The amount of DHP formed on the surfacewas found to be independent of the base layer thickness from 14 to75 nm. Pyrolysis-GC-MS measurements of the DHP revealed β-O-4and β-5 linkages. Mimicking lignification of plant cell wallson highly defined model films enables reproducible investigationsof structure–property relationships.Thin films of celluloseferulate were designed to study the formationof dehydrogenation polymers (DHPs) on anchor groups of the surface.Trimethylsilyl (TMS) cellulose ferulate with degree of substitutionvalues of 0.35 (ferulate) and 2.53 (TMS) was synthesized by sophisticatedpolysaccharide chemistry applying the Mitsunobu reaction. The biopolymerderivative was spin-coated into thin films to yield ferulate moietieson a smooth cellulose surface. Dehydrogenative polymerization of coniferylalcohol was performed in a Quartz crystal microbalance with a dissipationmonitoring device in the presence of H In the cell wall of woody plants, cellulose,hemicellulose, and lignin are the main components combined togetherin sophisticated hierarchical structures.4 and could also be tailored bychemical post-modification.6 Ultrathin films of cellulosewere applied as model systems and in field of advanced materials.7 Very smooth films are obtained by spin coatingor the Langmuir–Blodgett technique of trimethylsilyl (TMS)cellulose and subsequent regeneration with hydrochloric acid vapor.9 It is up to researchers to create more complex plant cell wall modelsbased on cellulose thin films, for example, by inclusion of lignificationprocesses.Nanometricthin films based on cellulose are predestinated forstudies on well-defined surfaces since they are suited for adsorptionstudies10 for the first time. Dehydrogenation polymers are typically obtained by oxidativecoupling of hydroxycinnamyl alcohols  applying a peroxidase with hydrogen peroxide. In subsequentstudies, reaction conditions were tuned including effects of polysaccharidematrices. For instance, pectin improves the dispersion of DHP in thecellulose network, enhancing the formation of aryl–aryl ethers.11 Cyclodextrin increases the β-O-4-content,whereas β-5-linkages are decreased.12 Moreover, the influence of hemicellulose on molecular and supramolecularstructures of DHPs synthesized in bulk was investigated.13 However, lignification experiments at interfacesusually do not allow a distinction between adsorption of DHP on thepolysaccharide surface and the formation of covalent bonds.Mimicking the lignification in plant cell walls bydehydrogenativepolymerization of monolignols was carried out in vitro by Freudenberg15 Wang et al. performedreal-time experiments on QCM-D, that is, dehydrogenative polymerizationof hydroxycinnamyl alcohols was carried out on gold and silica surfacesand films of cellulose nanocrystals.16 However,it would be advancing to use model surfaces with anchor groups, suchas ferulate moieties, present in plant cell walls, which could beartificially lignified.17Until now, the formation of synthetic lignin on planar surfacesis mostly limited to adsorption studies, for example, deposition ofDHP on hemicellulose films.In thepresent work, approaches from online monitoring by QCM-Dand polymerization of monolignols from a surface decorated with ferulatemoieties were combined. TMS cellulose ferulate was synthesized byadvanced synthetic chemistry utilizing the Mitsunobu reaction. Thesolubility of this biopolymer derivative in easily evaporable solventsallows tailoring of nanometric thin films by spin coating. Those syntheticmodel films enable defined artificial lignification to study structure–propertyrelationships.–1 (ABTS assay). TMS cellulose (DSNMR 2.85) was synthesized, according to a procedure by Kostaget al.18 applying N,N-dimethylacetamide (DMA)/LiCl as the solvent. Other chemicalswere purchased from Sigma-Aldrich, VWR, or Carl Roth and were usedwithout further treatment.Microcrystalline cellulose (Avicel PH-101)possessing a –1 was used for surface experiments.Silicon wafers  were purchased fromSi-Mat  and cut into 15 mm × 15 mm pieces.Quartz crystal microbalance with dissipation monitoring (QCM-D) sensorswith a gold layer (QSX301) were purchased from Quantum Design . Ultrapure water possessing a resistivity of 0.05 μScm–1 sample, 10 000 scans) in CDCl3 or dimethyl sulfoxide(DMSO-d6). Relaxation delay was set to 10 s for quantitativeevaluation of the 1H NMR experiments. Fourier transforminfrared (FTIR) spectra were recorded on a FTIR spectrometer Tensor27(Bruker Optics GmbH).NMR spectra were recorded on a BrukerAvance Neo 700 MHz SB instrument (up to 80 mg mLWater contact angles (CAs) were determinedwith a Dataphysics instrument  with the sessiledrop method and a drop volume of 3 μL. The photographic imageswere evaluated with Python software using the pyDSA package for dropshape analysis. Measurements were performed at room temperature andwere repeated at least 10 times.3N4  with a resonance frequency of 10 kHz and a force constantof 0.03 Nm–1. Images were processed using Gwyddionsoftware applying plane leveling, alignment of rows, correction ofhorizontal error lines, and fix zero. RMS (Sq) values were obtainedfrom statistical sizes of processed AFM images (10 μm ×10 μm).Atomic force microscopy (AFM)was performed in the contact modewith a TMX 2010 instrument . The images werescanned using cantilevers CSC12/Si–1. The relative resonant frequency(ΔF) and the relative dissipation factor (ΔD) of the third overtone (n = 3) were determinedin comparison to the zero values. The fundamental frequency of quartzcrystals is f0 ≈ 5 MHz, and thesensitivity constant C = 17.7 ng Hz–1 cm–2.QCM-D experiments were carried out with a Q-SenceE1 instrument at 23.0 °C, applying a flow rate of 100μL minF is changing less than 1 Hz in 10 min. The data files of individualmeasurements were stitched together to obtain the change in the frequency.Due to the rigidity of evenly distributed films, the Sauerbrey equationis valid, describing a linear relationship between frequency changeand adsorbed mass.19 Areal mass densityof the adsorbed film (mfilm) was calculatedfrom this Sauerbrey relationship using the following equation20To determine the layer thickness ofthin films, QCM-D sensors weremeasured in air in uncoated, spin-coated, and desilylated state. Themeasurements were performed until a stable baseline was obtained.A baseline is considered to be stable if Δn) is automatically consideredby software. Film thicknesses (d) were calculatedfrom mfilm and film densities (ρfilm) of cellulose and TMS cellulose films, according to theliterature.21The overtone number (–1) was introduced into the flow cell for 10 min. Subsequently, theexcess of loosely bound enzyme was removed by rinsing with H2O for 20 min. The polymerization was performed with an aqueous solutioncomposed of 20 mM H2O2 and 0.5 mg mL–1 coniferyl alcohol for 40 min. Afterward, the sensors were rinsedfor 30 min with H2O. ΔF was evaluatedat 40 and 110 min of the experiments to exclude bulk effects fromH2O2 and coniferyl alcohol. The Sauerbrey equationis not valid for viscoelastic layers that were formed by polymerizationof coniferyl alcohol. Monitoring ΔF and ΔD provides information about rigidity or softness of thefilms. The dissipation factor (D) is the ratio betweendissipated and stored energy in the oscillating system23Flow through QCM-Dexperiments were started by rinsing of the sensorswith ultrapure water for about 1 h to obtain a stable baseline whenrestarting the measurement. After 10 min, HRP  software was used to identify compounds by comparingspectra in the NIST MS library.Py-GC-MS was performed on an Agilent system(GC 7890 B/MSD 5977).Samples (about 300 μg) were yielded from silicon wafers by scrapingoff the film with a razor blade and pyrolyzed by a Multi-Shot PyrolyzerEGA/PY-3030D (Frontier Lab) at 450 °C. Separation was achievedby a ZB-5MS capillary column (30 m × 0.25 mm) with a temperatureprogram of 50–240 °C at 4 K minCellulose  was stirred in dry DMA (90 mL) for 2 h at 120 °C. Aftercooling the suspension to 90 °C, LiCl (5.4 g) was added, anda clear solution was obtained by continued stirring. Ferulic acid was added and dissolved completely in the solutionof cellulose. Afterward, triphenylphosphine  wasadded, and the resulting solution was cooled to 0 °C by an icebath. Subsequently, diisopropyl azodicarboxylate (DIAD)  was added dropwise under stirring. Furthermore, the reactionmixture was stirred at room temperature for 2 d. To yield the crudeproduct, the material was precipitated into methanol (1200 mL) andwas washed three times with methanol (600 mL), two times with water(600 mL), and finally with methanol (600 mL). The raw product wasdried under vacuum at 40 °C. For analytical purposes, the materialwas reprecipitated from DMSO in methanol.2), degree of substitution (DS) 0.35; 13CNMR : δ [ppm] = 164.3 (C=O),151.4, 149.9, 149.3, 148.0, 147.4, 146.3, 145.3, 141.4, 132.8, 125.5,123.5, 122.2, 117.1, 115.6, 114.1, 113.0, 112.3, 111.4, 102.9 (C1),80.3 (C4), 74.8 (C5/C3), 73.0 (C2), 63.3 (C6s), 60.2 (C6), 56.1, 55.2(OCH3).Cellulose ferulate was suspended in dry DMA (50 mL), and hexamethyldisilazane(HMDS)  and trimethylsilyl chloride (TMSCl)  were added. The reaction mixture was stirred for1 h at 80 °C, cooled to room temperature, and precipitated intodeionized water (1.2 L). The raw product was washed with water untilthe yellow color disappeared. Subsequently, the product was driedunder vacuum at 40 °C to yield about 5 g of solid. For furtherpurification, TMS cellulose ferulate was dissolved in ethyl acetate(70 mL), and insoluble impurities were removed by centrifugation.The clear solution was poured into aqueous sodium hydrogen carbonate(5 g in 1.2 L water), and the product was filtered off, washed withwater (four times 400 mL), and finally dried under vacuum at 40 °C.3), yield: 3.66 g , DSferulate 0.35, DSTMS 2.53; 1H NMR : δ [ppm] = 5.6–8.2(CHferulate), 2.5–5.0 , −1.0–1.0 (TMS).TMS cellulose ferulate (3 (0.5 g). Purification of thematerials was achieved