In this study, we developed an osteoplastic material based on collagen–fibronectin hydrogel impregnated with siRNA molecules targeting glycogen synthase kinase 3β (GSK3β), which inhibits the osteogenic differentiation of mesenchymal stem cells. The hydrogel impregnated with polyplexes containing siRNA GSK3β and polyethylenimine has been shown to have no cytotoxic effect: there was no statistically significant change in the cell’s viability after 7 days of incubation in its presence compared to the control group. On days 2 and 7, an increase in the level of expression of markers of osteogenic differentiation was observed, which confirms the osteoinductive qualities of the material. It has been demonstrated that the hydrogel maintains cell adhesion. Our results obtained in vitro indicate cytocompatibility and osteoinductive properties of collagen–fibronectin hydrogel impregnated with siRNA GSK3β molecules. Glycogen synthase kinase-3β (GSK3β) is one of the regulators of osteogenic differentiation that inhibits signaling of the its main pathways, BMP and WNT/β-catenin, by phosphorylating Smad1 and β-catenin molecules and inducing their subsequent ubiquitination and proteasomal degradation . FurtherGSK3β inhibitors due to their relatively small size. Lithium chloride (LiCl) was the first GSK3β inhibitor discovered and is still actively used in many studies. However, this molecule has several other biological targets that lead to undesirable phenomena and a fairly small therapeutic window . Most of the other small molecules are patented or are at the stage of preclinical studies. In addition, natural ways of regulating the activity of GSK3β in human cells using the mechanism of RNA interference are known.Small molecules are often used as SMAD7 gene transcript. microRNA-29a, directed at DKK1, Kremen2 and sFRP2; microRNA-218, the targeted genes of which are Dkk2, sFRP2 and Sost, as well as microRNA-335-5p, targeted at DKK1, contribute to osteogenic differentiation in vitro [PPAR-γ, Noggin, GNAS1, Chordin, GSK3β, B-cell lymphoma (Bcl)-like 2 (BCL2L2), and also genes involved in bone resorption processes—RANK, Plekho1 or Semaphorin 4d (Sema4d), promotes the osteogenic differentiation in in vitro or in vivo models [microRNA molecules participate in the positive and negative regulation of osteogenesis at all stages of signal transmission, affecting the mRNA of ligands, receptors, signaling molecules and transcription regulators involved in the implementation of cell differentiation. The use of microRNA-590-5p mimics and leads to the degradation of the in vitro ,5,6,7,8.o models ,11. However, the regulation of signaling pathways by means of the above-listed small molecules or microRNA molecules can lead to pleiotropic effects, since such compounds do not have a high selectivity and may have several targets at once ,13,14. TGSK3β gene expression at the mRNA level using siRNA molecules in order to increase the efficiency of osteogenic differentiation of mesenchymal precursors was offered for the first time. For this purpose, it is proposed to impregnate siRNA GSK3β molecules into a biocompatible polymer material for a targeted delivery to the bone defect area and prolonged release to ensure the long-term inhibition of the target gene expression.In this work, an approach based on decreasing the Collagen-fibronectin hydrogel was chosen as a promising carrier matrix for siRNA molecules. Xenogeneic collagen materials have a high biocompatibility, as opposed to materials based on other components of the extracellular matrix (ECM), such as hyaluronic acid, chondroitin sulfates and elastin .The aim of this investigation was to study the cytocompatibility and osteoinductive properties of the collagen–fibronectin hydrogel impregnated with siRNA GSK3β molecules to obtain an effective osteoplastic material.GSK3β mRNA using a siRNAfit program [For the purpose of the knockdown of  program , develop program . The siRPolyplexes, containing siRNA and linear polyethylenimine (PEI) with a molecular weight of 25 kDa, pH = 4.5  in a 1:3 ratio (µg siRNA: µg transfection agent) were formed in Dulbecco’s phosphate-buffered saline (DPBS)  according to the manufacturer’s instructions for 35–40 min at 37 °С .®, cat.PA100, “Imtek Ltd.”, Moscow, Russia) was mixed with human fibronectin  in a ratio of 1:3 by volume to obtain 100 μL. To impart osteoinductive properties to the material, polyplexes consisting of 300 pmol (1.5 pmol/μL) or 600 pmol (3 pmol/μL) siRNA GSK3β and PEI in a ratio of 1:3 (µg siRNA: µg PEI) in 50 μL DPBS  were added to the collagen–fibronectin mixture. A polymerization of hydrogels was performed for 2 h at 37 °C after the addition of 50 µL buffer ANB-12 pH = 11  to obtain 200 μL/sample. Finished samples of materials were stored at 4 °C for no more than 24 h.To obtain one sample of the material, a sterile neutral 10% porcine collagen type I solution at a concentration of 40 mg/mL  siRNA-B or polyplexes containing 60 pmol (0.3 pmol/µL) siRNA-B and PEI in a ratio of 1:3 (µg siRNA: µg PEI) were impregnated into collagen–fibronectin hydrogel at the gelation stage according to the procedure indicated in clause 2.3. The prepared hydrogels were placed in 96-well plates and infused with 200 µL of PBS-Tween  for 1 h, changing the solution for 3 h. Next, the solution, presumably containing siRNA-B, was incubated with 1.88 µg/mL of streptavidin conjugated with siRNA-B peroxidase for 15 min to form a biotin–streptavidin–peroxidase complex. The detection of the biotin–streptavidin–peroxidase complex was carried out by ELISA. The results were analyzed using an “EnSpire Multimode Plate Reader” .The human mesenchymal stem cells derived from human adipose tissue (hAD-MSCs) cultures at passages 2–3 were used in this work. hAD-MSCs cultures were obtained from tissues of healthy donors who signed an informed consent to participate in this study.2. The culture medium was changed every 3 days.The cells were cultured in a growth medium of Dulbecco’s modified Eagle’s medium (DMEM)  with 10% fetal bovine serum (FBS) , 100 mg/L amikacin , 4 mM L-glutamine , 10 µg/L FGF-2  and 2000 IU/L heparin sodium  at 37 °С and 5% СО® Space  and “CytoFLEX S” , and packages “FloMax® Software v2.3”  and “CytExpert Software v2.0” , respectively. Histogram overlays were performed with FlowJo™ Software v10.7.1 .Immunophenotyping of three hAD-MSCs cultures was performed to confirm the cellular phenotype. Immunophenotyping was performed using antibodies to a positive cell-surface markers of MSCs: CD29+ , CD44+ , CD90+ , and also “Human Mesenchymal Stem Cell Multi-Color Flow Kit” , including antibodies to positive and negative markers labeled with fluorescent dyes . To dete® 24-well permeable support with a pore diameter of 8 microns  in 1 mL of Opti-MEM medium with 5% FBS  for 24 h. As a negative control, hAD-MSCs were used, which were cultured in the equivalent to experimental groups’ amounts of Opti-MEM medium with 5% FBS.A total of 300 (1.5 pmol/μL) or 600 pmol (3 pmol/μL) siRNA-C as part of polyplexes, consisting of PEI in a ratio of 1:3 (µg siRNA: µg PEI), were added to the prepared collagen solution. Next, the mixture of collagen with siRNA were incubated in an atmosphere of ammonia vapor for 1 h. Upon completion of the polymerization process, the materials were rinsed with sterile distilled water and incubated with cells when the culture reached a monolayer confluency of 70–80% in Transwell® Space  flow cytometer and FloMax® Software v2.3  package. Files were concatenated and analyzed in FlowJo™ Software v10.7.1 .The efficiency of the cell transfection in suspension was evaluated using CyFlow® Multimode Plate Reader , the absorbance at 570 nm; the background value at 620 nm was subtracted.The cytotoxicity of collagen–fibronectin hydrogel, separately and impregnated with polyplexes containing 1.5 pmol/µL siRNA GSK3β on days 1, 4 and 7 after transfection, was examined using MTT assay  by quantTo assess the ability of the material to maintain cell adhesion, hAD-MSC cultures were incubated in the presence of collagen–fibronectin hydrogel separately, and they were impregnated with 1.5 pmol/µL siRNA GSK3β and PEI in a ratio of 1:3 (µg siRNA: µg PEI) for 24 h.To visualize the total number of cells on the surface of the materials, they were pre-stained with a vital red-fluorescent dye PKH26  for general cell membrane labeling according to the manufacturer’s instructions. A suspension of labeled cells was applied to samples of the material at a concentration of 1 million/mL and incubated for an hour in a 24-well plate in 100 µL FBS  for their attachment. Then, Opti-Mem medium with 5% FBS up to 1 mL was added to the wells and incubated under standard culture conditions. As a negative control, plate wells without the material were used. After 24 h, the medium for cell cultures was replaced with DMEM , containing 4 mM L-glutamine , 10% FBS, 100 mg/L amikacin .To visualize living and dead cells after 1 and 3 days of incubation, cells on the surface of materials were stained with Calcein AM dye  for 40 min and DAPI dye  for 10 min at a concentration of 1 µg/mL. Digital images of stained cells were obtained and processed using an inverted microscope, AG Axio Observer D1, with an AxioCam HRc camera and ZEN software v3.0 .GSK3β and osteogenic differentiation induction, hAD-MSCs were incubated in the presence of collagen–fibronectin hydrogel impregnated with polyplexes containing 1.5 pmol/μL of siRNA GSK3β and PEI in a 1:3 (µg siRNA: µg PEI) ratio. The incubation of the material was carried out when it reached a monolayer confluency of 70–80% in Transwell® 24-well permeable support with a pore diameter of 8 microns  in 1 mL of Opti-MEM medium with 5% FBS  for 24 h.In order for the knockdown of the gene hAD-MSCs and hAD-MSCs transfected with an equivalent amount of siRNA-C were used as negative controls, and hAD-MSCs transfected with an equivalent amount of siRNA GSK3β in the same culture plate were used as positive controls. The culture medium was replaced immediately after transfection and every 3 days with DMEM , containing 10% FBS, 4 mM of L-glutamine  and 100 mg/L of amikacin .GSK3β gene and osteogenic differentiation marker genes were evaluated on the 2nd and 7th days of the experiment.With a positive result in transfection after 24 h (the efficiency of siRNA-C molecule transfection is more than 90%), the expression of the GSK3β gene and osteogenic differentiation marker genes: ALPL , Runx2, BMP-2 (bone morphogenetic protein 2), OCN  and OPN (osteopontin). Target genes of the mRNA expression level were normalized by the average expression of the reference genes of GAPDH  and ACTB (β-actin) ; the synthesis of the first chain of total cDNA on RNA matrix was conducted using the “RevertAid kit” . A quantitative real-time PCR analysis was performed on a “CFX96 Touch™” amplifier  using intercalating “SYBR Green I” dye  and primers specific to the β-actin) .t-test when comparing 2 groups. A p-value <  0.05 was considered statistically significant. Data with a normal distribution are presented as average ± SD.The statistical processing of the data and graphing was performed using GraphPad Prism 8.0.1 . Intergroup differences were determined using the Holm–Šidák test when comparing 3 or more groups and the unpaired Student’s Osteoplastic material, based on collagen–fibronectin hydrogel and impregnated with siRNA GSK3β, was produced for the first time . The matFor all cell cultures obtained from human adipose tissue, an immunophenotype corresponding to hAD-MSCs was confirmed for positive  and negative cell-surface markers  . Below aCollagen–fibronectin hydrogel provided a prolonged release of siRNA molecules during 4 days of the experiment. An evaluation of the release of 0.3 pmol/mL of siRNA-B molecules and polyplexes containing 0.3 pmol/mL of siRNA-B and PEI (1:3) from collagen–fibronectin hydrogel showed that the release from polyplexes during the first 2 h of the experiment occurs on average 6.6 times faster than the release of siRNA-B molecules without a transfection agent. Within 24 h, 1.8 times more siRNA-B molecules in polyplexes were released from collagen–fibronectin hydrogel—34.27 ± 2.5%  more than siRNA-B molecules without a transfection agent—18.7 ± 2.38%. After 4 days, the number of released siRNA-B molecules was equal in both