This reactionis of fundamental interest for precise lattice patterning, however,is not well understood. Herein, using van der Waals density functionaltheory (vdW-DFT) incorporating spin-polarized calculations, we findthat O3 strongly physisorbs on graphene with a bindingenergy of −0.46 eV. It configures in a tilted position withthe two terminal O atoms centered above the neighboring graphene honeycombs.A dissociative chemisorption follows by surpassing an energy barrierof 0.75 eV and grafting an epoxy group on graphene reducing the energyof the system by 0.14 eV from the physisorbed state. Subsequent O3 chemisorption is preferred on the same honeycomb, yieldingtwo epoxy groups separated by a single C–C bridge. We showthat capturing the onset of spin in oxygen during chemisorption iscrucial. We verify this finding with experiments where an exponentialincrease in the density of epoxy groups as a function of reactiontemperature yields an energy barrier of 0.66 eV, in agreement withthe DFT prediction. These insights will help efforts to obtain precisepatterning of the graphene lattice.The exposure of graphene to O Epoxy groups alter the local density of statesof graphene, which can be used for stepwise tuning of band gap ofgraphene.12 When multiple epoxy groupsare grafted, they tend to form energy-minimizing clusters15 known to be responsible for the oxidative cleavage of graphiticlattice18 and the formation of vacancy defects.21 The formation of such defects, if controlled precisely, can beextremely attractive for application in molecular separation,24 enhanced water evaporation,25 sensing,27 sequencing,28 and power generation.29 In fact,exposure of graphene to O3 has become a promising way toprepare porous graphene membrane.32 However, the mechanismof the formation of the epoxy group from O3 is not wellunderstood. An accurate description of underlying energetics wouldallow one to model and predict the evolution of epoxy groups and relatedclusters and will help efforts to precisely pattern graphene.Controlled incorporation of defects ingraphene by surface functionalizationis attractive for modulating its electronic,3 with grapheneand carbon nanotubes are scarce, and report O3 physisorptionenergy in the range of −0.2 to −0.3 eV.35 The study of Lee and co-workers on the interaction of O3 with graphene is the most comprehensive report so far.36 They used the local spin density approximation(LSDA) to understand the reaction of O3 with graphene.They found that the O3 orientation on top of the graphenestrongly influences its physisorption. However, the reported physisorptionand chemisorption energies were small, −0.25 and −0.33eV, respectively. Such moderate binding energies do not explain thestrong affinity of O3 toward graphene or carbon nanotubes,which is observed by experiments at room temperature.38Theoretical studies on the interaction of O39 we show that O3 physisorption on graphene is quite strongcorresponding to a high binding energy of −0.46 eV. The interactionis uniquely maximized when O3 approaches graphene in atilted , face-downorientation and when the terminal O atoms of O3 are positioneddirectly above neighboring graphene honeycomb. Ozone then goes throughdissociative chemisorption to functionalize an epoxy group on graphenesurpassing an energy barrier of 0.75 eV. The resulting chemisorptionbinding energy is high, 0.60 eV. We show that the second chemisorptionevent preferentially takes place on the same honeycomb, and the resultingpair of epoxy groups is separated by a single C–C bridge. Finally,we carry out experiments probing reaction of O3 with graphene.Monitoring the coverage of the epoxy group on graphene as a functionof temperature, we calculate an energy barrier of 0.66 eV, in agreementwith the DFT calculations.Herein, using vdW-DFT calculations,3 adsorption on graphene, vdW-DFT calculations were performed usingthe Quantum ESPRESSO package.41 A supercell made of7 × 7 periodic unit cells of graphene was used to ensure thedecoupling of in-plane molecule–molecule interactions. TheBrillouin zone was sampled with uniform 4 × 4 × 1 unshiftedk-point grids. A vacuum region of 20 Å was used in the z-direction to avoid interactions among the periodic replicas.An energy cutoff of 90 Ry was used for the plane wave expansion ofthe wave functions. A kinetic energy cutoff of 720 Ry on the chargewas used together with ultrasoft pseudopotentials.43 In order to include the contributions of dispersion interactions,vdW-DF239 approximation was used. For comparison,some adsorption energies were also calculated with the more recent,semiempirical vdW-DF3 approximation.44 Convergencethresholds of 2 × 10–6 Ry and 10–4 Ry/Bohr for the total energy and forces, respectively, are used.To investigate O3 intoepoxy and oxygen, spin-polarized calculations were performed. Thereaction mechanisms and transition-state energy barriers were studiedwith climbing image nudged elastic band (NEB) calculations. The NEBsimulations were carried out using a 6 × 6 supercell and a uniform4 × 4 × 1 unshifted k-point grid to sample the Brillouinzone. The spin of the system is changed from the spin-neutral (O3) to a spin-polarized (epoxy + O2) at the transitionstate.For studying the dissociative chemisorption of O2 andH2 at 1000 °C for 30 min. The next step involved introducingCH4 and H2 gases with a 3:1 volume ratio intothe reactor to initiate graphene growth. The reactor was maintainedat 460 mTorr for 30 min during the growth process. Once the synthesiswas complete, the reactor was rapidly cooled down to room temperature.To synthesize single-layer graphene,low-pressure chemical vapor deposition was used on annealed Cu foilwith a thickness of 50 μm and a purity of 99.9% (Strem). TheCu foil was first annealed in an atmosphere of CO3 generator yielding O3 concentrationof 150 g/Nm3 in O2. The samples were heatedto temperatures ranging from 290 to 329 K for functionalization. Thereactor pressure was kept at 1 bar during each oxidation process.Following the functionalization, O3 was purged by flowingAr. Finally, the samples were transferred to the ultrahigh vacuum(UHV) chamber of X-ray photoemission spectroscopy (XPS) for the characterizationof the functional groups.Freshly synthesized graphenesamples were functionalized by placing them in a quartz tube connectedto an OHigh-resolution XPS measurements were conductedusing a Kratos Analytical Axis Supra instrument equipped with themonochromated Kα X-ray line of an aluminum anode. The pass energyand step size used in this study were 20 and 0.1 eV, respectively.To prevent charge buildup, the sample was ground during measurements.The binding energy data were used without any corrections, and dataprocessing was performed using CasaXPS software. Background subtractionwas performed