by filtration, washing three times with deionizedwater (100 mL), and drying under vacuum at 40 °C.Celluloseferulate (300 mg) was suspended in pyridine (5 mL), andacetic anhydride (5 mL) was added. The reaction mixture was stirredfor 24 h at 60 °C. Afterward, insoluble impurities were centrifugedoff. The product was yielded by precipitation into deionized water(150 mL) containing NaHCOOH, 13CNMR : δ [ppm] = 170.2 , 170.0 , 169.7 , 169.3 , 164.5 (C=O), 151.7(C–OMe), 146.3 (CH-9), 141.9 (C-OAc), 133.0 (C10), 123.5, 121.6,116.8 (CH-8/15/16), 111.5 (CH-11), 100.5 (C1′), 76.1 (C4),72.8 (C5), 72.5 (C3), 71.8 (C2), 62.0 (C6s), 56.1 (OCH3), 20.8 (CH3 Ac), 20.7 (CH3 ArOAc), 20.6 (CH3 Ac), 20.5 (CH3 Ac).Peracetylatedcellulose ferulate, FTIR: no νferulate value was calculated from integral intensities(I) of 1H NMR signals of CH-protons  and CH3-protons  with the following equationThe DSTMS value was calculated from integral intensities(I) of 1H NMR signals of CH3-protons  and CH-protons including DSferulate with the following equationThe DS2SO4 (96%) and 5 mL of H2O2 (35%)] for 1 h. Subsequently,surfaces were rinsed with ultrapure water and dried with nitrogengas. QCM-D crystals were cleaned with Radio Corporation of America(RCA) solution  at 75 °C for 15min and finally rinsed. The support materials were spin-coated at4000 rpm (acceleration 2500 rpm/s) for 60 s using a POLOS SPIN150i-NPPsingle substrate spin processor (Desktop Version) from SPS-EuropeB.V. . For cleavage of silyl groups, films weretreated with hydrochloric acid vapor (from 10 wt % HCl) in Petri dishesfor 15 min at room temperature. This procedure was adapted from theliterature describing desilylation of TMS cellulose films.8Silicon wafer pieces were pre-cleanedwith 2-propanol for 10 min in an ultrasonic bath, rinsed with water,and treated with piranha solution + is 27.5L mmol–1 cm–1 and was taken fromthe literature.25Activity of HRP insolution and on surface was determined by ABTS assay. The oxidationproduct [ABTS*]26 The reaction conditions were successfully transferred fromadvanced organic chemistry to polymer-analogous modifications. Thishighly selective esterification is tolerant regarding double bondsand phenolic hydroxyl groups. Thus, utilization of protecting groupsis not required. Moreover, drawbacks of Steglich esterification, thatis hardly removable dicyclohexylurea and poor solubility of products,could be avoided.Recently, we found a novel synthesis path for cellulose estersof hydroxycinnamic acids under Mitsunobu conditions.26 However, the low vapor pressure of DMSO limitsthe formation of nanometric films. In this regard, cellulose ferulatewas silylated with HMDS to enable processing in easily evaporableorganic solvents.To prepare thecellulosic thin films possessing ferulate moieties by spin coating,soluble derivatives are required. Therefore, the synthesis of DMSO-solublecellulose ferulate under Mitsunobu conditions was applied.1, 2) witha DS value of 0.35. In a further step, cellulose ferulate was convertedwith an excess of HMDS in the presence of TMSCl as the catalyst usingDMA as the solvent. This procedure was adapted from trimethylsilylationof pure cellulose.18 The TMS celluloseferulate (3) possessed DS values of 0.35 (ferulate) and2.53 (TMS). It was soluble in ethyl acetate and chloroform but insolublein acetone.In the first step, cellulose 1, 1 dissolv2) could be clearly revealed by FTIR and NMR spectroscopyand was published in detail previously.26 In the IR spectrum ,aliphatic −C–H vibrations are indicated at 2884 cm–1. The −C=O signal of the ester moietyappears at 1722 cm–1. Signals at 1632, 1594, and1510 cm–1 reveal aromatic structures. The 13C NMR spectrum clearly shows the linkage of ferulate to cellulosebackbone by the carbonyl signal at 164.3 ppm . The resonances of CH moieties from aromatic ringsand double bonds are detected between 111 and 152 ppm. Peaks of themodified anhydroglucose unit (C1–C6) are in typical range ofcellulose esters. The methoxy group is indicated at 56 ppm.Molecular structure of cellulose ferulate  shows a stronglydecreased OH stretching but increased aliphatic -C-H vibrations arisingfrom substitution with TMS groups. Moreover, a typical peak of theSi–CH3 vibration at 1250 cm–1 occurs.The 1H NMR spectrum shows CH moieties from aromatic ringsand double bonds at 5.6 to 8.2 ppm . Resonances of CH and OH groups arising from the modified anhydroglucoseunit and methoxy group are overlapping from 2.5 to 5.0 ppm. The maximumof a very strong TMS signal is at 0 ppm. 13C NMR spectrumof 3 does not provide additional information since theresolution is poor. However, the key resonances of substituents arevisible.The IR spectrum of trimethylsilylated cellulose ferulate  was calculated from the integral intensities of the 1H NMR spectrum of the peracetate.26 Calculationsare based on the ratio of CH moieties appearing in the low field from8.0 to 6.0 ppm and methyl groups of acetate in the high field from2.5 to 1.0 ppm. A complete acetylation of hydroxyl groups can be assumeddue to missing OH valence in the IR spectrum.The DSTMS value of TMS cellulose ferulate (3) was calculatedfrom the ratio of intensities of CH moieties (lowfield) and the TMS signal  was spin-coated followed by removal of silylgroups with HCl vapor (3 was dissolvedin ethyl acetate and spin-coated at defined rotation settings butvarying polymer concentrations. It turned out that smooth and uniformfilms can be obtained from solutions with a mass concentration (β)of 5 to 25 mg mL–1. The film obtained with β= 10 mg mL–1 was used in extended experiments andis termed Film 3, and its desilylated material is Film 2.To obtain nanometric films possessingferulate anchor groups for artificial lignin polymerization, TMS celluloseferulate (Cl vapor 1. In theF) and the change in the dissipation factor(ΔD). In general, dissipation is expressedas the ratio of dissipated and stored energy when the film on thesensor is subjected to the oscillating cycle  increaseswith increasing concentration. Thicknesses were calculated from mfilm and thin film density (ρfilm) from the literature. Assuming a film density of 1.5 g cm–3 for cellulose films,21 layer thickness(d) is in the range of 10 nm to 75 nm. A typicalexample for the calculation of areal mass density and thickness fromfrequency shifts ΔF can be considered in Film thicknesses of TMS cellulose ferulate filmsand cellulose ferulate films were determined by QCM-D measurementsin air using equilibrated values. The same sensors were measured beforeand after coating, as well as after desilylation. Individual baselinesof the measurements were stitched together to obtain frequency shifts(ΔD becamevisible for thicker films and its desilylated form (–1). Onthe one hand, increasing film thickness causes a more viscoelasticbehavior compared to a purely elastic thin layer. On the other hand,more hydrophilic cellulose ferulate films obtained by desilylationare containing adsorbed water. Those films are inherently softer thanTMS cellulose ferulate films. However, in practice, films can be consideredas rigid if ΔF/ΔD >25.24 The Sauerbrey relationship is validfor the whole data set of A small increase in dissipationΔFilm 2 and 3 is very similar. A few nanometricparticles are visible on both surfaces. Film 2 appearswith softer edges in the topography due to a condensed layer structureafter cleavage of TMS groups. However, RMS roughness did not changesignificantly from 4.2 nm to 2.7 nm.AFM measurements show smooth surfacesbefore and after desilylation4. The suFilm2 is higher due to remaining hydrophobic ferulate groups.According to the expectations, water CA is high (95°)forTMS cellulose ferulate films, possessing hydrophobic silyl groupsand also hydrophobic ferulate moieties. After cleavage of TMS groups,the water CA decreases to 43°. Compared to the water CA of apure cellulose film (25–30°), the value for Film 3 , OH stretching appears due to the formationof aliphatic and aromatic hydroxyl groups. The intensity of aliphatic−C–H vibrations is decreased, and in particular, Si–CH3 vibration at 1250 cm–1 disappears. Signalsarising from ferulate groups are still visible.The FTIR spectrum of 2O2 solutions catalyzed by HRP adsorbedon the surface. To underline the reactivity of anchor groups (ferulate)on the films, experiments were run in parallel with pure celluloselayers, obtained by regeneration of TMS cellulose films. The effectof the covalent attachment of artificial lignin to the surface vialignin-carbohydrate complex, that is ferulic acid ester, could bevisualized on silicon wafers.Oxidative polymerizationof coniferyl alcohol on cellulose ferulate films was carried out inaqueous H2O2 solutionswere placed on the films to perform polymerization. It could be alreadyseen by naked eye that a bright layer was formed on the celluloseferulate film. Images from light microscopy  show a uniform topography composedof nanometric globules. The supramolecular structures are also stableafter rinsing with water and blow drying. In contrast to celluloseferulate films, DHP are not tightly bound to pure cellulose films.The wafer appears to be optically similar to the bare cellulose filmafter rinsing.F and ΔD values over time. The inlet of HRP, coniferyl alcohol/H2O2, and rinsing steps are marked. The concentrations ofreagents applied for artificial lignification in a QCM-D instrumentwere already optimized by Wang et al.16 However, the focus of our study is to discover novel films for ligninpolymerization. Therefore, we used concentrations from their previouswork,16 that is 1 mg mL–1 HRP, 0.5 mg mL–1 coniferyl alcohol, and 20 mMH2O2. The adsorption of HRP on the surface couldbe detected but not quantified by QCM-D. ΔF is too small during adsorption and rinsing, which is compensatedby inherent baseline drifts. However, enzyme activity per area wasdetermined by ABTS assay and is 650 U m–2.For advanced real-time measurements, polymerization of coniferylalcohol on thin films was carried out on QCM-D sensors in a flow-throughexperiment. 