen as optimized sol–gel formulations (The addition of PS20 or PS80 in increasing concentrations led to a decreased turbidity and increased Tsol/gel . Formulaulations . The flow characteristics of the optimized sol–gels (F3 and F4) are classified according to the relationship between shear stress and shear rate ,16, and rheogram  below sh−1. These gels require a critical shear stress to break the structure of the gels to start flowing, with F4 having the highest yield stress of 316.5 Pa, followed by F3 at 247.5 Pa and Control 2 at 207.1 Pa. This suggests that PS80 increases the elasticity and mechanical strength of the gel more than PS20, which would be expected to enhance the retention time at the nasal mucosa [−1, the polymer chains disentangle along the direction of the shear, and the polymers start to flow steadily with an increasing shear rate . This is more likely due to the dehydration of the PPO block leading to the aggregation of orderly packed micelles at higher temperatures [The G′ modulus (storage modulus) quantifies the energy stored and recovered after deformation per cycle at a given frequency , and is eratures . The addF4 showed the highest elasticity at the nasal temperature compared to F3, whereas Control 2 showed the lowest . This maw/w was added to the sol–gel platform, stirred using a spatula for 10 s and immediately analyzed. The first endothermic peak (1) in the DSC thermogram . The release rates of clozapine from F3  and F4  were also shown to be significantly slower than that of the clozapine solution (337.9 ± 7.2 μg/cm2 h−1). These findings demonstrate that PS80 increased the rigidity of the formulation, leading to a lower amount of the drug and a slower rate of drug release from the polymer gel matrix at 34 °C. The increased residence time between clozapine and the nasal mucosa is desirable to prevent drug degradation via mucociliary clearance and the rapid turnover of mucus in the nasal cavity [l cavity .2 was highest in the Korsmeyer–Peppas model for both F3 and F4, and was therefore considered to be the best model , apparent permeability (p = 0.02), diffusion coefficient (p = 0.02) and cumulative clozapine permeated at 8 h (p = 0.002). Based on the data in The permeability parameters for the 0.1% clozapine solution (control), F3 and F4 are listed in p = 0.001), 6 h  and 8 h , but not at 2 h . This could be due to a lag phase, where no drug was permeated in the first 0.5 h and the controlled diffusion of clozapine from the polymer gel matrix occurred through the tissues 2 h post-application. The lower permeation of F4 may be attributed to its higher drug retention in the mucosal tissues.As shown in Clozapine has a low water solubility of 11.8 µg/mL at 25 °C ,39,40. T100PO65EO100) contains a PPO/PEO composition ratio of 0.34 and has a critical micelle concentration (CMC) of 0.7% w/v at 25 °C, 0.1% w/v at 30 °C and 0.025% w/v at 35 °C (11). P407 sol–gels are negative thermoresponsive polymers [P407  of PS20 is higher than PS80, which makes PS80 more favorable to the PPO units of P407.Oleic acid in PS80 has more carbon atoms, thus producing more hydrophobic interactions with the PPO core of P407. However, the double bond at C9 was reported to form weaker interactions with micelles due to steric hindrance . On the The addition of PS20 or PS80 markedly improved the drug loading, micellar packing, increased stabilization and mechanical strength of the P407-based sol–gel formulations. Formulation F4 was found to be more elastic than F3 at the nasal temperature due to the higher synergism of binary PS80-P407 mixed micelles, producing more thermodynamically stable and rigid micellar sol–gels than PS20-P407. The variations in encapsulation, drug release and drug permeation recorded between F3 and F4 did not consider the particle size, PDI and zeta potential, as they were not significantly different. Using the Korsmeyer–Peppas model, our results showed a non-Fickian, anomalous behavior release of clozapine from the sol–gel at 34 °C, which involved a combination of mechanisms, including polymer swelling and drug diffusion. The permeation of clozapine in F3 through the nasal mucosa tissue was shown to be better, with more of the drug retained within the tissue post-administration of F4. Therefore, binary PS20-P407 mixed micelles in a sol–gel system may be a preferable carrier system for intranasal clozapine delivery to the brain compared to binary PS80-P407 mixed micelles.2·2H2O), sodium chloride (NaCl) and methanol were of analytical grade and purchased from Sigma-Aldrich . Orthophosphoric acid was purchased from Thermo Fisher Scientific  and potassium dihydrogen orthophosphate anhydrous was purchased from Chem-Supply . Milli-Q water was used as a formulation vehicle.Clozapine (>98% purity) was purchased from Adooq Bioscience . P407, hydroxypropyl methyl cellulose (HPMC E4M) and glycerin were purchased from Sigma-Aldrich . PS20, PS80 and carbopol 934 NF were purchased from PCCA . Hydrochloric acid (HCl), sodium hydroxide (NaOH), potassium chloride (KCl), calcium chloride dihydrate  for 6 h at 2–8 °C. At the same time, 0.1% w/w clozapine was stirred in 1%, 3% or 5% w/w PS20 or PS80 at room temperature before being mixed with the polymer solution, and the final weight of the formulation was made up to 10 g with Milli-Q water and stirred thoroughly (400 rpm) overnight at 2–8 °C. The final sol–gel mixture was adjusted to pH 5.5 ± 0.2 with 0.1 M HCl or 0.1 N NaOH.The sol–gels were prepared using the cold method  as descrw/w P407 and stirred overnight (400 rpm) at 2–8 °C. Samples were centrifuged at 12,000 rpm at 4 °C for 45 min using a refrigerated centrifuge . The supernatant liquid was collected and filtered using a 0.45 µm, 25 mm PTFE Syringe Membrane Filter , and the concentration of clozapine was measured using HPLC [An excess amount of clozapine was added to 15.5% ing HPLC .2·2H2O 0.32 mg/mL with Milli-Q water, and was adjusted to pH 5.5 ± 0.1 with 0.1 M HCl and 0.1 N NaOH [SNF was prepared by dissolving KCl 1.29 mg/mL, NaCl 7.45 mg/mL and CaCl1 N NaOH ,48.sol/gel), G’ modulus, G’’ modulus and loss tangent (tan δ) of the sol–gels were evaluated using oscillatory measurements, with a temperature ramp between 8 °C to 40 °C and ramp rate of 5 °C/min at 1.0 Pa (stress) and 1.0 Hz (frequency). The dynamic viscosity of the sol–gels was measured using flow measurements, with a temperature ramp between 8 °C to 40 °C and ramp rate of 5 °C/min at 2.0 rad/s (angular velocity). The flow behavior of the sol–gels was evaluated at 4 °C (storage temperature) and 34 °C , with a flow ramp between 10 s−1 to 1000 s−1. The strength of the gels at nasal temperature was recorded using the G’ modulus as a measurement of stiffness. All measurements were carried out in triplicate and the resultant curves were generated directly from the manufacturer’s computer TRIOS software .Rheological evaluations of all sol–gel formulations were performed using a Discovery Hybrid Rheometer HR-3  with a 40 mm parallel plate geometry and a sample gap of 200 μm. The gelation temperature . The relative clarity of the sol–gels was measured in triplicate.The turbidity of the sol–gels when in liquid form was performed using a Hach TU5200 EPA Turbidimeter  measuring in nephelometric turbidity units (NTU) with a Class 2 laser product and 650 nm (EPA 0.43 mW) optical light source. The sol–gels were left at room temperature and any air bubbles were removed before evaluation. The scattered light was collected at a 90° angle to the incident light and 360° around the sample vial. The turbidity sensor was calibrated using three StablcalThe hydrodynamic particle diameter, PDI, and zeta potential of clozapine in the binary polysorbate/poloxamer mixed micelles were measured using dynamic light scattering (DLS) with Zetasizer Nano ZS . Diluted samples (1:10) were pipetted into disposable cuvettes for particle size and PDI measurements, and disposable DTS1070 folded zeta cells  for zeta potential measurements. The detection scattering angle was set at 173° (25 °C) with equilibration time at 60 s. Measurements were carried out in triplicate and recorded as the mean ± SD of three independent runs.v/v o-phosphoric acid) in the ratio of 65:35 (v/v). The flow rate was 0.2 mL/min and the retention time of clozapine was found to be 3.3 min. The concentration of clozapine was calculated using a standard calibration curve (R2: 0.9998) over the concentration range of 1–50 μg/mL. The lower limit of detection (LOD) was 0.03 μg/mL and the lower limit of quantification (LOQ) was 0.1 μg/mL.The concentration of clozapine was analyzed using a RP-HPLC equipment  equipped with a low pressure quaternary gradient pump, along with a dual wavelength UV detector (at 234 nm), auto sampler (10 μL injection volume) and column oven (maintained at 25 °C). The chromatographic data were processed using LC solution 1.24 SP1 software. The quantification analysis was performed under isocratic conditions with a C18 column . The mobile phase consisted of methanol and phosphate buffer  at −102 °C (50 Hz) and ≤20 mT for 24 h. The powdered sol–gels of approximately 4–5 mg were transferred into aluminum crucibles and the thermal analysis of the sol–gels was carried out using a Mettler Toledo TGA/DSC 2 equipment . Heating runs were performed under compressed air, with a heating rate of 10 °C/min over 25–600 °C (49). STARe software was used to generate the thermal curves by measuring heat output (DSC) and mass loss (TGA) as temperature is increased.X-ray diffraction was carried out to identify phases in the powdered sample, with data collected using a Bruker D8 Advance MKII