by using the Shirley method.3/graphene system was allowed to relax. O3 wasrelaxed in all directions to minimize the potential energy. C atomsin graphene were also relaxed to adjust their positions dependingon the interactions with O3. To extract the lowest energyO3/graphene configuration, we explored several O3 configurations and positions on top of the lattice   was the most optimal configurationthat minimized the energy as it allowed improved interaction for allthree O atoms. Therefore, we fixed the tilt angle to 79° forthe two tilted configurations. Finally, the relative distance betweenthe central O of O3 and C atoms in graphene honeycomb wasvaried along the xy plane to probe three most probablepositions  1b and twizontal) 1c and faizontal) 1d] confi3-graphenedistance (z) for the total nine configurations  are reported in z position of the O atom that is nearest to graphene. In all cases,as O3 is brought closer to graphene from 4 Å, theenergy of the system first decreases and then increases. This is attributedto the increasing attractive interactions at first followed by thedominance of the repulsive dispersion interactions. Irrespective ofthe three orientations, the minimum energy is always obtained forthe position 1 [red curve in the interaction energy plot, 3 aresurrounded by six C atoms of the honeycomb, which maximizes the interaction.In other words, the number of nearest neighbors (C atoms) for theterminal O atoms are highest in position 1, which ensures maximuminteraction. Therefore, position 1 is the most favorable.The interaction energy as a function of O3 is 3 Å away from the graphene lattice whilethe middle O is 3.3 Å away from the lattice (Supplementary Note S2). The resulting free energy correction is +0.09 eV,bringing the free energy of adsorption to values of −0.37 eV(vdW-DF2), −0.47 eV (vdW-DF3-OPT1), and −0.49 eV (vdW-DF3-OPT2).Finalconfigurations are ranked as following in the order of theirpreference at the most optimal position, i.e., position 1: tiltedΛ (−0.46 eV) > tilted V (−0.44 eV) > horizontal(−0.42 eV). Based on this, we assign the physisorbed stateto position 1 in a tilted Λ configuration with physisorptionbinding energy of −0.46 eV. In this position, the terminalO of O lattice 3a. When 35 and can explainthe observed strong affinity of O3 toward graphene. Weattribute our results to improved capture of long-range interactions,which allows maximization of interaction with all three O atoms. Thisperhaps also explains why the optimal configuration is close to planarwith a slight (11°) tilt. In general, nonlocal density functionals,including the vdW-DF2 functional, are considered promising to modelvdW interactions. They include nonlocal, long-range correlation interactionsdirectly in the form of the potential of the density functional. Todemonstrate the importance of vdW approximations, we also calculatedthe adsorption energy at the Perdew–Burke–Ernzerhof(PBE) level, which is less accurate in capturing the long-range interactions.PBE calculations indeed predicted a lower binding energy, −0.37eV .While it also predicted the Λ configuration, O3 isnot tilted  and the adsorptionheight is larger, 3.3 Å similar to what has been reported inthe literature.36 We also studied the effectof the supercell size. A 3 × 3 supercell used in the literatureis not large enough to completely remove the interactions betweenO3 molecules in neighboring images. Indeed, the bindingenergy increases and converges to −0.46 eV when the supercellsize is increased from 5 × 5 to 6 × 6 and then to 7 ×7 periodic unit cells . Takentogether, these findings underscore the importance of consideringnonlocal density functionals, optimal configuration (tilted Λin this case), and large enough supercell size for accurate modelingof physisorption interactions.The predicted physisorption energy in our study (−0.46 eV)is higher than reported by the literature3, we carried out climbingimage NEB calculations to understand the chemisorption of O3 on graphene . At this point, the net energy of the system startsto increase because of an increased repulsive interaction. The distancebetween the terminal O and C continues to decrease (images 4 and 5)until it reduces to 2.3 Å where a covalent O–C bond isformed (image 6). The net energy of the system is the highest at thispoint (0.3 eV). Therefore, we assign this as the transition statecorresponding to an energy barrier of 0.75 eV from the physisorbedstate.In the physisorbed tilted Λ configuration, terminalO atoms are closest to the graphene lattice with a distance of 3 Å.Chemisorption proceeds with the approach of a terminal O toward theC atom of the graphene lattice . When O2 is detached from the terminal O of O3, it prefers tobe magnetic (see discussion on spin-polarization later). Therefore,to accurately capture the system energy, we performed spin-polarizedcalculations after O2 detachment. The energy of the systemreduces significantly at this point. Subsequently, O2 driftsaways from the epoxy group (images 8 and 9) and physisorbs on topof the graphene lattice in a horizontal configuration (image 10).This reduces the energy of the system, compared to the physisorbedstate, by 0.14 eV. Therefore, we assign a chemisorption binding energyof −0.6 eV. With vdW-DF3, the chemisorption binding energywas found to be −0.69 eV (−0.66 eV) using the OPT1 (OPT2)variant of the functional. Moreover, the free energy correction is+0.12 eV (Supplementary Note S2), bringingthe free energy of adsorption to values of −0.48 eV (vdW-DF2),−0.57 eV (vdW-DF3-OPT1), −0.54 eV (vdW-DF3-OPT2).After reaching the transitionstate, the bond lengthbetween the central O of ONext, from the physisorbed Ographene 2. Detail3 is 3 Å away from the lattice . A projecteddensity of states (PDOS) analysis is provided in Figures S7 and S8 of the Supporting Information to confirmthe physisorbed/chemisorbed natures of the initial/final states, respectively.The physisorbed and chemisorbed states are comparedin  lattice 3a. In th lattice 3b. This 3) to a spin-polarized system (epoxy + O2). To reveal theimportance of the spin-polarized calculation at the transition state,we also carried out the NEB calculations using a spin-neutral system. In this case, chemisorptionis not favored compared to physisorption with an unfavorable bindingenergy. This underlines the importance of capturing the onset of spinin the O orbitals during the transition state for the chemisorptionof the complexes of O3.As discussed above, during the transition statefor chemisorption,the system undergoes a change from a spin-neutral system  is lower thanthat in the para configuration (1.0 Å).In the first configuration, the epoxy groups areseparated by asingle bridge site and are located in the same honeycomb. We referto this as the meta