experimental groups and amounted to 49.27% ± 13.12% for the group of siRNA-B molecules in polyplexes and 52.5 ± 18.3% for the group of siRNA-B molecules without a transfection agent . It can It is known that in the presence of PEI, plasmid DNA and siRNA molecules condense and compact, resulting in forming polyplexes with a common positive charge, capable of interacting with negatively charged heparan sulfates and other proteoglycans on the cell surface ,24,25,26The transfection efficiency of hAD-MSCs was 96.08 ± 2.25% and 98.19 ± 0.54%, respectively, during an incubation with hydrogels containing 1.5 and 3 pmol/µL siRNA-C. No statistically significant differences were detected when using these concentrations .For further experimental studies with the aim of impregnation in collagen–fibronectin hydrogel, a lower effective concentration of siRNA was chosen—1.5 pmol/μL.Collagen–fibronectin hydrogel separately and impregnated with 1.5 pmol/μL siRNA GSK3β molecules had no cytotoxic effect and maintained hAD-MSC adhesion. Cell viability in the presence of collagen–fibronectin hydrogel did not significantly differ from the values of the control group and amounted to 93.31 ± 1.59% on day 1, 102.37 ± 0.68% on day 4 and 97.99 ± 7.52% on day 7 of the experiment. Cell viability in the presence of collagen–fibronectin hydrogel impregnated with siRNA GSK3β also did not significantly differ from the values of the control group and amounted 95.06 ± 0.17% on day 1, 100.27 ± 5.23% on day 4 and 102.69 ± 3.13% on day 7 of the experiment .Most of the cells placed on the material acquired a spindle-shaped morphology and continued to be vital after a day . Thus, tGSK3β gene decreased by 63.3 ± 1.75%.On day 2 after incubation with collagen–fibronectin hydrogel impregnated with siRNA GSK3β molecules, the expression of Runx2 gene in the presence of collagen–fibronectin hydrogel impregnated with siRNA GSK3β molecules increased by 1.6 ± 0.3 times (p = 0.0392).The expression of ALPL by 2.3 ± 0.2 times (p < 0.0001), BMP-2 by 3.2 ± 1.4 times (p = 0.0136), OCN by 1.5 ± 0.6 times (no significant) and OPN by 3.6 ± 0.6 times (p < 0.0001)  .GSK3β gene remained at the level of 37.8 ± 4.32%. The expression of the Runx2 gene increased by 1.6 ± 0.5 times.On day 7 after incubation with collagen–fibronectin hydrogel impregnated with siRNA GSK3β molecules, the expression of ALPL by 20 ± 2.4 times (p < 0.0001), BMP-2 by 1.2 ± 0.1 times (no significant), OCN by 1.4 ± 0.4 times (no significant) and OPN by 3.2 ± 0.3 times (p < 0.0001) (The incubation of hAD-MSCs with collagen–fibronectin hydrogel impregnated with siRNA GSK3β molecules again led to an increase in the expression of gene  0.0001) .Runx2 gene increased by an average of 3.4 times and the expression of the remaining osteogenic differentiation marker genes increased 2–8-fold and 1.5–3-fold.Compared to the previous term, the expression of the GSK3β gene using gene-targeted siRNA molecules. The osteoinducing effect of collagen–fibronectin hydrogel impregnated with siRNA GSK3β at a concentration of 1.5 pmol/µL was observed, which indicates the preservation of functional activity of siRNA GSK3β molecules in the osteoplastic material.Thus, the efficiency of osteogenic differentiation in hAD-MSCs cultures can be increased by a knockdown of the Osteogenesis is a dynamic and complex process that begins as early as in the first month of fetal development. Its regulation involves many proteins included in different signaling pathways, including Wnt/β-catenin and BMP/SMAD, as well as TGF-β, which activates transcription factors Runx2 and Osterix, insulin/PI3K/Akt, Hedhehog and many others .GSK3β in vivo enhances the cartilage degeneration in a mouse model of posttraumatic osteoarthritis. This is due to an increase in the expression of MMP-10 and MMP-14 (the main collagenase activators) and a decrease in TIMP-3  [GSK3β by small molecule CHIR99021 is the intracellular accumulation of β-catenin, where it induces the gene transduction important for osteoblast differentiation, while at the same time suppresses the chondrocyte differentiation [GSK3 knockout leads to a serious decrease in the growth of the endochondral bone, as well as the premature remodeling of the growth plate. Articular cartilage remains unchanged at the same time [The inhibition of canases) . The conntiation . The GSKame time .GSK3β leads to an increase in the expression of osteogenic Alp, Runx2, Ocn and Col1a1 [GSK3 inhibitor AZD2858 increases the level of β-catenin after a short period of time. This drug may be effective as an anabolic strategy, since it has extensive bone-forming effects in predominantly trabecular areas [GSK3β inhibitor TDZD-8 reduced bone resorption [It is also known that the inhibition of d Col1a1 . One of d Col1a1 . The admd Col1a1 . The trear areas . Fracturar areas . Stem cear areas . In a mosorption . This wasorption .Prior studies have implicated the contribution of collagen and fibronectin to the proliferation of MSCs, their survival under stress and their good cell adhesion on the surface of such materials, as well as their osteogenic differentiation . Even coIn this work, using MTT assay, the great cytocompatibility of the collagen–fibronectin hydrogel separately impregnated with polyplexes containing siRNA GSK3β molecules was detected. The ability of materials to adhere cells on their surface is extremely important and a fundamental condition for their subsequent proliferation, migration and differentiation . The firPreviously, biomaterials impregnated with siRNA molecules aimed at osteogenic differentiation inhibitor genes were described in several papers, such as the article by Noggin et al. The use of such biomaterials led to the effective knockdown of target genes, induction of osteogenic differentiation and reduction of bone tissue regeneration time in vitro and in vivo models, respectively ,47,48. FWe have developed an osteoplastic material based on collagen–fibronectin hydrogel impregnated with siRNA GSK3β molecules; its osteoinducing effect has been confirmed in in vitro experiments. The material is capable of thermal curing at 37 °C for 2 h, and it has a gel-like consistency in the uncured state and a good plasticity in the hardened state. pH values of the material at the final stages of the thermal curing process were 7.3–7.4, which correspond to the pH of the internal environment of the human body. It is shown that all components and the integral composition of the osteoplastic material have a high biocompatibility. A high ability of adhesion and cell proliferation on the surface of the material has been demonstrated, which is important for ensuring its osteoconductive properties. In the prospect, the material can be recommended for use in the field of traumatology, dentistry, maxillofacial surgery and orthopedics.


Lead-based halide perovskites have recently showngreat potential in various applications such as solar cells, optoelectronics,and photocatalysis. Even though they show high performance, the hightoxicity of Pb2+ along with poor stability under ambientconditions restrains the application of these materials in photocatalysis.In this respect, we developed an in situ assembly strategy to fabricatethe lead-free double perovskite Cs2AgBiBr6 ona 2D bismuthene nanosheet prepared by a ligand-assisted reprecipitationmethod for a liquid-phase CO2 photocatalytic reductionreaction. The composite improved the production and selectivity ofthe eight-electron CH4 pathway compared with the two-electronCO pathway, storing more of the light energy harvested by the photocatalyst.The Cs2AgBiBr6/bismuthene composite shows aphotocatalytic activity of 1.49(±0.16) μmol g–1 h–1 CH4, 0.67(±0.14) μmolg–1 h–1 CO, and 0.75(±0.20)μmol g–1 h–1 H2, with a CH4 selectivity of 81(±1)% on an electronbasis with 1 sun. The improved performance is attributed to the enhancedcharge separation and suppressed electron–hole recombinationdue to good interfacial contact between the perovskite and bismuthenepromoted by the synthesis method.CO According tothe European Union Commission for climate action, CO2 into value-added chemicalfuels  is one of the mostpromising ideas to address global warming and store renewable energy.4 While the task of converting CO2 intofuel is scientifically challenging, it would have significant benefits,as it provides a way to use an abundant and renewable substance innature to produce fuels and feedstocks for which the current industryinfrastructure is already adapted for use. Recently, many techniquessuch as photoelectrochemical (PEC),5 thermocatalytic,6 biochemical,7 electrocatalytic,8 and photocatalytic9 techniques have made considerable advances in this area. Among them,photocatalytic CO2 conversion is an attractive and promisingtechnology because it offers a potentially affordable, carbon-freeroute to the synthesis of solar fuels.10The endergonicconversion of CO2 reductioninclude two-electron-reduction , four-electron-reduction (E° = −0.48 VSHE for HCHO), six-electron-reduction(E° = −0.38 VSHE for CH3OH) and eight-electron-reduction reactions (E° = −0.24 VSHE for CH4).11 Among those reaction processes, CH4, with an enthalpy of combustion of −890.03 kJ mol–1, can store the most solar energy into chemical energy for furtherutilization. However, most of the existing photocatalysts have insufficientreduction ability for the eight-electron CH4 productionpathway.13Generally, the routes for solar-driven CO14 H2 evolution,15 air purification,16 and CO2 reduction reactions.17 The photocatalyticCO2 reduction is often referred to as “artificialphotosynthesis” due to its potential for reducing CO2 on a semiconductor surface by utilizing the energy of the sun andprotons from water to produce hydrocarbons, thus mimicking the naturalprocess carried out by plants. It has great potential to produce value-addedfuels and provide an interesting route to tackle both climate changeand renewable energy production.18Semiconductor photocatalysts havebeen extensively reported inrecent years in many applications such as organic compound degradation,2 conversion to CH4 is not a straightforwardprocess, as there are thermodynamic and kinetic limitations to thereaction. An efficient semiconductor photocatalyst for this kind ofreaction should have a conduction band edge with a lower potentialthan that of the target reduction reaction, the ability to absorba wide energy range of solar irradiation, high mobility, and efficientseparation of the photogenerated charges.19The CO2 reduction due to their excellentproperties including tunable band structure, high absorption coefficient,and excellent charge separation efficiency.20 In recent years, the all-inorganic perovskite CsPbBr3 has attracted great attention for photocatalytic CO2 reduction.Hou et al. prepared CsPbBr3 quantum dots for photocatalyticreduction of CO2 to CO and CH4, with productionsof 4.3 and 1.5 μmol g–1 h–1, respectively.21Among many types of semiconductor materials for photocatalyticapplications, halide perovskites have recently emerged as promisingphotocatalysts for CO3, the use of a materialcomposed of toxic Pb2+ cationsis not sustainable, hindering its development and application.22 Alternatively, lead-free halide perovskiteshave recently been studied to overcome the toxicity problem. Examplesof lead-free perovskites are CsSnX3, CsSbX3,and various double perovskites such as Cs2AgBiBr6 (DP). They show a band gap of 1.8–2.2 eV and a long carrierrecombination lifetime. Among various Pb-free double perovskites,Cs2AgBiBr6 has shown better stability towardmoisture, air, heat, and light.23Despite the promisingphotocatalytic properties of CsPbBr2 reduction reaction. As a stable freestandingtwo-dimensional (2D) bismuth monolayer, bismuthene (Bi) has demonstratedhigh electrocatalytic efficiency for formate (HCOO–) production from the CO2 reduction reaction.24 In a recent work, 2D bismuthene has been usedas a functional interlayer between BiVO4 and NiFeOOH forenhanced oxygen-evolution photoanodes, significantly improving thePEC performance of photoanodes by optimizing the separation and mobilityof photoinduced charges.25Bismuth has recently been reported as a highly efficient electrocatalystfor the CO2AgBiBr6/bismuthene (DP/Bi) composites for photocatalyticCO2 reduction to CH4. Our approach promotesthe self-assembly of the