XRD X-ray diffractometer  equipped with a Cu source, a LynxEye detector and operated at 40 kV and 40 mA. Diffraction patterns were recorded by continuous scans from 5 to 45° 2Ɵ, with a step size of 0.04° and 15 rpm rotation at a scan rate of 0.4° s per step. The resulting patterns were imported into Diffrac EVA version 5.1, where phases were identified using the PDF-4 2020 ICDD database .w/w) was used as control, prepared in 1 mL 0.1 M HCl and made up to 10 g with Milli-Q water (adjusted to pH 5.5 with 0.1 N NaOH). The donor compartment was filled with 1.5 mL of the optimized sol–gel or clozapine solution, and the receiver compartment was filled with 12 mL of SNF. Snake skin dialysis membranes  with a 3.5 kDa MWCO were soaked in SNF for 15 min prior to experimentation. Membranes having effective areas of 1.5 × 1.5 cm2 exposed to the test formulations/solutions were then mounted between the donor cap and receiver body. The temperature of the chamber was kept at 34 ± 1 °C with standard stirring speed. Samples of 0.5 mL were collected from the receiver compartment at 0, 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h, and replaced with fresh SNF of equal volume after each sampling. The samples were placed in a −80 °C refrigerator until analysis using HPLC. The release study experiments were performed in triplicate.In vitro drug release studies were performed using a Logan DHC-6T vertical Franz diffusion apparatus . Clozapine solution , and the receiver compartment was filled with 12 mL of SNF to ensure sink conditions. The temperature of the chamber was kept at 34 ± 1 °C with standard stirring speed. The tissues were allowed to stabilize for 30 min prior to loading of the optimized sol–gels. Sample aliquots of 0.5 mL were collected from the receiver compartment at 0, 1, 2, 4, 6, 8 h and replaced with fresh SNF of equal volume after each sampling. The samples were placed in a −80 °C refrigerator until analysis using HPLC. The experiments were carried out in triplicate and the data were fitted into Fick’s second law of diffusion equation to determine the permeability parameters of clozapine in the sol–gels across the sheep nasal mucosa, where Ct is the cumulative drug permeated at time t, dC is concentration of clozapine in the donor chamber (0.5 mg), K is the partition coefficient (log P) of clozapine, L is the diffusion path length and D is the diffusion constant, which was calculated using Equation (3) [Ex vivo drug permeation of the optimized sol–gels was carried out using Franz diffusion apparatus using excised sheep nasal mucosal tissues ,50, whiction (3) ,51.(2)CappP, was calculated using Equation (4), and the steady state flux, Jss, was calculated using Equation (5), where S represents the cross-sectional area of flow.The apparent permeability coefficient, C0 is the initial concentration of clozapine released  and K0 is the zero-order constant.The in vitro and ex vivo drug release data were fitted to mathematical models, including zero-order, first-order, Hixson–Crowell and Korsmeyer–Peppas, to predict the kinetics and release mechanism of clozapine from the sol–gel polymer matrix . For zerK1 is the first-order constant.For first-order kinetics, Equation (7) was used to describe the release of clozapine from the polymer gel matrix, where HCK is the Hixson–Crowell constant.Equation (8) illustrates the release of clozapine using the Hixson–Crowell model, where F represents the fraction of drug released at time t, tM is the amount of drug released at time t, ∞M is the amount of drug released at infinity, mK is the Korsmeyer–Peppas constant and n is the diffusion or release component [n = 0.5 (Fickian Case I), the drug release is driven by diffusion, and when n = 1 (non-Fickian Case II), the drug release is governed by polymer swelling or relaxation of polymeric chains. When 0.5 < n < 1 , the drug release process is indicated by both diffusion and swelling of the polymer matrix. If n > 1 (non-Fickian Super Case II), the drug release is said to be due to the tension and breaking of the polymer [For Korsmeyer–Peppas kinetics, drug release can be described using Equation (9), where omponent . In this polymer .F = (Mt2) values were used to verify the release of clozapine from the sol–gel formulations, and the highest correlation coefficient was considered to be the best model.The obtained regression , room temperature (25 °C) and elevated temperature (40 °C). Phase separation was determined after refrigerated centrifugation  at one, two and three months for each temperature to observe for time-dependent changes in drug content .t-test to determine statistical differences between individual means. In all analyses, a two-tailed p-value < 0.05 denotes significance. All analyses were performed using GraphPad Prism v9.The data analysis was measured using