configuration 4a. In th3 will prefer to chemisorb wheneverit can overcome the energy barrier of 0.75 eV. The energy difference(0.14 eV) between the physisorbed and the chemisorbed states yieldsa large equilibrium constant  between the two statessuch that most of the sites on graphene would be in the chemisorbedstate at the equilibrium.The net lower energyof the chemisorbed state compared to the physisorbedstate indicates that Oc, can be predicted by the reciprocal of thechemisorption rate constant, kp→c, which in turn, can be approximatedby the Eyring equation .The chemisorption time scale, τequation 1:1where 3-functionalized graphene samples at various temperatures. The epoxy group coverage was analyzedby high-resolution XPS . For lower reaction temperatures (17 and23 °C), we used a longer (12 h) reaction time to obtain a clearC–O peak in XPS for quantitative analysis. For samples preparedat or above 34 °C, 1 h of reaction was enough to obtain a clearC–O peak. The resulting coverage of epoxy group, indicatedby the C 1s peak at 286.3 eV, was divided by the reaction time toobtain coverage rate, θ. We observed that θ increasedexponentially in the probed temperature range from 0.01 monolayer/hat 17 °C to 0.016, 0.071, 0.123, and 0.185 monolayer/h at 23,34, 43, and 56 °C, respectively  where most site on graphene are availablefor reaction, θ is expected to be proportional to the chemisorptionrate constant, kp→c, as per θ is driven by chemisorption whenever thechemisorption energybarrier, Ep→c from the temperature-dependent data on θ,we plotted ln θ versus 1/T (Ep→c of 0.66 eV, which agrees well with the vdW-DFT predictedenergy barrier of 0.75 eV.To extract rsus 1/T 5b. The o3 with grapheneusing vdW-DFT. We find that O3 strongly physisorbs witha binding energy of −0.46 eV, maximized by the interactionof all three O atoms in the titled configuration with the nearestO atom 3 Å away from graphene. Chemisorption follows this formingepoxy group on graphene. The small energy barrier of chemisorption,0.75 eV, makes O3 highly attractive to functionalize grapheneeven at room temperature, which is also demonstrated by experimentswhere a coverage rate of 0.071 monolayer/h is realized at 23 °C.From a computational point of view, we demonstrate the importanceof nonlocal density functionals, which captures long-range interactionsand improves the understanding of O3 sorption energeticsand related configurations. We also demonstrate the importance ofthe spin-polarized system at the onset of the transition state whereO2 dissociates from O3 to correctly capturethe chemisorption energy. These calculations highlight that O3 is extremely attractive to functionalize graphene even atroom temperature and can evolve as a popular tool to pattern the latticeof graphene for several applications relying on functionalized graphene.Overall, we investigate the reaction ofO


The superlattice obtained by aligning a monolayer graphene and boron nitride (BN) inherits from the hexagonal lattice a sixty degrees periodicity with the layer alignment. It implies that, in principle, the properties of the heterostructure must be identical for 0° and 60° of layer alignment. Here, we demonstrate, using dynamically rotatable van der Waals heterostructures, that the moiré superlattice formed in a bilayer graphene/BN has different electronic properties at 0° and 60° of alignment. Although the existence of these non-identical moiré twins is explained by different relaxation of the atomic structures for each alignment, the origin of the observed valley Hall effect remains to be explained. A simple Berry curvature argument is not sufficient to explain the 120° periodicity of this observation. Our results highlight the complexity of the interplay between mechanical and electronic properties in moiré structures and the importance of taking into account atomic structure relaxation to understand their electronic properties. Here, the authors report the unexpected observation of different electronic properties of bilayer graphene/boron nitride heterostructures at 0° and 60° twist angles, showing the complex interplay between lattice relaxation and the electronic properties of moiré structures. This is caused by the combination of two effects: (i) a long-wavelength geometric interference pattern, called a moiré pattern, which effectively acts as a periodic superlattice, and (ii) a local enlargement of the lattice constant of graphene to match the one of BN at the inner part of the moiré pattern, leading to a local commensurate state. Outside of the commensurate areas the accumulated stress, due to the stretching of the lattice constant, is released in the form of out-of-plane corrugations where the stacking order changes rapidly in space5. These corrugations have the same periodicity as the moiré pattern. For monolayer graphene, both the long-wavelength pattern and the commensurate state are observed every time one of the layers is rotated by sixty degrees.When the crystallographic alignment of monolayer graphene and BN is almost perfect (close to zero degrees between layers), the electronic, mechanical and optical properties of graphene are strongly modified5. In monolayer graphene/BN the breaking of inversion symmetry has been proposed as the origin of the opening of an energy gap at the charge neutrality point (CNP)6 and non-trivial quantum geometry characteristics of the electronic band structure7. However, very little is known about how the graphene/BN alignment affects systems with more than one layer, such as bilayer graphene.The commensurate state creates an imbalance of the interaction that the carbon atoms have with the BN substrate breaking the sublattice symmetry4 made of Bernal stacked bilayer graphene and BN. Our measurements reveal distinct behaviors for 0° and 60° which we attribute to different electronic band structures generated by different atomic displacements inside the moiré superlattice. However, the observation of the valley Hall effect, only present for 0° of alignment remains to be explained given that the current theoretical model fail to explain this hundred and twenty degrees periodicity.Here, we demonstrate that the electronic properties of the commensurate state in a bilayer graphene/BN heterostructure have a hundred and twenty degrees periodicity. We present experimental electron transport measurements in dynamically rotatable van der Waals heterostructures4, with the improvement of having a pre-shaped local graphite gate. The latter controls the carrier density only in the central area of our device, and has the same dimensions as the BN structure used to create the moiré pattern. It is important to mention that the bottom BN and graphene layers are intentionally misaligned, to more than 10°, to avoid the