perovskite nanoparticles on the bismuthenenanosheets, which influences perovskite nucleation and growth, forminghierarchical hexagonal plates. The DP/Bi heterostructure photocatalystsshowed significant improvement when compared to a pristine doubleperovskite. The photocatalytic CO2 reduction achieved 1.49(±0.16)μmol g–1 h–1 CH4, 0.67(±0.14) μmol g–1 h–1 CO, and 0.75(±0.20) μmol g–1 h–1 H2, with a CH4 selectivityof 81(±1)% on an electron basis with 1 sun of simulated light.The improved performance was attributed to enhanced charge separationand suppressed electron–hole recombination achieved througha good interfacial contact between the perovskite and bismuthene promotedby the synthesis method.In thiswork, we have adopted a ligand-assisted reprecipitation(LARP) method to successfully synthesize, for the first time, Cs3 , BiCl3 , NaBH4 , ethylacetate , oleic acid , 2-ethoxyethanol, N,N-dimethylformamide, ethanol , and methanol used in this work are of analytical grade andwere used without further purification.CsBr , AgBr , BiBr26 was adopted to synthesize Cs2AgBiBr6 nanoparticles as a reference. The LARP reaction is depictedin An optimized LARP protocol based on the work by Ng et al.24 BiCl3 (1.5 mmol) was dissolved in50 mL of 2-ethoxyethanol, followed by ultrasonication of the mixtureto form a uniform and transparent solution. The solution was vigorouslystirred for 30 min in an oil bath at 120 °C while N2 was bubbled into the solution to maintain an inert atmosphere. Thesolution was left to cool to room temperature. A 20 mL portion ofNaBH4 (60 mmol) was then added dropwise to the bismuthsolution, still under a nitrogen atmosphere, with stirring for 5 minunder these conditions. The resultant black precipitate was collectedby centrifugation and washed twice with DI water and ethanol, respectively.The obtained powder was dried in a vacuum oven at room temperaturefor 24 h and then stored under an N2 atmosphere for latercharacterization or application.Bismuthene nanosheetswere synthesized by a wet chemical method as reported by Yang et al.2AgBiBr6 nanoparticles onto bismuthene nanosheetsas illustrated in We adapted the LARP protocol to self-assembleCs2AgBiBr6 and its composites were calculated using the Scherrer equationon the peaks corresponding to the (022), (040), and (044) planes.X-ray photoelectron spectroscopy (XPS) analysis was done using a ThermoFisher K-Alpha spectrometer and monochromated Al Kα1 X-ray source (1593 eV), and the pass energy was set at 20 eV withconstant pass energy (CPE) mode. Furthermore, the binding energieswere referenced relative to the C (1s) C–C bond at 284.8 eV. The XPS data were processed using thesoftware Thermo Avantage version 5.9922. The morphology and crystallinityof the as-prepared samples were examined by high-resolution transmissionelectron microscopy (HRTEM) using a JEOL JEM-2100Plus microscope operatingat an accelerating voltage of 200 kV. The morphological aspects werealso examined by scanning electron microscopy (SEM) on a Zeiss AurigaCross Beam microscope. The light absorption of the as-prepared sampleswas measured by UV–visible diffuse reflectance spectroscopy(UV–vis DRS) on a Shimadzu 2600 spectrophotometer using anintegrating sphere for diffuse reflectance and BaSO4 asa standard. The baseline was set using pure BaSO4, andthen 10 mg of a sample was mixed with BaSO4 for analysis.Photocurrent measurements were carried out in a three-electrode PECcell with a quartz window, a working electrode, a Pt-wire counterelectrode, a nonaqueous Ag/AgCl reference electrode solution, and0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6) in acetonitrileas an electrolyte solution.27 A 300 W Xelamp equipped with an AM1.5G filter (LOT Quantum Design) was usedat 100 mW cm–2 (1 sun) which was chopped  with an automatic shutter. An externalpotential of +0.6 or −0.6 V vs the Ag/AgCl reference electrodewas applied with a potentiostat (Ivium CompactStat).Powder X-ray diffraction (XRD) wascarried out with a X’Pert PRO diffractometer by PANalyticaloperated at 40 kV voltage and 40 mA current using Cu Kα (λ= 0.15418 nm) radiation in the 2θ range 10–50°.The coherent diffraction domain sizes of Cs2 reduction tests were carried out under simulated solarirradiation at room temperature in a gastight 85 mL stainless steelreactor with a PTFE lining and a top quartz window. A 300 W Xe lampequipped with an AM1.5G filter was placed vertically above the reactor,and the light intensity was adjusted to 1 sun (100 mW cm–2). A 7 mg portion of a powder sample was dispersed in 35 mL of anhydrousmethanol and sonicated for 2 min to create a well-dispersed suspensionbefore loading into the photoreactor. Before irradiation, the photoreactorwas purged with He gas for 1 h to remove air from the system. Then,CO2 gas was bubbled through the suspension for 1 h at 30mL min–1 with continuous stirring to saturate thesystem. The gas inlet and outlet in the reactor were closed beforestarting the irradiation for 2 h under 1 simulated sun. The productswere analyzed by gas chromatography with He as a carrier gas and a barrier ionization discharge (BID)detector. The selectivity (S) of the products wascalculated on a total electron consumption basis, as given in 322N4CH, NCO, and N2H are the production rates of CH4, CO, and H2 in μmol g–1 h–1 and the coefficients 8, 2, and 2 are usedto account for the total electrons involved in the CO2 reductionto form CH4, CO, and H2, respectively. The apparentquantum efficiency (AQE) was measured using the same experimentalsetup, but with a 450 nm LED light source to obtain monochromaticlight. The AQE was calculated according to The photocatalyticCO2 reductionreaction, consisting of experiments (1) in darkness, (2) without photocatalyst,and (3) in He. In experiment 1 the same photocatalytic procedure wasperformed using DP/Bi0.5 as a photocatalyst without light irradiation.In experiment 2, anhydrous methanol was loaded into the photoreactorwithout any photocatalyst. In experiment 3 only He gas was used topurge the photoreactor containing the methanol suspension with DP/Bi0.5as a photocatalyst.A series of controlexperiments were carried out to confirm the CO2AgBiBr6 consistent with thestandard pattern of the Crystallography Open Database (COD) Card No.96-413-1245, with a = 11.2818 Å and space groupsymmetry Fm3̅m. The obtainedXRD patterns of DP show its characteristic diffraction at 13.7, 15.9,22.5, 26.4, 27.6, 31.9, 34.6, 35.7, 39.3, 45.7, 47.9, and 48.6°(2θ), corresponding to the (111), (020), (022), (131), (222),(040), (133), (042), (242), (044), (153), and (244) planes, respectively.Second, the XRD patterns of the few-layered bismuthene prepared bya wet chemical method were indexed with a hexagonal crystal structure(COD Card No. 96-500-0216), with a = b = 4.5488 Å, c = 11.8683 Å, and spacegroup R3®m. The obtained XRDpattern of bismuthene shows its characteristic diffraction peaks at22.7, 24.1, 27.4, 38.2, 39.9, 44.8, 46.2, and 48.9° (2θ),corresponding to the (003), (101), (102), (104), (110), (105), (113),and (202) planes, respectively. Finally, the XRD patterns of DP/Bicomposites all show the same cubic Cs2AgBiBr6 crystal phase, which indicates that the type of crystal phase isnot affected by the addition of bismuthene under this synthesis condition.No bismuthene peaks could be identified on the XRD patterns of thecomposites, confirming its good dispersion in the composite and itslow weight percentage in the composition. The XRD analysis confirmedthat the LARP method used to synthesize the DP and DP/Bi samples wasa successful approach, which we also found relatively easy to carryout and reproduce.The XRD patterns of the as-preparedDP nanoparticles, few-layeredbismuthene, and composites with varying contents of bismuthene (from0.5 to 2 wt %) are shown in 29 The domain size of the bare perovskite (DP) was 21.2(±4.1)nm. Therefore, the LARP method and oleic acid ligands prevented largedomain size formation but kept sizes above 10 nm, avoiding quantumsize effects that increase the band gap.30 The coherent diffraction domain size of the perovskite increasedwith the increasing bismuthene content, from 21.2(±4.1) nm forbare DP to 40.2(±4.0), 45.8(±5.7), and 48.7(±4.4) nmfor DP/Bi0.5, DP/Bi1, and DP/Bi2, respectively. This increase in domainsize is evidence that 2D bismuthene influenced the nucleation andgrowth of the perovskite crystals.The coherent diffraction domain size of the DPand DP/Bi compositeswere calculated using the Scherrer equation.2AgBiBr6, which is in agreement with the literature on the Cs2AgBiBr6 double perovskite.32 The Tauc equation33 was used to representthe solar absorption and evaluate the band gap (Eg) of Cs2AgBiBr6 in bare and composite samples. The plots of [F(R)hν]0.5 as a functionof the photon energy (hν) are shown in F(R), h, and ν arethe Kubelka–Munk function, Planck’s constant, and thelight frequency, respectively. The exponent 0.5 represents the indirectelectronic transition of the semiconductor Cs2AgBiBr6. The band gap of the samples was estimated by measuring theinterception of the extrapolation of the linear part of the curveand its baseline; accordingly, the estimated band gaps are 2.14, 2.27,2.28, and 2.28 eV for DP, DP/Bi0.5, DP/Bi1, and DP/Bi2, respectively.Similar band gap values are obtained with a variation within the errorof such measurement where scattering effects can have an influence.The same band gap values agree with the absence of quantum size effectsand the detection of the same crystalline phase by XRD . The TEM micrograph of bismuthene plane of the bismuthenephase. This is evidence of intimate contact between the perovskiteand bismuthene surfaces.To characterize the morphology of the samples,an SEM analysiswas conducted. smuthene5a shows ic phase 5d. Figuric phase 5f shows 36 As a surfactant it provides the steric stabilization of the nanoparticlesagainst van der Waals attractive interactions and thereby preventstheir agglomeration. It also limits the growth of nanoparticles andprevents an Ostwald ripening process from taking place, since itssurface layers act as a barrier to mass transfer.37 These characteristics may facilitate the process of self-assemblyof the nanoparticles in hierarchical structures.These bismuthene nanosheets obviously have a majorrole in thedirectionality of the self-assembly process of the perovskite nanoparticlesinto the hexagonal hierarchical structure. However, the exact mechanismof the directional self-assembly is not yet fully understood. It iswell established in the literature that the use of ligands such asoleic acid in the synthesis of nanoparticles enables better controlof the nanocrystal size and crystallization.good solvent (DMF) containing the oleic acidligand and undergo a swift change of solubility by several ordersof magnitude when injected into a poor solvent (ethylacetate), which results in the rapid nucleation and formation of theCs2AgBiBr6 nanoparticles. For the compositesynthesis, the bismuthene nanosheets are already well dispersed andexfoliated in the ethyl acetate solvent after 24 h of ultrasonication.When the perovskite precursor solution is injected into the bismuthene-ethylacetate dispersion, bismuthene nanosheets can act as nucleation sites,influencing the crystal growth and resulting in the hexagonal hierarchicalplate structures.For the synthesisof the bare perovskite, the precursors are dissolvedin a Figure S1) and of a representativeDP/Bi1 sample  indicate thepresence of Cs, Ag, Bi, Br, C, and O. In order to examine the chemical environmentand the oxidationstate of the atoms on the surface of the samples, X-ray photoelectronspectroscopy (XPS) characterizations were conducted. The survey spectraof the DP S1 and of7/2 and Bi 4f5/2 ofDP are located at binding energies of 159.00 and 164.33 eV, respectively.Beyond the two main peaks, assigned to the Bi3+ ions inthe perovskite, all of the composites exhibit two extra spin–orbitdoublets which are assigned to elemental bismuth Bi(0) from bismuthene.The XPS survey spectrum of bismuthene  indicates the presence of Bi, O, and C. The Bi 4f spectrumof bismuthene (7/2 and Bi(0) 4f5/2,38 respectively, while the peaks at 158.64and 163.95 eV were assigned to Bi–O 4f7/2 and Bi–O4f5/2 originating from the partially oxidized bismuthenesurface.39 It has been reported that the partial surfaceoxidation of