2O2 into the flow cell containing a cellulose ferulate-coatedsensorwith adsorbed HRP lead to a rapid change in frequency, indicatingthe increase in areal mass, that is the formation of DHP on the surface.For quantitative evaluation of the polymerization, the differenceof ΔF and ΔD at 40 minand 110 min (rinsing H2O) was considered. The shifts areindicated as ΔF110 – ΔF40 and ΔD110 – ΔD40 in FDHP and ΔDDHP hereafter. Thus, bulk effects from coniferyl alcohol and H2O2 could be removed; moreover, loosely attached DHP doesnot contribute to the areal mass. However, layers of DHP show pronouncedirreversible deformations under oscillating stress, that is, ΔDDHP increases up to 4 × 10–6, and the Sauerbrey equation is not valid (ΔF/ΔD < 25). A subsequent change from theinlet of coniferyl alcohol and H2O2 to rinsingwith H2O has only minor influence on ΔF and ΔD. There is just a small amount of DHPthat could be removed from the surface, and the remaining layer behavessoft. This is in accordance with the work of Wang et al.16 who also noticed that the Sauerbrey relationshipis not valid for DHP films. Thus, within this study, we did not calculateareal mass densities but compared ΔFDHP values. Nevertheless, the dependency of ΔFDHP from layer thickness of cellulose ferulate films indicatesan interesting polymerization behavior .The introduction of coniferyl alcohol/Hbehavior 7, bottomM images 4, right,Table S1) without ferulate groups was not reproducible,that is, ΔF was around −7 Hz or −80Hz depending on random adhesion . Successful lignification experiments of Wang et al.16 on films of cellulose nanocrystals can be explainedby residual amounts of hemicellulose and lignin from spruce softwoodpulp, which most likely provide anchor groups for polymerization.Thus, our study is not in conflict with previous work.Polymerization on pure cellulose films  during lignification due to the formationof a particulate topography. The size of nanospheres on the surfaceis in accordance with supramolecular structure of DHP. A general acceptedmechanism for the formation of DHP consists of four steps. First,modules are formed by polymerization of about 20 phenylpropanoid unitsfollowed by building up macromolecules with about 500 units,27 which are also called supermodules.28 Applying advanced microscopic techniques, anaggregation of these supermodules into globules and finally self-assemblinginto a colloidal crystal structure was found.29 The first two steps are based on the formation of covalent bonds,whereas the formation of supramolecular structures (step 3 and 4)is caused by hydrogen bonding and van der Waals interactions.30Surfaceroughness (RMS) of thin films increased from 2.7 to 41nm ,which is the dominant product of the primary pyrolysis reaction (200–400°C) of β-O-4 lignin models under H-donation.32 This is consistent with results for DHP obtainedby Zutropfverfahren (ZT) where β-O-4 linkages are predominant.33 The products of Zulaufverfahren are rich in5–5 structures. As the immobilized HRP is continuously suppliedwith the substrate, in this study, the reaction conditions can beconsidered as variation of the ZT procedure.16To investigate the molecularstructure of artificial lignin in detail, n), dihydroconiferyl alcohol (l), isoeugenol (h), and 4-vinylguaiacol (e) could be found too.34 Moreover, other evaporable typical lignin pyrolysisproducts, for example, guaiacylacetone (k), acetovanillone(j), vanillin (g), eugenol (f), 4-ethylguaiacol (d), 4-methylguaiacol (b), and guaiacol (a) were detected.35 In addition to the β-O-4 motive, β-5 linkageswere evidenced by pyrolysis product (i) arising fromphenylcoumaran units. Catechol (c) is a product of secondarypyrolysis reactions (>400 °C), changing the aromatic substitutionpattern.32The products of typical side chainconversion of coniferyl alcoholin primary pyrolysis reactions such as coniferyl aldehyde  and 2.53(TMS) was obtained by innovative Mitsunobu chemistry and subsequentconversion with HMDS. Silylation was required for spin coating ofthe cellulose derivative from easily evaporable solvents, for example,ethyl acetate. The obtained thin films could be desilylated by hydrochloricacid vapor to yield ferulate moieties on a cellulose surface. Thinfilms were characterized by FTIR spectroscopy, QCM-D, AFM, and goniometry.Layer thickness could be adjusted from 10 to 75 nm by varying massconcentrations of the polymer solution.–1) was carried outin the presence of H2O2 (20 mM) and catalyzedby HRP (650 U m–2) adsorbed on the surface. Thisprocedure can be considered as variationof the ZT in a QCM-D device to allow online monitoring. The frequencyshifts caused by formation of DHP on the sensors indicate an independencyfrom base layer thickness in the range from 14 nm to 75 nm. Thus,lignification seems to take place only at ferulate groups of the toplayer of the film but not inside the material. A referential experimenton a cellulose film without ferulate groups showed loose attachmentof DHP on the surface. QCM-D measurements on pure cellulose layerswere not reproducible since deposited material could be rinsed off.Dehydrogenative polymerizationof coniferyl alcohol (0.5 mg mLAFM images show a supramolecular DHP layer composed of nanosphereswith a diameter of about 300 nm on the cellulosic films. The molecularstructure of DHP was investigated by Py-GC-MS to evidence β-O-4and β-5 linkages. Based on a very uniform and adjustable molecularand supramolecular structure of the model films, biomimetic lignificationexperiments will be performed in future to discover structure–propertyrelationships. Cellulose ferulate films will open new avenues to tailoradvanced materials. Moreover, highly defined DHP layers could serveas sensors for ligninolytic enzymes.


Staphylococcus aureus (MRSA) is a major bacterial pathogen that causes hospital- and community-acquired infections. Owing to its multidrug resistance, it is imperative to develop new antimicrobial agents to treat MRSA infections. In this study, using genome mining analysis and a culture-based screening method to detect bacteriocin activity, we screened a strain, Bacillus sp. TL12, which harbored a putative leaderless bacteriocin gene cluster (bac gene cluster) and exhibited potent anti-MRSA activity. The antimicrobial agents, products of the bac gene cluster, were purified and identified as four novel leaderless bacteriocins: bacin A1, A2, A3, and A4. Bacin A2 was evaluated as a representative antimicrobial agent and showed remarkable antimicrobial activity against S. aureus, MRSA, and the foodborne pathogens Listeria monocytogenes and Bacillus cereus. Mechanistic experiments revealed that bacin A2 damaged cell membranes and exhibited bactericidal activity against MRSA. Bacin A2 effectively inhibited the formation of S. aureus and MRSA biofilms (>0.5× MIC) and killed the cells in their established biofilms (>4× MIC). The hemolytic and NIH/3T3 cytotoxicity assay results for bacin A2 confirmed its biosafety. Thus, bacins have potential as alternative antimicrobial agents for treating MRSA infections.Methicillin-resistant IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that is difficult to treat because of its resistance to several widely used antibiotics. The present study aimed to identify novel anti-MRSA bacteriocins in a prominent producer of bacteriocins, Bacillus cereus group. Four novel leaderless bacteriocins, bacin A1, A2, A3, and A4, which show potent bactericidal effect against S. aureus and MRSA, were identified in Bacillus sp. TL12. Moreover, bacins inhibited biofilm formation and killed cells in the established biofilms of S. aureus and MRSA. These findings suggest that bacins are promising alternatives to treat MRSA infections. Drug-resistant pathogens continue to be an important and growing threat to human health and economic development –3. The C–Staphylococcus aureus is a commensal bacteria of the skin and nasal flora in approximately 30% of healthy humans, and it is also an important opportunistic pathogen that causes skin and soft tissue infections, bacteremia, osteomyelitis, septic arthritis, pneumonia, endocarditis, and device-related infections , which evolved from S. aureus after acquisition of the mecA gene from other microorganisms via horizontal gene transfer  and human pathogens   , 23–29. B. cereus group strains that produce novel bacteriocins with anti-MRSA activity , and the antimicrobial activity of their supernatants against the MRSA standard strain ATCC 43300 was tested. Of these, 502 strains were predicted to contain novel bacteriocin synthesis gene clusters, and the types of bacteriocins included lantipeptides, lasso peptides, sactibiotics, linear azol(in)e-containing peptides, thiopeptides, glycocins, circular bacteriocins, leaderless bacteriocins, and class III bacteriocins (data not shown). Further anti-MRSA activity testing showed that the supernatants of 56 strains exhibited antimicrobial activity against MRSA .A two-step approach was used to screen the activity . In stepBacillus sp. TL12, contained a putative leaderless bacteriocin gene cluster (termed the bac gene cluster) and showed the highest anti-MRSA activity , which exhibited 45 to 47% identities to LnqZ, the precursor peptide of leaderless bacteriocin, lacticin Z  was separated by reverse-phase high-performance liquid chromatography (RP-HPLC). As shown in The antimicrobial substance in the culture supernatant (grown in LB medium for 10 h) of bac gene cluster—the four novel leaderless bacteriocins bacin A1, A2, A3, and A4 and their N-terminal amino acid is formylmethionine (fM).To further verify the speculation on the structure of four antimicrobial substances, we took compound 4 as a representative and performed amino acid sequencing and tandem mass spectrometry (MS/MS) analysis. No amino acid residues were obtained by N-terminal amino acid sequencing (Edman degradation) of compound 4 (data not shown). MS/MS analysis showed that the molecular masses of a series of