formation of a double moiré10. The carrier density of the external parts of graphene is tuned by the global Si gate, acting effectively as a tunable contact resistance11. The angular alignment of the bilayer graphene/BN heterostructure is controlled in situ by means of a pre-shaped BN handle deposited on top of graphene. This handle can be rotated by applying a lateral force with the tip of an atomic force microscope (AFM), Fig. 2V−1s−1 for intermediate densities ± 0.65 x 1012 cm−2 at T < 10 K. The mean free path was calculated to be between 1.2 μm and 2 μm for the same carrier density and temperature range . In this case all occupied states in the valence band contribute to the valley Hall effect23. Using the saturation value of the non-local resistance, dashed horizontal line of Fig. lv = 1.5 μm, in good agreement with previous reports15.Since the Fermi energy is at the CNP, and the temperature of the system is much lower than the energy gap, the valley Hall conductivity is maximal having a value of entclass1pt{minimaπ/2. For T > 40 K we do not have a clear picture of why we observe a nearly quadratic behavior, we attribute this to a mixture of regimes where both the valley Hall effect and ohmic response compete. This regime needs more experimental and theoretical investigation.This behavior, where the non-local signal is independent of the local response, is characteristic of a system where the valley conductivity is larger than the local conductivity, which implies a fully developed valley Hall effect, where the Hall angle becomes RNL ∝ ρ0.9. This behavior is consistent with an ohmic contribution RNL = 4ρeπL/W−/π, and can be adjusted by using only the geometry of our sample, as is expected given its van der Pauw geometry. In contrast with the 0° of alignment this linear behavior can be observed over the full temperature range. This striking difference of the development of the valley Hall effect at 0° but absent at 60° reveals that the consequences of the moiré patterns for the two alignments are non-identical. Changing the alignment further to 120° restores the signatures of the valley Hall effect  and that it will modify the trigonal warping of the electronic band structure. Further combinations of scanning gates and electron transport will be necessary to clarify our observation.Interestingly, neither the valley Hall effect, observed at 0°, or the ohmic behavior, observed at 60°, are reproduced in the fully misaligned case, 30°, Fig. 26, the presence of misaligned hBN substrate induces a small crystal field (≈15 meV/nm) in bilayer graphene, leading to a small correction on the simulated bandgap. This correction was added in calculations of Fig. Dxy, clearly shows that, for the layer that is closer to the BN (layer 1), there is an almost circular symmetry around the center of each moiré superlattice (marked by the pink dashed lines). On the other hand, the second layer shows a breaking of this symmetry into a 2π/3 rotational symmetry. The in-plane atomic displacement of the second layer is also at least one order of magnitude smaller than for the first layer. Additionally, we can see that the in-plane atomic displacement on the second layer is larger in the case of 0°. The differences in the stretching of each layer results in the spatial variation of stacking structure of the bilayer graphene  as illustrated in Supplementary Fig. To understand why these two angular alignments give rise to different behaviors we investigated the in-plane atomic structural relaxation of each layer, Fig. 6. Then the carbon atoms of layer 2 will be sitting on nitrogen atoms. We can see in Fig. The difference of the in-plane atomic structure relaxations for 0° and 60° can be traced back to the Bernal stacked configuration, see Fig. 5. These corrugations are transmitted to the second layer and can be observed in the height sensor of our AFM measurements (PeakForce mode) of a different bilayer graphene/BN aligned sample, Fig. λ ~ 14 nm is clearly observed. Note that the asymmetry on the AFM image and large value on the height of the deformation is an artifact given by the width of the AFM tip being comparable to the size of the features we want to measure (~5 nm). This confirms the existence of a commensurate state and the transmission of the corrugations to the second layer in aligned bilayer graphene/BN heterostructures, supporting our numerical simulations.As we mentioned before, the stress generated by the commensurate state is released in the form of corrugations, as in the case of monolayer graphene27 this represents an unintentional displacement field of ~0.1 V/nm. This is not surprising since our devices do not have a top gate to screen external doping deposited on top of the device. Taking into account this unintentional doping and the atomic structure relaxation we calculated the electronic band structures for 0° and 60°, Fig. The different atomic structure relaxation of the layers results in different electronic band structures for 0° and 60° of alignment  on graphene, our simulations consider a simplified atomic system consisting of a bilayer graphene coupled to a flat monolayer h-BN. The lattice mismatch (~1.8%) between BN and graphene is taken into account. The atomic structure is optimized until all force components are <1 meV/atom. After relaxation, the electronic structure is computed by constructing and solving the corresponding pz tight-binding Hamiltonian36. The large tight-binding Hamiltonian matrix of moiré structures is diagonalized using conventional eigenvalue calculations of large-sparse matrices.The atomic-structure relaxation of h-BN/graphene layers is obtained using molecular dynamics with classical potentials. In particular, intra-layer forces are computed using the optimized Tersoff and Brenner potentialsSupplementary InformationPeer Review File