bismuthene works as a passivation layer that actuallyprevents further inner degradation, in the same way that aluminumoxide prevents the oxidation of deeper aluminum.405/2 and Bi(0) 4f7/2 for the composites are shifted in comparison to those forthe bismuthene sample, reaching a shift by ca. 1.46 and 1.26 eV forBi(0) 4f5/2 and Bi(0) 4f7/2 in DP/Bi2, respectively,indicating interfacial interactions between the Bi atoms in bismutheneand the DP nanoparticles.34The high-resolution Bi 4f spectra of Bi, DP and DP/Bicompositesare shown in smuthene S3 indicasmuthene S3 indica3+ 4f7/2 andBi3+ 4f5/2 peaks  were deconvoluted into three peaks. The main peak at 284.8 eV wasassigned to the aliphatic C–C bond from the residual oleicacid used in the synthesis of the samples and was used to calibratethe XPS spectra. The 286.38 and 289.38 eV peaks in DP confirmed thepresence of the C–O and O=C–O bonds, respectively,from oleic acid. The position of the peaks for the C–O andO=C–O bonds in the composites can be observed in Figure S5) wasassigned to the C–C bond from adventitious carbon and was usedas a reference for binding energy values./2 peaks 6a both s/2 peaks 6c shifteAg 3d3/2 6d shifteBr 3d3/2 6e exhibiEf) XPS was used to determine the energy gapbetween the VB edge and the Fermi level (Ef) 7a. ThevEf and the vacuum level, wasmeasured to be 4.70 eV . These results suggest that the DPnanoparticles have a suitable CB edge potential with a sufficientlyshallow potential to allow them to overcome the electrochemical potentialto form multielectron reduced species from CO2, such asCH4  and CO .41The as-prepared Cs2 reduction reaction,the photoelectrochemical (PEC) behavior of the samples was evaluatedby measuring their photocurrent response. Since the mobility and efficient separationof the photogeneratedcharges play a pivotal role in the CO–6 A cm–2 and an increasing photocurrent responsewith growing bismuthene content at a negative applied bias under illumination. –2.The linear sweeps from −0.6 to +0.6 V vsAg/AgCl in thedark show a very small current density in the range of 10–2 at −0.4 V (vs Ag/AgCl) for DP/Bi0.5,DP/Bi1, and DP/Bi2, respectively. These results show that the PECperformance is significantly improved by the presence of bismuthene,with DP/Bi2 reaching 3 times higher photocurrent density comparedto bare DP, suggesting that bismuthene improved the separation ofphotoexcited electrons and holes in the perovskite.42 These results are in agreement with the interfacial interactionsobserved by XPS in 4/bismuthene/NiFeOOH composite photoanodes for PEC water oxidation,where composites with bismuthene showed a much higher current densitycompared to the bare BiVO4 photoanode due to improved bandbending.25 Bismuthene, therefore, increasesthe separation and extraction of photogenerated charges.Thecurrent density increased with the increase of the bismutheneamount in the composites, measuring 13.2, 24.3, and 32.3 μAcm2 reduction reaction was evaluated in the liquid phase usingmethanol as a hole scavenger and proton source. The reaction was carriedout under simulated solar light, 1 sun (100 mW cm –2) with no cutoff filter, and the results are presented in 2, CO, and CH4 gases in μmol g–1 h–1 (2AgBiBr6 (DP) nanoparticles are 0.83(±0.13)μmol g–1 h–1 CH4, 0.43(±0.06) μmol g–1 h–1 CO, and 0.35(±0.05) μmol g–1 h–1 H2.Finally,the photocatalytic performance of the samples in the CO g–1 h–1 9a. The p4 productionby 65% in comparison with pure DP, while CO and H2 productionsremained constant with production rates of 1.37(±0.06) μmolg–1 h–1 CH4, 0.43(±0.02)μmol g–1 h–1 CO and 0.35(±0.04)μmol g–1 h–1 H2. When the bismuthene content was increased in DP/Bi1, the CH4 production had a slight increase of 8% compared with DP/Bi0.5,while CO and H2 production rates increased by 55% and 114%,respectively, reaching the values of 1.49(±0.16) μmol g–1 h–1 CH4, 0.67(±0.14)μmol g–1 h–1 CO, and 0.75(±0.20)μmol g–1 h–1 H2. When the bismuthene content was further increased in DP/Bi2, theCH4 production rate decreased by 63% compared with DP/Bi1,but CO and H2 productions slightly increased by 7% and9%, respectively, showing a production of 0.94(±0.23) μmolg–1 h–1 CH4, 0.72(±0.05)μmol g–1 h–1 CO, and 0.82±0.03) μmol g–1 h–1 H2.The DP/Bi0.5 composite increased the CH4 and H2 was calculatedusing 4, 87(±5)%, comparedwith 81(±7)% CH4 selectivity for pristine DP. Thecomposite DP/Bi1 showed the same selectivity for CH4 asDP of 81(±1)%, and as the bismuthene content increased in DP/Bi2,the CH4 selectivity decreased to 71(±1)%.The selectivity of CO, CH4, two-electron formationof CO, and two-electron formation of H2. Cs2AgBiBr6 (DP) showed a photocatalytic activity of 8.19μmol e– g–1 h–1, DP/Bi0.5 of 12.53 μmol e– g–1 h–1, DP/Bi1 of 14.79 μmol e– g–1 h–1, and DP/Bi2 of 10.61μmol e– g–1 h–1. These results show that DP/Bi1 had higher overall photocatalyticactivity compared to the other samples and confirm that the photocatalyticactivity increases with increasing bismuthene content. This is consistentwith the higher current density and improved separation of the photogeneratedcharges in the composites compared with the pristine perovskite  in darkness,(2) without photocatalyst, and (3) in He. No detectable products werefound in the experiments carried out in the dark or without photocatalyst,indicating that the CO2 reduction reaction takes placeas a light-driven catalytic process involving the photocatalysts.442. In the reaction with He replacingCO2, small amounts of H2 (0.22 ± 0.10 μmolg–1 h–1) were detected, whichcan be attributed to the photocatalytic dehydrogenation of methanol.46 Trace amounts of CO and CH4, 0.14 ± 0.02 and 0.22± 0.04 μmol g–1 h–1, respectively, were also detected, presumably from the slight decompositionof the organic ligands capping the photocatalysts and the presenceof preadsorbed CO2 on the photocatalyst. The drastic dropin production rates of carbon products in the reaction without CO2 indicates that the overall reaction involves CO2 reduction and methanol oxidation cycles to produce CH4, and CO, whereas H2 is formed in a parallel competitivemethanol reduction reaction.The photocatalytic CO2 reduction, two sequential runs of 2 h each were performedon a representative DP/Bi1 sample . After one cycle, the production rates change from 1.24 to 0.03μmol g–1 h–1 CH4, from 0.55 to 0.03 μmol g–1 h–1 CO, and from 0.61 to 0.05 μmol g–1 h–1 H2. The XRD patterns and SEM micrographsof DP/Bi1 before and after two cycling tests  show that there is some crystal structureand morphological change during the photocatalytic reaction. The XRDpatterns for the sample after the photocatalytic reaction show narrowercubic Cs2AgBiBr6 peaks along with minor secondarypeaks, which correspond to the ternary phase Cs3Bi2Br9 (COD 96-210-6377). These changes indicate that,during the photocatalytic reaction, part of Cs2AgBiBr6 sintered into larger diffraction domains and another partdecomposed into Cs3Bi2Br9, resultingin a drop in photocatalytic activity after the first run test. Therefore,these composites would benefit from strategies to improve their stability,such as using reduced graphene oxide,22 nitrogen-doped carbon47 or Ce-UiO-66-H48 as a protective layer, which would give it amore hydrophobic character, which will be addressed in future work.To assess the stability of thecomposite in the photocatalyticCO4 productionof the DP/Bi1 sample was calculated to describe the efficiency ofthe photocatalysts in absorbing incident photons and generating photoinducedcharges that react to produce solar fuels. The calculated AQE forDP/Bi1 was 0.0012% at 450 nm.The apparent quantum efficiency (AQE) of CH4, CO, and H2 were producedvia the following reactionmechanism: Cs2AgBiBr6 is photoexcited, generatingholes (h+) that migrate to its surfaceand electrons (e–) that migrateand are collected on the bismuthene nanosheetsFrom these observations, it canbe inferred that CH+, oxidizemethanol to produce hydroxymethyl radical, •CH2OH, and H+ 49The photogeneratedholes, h2 to produce CO502 to produce CH450+, originating from the methanoloxidation, producing H241At this point, parallelreduction reactions can take place, suchas the two-electron reduction of CO4 and not altering CO and H2 production. As thebismuthene content was increased in DP/Bi1, 2, with theCH4 production remaining almost unchanged. When the bismuthenecontent was further increased in DP/Bi2, CH4 productiondecreased, and CO and H2 production remained almost unchanged.This can be explained by the bismuthene having assisted the separationof photogenerated electron–hole pairs from the perovskite,thereby providing a higher availability of electrons for the multielectronreduction of CO2 to CH4. However, the higherconcentration of bismuthene in the composites increased the selectivitytoward CO and H2 production.The results indicatethat the low concentration of bismuthene inthe composites, as in DP/Bi0.5, favored 2AgBiBr6 doubleperovskite (DP) nanoparticles and their composites with bismuthene(Bi) by a simple and fast ligand-assisted reprecipitation method.The resulting DP/Bi composites exhibited significantly improved photocatalyticactivity for CO2 reduction using methanol as a hole scavenger,showing a CH4 production of up to 1.49(±0.16) μmolg–1 h–1 (under 1 sun) with a CH4 selectivity of 81(±1)% for an optimized DP/Bi compositewith 1 wt % bismuthene. This result was 80% higher than that for Cs2AgBiBr6 nanoparticles. The higher performance ofthe DP/Bi composite for photocatalytic CO2 reduction showsthat the synthesis method adopted in this work involving a ligand-assistedreprecipitation method proved to be efficient in creating a good interfacialcontact between the Cs2AgBiBr6 and bismuthene,which promotes efficient photoinduced charge carrier separation and,consequently, a lower recombination of electron–hole pairs.This was confirmed by photoelectrochemical measurements. The improvedoptical and electronic properties of the composites allowed a higherabsorption of the light energy and facilitated migration of the electronsto the bismuthene layers and holes to the Cs2AgBiBr6 surface, thereby improving its overall photocatalytic activity.This strategy shows great potential for creating two-dimensional Pb-freehalide perovskites on a larger scale with improved CH4 selectivityfor solar fuels production.We successfully synthesized Cs


DMD mutations. The VENUS contains a modified enhanced green fluorescence protein (EGFP) gene, in which expression was restored upon the CRISPR-mediated correction of DMD loss-of-function mutations. We observed that the highest editing efficiency was achieved by NHBEJ (74–77%), followed by HDR (21–24%) and PE2 (1.5%) in HEK293T VENUS reporter cells. A similar HDR (23%) and PE2 (1.1%) correction efficiency is achieved in fibroblast VENUS cells. With PE3 (PE2 plus nicking gRNA), the c.7893delC correction efficiency was increased 3-fold. Furthermore, an approximately 31% correction efficiency of the endogenous DMD: c.7893delC is achieved in the FACS-enriched HDR-edited VENUS EGFP+ patient fibroblasts. We demonstrated that a highly efficient correction of DMD loss-of-function mutations in patient cells can be achieved by several means of CRISPR gene editing.Recent progress in CRISPR gene editing tools has substantially increased the opportunities for curing devastating genetic diseases. Here we compare in-frame deletion by CRISPR-based non-homologous blunt end joining (NHBEJ), homology-directed repair (HDR), and prime editing -based correction of two Duchenne Muscular Dystrophy (DMD) loss-of-function mutations (c.5533G>T and c.7893delC). To enable accurate and rapid evaluation of editing efficiency, we generated a genomically integrated synthetic reporter system (VENUS) carrying the  Since the first report in 2012, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associate protein 9 (Cas9) gene editing tools have been largely expanded . We defihttps://www.ncbi.nlm.nih.gov/clinvar/ (accessed on 21 February 2023)). Over the last decade, two types of CRISPR-derived technologies have been developed for site-specific editing that are independent of DSB and DNA repair templates. By fusing an engineered deaminase to the Cas9 in which DNA nuclease domains are partially  or completely  inactivated, a serial of so-called base editing technologies , cytosine base editors (CBE), and