fragment ions of compound 4  were approximately 28 Da higher than those of the corresponding fragment ions of BacA2 . To sum S. aureus ATCC 6538 , S. aureus ATCC 12600 , S. aureus ATCC 43300 (MRSA) , S. epidermidis CMCC 26069 , B. cereus ATCC 14579 , B. cereus CMCC 63303 , L. monocytogenes ATCC 19111 , and L. monocytogenes ATCC 19115 ; exhibited modest antimicrobial activity against Streptococcus pyogenes ATCC 19615 , E. faecalis ATCC 29212 , and E. faecalis ATCC 51299 (VRE) ; and had no antimicrobial effect on all tested Gram-negative strains even at the high concentration of 128 μM   . Scannin6 mg/mL) . In concS. aureus ATCC 6538 and MRSA ATCC 43300 were examined by crystal violet (CV) assay. As shown in S. aureus ATCC 6538 and MRSA ATCC 43300 were not affected (P > 0.05); at 1/2× MIC of bacin A2, the growth and biofilm formation of S. aureus ATCC 6538 and MRSA ATCC 43300 were all significantly inhibited compared to the untreated control (P < 0.05); and at 1× MIC and 2× MIC of bacin A2 or 5 μM Nisin A (positive control), the growth of S. aureus ATCC 6538 and MRSA ATCC 43300 was completely prevented with no biofilm formation (P < 0.0001). These results demonstrated that bacin A2 could effectively inhibit the biofilm formation of S. aureus and MRSA.The effects of bacin A2 on biofilm formation of S. aureus ATCC 6538 and MRSA ATCC 43300 were examined by the direct method (viable counting) and indirect method -2H-tetrazolium-5-carboxanilide salt [XTT] assay). As shown in P < 0.0001), and no XTT conversion was observed under 8× MIC of bacin A2 or 50 μM Nisin A (positive control) (P < 0.0001). The results of the viable count method correlated with the results of XTT assay, and the number of live S. aureus cells in biofilms was decreased by more than 3-log at 4× MIC of bacin A2; no viable cells were detected at a high concentration of bacin A2 (8× MIC) or 50 μM Nisin A (positive control) (S. aureus and MRSA cells in preformed biofilms.The effects of bacin A2 on 1-day-old preformed biofilms of control)  and D. TP < 0.0001) (P < 0.0001)  . The cyt 0.0001) .B. cereus group strains that produce novel bacteriocins with anti-MRSA activity and further identify these novel bacteriocins. Considering that a dozen bacteriocins have been reported in the B. cereus group strains, this approach could exclude the strains expressing only previously reported bacteriocin synthetic gene clusters to reduce the workload and improve screening efficiency  and diarrheal enterotoxins and cause a self-limiting gastrointestinal disorder (vomiting and diarrhea)  and causes gastroenteritis, systemic listeriosis, abortion in pregnant women, and neonatal listeriosis is fetuses  and enzymes  and cause severe cramping and vomiting with or without diarrhea  , 35. L.  fetuses . In addidiarrhea –39. TherS. aureus ATCC 43300, as the indicator strain. The growth and time-kill curve assays revealed that bacin A2 is a bactericidal substance. SEM analysis revealed that the cell morphology becomes irregular and even cleaved when treated with 2× MIC or 4× MIC of bacin A2. Moreover, efflux of intracellular potassium ions could be detected after the treatment of bacin A2. Combining the high isoelectric points (pI 10.26) and net positive charge (+3) at physiological pH of bacin A2  that adhere to living or nonliving surfaces . S. auree system , 42. Thebiofilms , and we l agents –45. WhenStaphylococcus pseudintermedius . Furthermore, it showed low hemolytic activity and cytotoxicity even at high concentrations (16 and 64 μM). This demonstrated that bacins are antimicrobial substances with biosafety, which can be further explored for practical application in health care and industry.Bacteriocins are important virulence factors in some bacterial pathogens and show antimicrobial activity along with hemolysis and/or cytotoxicity. Examples include BacSp222 of ermedius , cytolysfaecalis , and strtococcus . TherefoB. cereus group for bacteriocin production, which were isolated from various types of soil samples, including forest, riverside, farmland, and wetland from different regions of China by a classical heat treatment method and preserved in our laboratory . The genome of these strains was sequenced by Wuhan Grandomics Biological Technology Co. using the Illumina HiSeq X 10 platform, and sequence reads were assembled through PGCGAP 1.0.34 (https://liaochenlanruo.fun/pgcgap/index-v1.0.34.html). Species identification of these strains was performed using a 16S rRNA gene sequence and comparative genome analyses . The species of these strains included B. albus, B. bombysepticus, B. cereus, B. mycoides, B. thuringiensis, B. toyonensis, B. pseudomycoides, B. weihenstephanensis, and some other unidentified species (Table S1).We screened a total of 1,056 strains of the 6 cells was spread onto plates. The wells were punched and filled up with the supernatants. The plates were incubated at 4°C for 2 h and then at 30°C for 16 h. The diameter of the inhibition zone was measured to assess the antimicrobial activity of supernatant during the growth. The supernatants with antimicrobial activities were then incubated with mixed proteases (trypsin and α-chymotrypsin at concentrations of 1 mg/mL) at 37°C for 3 h. Residual antimicrobial activity was measured using the agar well diffusion method as described earlier.The screening strategy for novel bacteriocin-producing strains with anti-MRSA activity was divided into two steps . In stepbac gene cluster) and could produce anti-MRSA substances that could be degraded by mixed proteases (OL774512.1) had high homology with different strains of the B. cereus group, such as B. anthracis BJHS4 (99.9%), B. cereus D2-5 (99.9%), B. mycoides strain S20704 (99.9%), and B. thuringiensis FDAARGOS_792 (99.8%). It was impossible to confirm the species with just 16S rRNA gene sequence analysis. The average nucleotide identity and digital DNA-DNA hybridization between genome sequences of the TL12 strain and the most closely related strain, B. pseudomycoides BTZ, were 89.72 and 51.3%, respectively, both of which were lower than the proposed boundary average nucleotide identity value (95 to 96%) and digital DNA-DNA hybridization value (70%) for distinguishing species , two putative membrane proteins with bPH2 domains (bacM1 and bacM2), a HlyD family secretion protein (bacT), and a protein with an ATP-binding conserved domain (bacE)  . The amin (bacE) , and theBacillus sp. TL12 was activated overnight in LB medium, and the culture (1 mL) was transferred into LB medium (100 mL) and incubated at 30°C for 10 h (OD600 = 4.0) with shaking at 200 rpm. The supernatant (5 liters) was collected by centrifugation at 10,000 × g for 10 min and then incubated with 500 g Amberlite XAD-7 resin  at 4°C for 24 h. The resin was washed sequentially with 3 liters double-distilled water (ddH2O) and 1.5 liters 30% (vol/vol) ethanol, and the adsorbed antimicrobial agents were eluted by 1 liter 80% (vol/vol) ethanol (pH 2.0). The eluate was condensed using a rotary vacuum evaporator at 45°C and freeze-dried with a vacuum freeze dryer. The obtained powder was dissolved in 5 mL ddH2O and centrifuged at 10,000 × g for 10 min. The obtained supernatant was referred to as the antimicrobial CE and was further purified by the reverse-phase high-performance liquid chromatography (Dionex Ultimate 3000 system). The mobile phase was Milli-Q water (0.1% trifluoroacetic acid) and acetonitrile. After 50 μL CE was loaded onto the C4 column (Vydamas VD0128), elution was performed with a linear gradient of 10 to 90% acetonitrile within 60 min at a 1 mL/min flow rate. The absorbance of the eluent was monitored at 220 nm. Subsequently, the antimicrobial activity of each of the fractions was tested against the indicator strain MRSA ATCC 43300 by the agar well diffusion method as described earlier. If two or more fractions had antimicrobial activities, their antimicrobial activity after mixing them was also tested to detect their synergistic antimicrobial activity. Each fraction containing the antimicrobial substance was manually collected 100 times and lyophilized into a powder. The acquired powder was dissolved in ddH2O and repeatedly purified with the above HPLC procedure five times to acquire the high purity of antimicrobial substance (>99%).Bacillus sp. TL12 was performed by the Edman degradation method using a PPSQ-33A protein sequencer . The exact mass of antimicrobial substances was performed on the Agilent 6540 ultrahigh definition accurate-mass quadrupole time-of-flight LC/MS system. MS conditions were adjusted to the following conditions: capillary temperature, 350°C; nebulizer pressure, 35 lb/in2; drying gas, 9 liters/min; and source voltage, 3.5 kV. The tandem mass spectrometry (MS/MS) spectrum analysis of antimicrobial substance was applied for fragmentation analysis of peptides, and the target ion was fragmented by adding a voltage of between 30 and 50 V.Amino acid sequencing of antimicrobial substance of 6 CFU/mL. Aliquots of 50 μL of the bacterial suspension and an equal volume 50 μL of bacin A2 solution were added to a 96-well microtiter plate and incubated at appropriate temperature for 18 h. We used the corresponding culture medium to dissolve and dilute bacin A2 according to different indicator bacteria  and TSB medium were used as positive and negative controls, respectively. Nisin A was purified from commercial nisin A product (>900 IU/mg)  as described previously (600) was assessed using a microplate reader. The biofilm assay was performed by discarding the culture supernatants, washing twice with phosphate-buffered saline (PBS), fixing with methanol for 20 min, and staining with 0.1% CV for 30 min. Subsequently, the CV was removed, and the wells were washed with water. Biofilm formation was quantified by the addition of 200 μL of 95% ethanol to each CV-stained biofilm cells, and the absorbance of solution at 570 nm was measured using a microplate reader.The effect of bacin A2 on ications . Briefly5 CFU/mL of S. aureus ATCC 6538 and MRSA ATCC 43300 were respectively incubated into the wells of microtiter plates. After incubation at 37°C for 24 h, the culture supernatant from each well was removed, and the wells were washed twice with PBS. Bacin A2 (200 μL of various MICs) dissolved in TSB was added and incubated with the formed biofilm. After incubation at 37°C for 24 h, the supernatant in the wells was removed and washed once with PBS, and the bacin A2-treated biofilm was evaluated for viability using XTT assay as described previously  and TSB medium were used as positive and negative controls, respectively.To assess the effect of bacin on 1-day-old biofilm, 200 μL of 5 × 10eviously . A 200 μ(i) Time-dependent killing of MRSA by bacin A2. An overnight culture of MRSA ATCC 43300 was diluted with NB medium to an OD600 of 0.4 and incubated with bacin A2 at final concentrations of 0×, 1/2×, 1×, 2×, 4×, and 8× MIC at 37°C and 200 rpm. Bacin A2 was dissolved and diluted with NB medium. The absorbance of the culture at 600 nm was measured hourly to monitor the cell growth. NB medium was used as the negative control. After incubation for 3 h, each culture was photographed.