Kampo medicine is widely used in Japan; however, most physicians and pharmacists have insufficient knowledge and experience in it. Although a chatbot-style system using machine learning and natural language processing has been used in some clinical settings and proven useful, the system developed specifically for the Japanese language using this method has not been validated by research. The purpose of this study is to develop a novel drug information provision system for Kampo medicines using a natural language classifier® (NLC®) based on IBM Watson.The target Kampo formulas were 33 formulas listed in the 17th revision of the Japanese Pharmacopoeia. The information included in the system comes from the package inserts of Kampo medicines, Manuals for Management of Individual Serious Adverse Drug Reactions, and data on off-label usage. The system developed in this study classifies questions about the drug information of Kampo formulas input by natural language into preset questions and outputs preset answers for the questions. The system uses morphological analysis, synonym conversion by thesaurus, and NLC®. We fine-tuned the information registered into NLC® and increased the thesaurus. To validate the system, 900 validation questions were provided by six pharmacists who were classified into high or low levels of knowledge and experience of Kampo medicines and three pharmacy students.The precision, recall, and F-measure of the system performance were 0.986, 0.915, and 0.949, respectively. The results were stable even with differences in the amount of expertise of the question authors.We developed a system using natural language classification that can give appropriate answers to most of the validation questions.The online version contains supplementary material available at 10.1186/s12911-023-02230-3. The widespread use of the Internet has enabled instant access to vast amounts of information related to medications; however, it is necessary to assess the correctness of the information that is obtained . In addiIn Japan, traditional Kampo medicines (漢方薬) are widely used –7. TradiCenters for drug information, where experts are available to answer questions, have been established in each country to help healthcare providers obtain reliable drug information , 14, 15.The purpose of this study is to develop a drug information provision system for Kampo medicines using an NLC® based on IBM Watson. The system developed in this study is a prototype built on limited basic information, and it is highly versatile in actual clinical practice. The performance of this system was evaluated with 900 validation questions provided by six pharmacists (classified into high or low levels of knowledge and experience with Kampo medicines) and three pharmacy students.The target Kampo formulas were 33 prescriptions listed in the 17th revision of the Japanese Pharmacopoeia . The infThe system developed in this study classifies natural-language input questions about the drug information of Kampo formulas into preset questions and then outputs the respective preset answers for these questions Fig. . For devNLC® is IBM’s application programming interface that applies deep learning to NLP to understand the intent of input questions and classify them to predefined answers . The claThe following two steps were necessary in developing the system:indications of Kakkonto ”, “I want to know the treatment of pseudohyperaldosteronism”); questions in which the name of the answer section was not directly included ; and questions that required the system to refer to one section of multiple medications throughout . The total number of answers in the whole NLC® was 2,587.We created and registered answers and corresponding training data questions to the NLC® based on the data from the information sources using the following procedure: Before creating the questions, we divided information on the package inserts and the Manuals into 13 and 18 sections, respectively, and used the information written there as the answers. We instructed the QA authors to prepare three types of questions: questions in which the name of the answer section was directly included in the question  and six downstream NLCs® (Category NLC®) Fig. . Users cThe NLC® calculates the confidence rate in the answer to the input question. The calculation method of the confidence rate is not disclosed, but it considers the similarity between the input question and the registered question. It is designed such that the sum of all the confidence rates of the candidate’s answers will total to 100%. Our system allows setting a threshold value of a confidence rate. The system answers “I do not have an answer” if the confidence rate is below the threshold value. When users input a question for which no question is registered, our system cannot give a correct answer and outputs a wrong answer. This is because the system outputs the registered answer with the highest confidence rate. If a user asks a question that has a registered answer in the system, it will find a registered question with a high degree of similarity that will likely have a higher confidence rate. In contrast, if a user question has no registered answer, the likelihood of finding a registered question with a high degree of similarity is low and the confidence rate is likely to be low. When the system receives user questions for which no registered answers have been input and the confidence rate is below a certain threshold level, having the system say “I do not have an answer” decreases the likelihood of wrong answers being output.In system development using NLC®, it is time-consuming to increase the number of registered questions corresponding to one answer. Registered questions are created by humans, so the more data the system has, the more time-consuming this becomes. To solve this problem, we created a thesaurus. Fluctuations in natural language expressions can be absorbed by NLC®. However, if we create a system using only NLC®, questions with many synonyms need to be registered, the number of questions will be huge, and individual confidence rates will decrease. Therefore, we decided to perform synonym conversion before questions are input to the NLC®. In the thesaurus, 369 keywords corresponded to 1,429 synonyms. The system used the thesaurus to convert the words in the user questions into keywords. The thesaurus was created in the same way as the questions and answers registered with NLC®. It was constructed by three pharmacists  and seven pharmacy students. First, we extracted words from the package inserts and the Manuals. Colloquial expressions describing symptoms were extracted from a book in Japanese regarding patients’ subjective descriptions of side effects [For the system to perform optimally, the answers to the questions entered into the system must match the answers given by the experts after reading the questions. To ensure that the system was optimized, we created multiple questions that might be asked in clinical settings and input them to check whether the system found the correct answers. If it did not, we rechecked the questions in the NLC® and the synonym dictionary. In addition, the developers discussed why the correct answer could not be derived. We modified the words of the registered questions in NLC® and expanded the thesaurus several times by increasing the number of phrasings of questions in the NLC®, registering words used in the questions to the synonym dictionary, and registering words in the thesaurus as one of the wordings of the questions in the NLC®.Validation was conducted to investigate the performance of the system. The 900 validation questions were based on what pharmacists may want to