glycosylase base editors (GBE)) has been developed for inducing DNA substitution (transitions or transversions) [Another DNA repair mechanism is homology-directed repair (HDR). Compared to NHEJ and MMEJ, HDR efficiency is much lower . In our ersions) ,6,7. Theersions) . Unlike DMD gene , which encodes the dystrophin protein. Dystrophin is part of the dystrophin–glycoprotein complex and acts as a link between the actin cytoskeleton and the extracellular matrix in muscle cells, providing membrane stability. To date, many disease-causing mutations  have been detected in the DMD gene. Certain regions of the DMD gene were found to be mutational “hotspots”. Around 60% of DMD cases were caused by mutations located in/around exons 45 to 55. Exons 2–10 are also among the highly mutated regions [DMD mutations result in a version of dystrophin that does not function correctly or the loss of dystrophin expression entirely, ultimately causing DMD. Currently, there is no cure for DMD. However, to restore muscular function in DMD patients, numerous treatment approaches have been explored, including using CRISPR editing to correct the DMD mutation and restore dystrophin expression [Duchenne Muscular Dystrophy (DMD) is an X-linked recessive and muscle-wasting disorder, which is the most prevalent childhood muscular dystrophy and affects approximately 1 in 5000 male newborns worldwide . DMD is pression .Several methods have been developed for analyzing CRISPR-induced indels in cells. For a complete overview of these methods, we refer readers to a recent review by Eric and colleagues . PreviouPCR primers and VENUS oligonucleotides are ordered from Merck KGaA, Darmstadt, Germany. The ssODN (single-stranded oligo deoxynucleotides) for HDR and pegRNA are ordered from Integrated DNA Technologies . Chemically modified synthetic CRISPR gRNA is ordered from Synthego, Redwood City, CA, USA.For the VENUS-DMD:c.5533G>T vector, 42 base pairs of the DMD coding sequencing (reading frame from +1), including the DMD:c.5533G>T, were selected. For the VENUS-DMD:c.7893delC vector, 41 base pairs of the DMD coding sequencing (reading frame from +1), including the DMD:c.7893delC, were selected. A “5′-CTAGgg” and a “5′-CTAG” overhang sequence was added to the sense and antisense VENUS oligonucleotides, respectively. A conventional VENUS oligo design sheet is provided in EGFP) gene. To generate the VENUS reporter vector, pCCL-PGK-EGFP-IRES-Puro was digested with XbaI  followed by Calf Intestinal (CIP) alkaline phosphatase treatment to remove 5′-phosphate groups from the linearized plasmid. The linearized plasmid was purified with 1% agarose gel.A dicistronic lentiviral vector pCCL-PGK-EGFP-IRES-Puro (a gift kindly provided by Prof. Jacob Giehm Mikkelsen from Aarhus University), which contains an XbaI cloning site between the ATG start codon and another coding sequencing of the enhanced green fluorescence protein  was mixed in a 2 µL 10× NEB buffer 2 and 16 µL ddHE. coli cells and grown in an LB agar plate with Ampicillin overnight.For ligation of the double-stranded VENUS DNA into the linearized pCCL-PGK-EGFP-IRES-Puro vector, a 200 ng backbone and 2 µL of annealed VENUS DNA were ligated with 1 µL of T4 ligase  in a 1 µL T4 ligase buffer in a 10 µL reaction. Ligation was performed at 16 °C for 4 h. After ligation, a 10 µL ligation product was transformed into 50 µL competent E. coli colonies carrying the correct VENUS plasmid. We used the VENUS sense oligonucleotide as a forward primer and an internal primer located in the antisense strand of the EGFP coding region as a reverse primer , 0.6 µL forward (5 µM), 0.6 µL reverse primer (5 µM), 0.3 µL dNTP (10 mM), 1.5 µL 10× Dream Taq buffer, and ddH2O. PCR was performed with a program of one cycle at 94 °C for 2 min, 35 cycles at 94 °C for 20 s, 62 °C for 30 s, and 72 °C for 40 s, and 1 cycle at 72 °C for 7 min. PCR-positive colonies were further grown in an LB medium and the VENUS plasmid was purified by a PureLink Hipure Plasmid Maxiprep kit , followed by Sanger sequencing to validate the correct insert.We adopted a PCR screening strategy to screen for e primer . Each PC6 HEK293T cells were seeded in a P10 Petri dish, which reached approximately 70% confluence on the day of transfection. For each lentivirus packaging transfection, each transfection mix contains 66 µL PEI (1 µg/µL), 8.6 µg VENUS plasmid, 2.5 µg pMD.2G, 8.6 µg pRRE, and 2 µg pRSV-REV in 1 mL OptiMEM. The transfection cocktail was mixed well by gently pipetting, incubating at room temperature for 15 min, and then adding it into a 10 cm dish dropwise. After 48 hr of transfection, the cell culture medium was collected, and cell debris was removed with a 0.45 µM filter. Polybrene was added to the crude lentivirus supernatant to the final concentration of 8 µg/mL, which was aliquoted into 1.5 mL EP tubes (1 mL per tube) and stored at −80 °C before use.HEK293T cells were cultured in a DMEM Medium with 10% FBS (D10). One day before transfection, 2 × 105). At a cell confluency of 50–70% (approximately 24 h after cell seeding), cells were changed to a fresh DMEM medium with 10% FBS (D10) and 8 μg/mL of polybrene, and 200 µL of a crude virus was added to each well. After 48 h of transduction, cells were changed to a D10 medium with 1 µg/mL of puromycin (D10-Puro1), hereafter refreshed with a D10-Puro1 medium every two days. Cell death was observed three days after culturing cells in the D10-Puro1 medium. When all cells in the un-transduced control group were dead, Puro-resistant VENUS-transduced cells were cultured in D10-Puro0.5 (0.5 µg/mL puromycin) for maintenance and expansion.A wild type HEK293T cell and DMD fibroblasts were seeded into a 6-well plate . Fibroblast with VENUS was grown in DMEM F-12 with 20% FBS, 1% P/S, bFGF (5 ng/mL), and 0.5 µg/mL puromycin. All cells were passaged when confluency reached 80%.We used a Lonza 4D nucleofection for all CRISPR nucleofection. All gRNAs were dissolved in nuclease-free water to a concentration of 100 pmol/µL.NHBEJ: 0.6 µL Cas9 protein (IDT), 0.6 µL Sp1 gRNA, 0.6 µL Sp2 gRNA, and 16.2 µL Opti-MEM were mixed in an EP tube and incubated at room temperature for 15 min. Then, 200,000 cells were resuspended with the 18 µL ribonucleoprotein (RNP) mixture and transferred to a 16-well nucleofection chip (Lonza). Nucleofection was performed with the X channel CM138 program.HDR: RNP was prepared as NHBEJ, except for 0.3 µL ssODN (100 pmol/µL), which was added to the RNP mixture after 10 min of incubation at R.T.http://n2t.net/addgene:132775/ (accessed on 15 March 2022); RRID:Addgene_132775). For each nucleofection, 0.8 µL pegRNA (100 pmol/µL) and 2 µL PE2 mRNA (1 µg/µL) were mixed with Opti-MEM to a total volume of 18 µL. PE3 uses the same 0.8 µL pegRNA (100 pmol/µL) and 2 µL PE2 mRNA (1 µg/µL) with PE2 and was then mixed with another 0.8 µL nicked gRNA (100 pmol/µL) in Opti-MEM to a total volume of 18 µL.PE: PE2 mRNA was generated from the pCMV-PE2 plasmid using an in vitro transcription kit (see below). The pCMV-PE2 was a gift from David Liu  in a total volume of 50 µL and purified with a PCR clean-up kit . IVT was performed using the MEGAscript™ T7 Transcription Kit (AM1334). Each reaction contains an 11 µL dNTP mixture (2 µL ATP (75 mM), 2 µL CTP (75 mM), 2 µL UTP (75 mM), 2 µL GTP (15 mM), and 3 µL Clean Cap), 2 µL 10× reaction buffer, 1 µg linearized DNA template, 2 µL enzyme mix, and NF-H2O, 5 µL MnCl2 (25 mM), and 2 µL E-PAP enzyme was added to IVT product, mixed well, and incubated at 37 °C for 1 h. One µL TURBO DNase was then added to the reaction and incubated for 15 min to remove the DNA template.Following the IVT reaction, polyA was added to the mRNA using a Poly(A) Tailing kit. The reaction mixture containing the 10 µL 5X E-PAP buffer, 5 µL ATP (10 mM), 8 µL NF-H2O, and stored at −80 °C.The IVT mRNA was purified by lithium chloride precipitation. A total of 30 μL of nuclease-free water and 30 μL of a LiCl precipitation solution were added to the IVT reaction. The reaction was mixed well by pipetting gently and placed at −20 °C for at least 30 min. After that, the reaction mixture was centrifuged at 4 °C for 15 min at maximum speed. The supernatant was carefully removed without disturbing the RNA pellet. The RNA pellet was washed once with 1 mL 70% ethanol, resuspended in 30 μL of nuclease-free (NF) HFluorescence imaging was performed using the ZOE Fluorescent Cell Imager, and three fields of view were randomly selected for each group.After 72 h of nucleofection, cells were collected for flow cytometry  to analyze the percentage of GFP positive cells. Data analysis was performed using the NovoCyte analyzer and Quanteon analyzer, and fluorescence channels were selected for GFP wavelengths. Each sample was assayed at 100 µL and the flow analysis results were analyzed using NovoExpress. At least 10,000 cells were analyzed for each experimental replicate.Cell sorting was performed using a 4 Laser FACS Aria III. The fluorescence channel was selected to the fluorescence wavelength of GFP, and all the cells expressing green fluorescence were sorted out and inoculated in 48-well plates for further expansion.2O to total volume as 50 µL) and a PCR program .Genomic DNA was purified from cells using the NucleoSpin DNA RapidLyse kit (#740100.50). A total of 100 ng of genomic DNA was used as a template for PCR reactions using Dream Taq . Each PCR reaction contains 0.08 µL Dream Taq, with 0.6 µL forward and 0.6 µL reverse primer (5 µM), 0.3 µL dNTP (10 mM), 1.5 µL 10× Dream Taq buffer, 3 µL Betaine (5 M), and 15 µL reaction volume. The PCR program used for VENUS genotyping is 94 °C for 2 min, 35 cycles at 94 °C for 20 s, 62 °C for 30 s, and 72 °C for 40 s, and 1 cycle at 72 °C for 7 min. Genotyping of the endogenous DMD locus in the fibroblasts was performed with pfx polymerase .t-test were used to compare differences between groups. SnapGene Viewer was used to visualize Sanger sequencing results, and the web tools ICE Analysis  and DECODR [https://decodr.org/, accessed on 1 September 2022) were used for genotype percentage analysis of Sanger data.All experiments in this project were performed in three replicates. The data from the FCM in this project were analyzed using NovoExpress software. Prism 7 was used to analyze the data to make graphs and dot plots. The editing efficiency of different editing methods in the VENUS system was calculated using the percentage of cells with green fluorescence detected by flow analysis. One-way ANOVA and Student’s d DECODR  AnalysisTo enable the accurate and rapid quantification of LOF correction efficiency, we first developed a complementary reporter system, which can be used for the rapid and sensitive quantification of recoding efficiency to restore loss-of-function (LOF) mutations (nonsense mutation and frameshift indel mutations) in cells. To this end, we generated an engineered-enhanced green fluorescent protein (EGFP) reporter vector encompassing a user-desired LOF mutation in its 5′ coding region A, hereafWe next evaluated three CRISPR-based methods for the correction of DMD LOF mutations using the VENUS system A. NHBEJ p < 0.0001, one-way ANOVA) than HDR  and PE2 .Instead of using episomal plasmids, we first stably integrated the VENUS reporter vector into the genome of HEK293T cells by lentiviral transduction and puromycin selection A. This ep = 0.0002 (ANOVA)) for the DMD.c7893delC (pegRNA2) (We next conducted similar experiments to restore the reading frame caused by the DMD.c7893delC mutation in the HEK239T VENUS-DMD.c7893delC reporter cells. 