(ii) Potassium efflux. MRSA ATCC 43300 was cultured overnight, harvested using centrifugation method , washed, and resuspended in normal saline to an OD600 of 0.6. Subsequently, 1-mL aliquots were incubated with 2× and 4× MIC of bacin A2 at 30°C, and the total potassium ions (K+) in the supernatant were measured hourly using an HI96750 potassium photometer. Bacin A2 was dissolved and diluted with normal saline. Normal saline was used as the negative control.(iii) SEM. Bacin A2 was dissolved and diluted with NB medium and was incubated with MRSA ATCC 43300 (OD600 = 0.4) to final concentrations of 2× and 4× MIC at 37°C for 3 h with gentle shaking. NB medium was used as the negative control. The cells were corrected by centrifugation , washed three times with PBS , and fixed with 2.5% glutaraldehyde for 12 h. The cells were dehydrated using gradient alcohol solutions (10 to 100%) for 20 min each, treated with isoamyl acetate for 20 min, dried by a vacuum freeze dryer for 12 h, and sputter-coated with gold. The images were observed and acquired with SEM .(iv) Analysis of hemolytic activity. Defibrillated sheep blood was centrifuged at 1,000 × g for 5 min, and the acquired blood cells were washed three times with PBS. The cells were resuspended with PBS and added to centrifugation tubes. Bacin A2 was dissolved in PBS and added to the tubes at final concentrations of 1, 16, and 64 μM. After incubation for 2 h at 37°C, the cells were centrifuged at 1,000 × g for 5 min. The supernatant was added to a 96-well microtiter plate, and the absorbance of each well was measured at 540-nm wavelength. PBS was taken as the negative control (0% hemolysis), and 1% Triton X-100 was taken as the positive control (100% hemolysis).(v) Analysis of cytotoxicity. The cell counting kit 8 (CCK-8) assay was used to determine the cytotoxicity of bacin A2. Exponentially growing NIH/3T3 mouse embryonic fibroblast cells were added to a 96-well microtiter plate (5 × 103 cells/well), and the plate was incubated at 37°C for 24 h. The medium was replaced with fresh medium  containing bacin A2 at final concentrations of 1, 16, and 64 μM. After 8 h of incubation at 37°C, CCK-8 solution  was added to the wells, and the plate was incubated at 37°C for 3 h. Finally, the absorbance of each well was measured at 450-nm wavelength. PBS was taken as the negative control, and paclitaxel (10 μg/mL) was taken as the positive control.P values) between pairwise comparison with the control group were analyzed by Student’s t test, and P ≤ 0.05 was considered statistically significant. Asterisk ratings indicate statistically significant differences: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001.All experiments were performed in triplicate, and the data were statistically analyzed using SPSS 26.0. All data are presented as the means ± standard deviation. Significant differences (Bacillus sp. TL12 were submitted to GenBank with the accession numbers OL774512.1 and JAJJBY000000000.1, respectively. The nucleotide sequences of the bac gene cluster were submitted to GenBank under the accession number OL456173.1.The 16S rRNA and genome sequence of


The occurrence of abnormal organ 18FDG uptake, deemed to be due to ICPI-related organ inflammation, was collected.Data from patients with IIIB/IV NSCLC included in two different prospective trials were analyzed. Interim1 and PETInterim2 revealed the occurrence of at least one ICPI-induced organ inflammation in 72.8% of patients, including midgut/hindgut inflammation (33.7%), gastritis (21.7%), thyroiditis (18.5%), pneumonitis (17.4%), and other organ inflammations (9.8%). iPERCIST tumor response was associated with improved progression-free survival (p < 0.001). iPERCIST tumor response and immuno-induced gastritis assessed on PET were both associated with improved overall survival (OS) (p < 0.001 and p = 0.032). Combining these two independent variables, we built a model predicting patients’ 2-year OS with a sensitivity of 80.3% and a specificity of 69.2% (AUC = 72.7). Validation cohort : Immuno-induced gastritis (19.6% of patients) was associated with improved OS (p = 0.04). The model built previously predicted 2-year OS with a sensitivity and specificity of 72.0% and 63.6% (AUC = 70.7) and 3-year OS with a sensitivity and specificity of 69.2% and 80.0% (AUC = 78.2).Exploratory cohort : PET18FDG PET in around 20% of patients with NSCLC treated with ICPI is a novel and reproducible imaging biomarker of improved OS.Immuno-induced gastritis revealed by early interim The online version contains supplementary material available at 10.1007/s00259-022-05788-8. The recent use of immune checkpoint inhibitors (ICPIs) in metastatic non-small cell lung cancer (NSCLC) has demonstrated high improvement in patients’ outcomes and has become a standard of care in the first-line setting of metastatic NSCLC for patients without oncogenic driver mutations , 2. NoneBy activating the immune system, ICPIs can lead to various inflammatory adverse reactions, termed immune-related adverse events (irAEs) , 6. A va18FDG) positron emission tomography (PET)/computed tomography (CT) is recommended for the pre-therapeutic staging of NSCLC [18FDG PET performed in the setting of ICPI response evaluation. Even if they do not necessarily cause symptoms, these ICPI-induced organ inflammations are usually reported for the oncologist to ensure close clinical monitoring and, in some patients, therapeutic management of irAEs.Due to its high sensitivity for lesion detection, fluorine-18-fluorodeoxyglucose  and Genova . Both studies were approved by the concerned ethics committee and regulatory agencies and informed consent was obtained from all included patients . The present retrospective ancillary study was approved by the French INDS : MR 2,610,080,620.To identify and externally validate 18FDG PET scan (> 9 mmol/l); (6) no measurable lesion by PERCIST V1.0.From February 2017 to April 2020, 112 consecutive patients scheduled to initiate anti-PD1/PD-L1 therapy as their first or later systemic treatment for metastatic NSCLC were prospectively evaluated in an open, uncontrolled, and non-randomized current-care study in Centre Antoine Lacassagne, Nice. The inclusion criteria were as follows: (1) pathologically proven stage IIIB or IV NSCLC, irrespective of the histologic subtype; (2) an indication to start ICPI in monotherapy and in first or later line; (3) ECOG performance status of 0 to 2; (4) age of at least 18 years. The exclusion criteria were as follows: (1) clinical or biological contraindication for immune checkpoint inhibitors; (2) evidence of concurrent cancer; (3) vulnerable patients as defined in Article L1121-5 to -8 of the French Public Health Code; (4) refusal of written consent; (5) high glycemia at baseline 18FDG PET scans were performed within 2 months before the start of ICPIs (PETBaseline), after 7 weeks (PETInterim1), and after 3 months (PETInterim2) of treatment.Patients received one of three possible treatment regimens: either pembrolizumab administered intravenously at a standard dose of 2 mg/kg or 200 mg every 3 weeks, nivolumab at a standard dose of 240 mg every 2 weeks, or atezolizumab at a standard dose of 1200 mg every 3 weeks. Interim1, the clinical status of the patients was considered. If no clinical worsening was observed, the treatment was continued until PETInterim2, based on the previously described immune-related atypical response patterns [In case of progressive disease on PETpatterns –16, 18.18FDG. A low-dose attenuation CT acquisition  was performed 60 ± 5 min after the administration of 18FDG, followed by an inspiratory chest-restricted diagnostic CT . Lastly, a diagnostic CT acquisition was done from the vertex of skull to mid-thigh  after a venous injection of iodinated contrast agent in the absence of allergy or renal impairment. The same imaging system and the same acquisition parameters (duration and delay from injection) were used for baseline and post-treatment studies.Two different PET/CT imaging systems were used: a Biograph mCT PET/CT scanner from February 2017 to September 2019 and a Biograph Vision 600 PET/CT scanner from September 2019 to April 2020 . Both PET systems fulfilled the EARL accreditation specifications for FDG PET/CT tumor imaging. Patients were instructed to fast for at least 6 h before the intravenous injection of 3 MBq/kg (Biograph mCT PET/CT) or 2.5 MBq/kg (Biograph Vision 600 PET/CT) of From May 2015 to April 2016, 49 patients with advanced pre-treated NSCLC were prospectively enrolled in a translational research trial at the Lung Cancer Unit of the IRCCS Policlinico San Martino. The trial was an ancillary single-institution study conducted within the expanded-access program for nivolumab (NCT02475382). Accordingly, the specific study design was approved by the research committee of Regione Liguria.18FDG PET scan (> 160 mg/dL).The inclusion criteria were as follows: (1) histologically or cytologically confirmed NSCLC; (2) a clinical stage of IIIB or IV ; (3) at least one previous line of therapy; (4) at least one measurable lesion by RECIST 1.1; (5) ECOG performance status 0 to 2; (6) age of at least 18 years. The exclusion criteria were (1) meningeal carcinomatosis; (2) active autoimmune disease or a syndrome requiring daily steroid treatment; (3) a previous line of therapy with ICPIs; (4) the administration of a live attenuated vaccine within the 30 days before the first nivolumab administration; (5) untreated brain metastases or brain metastase(s) proven to be evolutive less than 2 weeks before nivolumab initiation; and (6) high glycaemia at baseline 18FDG PET scans were performed within 30 days before the start of nivolumab (PETBaseline) and repeated after 8 weeks  of treatment (PETInterim1). If the patient did not demonstrate a clinical worsening, treatment was continued and a subsequent PET/CT evaluation (PETInterim2) was performed after 2 additional cycles  for patients with a progressive disease on PETInterim1, or after 4 additional cycles  for patients with stable disease or partial response on PETInterim1.Patients received nivolumab at a standard dose of 3 mg/kg every 2 weeks. 