know about using Kampo medicine in a clinical setting, including indications, adverse reactions, and drug interactions. Nine additional people (pharmacists and pharmacy students) who had not participated in any of the previous steps prepared 100 validation questions each. To ensure that the performance of the system was not biased by the pharmacists’ knowledge and experience of Kampo medicines, the nine question-authors were divided based on their knowledge into three groups of three people each: (1) pharmacists with advanced knowledge and experience of Kampo medicines (Advanced group); (2) pharmacists with basic knowledge and experience of Kampo medicines (Basic group); and (3) pharmacy students after practical clinical training based on previous studies . The phaTo confirm the threshold of the confidence rate of the system, we drew a receiver operating characteristic (ROC) curve using a threshold. The threshold value at which the value of “Sensitivity-(1-Specificity)” is maximized was obtained. The system was set to answer “I do not have an answer” if the confidence rate was less than this threshold. Additionally, four thresholds with confidence rates of 50%, 60%, 70%, and 61%, derived from the ROC curve analysis, were considered.The validation questions were classified by the administrator based on whether the question had a correct answer in the system. The questions were put into the system and the confidence rate and answers  were recorded. After all 900 questions were input, the precision, recall, and F-measure were calculated for all validation questions and each group.The true positive (TP), false negative (FN), false positive (FP), and true negative (TN) were calculated as follows: TP was defined as the number of questions for which there was a correct answer in the system and the system gave the correct answer. FN was defined as the number of questions for which there was a correct answer in the system, but the system could not give the correct answer or replied with “I do not have an answer.“ FP was defined as the number of questions that had no correct answer in the system and the system should answer “I do not have an answer” but the system gave some wrong answer. TN was defined as the number of questions for which there was no correct answer in the system and the system was able to answer “I do not have an answer.”Precision is the quotient of TP divided by the sum of the TP and FP. Recall is the quotient of TP divided by the sum of TP and FN. The F-measure is the harmonic mean of precision and recall.The system was completed in July 2021.Table If there is an answer in the system to the question (Yes) and the system answer is the expected one (〇), the system answer is judged as correct (TP). If there is an answer in the system to the question (Yes) but the system answer is unexpected (×) or “I do not have an answer” (△), the result is judged as incorrect (FN). If there is no answer to the question in the system (No) and the system answer is “I do not have an answer” (△), the result is judged as correct (TN). If there is no answer in the system to the question (No) but the system gives another one (×), the result is judged as incorrect (FP).To confirm the threshold of confidence rate, we drew an ROC curve Fig. . The besThe results of the system are shown in Fig. We developed a novel drug information provision system for Kampo medicines using NLP. The developed system showed high performance with precision, recall, and F-measures of approximately 0.9 for all questions created by pharmacists regardless of their level of expertise in Kampo medicines. This NLC-based chatbot system is considered usable by pharmacists with any level of knowledge and experience because of its superior performance.Although Google search is the information search tool most frequently used by medical students  and is aNone of the previous studies that used IBM Watson® to develop a system to provide drug information validated the resulting system , 20. In Moreover, there have been no previous studies using the same Japanese language NLP used in this system. Previous studies using other Japanese language NLPs were meant to detect adverse reactions in electronic medical records, drug histories, articles, and social media –46. The The system worked well at the threshold value of 61% based on the ROC curve, with acceptably high levels of precision, recall, and F-measure. If we increased the threshold of the confidence rate, the system answered questions with “I do not have an answer.” This increased the number of FNs and decreased the recall because FN is included in the denominator. Contrarily, the precision values tended to become higher with the higher threshold of confidence rate because FP included in the denominator was decreased. These differences were, however, not significant.The two main reasons that cause the system to give incorrect results even for the validation questions with correct answers are Table  problemsAdditionally, ambiguous or indirect wording of a validation question may lead to a wrong answer including FN or FP depending on the threshold of the confidence rate. For example, the validation question, “Can I concomitantly use Hochuekkito in patients who are taking antihypertensive agents?” or “How many grams of licorice can I take in a day when I take Yokukansan?” did not lead to a correct answer because there is no yes/no answer or such direct description on the official information we referred to in the present study.The process inside the NLC® is a black box; hence, similar questions could not be fixed to lead to correct answers. Watson® has knowledge originally trained by IBM and then trained further with data we provided. Because the learning model is created by integrating those data, the user cannot completely control Watson®’s behavior. As an example, consider “Misclassification by NLC®”: even if two questions and answers are learned that are distinguishable from our point of view, there is a possibility that Watson® will interpret them as identical questions and answers with the same meaning. To solve this problem, we are currently developing another artificial intelligence engine that enables us to expand the corpus and control. We hope to use the corpus created in this study to evaluate the usefulness of the newly developed QA system. Our data set is also available from the authors upon reasonable request so that other researchers can evaluate other QA systems.Some validation questions and answers covered more than one category or Kampo formula name; the information on the two medications was in different categories. NLC® cannot classify questions, and it cannot answer questions about information outside the system if it is asked as a closed question. To solve this