77.4 ± 0.3% and 25.0 ± 0.5% of the HEK239T VENUS-DMD.c7893delC were EGFP+ 72 h after NHBEJ (Sp1 + Sp2) and HDR (Sp4 + ssODN2) editing, respectively . This ispegRNA2) B,C.Since EGFP expression only reflects the correction of the nonsense mutation for DMD:c.5533G>T and reading frame restoring for DMD:c.7893delC, we next investigated the CRISPR-mediated editing at the VENUS site by target PCR amplification and Sanger sequencing. For NHBEJ-edited cells, we analyzed all cells without a FACS enrichment of EGFP+ cells. Upon successful NHBEJ editing with the Sp1 and Sp2 gRNAs, 87bp and 86bp are deleted from the coding region of the VENUS-DMD:c.5533G>T and VENUS-DMD:c.7893delC reporter genes, respectively . Sanger In the EGFP+ HEK293T VENUS-DMD:c.7893delC cells, all VENUS loci are edited after HDR with Sp4 gRNA and ssODN2. However, we detected two alleles from Sanger sequencing B. The maCollectively, our results show that VENUS is a sensitive method for analyzing NHBEJ, HDR, and PE2-mediated correction of LOF mutations in cells. Our results also highlight the different editing rates between CRISPR editing methods.In addition to its utilities as tools for evaluating CRISPR editing efficiency, fluorescence reporter vectors with surrogate editing sites have been used to enrich edited cells ,19,20,21EGFP+ fibroblasts were detected by fluorescence microscopy and FACS three days after the HDR and PE2 editing A,B. QuanOur results showed that the VENUS system provides an alternative and attractive strategy for the rapid assessment of the CRISPR-based correction of LOF mutations in cells. We observed that the CRISPR editing efficiency obtained in the exogenous VENUS reporter locus is higher than the corresponding endogenous site, which might be attributed to the epigenetic and chromatin accessibility effects. A direct comparison of the editing efficiency obtained in both reporter and endogenous loci might provide important insights into the impact of chromatin structure on CRISPR activity, and most importantly aid the further development of better CRISPR activity prediction tools. However, it should be further addressed in future studies. One limitation of the VENUS reporter system is its limited insertion size of the synthetic fragment by ligation, which could be improved by using, e.g., synthetic LOF reporter genes. However, the VENUS reporter system could probably be used as a conventional tool for assessing the effect of different modifications to the HDR template, as well as pegRNA  on CRISPR gene editing. Additionally, as demonstrated by us and several other groups, there is a good correlation of CRISPR on-target efficiency (Pearson’s R above 0.7) between the reporter and endogenous target sites ,24,26,27Although not demonstrated in this study, the restoration of DMD expression can be achieved by reprogramming the CRISPR-edited fibroblasts with transdifferentiating . ConsistIn this study, we obtained over 20% editing efficiency with HDR, which is considerably high for cells. However, this should be further evaluated in muscle tissues and is not suitable for in vivo DMD therapy, as the HDR pathway is inactive in muscle tissues. Many other factors could affect HDR efficiency in cells, such as the length of the ssODN, modifications of the nucleotides and the gRNA, form, and the time of delivering the Cas9, gRNA, and ssODN . Likewis


WT, p38βWT, or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α−/−, and p38β−/− MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38β suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38β signaling and on the specific targets of these two kinases.The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38β are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38β, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38α The MAPKs are a family of structurally homologous kinases with partially overlapping substrate repertoires, tissue distribution, and biological function. In mammals, there are four major MAPK subfamilies, ERK ERK1, ERK2), JNK JNK1-3), ERK5/BMK, and p38 . These MAPKs regulate critical cellular processes, including proliferation, apoptosis, and embryogenesis  in the mutant p38 relative to the wild-type p38s  relative to in the p38β−/− MEFs (HSP27 is a known substrate of MAPKAPK2 (MK2) and MAPAPK3 (MK3) ,63. The g p38αWT . This phg p38αWT C. Intere−/− MEFs . This obp-value lower than 0.01  in the KEGG database , was difWT, which was itself activated by constitutively active MKK6EE . We assume that this phenomenon was a consequence of negative feedback responses, which could be imposed by enhancing phosphatase activities, or by allosteric factors. The strong feedback implies that chronic p38 activity also induces cellular stress, as was learned from the effects of the active p38s on the cytoskeleton. These changes in the phosphoproteome are possibly interesting since they include many sites that are modulated in a fashion similar to chronic inflammation and cellular stresses. Analysis of the proteome and phosphoproteome of MEFs, in comparison to those of p38αormed by  in respoormed by  in respoormed by  or by foormed by . Furtherormed by ,71 in whormed by . In addiormed by . Very usormed by , UniProtormed by , and Phoormed by ,61. The ormed by  is indicD176A+F327S affected the phosphoproteome more intensely than its counterpart p38βD176A. Second, the phosphorylation of HSP27-Ser82 was upregulated only in cells expressing the intrinsically active variant of p38α. HSP27 is known to maintain cytoskeletal integrity, in part via MK2 or MK5 [An interesting observation is the differential phosphorylations due to the presence of the intrinsically active expression of p38α or p38β. First, expression of the intrinsically active variant p38α2 or MK5 , and to 2 or MK5 ,76,77. TA subset of cancer-related proteins was more strongly phosphorylated in cells expressing intrinsically active p38α than in cells expressing active p38β. Among these affected proteins were adenomatous polyposis coli protein (APC), histone deacetylase 2, BCL2-associated agonist of cell death, and transformation-related protein 53 (p53) ,78. In tHuman p53 is known to be phosphorylated by p38 on Ser15, Ser33, Ser46, and Ser392 but not on Ser315 and Ser313. The phosphorylation site on Ser315  was already described as a phosphorylation site by other kinases, such as Aurora kinase, GSK3β, CDK1, and CDK9, but not by p38s (according to the UniProt database) ,80. Our The p38 MAP kinases and their signaling cascades have been studied extensively, mostly due to their role in mediating different stresses, inflammatory responses, and cancer ,12,13. AKO and p38βKO MEFs [2 at 37 °C. Proliferation rates were calculated based on cell counting on consecutive days by TC20 Automated Cell Counter  and methylene blue staining.Mouse embryonic fibroblasts (MEFs), p38αβKO MEFs , and HEKWT, p38βWT, constitutively active p38αD176A+F327S or p38βD176A [MEFs were transduced with retroviral vectors containing the cDNAs encoding p38α38βD176A ,41,48, oTo produce the viral vectors, HEK-293 cells were co-transfected by the calcium phosphate method with the retroviral vector pMigr containing the indicated p38 variants and the packaging plasmid PCL-ECO in a 1:1 ratio. Virus-containing supernatants were collected at 48 and 72 h post transfection, filtered through a 45 µm filter, supplemented with polybrene at a final concentration of 8 µg/mL, and added to MEF cells plated 24 h before the procedure. Infection efficiencies were assessed by fluorescent microscopy or FACS analysis. The cells were then sorted using the FACSAria IIIU Cell Sorter , and EGFP-positive cells were collected and expanded.MEFs were plated one day before treatment. Before treatment, the media containing the light, medium, and heavy amino acids were replaced with fresh media, with or without 150 nM anisomycin . After 5 min of incubation with the anisomycin, the proteins were collected for phosphopeptides enrichment.D176A+F327S and p38βD176A were labeled with the heavy forms of lysine  and arginine , the wild-type-p38α- and p38β-transfected cells were labeled with the light forms of lysine  and arginine , and the empty-vector-transfected MEFs were labeled with the medium form of lysine  and arginine  .For SILAC analysis , the intThe SILAC-labeled cells were grown in 15 cm culture plates, and 3 mg of each light, medium, and heavy amino acid labeled protein extract was mixed for proteomics analysis and phosphopeptides enrichment (separate to the transfected p38α and the p38β variants). The proteins from the different cell lines were extracted from the plates with lysis buffer composed of 8 M urea, 75 mM NaCl, 1 mM NaF, 50 mM Tris pH 8, 1 mM β-glycerolphosphate, 1 mM Na orthovanadate, 1 mM Na pyrophosphate, protease inhibitor cocktail , and 1 mM PMSF.Before tryptic digestion, disulfides were reduced by incubation with 2.8 mM DTT (Sigma) for 30 min at 60 °C and carbamidomethylated with 8.8 mM iodoacetamide (Sigma) for 30 min at room temperature in the dark. Trypsin  was added in a ratio of 1:50 to the proteins and incubated overnight at 37 °C. A second dose of trypsin was added in the morning and incubated for four more hours. The samples were then acidified with 0.1% TFA and desalted with Sep-Pak C18 reversed-phase cartridges .2PO4 (pH 2.65) and 30% acetonitrile over 92 min at a flow rate of 0.5 mL/min. The elution was divided into 10 fractions, which were then affinity enriched for phosphopeptides.Peptides were dissolved in 0.1% TFA and loaded on a 1 mL Resource S Column  on a Jasco LC-Net II/ADC HPLC. Peptides were eluted with a KCl gradient from 0 to 350 mM containing 7 mM KH2)  was added to a 100 µL loading solution  in 80% acetonitrile and 0.1% TFA) and incubated with shaking for 10 min at room temperature. Loading solution was added to the fractions in a ratio of 1:6. Then, the TiO2 beads were transferred to the samples and shaken for 30 min at room temperature. The beads were washed with 1 mL washing solution #1  and 1 mL washing solution #2 . The beads were transferred to C8 Stage-Tips [4OH) followed by 100 µL elution buffer #2  combined into one tube. The eluate was dried in a vacuum centrifuge  and desalted on C18 Stage-Tips.A 5 mg amount of Titansphere chromatography material  target value of 105 with a maximum injection time of 100 ms. Fragmentation was performed for charge states of 2 to 7. The peptide match option was set to Preferred. The normalized collision energy was set to 25%, and MS/MS resolution was 17,500 at 200 m/z. Fragmented m/z values were dynamically excluded from further selection for 20 s.Mass spectrometry analysis was performed on a Q Exactive Plus Mass Spectrometer  using the positive ion mode and full MS scan repetitively followed by higher-energy collision dissociation (HCD) of the 10 most dominant ions selected from the full MS scans. Reversed-phase chromatography was performed with home-made, about 30 cm long, 75 μm inner diameter capillaries packed with 3.5 μm silica ReproSil-Pur C18-AQ resin , as previously described . PeptideThe MS data were interpreted by MaxQuant software version 1.5.0.25 , and theWestern blot analyses were performed with the following antibodies: anti-HA ; p38α (sc-535) and p38β (sc-390984) ; phospho-p38 MAP kinase (Thr180/Tyr182) ; actin . The immune detection was performed by peroxidase-conjugated secondary antibodies , and signals were developed using WesternBright ECL .Escherichia coli Rosetta strain . For expression of p53, we used a pREST plasmid  encoding a fusion protein modified in frame with N-terminal hexahistidine and 94–360 residues of the human p53, followed by the B. stearothermophilus dihydrolipoyl reductase sequence (2–85 aa). This plasmid was constructed in the laboratory of Alan Fersht, Cambridge, UK, and kindly provided to us by Gad Shuster at the Faculty of Biology, Technion, Israel. The 6xHis-p53 protein was purified from E. coli using metal affinity chromatography with Ni–nitrilotriacetic acid (Ni-NTA)-loaded beads . Protein concentrations were determined by the Bradford method.For bacterial expression of the p38 proteins, the pET15b and pET28a vectors N-terminally tagged with hexahistidine were used. Protein expression was induced in the EE) to activate the different p38s. To initialize the reaction, 45 μL of the reaction mixture containing 20 mM MgCl2, 20 mM 2-glycerolphosphate, 0.1 mM Na3VO4, 1 mM dithiothreitol (DTT), 50 μM ATP, 25 mM HEPES, and 40 μg of p53, pH 7.5, was added to 5 μL of activated p38 enzyme (0.2 μg of purified p38) for 10 min at 30 °C, with agitation, and were terminated by transfer to ice. Next, 5 μg total protein of the reaction mix, containing the wild-type p38α (p38αWT), p38αD176A+F327S, wild-type p38β (p38βWT), or p38βD176A proteins and p53 were digested with trypsin to detect phosphorylation on p53 by LC-MS/MS analysis.Purified p38 proteins were incubated with an active mutant of MKK6 in which both Ser207 and Thr211 were mutated to Glu  and 1:1000 Hoechst for 60 min. The slides were stored in PBS at 4 °C until use. The confocal images were taken by Confocal LSM 510 META Microscope .