18FDG. The PET/CT fulfilled the EARL accreditation program. No cross-validation or comparison of the recovery coefficient with the PET/CT of Nice was performed for this study.A Biograph 16 PET/CT imaging system was used for all patients . Patients were instructed to fast overnight before the intravenous injection of 300–400 MBq of peak), were calculated in regions of interest placed on the site of the tumors or organ inflammations.Peak standardized uptake values, normalized by body weight . These diffuse inflammatory organ uptakes, deemed to be induced by ICPI, were collected separately for each organ .The occurrence of pathological organ The iPERCIST criteria were used for the interpretation of the PET scans .Patients were followed with regular clinical evaluations and standard of care imaging  was the primary endpoint of the study. OS was recorded and defined as the time from initiation of ICPI to death from any cause.Progression-free survival (PFS) was the second endpoint of the study and defined as the time from the initiation of ICPI to confirmed tumor progression or death. Tumor progression needed to be confirmed by a multi-disciplinary tumor board, confronting the patient’s clinical status, PET/CT results, and brain MRI results. Confirmed tumor progression necessarily implied the decision to definitively stop the treatment. For PFS, we did not take the time of first evidence of tumor progression on PET/CT due pseudo-progression or dissociated response patterns .t = 24 months was evaluated using the area under the ROC curve (AUC). Risk groups for predictive models were obtained using the threshold value to obtain the best compromise between sensitivity and specificity. A p value of < 0.05 was considered statistically significant and all tests were two-sided. All statistical analyses were performed with R.3.5.2 software on Windows® and the survMisc, timeROC, and Survminer packages.Categorical data are shown as counts and percentages, and continuous variables as minimum, median, maximum, and means with standard deviations. Available case analysis was the approach used for missing data. OS and PFS were evaluated using the Kaplan–Meier method and estimated with 95% confidence interval (95%CI). Median follow-up with a 95% confidence interval was calculated with the reverse Kaplan–Meier method. The log-rank test was used for the univariate analysis of PFS and OS. For OS, a predictive model was built, using a multivariable Cox regression method by following a step-by-step descending variable selection procedure using the AIC criterion. The proportional hazard assumption was checked using statistical tests and graphical diagnostics based on the scaled Schoenfeld residuals . A classInterim1 being waved, due to hyper-progression (n = 9) or severe treatment toxicity (n = 1). Four patients were excluded for other reasons: no target lesion on PET (n = 1), PETInterim1 cancelation, or wrong timing (n = 3).One hundred and twelve patients were prospectively evaluated. Six patients were excluded because ICPI was finally not initiated. Ten patients were excluded because the treatment was stopped in the very first weeks of treatment, with PETThe ninety-two remaining patients could therefore be included in this study. Mean patient age was 65.3 ± 10.0 years. All patients had a locally advanced or metastatic NSCLC, with the pathological type being a squamous cell carcinoma in 17.4% (16/92), adenocarcinoma in 78.3% (72/92), and carcinoma “not otherwise specified” (NOS) in 4.3% (4/92) of patients. 54.3% (50/92) of patients were treated with pembrolizumab, mostly in first line of the metastatic setting, and 42.4% (39/92) of patients were treated with nivolumab, mostly in second line. The 3.3% (3/92) of remaining patients were treated with atezolizumab.Interim1 was 49.1 ± 6.1 days. Mean time between introduction of immunotherapy and PETInterim2 was 97.3 ± 14.4 days.Mean time between baseline PET and introduction of immunotherapy was 15.1 ± 16.1 days. Mean time between introduction of immunotherapy and PETPatients’ median follow-up time was 25.3 months (CI 95%: 20.5–28.3). Patients’ median PFS was 14.3 months (CI 95%: 7.1–22.7) PERCIST criteria (Table n = 27/92).29.3% of patients had a metabolic complete or partial response (n = 6/92).6.6% of patients had a stable metabolic disease (n = 59/92).64.1% of patients had an (unconfirmed) metabolic progressive disease (Tumor response on PETInterim2: iPERCIST criteria (Table Interim1.44.0% (33/75) of patients had a complete or partial metabolic response, 13 of them having initially shown a pseudo-progression on PET5.3% (4/75) of patients had a stable metabolic disease.13.3% (10/75) of patients had a first, unconfirmed, progressive metabolic disease.37.3% (28/75) of patients had a confirmed metabolic progressive disease (cPD), corresponding to patients with 2 consecutive PERCIST progressive diseases (including 11/28 patients with dissociated evolution of lesions).Tumor response on PETia Table :44.0% .The occurrence of at least one site of organ inflammation after introduction of ICPI was observed on PETMidgut/hindgut inflammation in 33.7% (31/92) of patients.Gastritis  in 21.7% (20/92) of patients.Thyroiditis in 18.5% (17/92) of patients.Pneumonitis in 17.4% (16/92) of patients.Other organ inflammation in 9.8% (9/92) of patients: pancreatitis, ENT inflammation, arthritis, esophagitis, mesenteric panniculitis, pleuritis.The types of ICPI-induced organ inflammation observed were as follows:Clinico-biological variablesAssociation with PFS:PET imaging variablesNone of clinical characteristic assessed  was significantly associated with PFS, including PD-L1 tumor expression.Interim 1 and 2, was significantly associated with improved PFS .Tumor metabolic response, using iPERCIST criteria on PETInterim1 and/or PETInterim2 was not significantly associated with PFS, either on a global or a site-per-site analysis.Occurrence of ICPI-induced organ inflammation on PETClinico-biological variablesAssociation with OS:Among the clinical and biological patients’ characteristics assessed , none was significantly associated with OS.p = 0.08).Imaging variables Fig. Patients with a higher ECOG performance status tended to have a worse OS but this was not statistically significant . Patients with a tumor response/stability on PETInterim1 had a 2.5-fold improved OS compared to patients with tumor progression (2-year OS = 73% vs 29%).Tumor metabolic response, using iPERCIST criteria on PETInterim1 and/or PETInterim2 was significantly associated with improved OS (p = 0.026). Patients with early gastritis occurrence had a twofold improved OS compared to patients without it (2-year OS = 75% vs 37%). The other sites of ICPI-induced inflammation  were not significantly associated with OS.Immunotherapy-induced gastritis on PETp < 0.001).Combining iPERCIST criteria and immunotherapy-induced gastritis occurrence in a sub-group analysis, the 2-year OS rate was significantly different between the 4 sub-groups of patients (Interim1) and immune-induced gastritis (on PETInterim1/2) as two complementary variables (Supplementary Table p < 0.001)  Fig. .Fig. 3PaInterim1 cancelation or wrong timing. Forty-five remaining patients were evaluated in this study. Patients’ characteristics are indicated in Table Forty-nine patients were prospectively included. Four patients were excluded because of PETInterim1 was 52.7 ± 7.9 days. Mean time between introduction of immunotherapy and PETInterim2 was 98.3 ± 19.0 days.Mean delay between baseline PET and initiation of immunotherapy was 17.1 ± 13.3 days. Mean time between introduction of immunotherapy and PETInterim1 or PETInterim2 in 86.7% of patients (39/45).Patients’ median follow-up was 56.6 months (CI 95% = 55.2–NA); Patients’ median progression-free survival was 5.9 months (CI 95% = 3.2–8.7) .As observed in the exploratory cohort from Nice, iPERCIST tumor response on PET OS Fig. . PatientInterim1 and/or PETInterim2 had a 2.7-fold improved OS compared to patients without it   were not significantly associated with OS.Finally, the prognostic model obtained in the cohort from Nice, including iPERCIST response and immune-induced gastritis criteria, was assessed in this independent validation cohort, using the optimal threshold (= − 0.19) defined in the exploratory cohort. The AUC of ROC curve to predict 2-year OS was equal to 70.7 (CI 95% = 78.1–88.3) with a sensitivity of 72.0% and a specificity of 63.6%.Having a longer follow-up in this validation cohort, we also assessed the prognostic value of the same model to predict 3-year OS: the AUC was equal to 78.2 (CI 95% = 57.1–99.2) with a sensitivity of 69.2% and a specificity of 80.0%.The