problem, we plan to adopt a keyword matching system that searches for keywords in the question text; if the keywords are included in the text, the system will display responses tied to predefined keywords. By including a keyword matching system, it will be possible to display the list of indications based on the keyword “indications” and treat the query as an open question on indications for the Kampo formula. Question text structure and ambiguous questions are difficult even for experts to answer. Thus, there are limitations to answering these questions using an NLC®-based system.This study had several limitations that should be addressed in future studies. First, the system we developed can only answer registered questions. The system administrator must add new questions and answers whenever new questions come in. As with information retrieval on the Internet, prior studies have shown that it is necessary for application users to have the ability to judge whether the application is providing the correct information , 47. OurWe developed a novel drug information provision system for Kampo medicines using NLP. The precision, recall, and F-measure were 0.986, 0.915, and 0.949, respectively. We developed a system that gives appropriate answers to questions using natural language, assuming a clinical situation. We plan to confirm the results in the clinical field in the future. Other sources, such as the classic textbooks, will also be included.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4


Excellent dispersion and uniform distribution of the 2 inclusion. Among the coating systems, throughout the immersion time, the graphene/Globally, researchers have devoted consistent efforts to producing excellent coating properties since coating plays an essential role in enhancing electrochemical performance and surface quality. In this study, TiO Corrosion is defined as the process of loss and destruction due to the chemical or electrochemical interaction between the metal substances and corrosive environments, which have resulted in the disintegration and deterioration of metal or their alloy materials ,2,3. TheIn order to control the corrosion-promotion activity of graphene, it is essential to obtain an insight into the mechanism of the corrosion-promotion occurrence . PreviouFurthermore, organic coatings can be easily fabricated on metal substrates, thus playing an essential role in inhibiting the corrosion process . For insxibility ,16. In axibility . In othexibility .In the year 2004, a new generation material, graphene, was first isolated by simple mechanical exfoliation . Graphenon atoms ,22,23. Fectively . Besidesectively . Moreoveectively . Furtherectively . To suppectively . Here, iectively ,15. For ectively . Here, iAt a very low weight percentage, the graphene and GO nanoparticles can be well dispersed and exfoliated in the polymeric matrix due to their high surface area and strong Van der Waals interactions . Howevercoatings . On the atalysis ,39. For ectively . Moreoveectively ,41,42. Bectively . Furthered water . Moreoveed water ,45. Henccoatings .Therefore, in this communication, the organic-inorganic based nanocomposite coating systems consisting of the acrylic-epoxy polymeric matrix, PDMS, 1 wt.% of graphene along with different loading rates of Without any further purification, all the listed chemicals were used as received from the suppliers to fabricate the hybrid polymer nanocomposite coatings. Acrylic polyol resin, denoted as A and its curing agent aliphatic poly-isocyanate resin (NCO) were purchased from Synthese, Selangor, Malaysia and Bayer Material Science, Germany, respectively. Epoxy resin, denoted as E, produced from bisphenol A and epichlorohydrin, was purchased from ASA Chemicals, Malaysia. This preparation was conducted to further investigate the effects of graphene nanofillers along with the inclusion of −1 at a resolution of 4 cm−1 and the data was recorded after 32 scans.FTIR analysis was performed to observe cross-linking and qualitative investigations. ATR-Nicolet iS10 Spectrometer  was utilized for this analysis in the range of 4000 to 400 cmBy using Mettler Toledo TGA Q500 equipment that runs dynamically from 30 °C to 800 °C at a heating rate of 10 °C/min under nitrogen gas with a flow rate of 20 mL/min, the TGA tests were employed to evaluate the thermal stability of the hybrid nanocomposite coating systems. The TGA apparatus was purchased from Perkin Elmer, Pyris Diamond, USA and all the TGA thermograms were analyzed by the TARe software, TA Universal Analysis, Version 4.7A.The UV-3101PC  UV-vis spectrometer in transmission mode at a wavelength range of 200 nm to 800 nm on medium-speed scanning was employed to analyze the degree of transparency of the coatings coated on the glass substrates. The objective of this analysis was to evaluate the percentage changes in film transparency after the inclusion of The WCA test was carried out to evaluate the hydrophobic performance of the developed nanocomposite coatings by using an optical contact angle (OCA) 15EC instrument. At different locations, 5 droplets of 5 µL distilled water were dispensed on the surface of the specimens, and the images of static WCAs were captured, respectively. An average of three readings were recorded, and the highest value was reported with less than 2° as a measurement error.The effect of the graphene/The anticorrosion properties of the composite coatings were analyzed by EIS. Gamry potentiostat  was used, and the sinusoidal voltage amplitude was set at 10 mV. Herein, a three-electrode cell system was used for investigation for 90 days, and the electrolyte was 3.5 wt.% NaCl. The electrochemical cell was developed by placing a 2.0 cm inner diameter poly (vinyl chloride) (PVC) tube vertically on the coated substrate surface. On the other hand, the uncoated section was a working electrode, while the platinum as a counter electrode, and the saturated calomel electrode (SCE) was a reference electrode. In the frequency range of 100 kHz to 10 mHz, the EIS analysis was carried out after placing the electrochemical cell in a Faraday cage to minimize the noise.The FESEM was utilized to examine the surface morphology and the homogenous distribution of the nanoparticles within the polymer matrix. All the free film nanocomposite samples were coated with platinum by using a platinum sputter coater  in order to minimize the charging effects. The FEI Quanta 450 FEG with SDD EDS detector up to 5 KV as accelerating voltage in the presence of low vacuum was utilized to characterize the coating films.The FTIR spectrometer was used to identify the appearance of the chemical functional groups of the graphene/400 cm−1 . The banectively . As a re280 cm−1 ,48. For ibration . In addi bending . Also, t680 