Excellent operational stability, repeatability and interference-capability were displayed by the modified electrode. Furthermore, the practical applicability of the sensor was tested in commercially available waters with excellent results.The importance of nitrite ions has long been recognized due to their extensive use in environmental chemistry and public health. The growing use of nitrogen fertilizers and additives containing nitrite in processed food items has increased exposure and, as a result, generated concerns about potential harmful health consequences. This work presents the development of an electrochemical sensor based on graphene/glassy carbon electrode (EGr/GC) with applicability in trace level detection of nitrite in water samples. According to the structural characterization of the exfoliated material, it appears as a mixture of graphene oxide , few-layers graphene  and multi-layers graphene  and exhibits remarkable enhanced sensing response towards nitrite compared to the bare electrode (three orders of magnitude higher). The EGr/GC sensor demonstrated a linear range between 3 × 10 Sodium acetate anhydrous  was purchased from ChimReactiv SRL . N,N-dimethylformamide (DMF) was acquired from Fluka Chemie GmbH , while boric acid  was provided by Andra Chim SRL . The MQuant® Nitrite Test  was employed for UV-Vis investigation of nitrite solutions. Deionized water with a resistivity of at least 18.2 MΩ × cm was used to prepare all solutions.The chemicals used during the experiments were of analytical grade and were not further purified before usage. Graphite rods ; potassium chloride ; sodium dihydrogen phosphate  equipped with a cold field emission gun (CSEG). The structural characterization of graphene was performed by X-ray powder diffraction (XRD), with a Bruker D8 Advance Diffractometer  equipped with a LYNXEYE detector (λ = 1.5406 Å). After the deconvolution of the recorded pattern, some parameters of graphene samples were determined: the graphene crystallite size (D), the interlayer distance (d) and the number of layers (n). The graphene Raman spectrum was recorded with an NTEGRA Spectra platform, placed on a NEWPORT RS4000 optical table and equipped with a SOLAR TII confocal Raman spectrometer coupled with an Olympus IX71 microscope in two different configurations . A Christ-Alpha 1-4 LSC freeze-drier  was employed for drying the sample obtained after electrochemical exfoliation of graphite rods.2 area) and the reference was an Ag/AgCl electrode.For electrochemical measurements  a Potentiostat/Galvanostat instrument PGSTAT-302N  coupled with a personal computer was employed. The working electrode was either bare glassy carbon electrode (3 mm diameter), or that modified with graphene. The counter electrode was a large platinum sheet (2 cm4)2SO4 + 0.1 M H3BO3 + 0.05 M NaCl), two graphite rods (anode and cathode) were immersed and connected to the exfoliation system. The solution temperature was kept at around 10 °C with a thermostat and the time parameters for current pulse exfoliation were set before starting the graphene synthesis: current pulse duration 0.8 s; pause between two current pulses 0.2 s  (0.2 M KCl supporting electrolyte). Using the Randles–Ševcik equation [p versus υ1/2 plot, the active area was calculated (see p versus υ1/2 plot was fit by the following linear regression equation: Ip = 2.11 × 10−7 + 3.11 × 10−5 × υ1/2 (R2 = 0.996) and, from the corresponding slope, the EGr/GC area was determined to be 0.049 cm2. As expected, the value was considerably larger than that obtained for bare GC electrode (0.028 cm2), due to the porous morphology of the graphene layer.Before the investigation of the electro-catalytic properties of EGr/GC electrode towards NOequation  and, resated see . The Ip 2− oxidation (10−3 M). The influence was studied by cyclic voltammetry, within the 3.6–8.0 pH range. As can be seen in p), its value increased with the increase of pH up to pH 5.0, then began to decrease (see p) strongly decreased in acidic solution (pH 3.6–pH 5.0), then kept an almost constant value between pH 6.0 and pH 8.0 (see 2) due to their low pKa value (3.3) [Next, the experiments were focused on studying the effect of the pH solution on the electrochemical response of the EGr/GC electrode towards NOease see a. On the 8.0 see b. Accordue (3.3)  and so p2− oxidation is an irreversible process (no peak in the reverse scan), the Laviron equation [p, with the natural logarithm of the scan rate, lnυ, for irreversible reactions (oxidation) when employing the linear sweep voltammetry technique.aα is the charge transfer coefficient, sk is the standard rate constant of the surface reaction, n is the number of electrons involved in the reaction and E0′ is the formal potential.Since the NOequation  may be u−3 M NaNO2 at various scanning rates, from 2 to 100 mV/s. Based on the Ep vs. lnυ plot and the linear regression equation (Ep = 0.899 + 0.023 × lnυ), we determined the value of (1−αa)n from the corresponding slope ):LSVs were recorded in pH 5.0 acetate solution containing 10ng slope . Assumina et al. , the ele2− oxidation, CV measurements were recorded with bare and graphene-modified electrodes and the results are presented in −3 M NaNO2 in pH 5.0 acetate buffers; 10 mV/s scan rate). It is worth mentioning that, in the case of the bare GC electrode (blue curve), the oxidation peak is small and broad and appears at very high potential (+1.18 V). In contrast, the EGr/GC electrode exhibits a considerably higher electrochemical peak (three times higher) and the peak potential is shifted towards lower values (+0.8 V). Such behavior confirms that the graphene layer has an important role in promoting the electron transfer from the solution containing NO2− anions to the glassy-carbon surface.In order to evidence the electro-catalytic properties of the EGr/GC electrode towards NO2− detection were determined by employing the SWV technique. This technique is more sensitive in comparison with the classical cyclic voltammetry, due to the elimination of the capacitive current. −7–10−3 M NaNO2; pH 5.0 acetate; 10 mV/s scan rate). The inset of the figure shows the signals recorded at very low concentrations . In these cases, the peak potential is slightly shifted towards higher potentials (+0.88 V), which is a normal behavior for an irreversible process.Next, the electrochemical parameters for NOp = 6.16 × 10−7 + 0.018 × C (R2 = 0.988) for EGr/GC; and Ip = −1.62 × 10−8 + 9 × 10−4 × C (R2 = 0.916) for bare GC. For the graphene-modified electrode, the linear range was 3 × 10−7–10−3 M, the sensitivity was determined to be 0.018 A/M, the limit of quantification (LOQ) was 3 × 10−7 M and the limit of detection (LOD) was 9.9 × 10−8 M. As expected, the bare GC electrode had considerably lower performances, such as: linear range: 6 × 10−5–10−3 M; sensitivity 9 × 10−4 A/M; limit of quantification (LOQ) 6 × 10−5 M; and limit of detection (LOD) 1.82 × 10−5 M.The corresponding calibration plot is presented in −7 to 4 × 10−4 M NaNO2, the limit of quantification (LOQ) was 3 × 10−7 M, the sensitivity was 0.018 A/M and the detection limit (LOD) was 9.09 × 10−8 M. The detection limit was calculated by dividing the limit of quantification by 3.3. For plotting of the calibration curves, we employed background subtracted signals. As observed in the case of the SW technique, the sensitivity of the EGr/GC electrode was two orders of magnitude higher than that of the bare GC, demonstrating the advantages of using graphene-modified electrodes in the electrochemical detection of nitrite ions.Similar results were obtained by employing the amperometric technique and the recorded signals and calibration plots are presented in The performances of the EGr/GC electrode towards nitrite detection were compared with those of other modified electrodes reported in the literature . With fe2− detection different interfering species were employed 2 had no effect on the electrochemical signal of the NO2− specie. NaCl slightly decreased the NO2− signal, while ascorbic acid increased the signal.In order to prove the selectivity of the EGr/GC electrode towards NOemployed . The amp2+ (73.65); Mg2+ (3.101); Na+ (8.655); HCO3 (250.1); SO4− (<40); Cl− (16.379); K+ (1.078); and NO2− (<0.05). The following procedure was employed to determine the NO2− concentration from the water. First, the water pH was brought to pH 5.0, after the addition of the corresponding amounts of acetic acid (0.2 M) and sodium acetate (0.2 M). The electrochemical signal of NO2− was recorded in the mineral water solution and its concentration was denoted as Cx1. Next, four beakers were filled with mineral water and increasing volumes  of NaNO2 stock solution (10−3 M). The final volume in each beaker was 10 mL and the added NaNO2 concentrations were denoted as C1, C2 and C3, respectively. The corresponding SW electrochemical signals were also recorded and the peak current was represented as a function of NaNO2 added concentrations .Based on the fact that the electrode exhibited excellent electro-catalytic properties, it was further used for the determination of nitrite in mineral waters. Two commercial waters were employed, bought from a local supermarket. According to the label, the first one contains the minerals : Catrations a,b. In t+ (2); K+ (1.4); Mg2+ (2.4); Ca2+ (9.2); F− (<0.03); Cl− (<5); SO4− (6.2); HCO3 (42.7); NO2− (<0.01); NO3 (4.29).The same procedure was applied in the case of the second mineral water, which contained the following minerals : Nax2 was determined to be 9.3 × 10−7 M, which corresponds to 0.04 mg/L NO2−.The SW voltammograms can be seen in 2 solution has no absorption peak in the visible range, the MQuant® Nitrite Test (Merck) was employed. The test is based on the reaction between nitrite and sulphanilic acid, which forms a diazonium cation. The cation subsequently couples to the aromatic amine 1-naphthylamine and produces a red-violet azo dye with absorption at λmax = 540 nm. In the case of the first mineral water, the spectrophotometric method indicated that Cx1(UV) = 6.21 × 10−7 M, while for the second mineral water Cx2(UV) = 7 × 10−7 M 2SO4 + 0.1 M H3BO3 + 0.05 M NaCl. After preparation, the optimum volume (10 µL) of graphene dispersed in DMF (2 mg/mL concentration) was deposited on top of a glassy carbon electrode (EGr/GC) and then tested towards nitrite detection, in laboratory solutions and real samples. The reported results indicated that the developed graphene-based electrochemical sensor is a reliable and highly sensitive tool for nitrite assay in waters, since it displayed excellent stability and reproducibility, exhibiting a low limit of detection (9.09 × 10−8 M). Since the developed sensing platform is able to detect, with a high degree of accuracy and reliability low nitrite concentrations, it may be further used to monitor nitrite levels in both industrial and environmental conditions and provide a necessary tool in the prevention of water pollution.A graphene sample was prepared by electrochemical exfoliation of graphite rods in an electrochemical cell filled with the appropriate solution: 0.05 M (NH