OS curves, distinguishing poor and good prognostic groups using this model, are given in Fig. 18FDG uptake. Assessment of adverse inflammatory events following the introduction of ICPIs on 18FDG PET/CT imaging may precede clinical symptoms, potentially leading to the earlier therapeutic management of irAEs. However, the utility of 18FDG PET/CT in the follow-up of patients, particularly for the detection of inflammatory adverse events, has been poorly investigated up to now. In a recent study including patients with non-metastatic bladder cancer treated with neoadjuvant pembrolizumab, 18FDG PET/CT showed ICPI-induced organ inflammation in 39% of patients, corresponding to grade 1 or 2 clinically detectable irAEs in 45% of cases [n = 53) wherein 18FDG PET/CT detected 74% of irAE in patients treated with anti-PD1 [Tumors and activated inflammatory cells both have increased expression of glucose transporters and glycolysis, leading to increased of cases . Mekki eanti-PD1 .18FDG PET/CT detects many ICPI-induced organ inflammation occurring during the first 3 months after ICPI initiation . This rate of inflammatory FDG uptake, either symptomatic or not, is very high, but in accordance with the also high incidence of irAE occurrence, described in a recent meta-analysis [In our study, analysis . In NSCLanalysis , 25.18FDG PET is not necessarily symptomatic. Secondly, only irAEs managed with specific treatments are usually reported in clinical trials, without considering irAE of grade 1. Lastly, some irAEs are clinically hard to diagnose due to their pleomorphic presentations, such as asthenia and myalgia [However, direct comparisons between the rates and types of clinical irAEs and inflammatory tissue uptake detected on PET cannot be done. Firstly, ICPI-induced organ inflammation assessed on  myalgia .Thus far, research on predictive biomarkers of ICPI efficacy has focused on tumor or micro-environmental biological parameters. Clinical biomarkers have been less studied. Previous studies have suggested that the occurrence of symptomatic irAEs is associated with a more favorable outcome to PD-1 inhibitors, probably due to cross-reactivity between tumor and host tissues , 27–31. 18FDG PET has been less investigated than clinical irAEs. As a previous study conducted on a small cohort of 25 patients with metastatic melanoma [18FDG PET/CT performed in the early patients’ follow-up. But on a per-organ analysis, the occurrence of a gastritis during the first 3 months of ICPI treatment was significantly associated with improved OS (but not PFS) in the exploratory cohort. This discrepancy between OS and PFS is common when evaluating immunotherapy and may be explained by the difficulty of determining a reliable PFS in that setting. Patients with an immune-induced gastritis had a twofold improved 2-year OS compared to patients without it. This result was confirmed in the external validation cohort, in a different hospital and using a different PET/CT system. This independent validation was important to avoid false biomarker discovery and confirm that immuno-induced gastritis on PET is a reproducible predictive biomarker of improved clinical outcome. Moreover, combining tumor response (iPERCIST criteria) and gastritis occurrence can more accurately predict patient’s overall survival, as demonstrated by the predictive model. This model could identify long-term survivors with immunotherapy in advanced NSCLC (3-year OS) with a 69% sensitivity and 80% specificity in the external validation cohort. From a clinical perspective, ICPI-induced gastritis could be an additional argument for ICPI efficacy in case of doubtful or atypical response patterns on 18FDG PET/CT, such as dissociated response or pseudo-progression, often observed in the monitoring of patients treated with ICPI [The prognostic value of immune-induced inflammatory organ uptake assessed with melanoma , we did ith ICPI , 33.n = 205) has described the incidence of ICPI-gastritis at 5.4% of patients receiving immunotherapy for melanoma [Because gastritis symptoms can be insidious, overlapping with the common symptoms of metastatic cancer, immuno-induced gastritis may frequently be under-diagnosed without endoscopic confirmation . The curmelanoma .18FDG PET/CT case reports [The occurrence of diffuse uptake of the gastric wall during ICPI treatment, related to immune-induced gastritis, has been mentioned in few previous  reports , 32, 38. reports . In thisFaecalibacterium, confer sensitivity to ICPI, also increasing the risk of irAEs [Helicobacter pylori (HP) can induce PD-L1 expression on gastric epithelial cells [There is currently no clear physio-pathological explanation of the association between immune-induced gastritis and improved outcome. The patient’s microbiota may be an interesting first track to explore this association. Indeed, the role of microbiota in regulating response and toxicities to ICPIs in cancer is increasingly recognized , 40, as of irAEs . Recent of irAEs . Interesal cells  and, in al cells . Similaral cells . These d18FDG PET/CT was not significantly associated with patient’s outcome. One explanation may be that we collected the occurrence of ICPI-induced organ inflammation only in a binary manner (occurrence versus nonoccurrence), without quantifying the magnitude of the intensity and extent of inflammatory uptake assessed on PETInterim1/2. It could have been relevant to quantify the severity of 18FDG uptake to distinguish patients with minor and major inflammatory involvement of the midgut/hindgut. This quantitative work could be the subject of a future specific ancillary study.Despite the well-described role of the gut microbiome (assessed from stool samples) in shaping systemic immune responses, the occurrence of ICPI-induced midgut/hindgut inflammation on This study has some limitations. Firstly, although most irAEs occur within the first 3 months of treatment, the 3-month follow-up may be too short to detect some types of delayed irAEs such as nephritis , 27. SecInterim1), tumor escape and progression occurred earlier in the more heavily treated cohort, prior to ICPI, with a median PFS of 14.3 months (Nice) vs 5.9 months (Genova).Finally, significant differences in survival were observed between the exploratory and validation cohorts. This may be explained by differences in the clinical characteristics of the two cohorts. Patients from Nice mainly received ICPI in the first and second lines, whereas two-thirds of Genova patients were treated in second or later lines. Although the early objective response rate to ICPI did not differ significantly between the 2 cohorts PERCIST criteria. Regarding the clinical relevance of this finding, ICPI-induced gastritis could be an additional argument for ICPI efficacy in case of doubtful or atypical response patterns on 18FDG PET/CT, such as dissociated response or pseudo-progression.Beyond the standard assessment of metastatic lung cancer response to ICPIs, Supplementary file1 (DOCX 1875 KB)Below is the link to the electronic supplementary material.


E. cognata leaves following a hydrothermal route. The phyto-fueled ZnO@PdO/Pd nanomaterial was fabricated into a supercapacitor electrode and was scrutinized by galvanostatic charge–discharge, electrochemical impedance spectroscopy and cyclic voltammetry to evaluate its energy storage potential, and transport of electrons and conductivity. Substantial specific capacitance i.e., 178 F g−1 was obtained in the current study in aKOH electrolyte solution. A specific energy density of 3.7 W h Kg−1 was measured using the charge–discharge data. A high power density of 3718 W Kg−1 was observed for the ZnO@PdO/Pd electrode. Furthermore, the observed low internal resistance of 0.4 Ω suggested effective electron- and ion diffusion. Thus, the superb electrochemical behavior of the ZnO@PdO/Pd nanocomposite was exposed, as verified by the significant redox behavior shown by cyclic voltammetry and galvanostatic charge–discharge.In recent times, tremendous efforts have been devoted to the efficient and cost-effective advancements of electrochemically active metal oxide nanomaterials. Here, we have synthesized a facile nanomaterial of ZnO@PdO/Pd by employing extracted fuel from  In recent times, tremendous efforts have been devoted to the efficient and cost-effective advancements of electrochemically active metal oxide nanomaterials. Then, the next category of nanomaterials is transition metal oxide nanomaterials used in the fabrication of pseudocapacitors, which present outstanding specific capacitance and energy density.17,18 An electrical double layer capacitor shows charge storage by reversible ion adsorption at the surface of the electrode and electrolyte interfaces, while charge storage shown by pseudocapacitors is via Faradaic-redox reactions occurring at the electrode surface. Pseudocapacitors due to their fast reactions exhibit higher energy density and higher specific capacitance compared to EDLCs. In pseudocapacitors, electrons are passed on the valence band of the anode species or a redox cathode. Therefore, pseudocapacitors rest on the nature and the structure of the electrode material.18–20Numerous studies have been carried out on diverse nanomaterials with higher electro-activity to adapt the efficiency of supercapacitors.Euphorbia cognata Boiss, and evaluated its possibility for energy storage devices.5 Phyto-mediated ZnO NPs revealed a specific capacitance of 86 F g−1 at 2 mV s−1.5 Then, we have synthesized and reported the phyto-assisted ZnO–Co3O4 as an electrode material for supercapacitors, and this electrode material showed a capacitance of 165 F g−1.1 Another, ZnO-based electrode material, ZnO@NiO, was synthesized via the same biogenic route using the leaf extract of Euphorbia cognata Boiss.3Among various pseudocapacitor electrode materials, transition metal oxides (TMOs) with numerous valences are considered as the best electrode materials for pseudocapacitors, offering an enhanced oxidation states for the effective redox ion transfer. Among TMOs, ZnO is one of the most suitable materials for pseudocapacitor applications due to its higher electrochemical activity, lower cost and greener nature. Recently, we synthesized ZnO NPs using the organic fuel prepared from leaf extract of Euphorbia cognata Boiss (hereafter written as ECBE) was a significant reagent for the synthesis of metal oxides nanomaterials.1,3,5,21,22 Motivated from the success of pervious projects of ECBE-based nanomaterials, we have employed the same ECBE-based synthesis strategy to synthesize a facile nanomaterial of ZnO@PdO/Pd without using any chemical reagent. The ZnO-based nanomaterial possessed high crystallinity, and thus possessed good supercapacitive properties.1–5 In this study, supercapacitive properties of the facile ZnO@PdO/Pd nanomaterial have been investigated by galvanostatic charge–discharge (GCD), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques in a non-toxic and lower cost aqueous electrolyte, and it showed considerable specific capacitance.Thus, the reported investigations vividly showed that the leaf extract of −1) to lower (2 mV s−1) and under numerous current densities (0.5–30 A g−1). The overall electrochemical results revealed that the ZnO@PdO/Pd nanomaterial was a capable material useful as an electrode for supercapacitor applications.The present study is first a comprehensive exploration carried out on the preparation of a phyto fuel (PF)-doped mixed metal oxide (MMO) nanostructure, and the recognition of phyto-stabilizing agents (PSAs) in the obtained product along with the exploration of its electrochemical performance related to supercapacitors. The electrochemical properties of the as obtained novel nano-product were tested at various scan rates ranging from higher (300 mV s2.2CCH3)2 (H2O)2] and palladium acetate [(Pd(CH3COO)2)], ethanol and methanol (C2H5OH) were bought from Sigma Aldrich, Germany. The electrode material fabrication was carried out using acetylene black, polyvinylidene (PVDF) as well as N-methyl pyrrolidinone (NMP) bought from Sigma Aldrich, Germany. Fresh leaves of E. cognata (EC) were obtained from Rawalakot area  of AJK, Pakistan.Zinc acetate  solution was prepared by taking 898 mg (4 moles) of [(Pd(CH3COO)2)] into 100 mL of DI water. This sonicated suspension of ZnO NPs was added into the prepared 40 mM solution of [(Pd(CH3COO)2)] in a ratio of 8:2 via a stirring (550 rpm) treatment for about 15 min along with temperature upsurged up to 70 °C. Moreover, 2.5 mL of ECBE was also mixed in this mixture along with stirring for another 1 h. The mixture was then evaporated within an oven for all night, and then dried in a furnace at 450 °C for 4–5 h. The resultant material was ground, saved in Eppendorfs, and labelled as ZnO@PdO/Pd.Hereafter, 40 mM [ to study the bandgap. OGFs were monitored via FT-IR spectroscopy . Presence of organic species was confirmed by GCMS QP5050. Phases were identified by XRD . The shape and type of elements present were determined by SEM  along with EDX . Similarly, investigations on the surface chemistry were accomplished via a Kratos-Axis Ultra spectrometer (XPS).ZnO@PdO/Pd was analysed 3.1.N-methyl pyrrolidinone, a slurry encompassing ZnO@PdO/Pd, AB, as well as PVDF in 8 : 1 : 1 was prepared, which was then dispersed over porous Ni foam at 60 °C for 10 h under vacuum. The electrode material was examined electrochemically via CV, GCD, and EIS. To carry out these studies, an electrochemical work station was produced having a three-electrode system along with a working electrode comprising a ternary nanocomposite-adapted Ni foam, a platinum wire counter electrode and saturated calomel as the reference electrode. Electrochemical analyses were carried out via KOH (3 M) by CV at varying scanning rates ranging 2–300 mV s−1. Galvanostatic charge discharge (GCD) was altered by fluctuating the current densities (CD) i.e., 0.5–30 A g−1. EIS was directed for sample utilizing frequency of 50 mHz to 10 kHz.In order to fabricate the resultant product, within 4.4.1.23–28 In the current study, a facile nanomaterial has been synthesized for investigating its electrochemical behaviour. Before employing ZnO@PdO/Pd for fabrication of an electrode, it was analysed for its compositional and morphological properties. Initially, PF-assisted ZnO@PdO/Pd was examined by FTIR spectroscopy and GC-MS for obtaining the presence of PSAs.In the literature, numerous plant-mediated nanomaterials have been synthesized.−1, given by the N–H wag along with the C–H oop, matching to aromatics as well as 1° and 2° amines. These vibration peaks were associated with the PF-doped ZnO@PdO/Pd material. Inset shown in −1.39,54 Thus, aromatics and 1° and 2° amines behaved as SAs in the as-prepared product.8H11N), benzenemethanol (C7H8O), benzeneethanamine along with octodrine (C8H19N), connected to PdO/Pd-doped ZnO, respectively.GC-MS of our tested sample is presented in θ) = 31.97, 34.65, 36.465, 47.751, 56.79, 63.06, 66.63, 69.24, and 77.128, relating to (100), (002), (101), (102), (110), (103), (200), (201) and (202) miller index planes. According to ICSD 00-036-1451, the crystal system is hexagonal for ZnO, the space group is P63mc and space group number is 186. The unit cell parameters are as follows;θ) = 34.082, 42.184, 54.97, 60.45, 71.61, and 85.81 corresponding to hkl planes of (101), (110), (112), (103), (211), and (114) (ICSD 00-041-1107). According to the ICSD 00-041-1107 crystal system, PdO is tetragonal, its space group is P42/mmc, space group number is 131, and the unit cell parameters are a (Å): 3.0456, b (Å): 3.0456, c (Å): 3.3387, alpha (°): 90, beta (°): 90, gamma (°): 90. However, the formation of cubic Pd (●) was confirmed by peaks at 2 theta (θ) = 40.3601 (111), 46.89 (200), 68.22 (220), 82.35 (311) and 86.06 (222) with ICSD 00-005-0681. However, the formation of cubic Pd (●) was confirmed by peaks at 2 theta (θ) = 40.3601 (111), 46.89 (200), 68.22 (220), 82.35 (311) and 86.06 (222) with ICSD 00-005-0681. ICSD 00-005-0681 states that the crystal system of Pd is cubic, space group is Fm3m, space group number is 225, while unit cell parameters are a (Å): 3.8898, b (Å): 3.8898, c (Å): 3.8898, Alpha, Beta and Gamma is same as for PdO. Moreover, the crystallite size shown by ZnO@PdO/Pd was 27–29 nm as calculated by the Debye Scherrer's.71Moreover, XRD revealed the PdO (♦) at 2 theta  relatable to the ZnO spectra.42 Pd 3d spectra indicate that Pd 3d5/2 and Pd 3d 3/2 also have Pd2+ on 342.2 BE  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O, C–O, OH and H–C–O.29,42 The C 1s spectrum shows OFGs, i.e., C–O, COO, O–CO, CO, C–C, C–H and CC.29,42,64 The energy region in C 1s is higher than 290 eV because of sp3 hybridized (C–C).64 Thus it is fruitfully expressed by XPS that the functionalization of ZnO@PdO@Pd by OFGs is achieved in a good way.The Zn 2p spectrum  shows tw342.2 BE . The O1s342.2 BE  indicate1,3,5,23–25FE-SEM pictures of ZnO@PdO/Pd displayed in 4.2.Apart from compositional and morphological analysis, the facile fabricated material was further investigated for its optical direct bandgap energy (eV). For this, a very diluted and transparent aqueous suspension of the as-synthesized nanomaterial was scanned by UV-Vis at 200–800 nm. The subsequent spectrum is displayed in i.e., 2.25 eV, which made the transference of electrons quite easy with greater CD making it promising for capacitance.29–31,43,444.3.72–74 Moreover, it can ominously lower the BGE of ZnO, resulting in a boosted electrochemical behavior  and cathodic peaks (CP) altered their intensity, as well as the positions. AP showed an increase in CD (peak), while moving to high potentials. However, CP showed a decline in CD showing a shift to low potentials. These results were in agreement with those obtained by Khan et al.,32 Duraisamy et al.34 and Pramanik et al.35 Reasons behind these findings were kinetic irreversibility as well as electric polarization given by ions of electrolyte at the surface of the electrode with higher SR.33,36–40 This changed the CV curves shape to a rectangular form with more SR. SC values shown by the ZnO@PdO/Pd electrode are noted at different SR  along with PD (W kg−1)   . Fig. 9b O3 ref.  and 69 a . Fig. 9Ri) as well as the charge transfer resistance (Rct), as shown in Rct. Intercept  shown at higher frequencies was because of Ri. Zw).1,29,30 Lower Ri of 0.4 Ω shown by our prepared ZnO@PdO/Pd confirmed the high speed movement of ions as well as electrons, and this low value is because of more C and O consisting OFGs at the electrode surface, thus creating more sites for the transportation of electrons and ions giving a rise to improved conductivity.50–55,67–70 The semi-circle arc shown in Zw as well as a minor semicircle present along with it. EIS measurements were also carried out to evaluate the internal resistance (Ri and Rct showed a superb redox behavior (CV as well as GCD), verifying that the fabricated ZnO@PdO/Pd nanostructure can be fruitfully employed as a supercapacitor.These electrochemical examinations including the low 5.8H11N, C7H8O, and C8H19N). Consequently, ZnO@PdO/Pd displayed more active sites, thus facilitating the effective diffusion of electrons as well as ions. An enhanced specific capacitance and energy density was obtained for the fabricated electrode due to the combination of ZnO and PdO. The significant redox behavior was noticed in the fabricated electrode, as depicted via CV along with GCD, revealing that it had a considerable potential for energy storage devices. Thus, current investigation presented the excellent potential of ZnO@PdO/Pd for pseudocapacitance.We have successfully synthesized the facile ZnO@PdO/Pd nanomaterial demonstrating its pretrial utility for energy storage. The ZnO@PdO/Pd nanomaterial revealed a spherical shape and less agglomeration. XPS analysis and GCMS unveiled ZnO@PdO/Pd surface functionalization by phyto compounds (CThere are no conflicts of interest among the authors.