Mo loading was about 7 wt.%. Catalysts and supports were characterized by different physical-chemical methods . Hydroprocessing was performed at a temperature of 250–350 °C, hydrogen pressure of 3.0–5.0 MPa, liquid hourly space velocity (LHSV) of 36 h−1, and an H2/feed ratio of 600 Nm3/m3. Complete conversion of oxygen-containing compounds was achieved at 310 °C in the presence of MoS2/Al2O3-zeolite catalysts; the selectivity for the conversion of methyl palmitate via the ‘direct’ hydrodeoxygenation (HDO) route was over 85%. The yield of iso-alkanes gradually increases in order: MoS2/Al2O3 < MoS2/Al2O3-ZSM-12 < MoS2/Al2O3-ZSM-5 < MoS2/Al2O3-SAPO-11 < MoS2/Al2O3-ZSM-22. The sample MoS2/Al2O3-ZSM-22 demonstrated the highest yield of iso-alkanes (40%). The hydroisomerization activity of the catalysts was in good correlation with the concentration of Brønsted acid sites in the synthesized supports.A series of bifunctional catalysts, MoS One of the approaches to solving this problem in the transport sector is the use of renewable bio-resources to obtain biofuel components that are not inferior in quality to products of petroleum origin Cl, Sigma-Aldrich, 97%, abbreviated [MTEA]Cl), sodium hydroxide , and ammonium nitrate . Solution A, consisting of 12.6 g of distilled water, 0.4 g of Al2(SO4)3·18H2O, 1 g of NaOH, and 3.3 g [MTEA]Cl used as a template, was stirred until all of the components became completely dissolved. Solution B, consisting of 25.2 g of a 40% (wt.) colloidal solution of silicon dioxide of the brand LUDOX HS-40 and 10.1 g of distilled water, was stirred until the reaction mixture was homogeneous. Solution A was dropped into solution B and stirred gently. The gel was poured into a Teflon liner and placed into the autoclave, which was heated at 155 °C for 120 h. The product was filtered off, washed with distilled water, dried at 110 °C for 12 h, and calcined at 550 °C for 10 h (heating rate 1 deg∙min−1). To obtain the H-form of zeolite, the synthesized material was treated 3 times with a 1 M aqueous solution of NH4NO3 at 80 °C for 17 h. The solid product was filtered off, washed with distilled water, dried at 110 °C for 12 h, and calcined at 550 °C for 8 h (heating rate 1 deg∙min−1).The synthesis of zeolite ZSM-12 was carried out using the following reagents: a colloidal solution of silicon dioxide LUDOX HS-40 , aluminum sulfate octadecahydrate . Zeolite-containing granular supports were prepared by mixing pseudoboehmite  and zeolite powders, followed by peptization with nitric acid, and then piston extrusion through a trefoil-shaped die. After extruding, support granules were dried at 110 °C for 12 h and then calcined at 550 °C in air flow for 6 h. Zeolite content was 30 wt.% in all calcined composite supports. Synthesized supports were denoted as Al2O3-ZSM-5, Al2O3-ZSM-12, Al2O3-ZSM-22, and Al2O3-SAPO-11.Alumina support was prepared by HNO4)6Mo7O24 4H2O from Vekton, Saint Petersburg, Russia) and citric acid monohydrate . Mo content was about 7.0 wt.% after calcination of the catalysts at 550 °C for 4 h.Mo catalysts were prepared by incipient wetness impregnation of synthesized alumina and zeolite-containing extrudates by aqua solution containing ammonium heptamolybdate .The elemental analysis was performed using inductively coupled plasma atomic emission spectroscopy (ICPAES) on Optima 4300 DV . The Mo content was determined after calcination of the catalysts at 550 °C for 4 h.X-ray powder diffraction (XRD) patterns of supports and catalysts were obtained with an instrument STOE STADI MP  with a detector MYTHEN2 1K using MoKα radiation (wavelength λ = 0.7093Å). The measurements were carried out in a range of 2θ from 2 to 40°, with a scanning step of 0.015°.2O3-zeolite supports and pure Al2O3 was characterized by FTIR spectroscopy of adsorbed carbon monoxide. FTIR spectra were recorded on a Shimadzu FTIR-8300 spectrometer  within the spectral range of 700–6000 cm−1, resolution of 4 cm−1, and 300 scans for signal accumulation. The powder samples were pressed into thin, self-supporting wafers of 0.010–0.012 g × cm−2 density and pretreated in a home-made IR cell at 500 °C for 2 h under a dynamic vacuum of less than 10−3 Pa. In the presented spectra, the absorbance was normalized to sample wafer density. CO was introduced at liquid nitrogen temperature in doses from a low pressure of 0.1 mbar up to an equilibrium pressure of 10 mbar. The concentration of Brønsted acid sites (BAS) was determined from the integral intensity of the bands assigned to hydrogen-bonded complexes of CO molecules with the OH groups using the following molar integral absorption coefficient values: A0 = 54 cm/μmol for the complexes with νOH...CO ~ 3280–3380 cm−1 and A0 = 27 cm/μmol for the complexes with νOH...CO 3500 cm−1 [The acidity of Al500 cm−1 .The morphology of supports was studied using a Hitachi Regulus SU8230 FESEM scanning electron microscope  with an accelerating voltage of 2 and 5 kV in the modes of secondary (SE) and backscattered (BSE) electrons using an upper (U) detector, which makes it possible to obtain microscopic images in phase and topographic contrasts. The study of the chemical composition was also carried out on a Hitachi Regulus SU8230 FESEM scanning electron microscope  with an accelerating voltage of 20 kV. The device is equipped with an AztecLive  energy-dispersive X-ray characteristic spectrometer (EDX) with a semiconductor Si detector with an energy resolution of 128 eV.The morphology of the sulfide phase of the catalysts after hydroprocessing was studied by high-resolution transmission electron microscopy (HRTEM) using a ThemisZ electron microscope  with an accelerating voltage of 200 kV and a limiting resolution of 0.07 nm. Images were recorded using a Ceta 16 CCD array . The instrument is equipped with a SuperX  energy-dispersive characteristic X-ray spectrometer (EDX) with a semiconductor Si detector with an energy resolution of 128 eV. To obtain statistical information, the structural parameters of ca. 500 particles were measured.2 pressure—3.5 MPa, H2/feed ratio—300 Nm3/m3, and LHSV—20 h−1. Sulfidation was performed at a temperature of 340 °C for 4 h with a heating rate of 25 °C/h.The catalytic experiments were performed using an experimental setup with a trickle-bed reactor with an inner diameter of 12 mm and a length of 370 mm. In each experiment, 0.5 mL of catalyst (0.25–0.50 mm size fraction) was diluted with inert material, carborundum (0.1–0.25 mm size fraction), in a 1:8 volume ratio. Prior to the catalytic experiments, the catalysts were activated by in-situ sulfidation with dimethyl disulfide in dodecane (0.6 wt.% sulfur) at H2 pressure of 3.0 and 5.0 MPa, H2/feed ratio of 600 Nm3/m3, and LHSV of 36 h−1. The feed was 10 wt.% of methyl palmitate (1.17 wt.% O) with dimethyl disulfide (0.6 wt.% sulfur) in dodecane. Dimethyl disulfide was added to the feedstock to maintain the sulfide form of the active component (MoS2). The duration of each step was 6 h.Hydroprocessing of methyl palmitate was carried out at a temperature range of 250–350 °C, H2/feed ratio of 600 Nm3/m3, and LHSV of 36 h−1).To check catalyst stability, oxygen conversion was compared in the first and last stages carried out in the same conditions in each experiment  conversion were analyzed using an Agilent 6890N gas chromatograph  equipped with a flame ionization detector and an HP-1MS quartz capillary column (30 m × 0.32 mm × 1 μm). Methyl palmitate conversion was calculated as follows (1):The total oxygen content in liquid samples was determined using a Vario EL Cube elemental CHNSO analyzer .Oxygen conversion was calculated as (2):The gas phase during the MP hydroprocessing was analyzed online using a gas chromatograph Chromos 1000  equipped with a methanator and a flame ionization detector.16C is the content of 16C alkanes  in the final product, and 15C is the content of 15C alkanes  in the final product.The selectivity of the ‘direct’ HDO route (HDO selectivity) was calculated as follows (3):16i-C and i-C15 are the contents of iso-C16 and iso-C15 alkanes in the final product at complete oxygen conversion; The yield of iso-alkanes was calculated as follows (4):2O3) and corresponding sulfide Mo-containing catalysts were characterized by XRD, HRTEM, and SEM. According to XRD data, the structure of zeolites was preserved in synthesized supports and catalysts. The uniform distribution of zeolite crystallites in composite materials  was confirmed by SEM-EDX. 100% conversion of oxygen was observed at 310 °C over sulfided Mo/Al2O3-zeolite catalysts in the hydroprocessing of methyl palmitate. A temperature rise from 310 to 350 °C resulted in a decrease in HDO selectivity. It was found that the addition of zeolite to alumina has a slight influence on MP conversion, but the effect on the conversion of oxygen-containing compounds is greater. The activity of MoS2/Al2O3-zeolite catalysts in the production of isomerized alkanes in MP hydroconversion is in good correlation with the concentration of Brønsted acid sites. The yield of iso-alkanes in the hydroisomerization of MP increased in the following order: Al2O3 < Al2O3-ZSM-12 < Al2O3-ZSM-5 < Al2O3-SAPO-11 < Al2O3-ZSM-22. The yield of iso-alkanes was affected by temperature and hydrogen pressure. An increase in temperature and pressure resulted in a decrease in iso-alkane yield. This observation can probably be explained by the reaction mechanism under the given reaction conditions. Iso-alkanes are formed from intermediate products of methyl palmitate HDO, not from alkanes.Synthesized composite zeolite-containing supports (30 wt.% zeolite and 70% Al