cm−1 ,50,51. Aectively ,50,51. Fing band ,52. Here2 which is evidenced by the increase in the residue values. In other words, over the temperature range, some changes at each degradation occurred with the increment of the loading rates of coatings . Howeverarticles ,55. WithIn this study, the UV-Vis spectroscopy was used to evaluate the optical properties of the graphene/adiation . Alternao 800 nm . Howeverstigated . Despiteng films . In othee system . From Fie system ,60.As illustrated in o 99.75° . Interesectively . Furthervaluated . Here, ivaluated . Figure In addition, it can be clearly seen that the concentration of ted film .Interestingly, it is noteworthy to mention that at the highest hobicity . Furtherhobicity . To suppo of 1:9 . Alternaely 100° . In shorThe adhesion of the coating film to the metal substrate is considered to be an important factor in order to develop an intact coating system with excellent overall performance. In this analysis, the adhesion properties of the graphene/The corrosion resistance of the graphene-based hybrid polymer nanocomposite coating systems with different inclusion rates of Due to the good corrosion resistance of As the exposure time elapsed, no significant change or bend was observed in the slope of the Bode plot at low-frequency region for all the coating systems after 30 days of immersion, whereby the Interestingly, the After 90 days of immersion, as depicted in Interestingly, after 30 days of immersion, both However, after 90 days of immersion, The surface morphologies of all the graphene/ process . As mentIn general, the graphene nanoplatelets consist of an irregular shape, whereas the (<50 µm) . Additioeristics .Furthermore, the coating samples containing 0.5 wt.%, 1 wt.%, and 2 wt.% of At different loading ratios, the


Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG ( Human immunodeficiency virus-1 (HIV-1), which causes acquired immunodeficiency syndrome (AIDS), was discovered in the early 1980s, and since then, it became a global pandemic , includiOver a hundred types of human papilloma virus (HPV) were already known and the HPV genotypes 16 and 18 are defined to be the cause for about 70% of women cervical cancer all over the world ,10,11. RIn our prior review papers, titled design concepts of VLP-based HIV-1 and HPV vaccines, we mentioned that papillomavirus VLPs, defined as non-enveloped VLPs, could play a functional role as delivery vehicles to present HIV-1 neutralizing antibody or CTL epitopes ,14. ThisThe recombinant HPV16 L1 proteins were successfully produced in insect cells ,25, yeasUltracentrifugal approaches, such as sucrose cushion (SC) or cesium chloride (CsCl) density gradients, were widely used to isolate the HPV6 L1 VLPs previously because the large VLP mass  was separated to most of contaminants ,33,34,35In this study, the HPV 16 L1 protein acted as the structural vaccine scaffold, and the P18I10 peptide was selected as HIV-1 immunogen and inserted into DE loop of HPV16 L1 protein. The immunodominant P18I10 CTL epitope comprising 10 amino acids (residues 311–320: RGPGRAFVTI) is derived from the third variable domain (V3) of the HIV-1 Env gp120 ,44. The The chimeric L1:P18I10 DNA coding sequence was cloned into pFastBac1 and pcDNA3.1 plasmid DNA expression vector, respectively, as shown in We used immunofluorescence staining to determine the expression of L1:P18I10 proteins and evaluate the polyethylenimine (PEI) or baculovirus-mediated transfection efficiency from day 0 to day 4 D,E. The Since frequency and intensity of L1 signals detected by immunofluorescence did not directly correlate with L1:P18I10 protein expression level in the host cells, we further quantified L1:P18I10 capsid proteins by Western blot analysis F,G. L1:P8 293F cells) was approximately ~39% lower than BEVS/IC system (137.87 µg per 1 × 108 Sf9 cells) in day 4 post-transfection. As shown in 8 293F cells) could be higher than BEVS/IC system (160 µg per 1 × 108 Sf9 cells) in day 4 post-transfection  containing approximately 2% of L1:P18I10 VLPs produced from 293F expression system was loaded into the size exclusion chromatography (SEC), heparin affinity chromatography (H-AC), and ion exchange chromatography (IEX) columns, respectively C–F. As sAlthough an application note of the general electric (GE) company indicated that disassembled HPV16 L1 monomers can bind to anion exchange chromatography media (AEC) at pH 8.5 , we obseBy following previous studies ,35,48, LBased on optimized chromatographic parameters obtained from the previous section, we designed a capture, intermediate purification and polishing (CiPP) chromatographic strategy to purify 293F cell-derived L1:P18I10 VLPs in flow-through mode using CEC, SEC, and H-AC C. Most oIn order to assess in vitro stability of purified L1:P18I10 VLPs, we performed non-reducing SDS-PAGE to evaluate disulfide cross-linking of L1:P18I10 capsid proteins A. It is In order to demonstrate that purified L1:P18I10 proteins from both methods are able to self-assemble to icosahedral particles, we further performed molecular mass assessment. As shown in Transmission electron microscopy (TEM) was used to examine the morphologic conformation of purified L1:P18I10 VLPs. As shown in The chromatography-purified L1:P18I10 proteins can also in vitro self-assemble into VLPs in a diameter of around 50–60 nm B. The moTo confirm whether HIV-1 P18I10 epitopes were expressed on chimeric HPV-16 L1 capsid proteins, we first extrapolated actual concentration of purified L1:P18I10 VLPs from a standard curve of commercial HPV16 L1 VLPs A. Equal p = 0.6409). Moreover, the L1:P18I10 VLP-induced IgG in mice sera was able to bind L1:P18I10 VLPs, but not HPV16 L1 VLPs (p < 0.01%). One group of mouse was immunized with only PBS buffer as a naïve group  to set up the cutpoint. Our immunogenicity data revealed that L1:P18I10 VLPs induced high anti-HPV16 L1, but lower anti-HIV-1 P18I10 antibodies (around 0.15 at MRD 1:50). After statistical analysis, the OD value for P18I10 induced IgG was significant from the naive PBS group. Therefore, we thought there is no pre-existing antibody against P18I10. Many other critical factors that could affect the immunogenicity of L1:P18I10 VLPs should be also considered, such as immunogen insertion site among different loops of VLPs, dose, prime-boost intervals, and administration route, etc. Overall, these results suggested that chimeric L1:P18I10 VLP-immunized mice produced almost the same level of anti-L1 IgG as Gardasil-9-immunized mice, and also elicited low anti-P18I10 binding antibodies.To evaluate whether chimeric L1:P18I10 VLPs induce HPV-16 L1 and HIV-1 P18I10-specific humoral and cell-mediated immune responses in BALB/c mice, the immunization schedule was designed as shown in  L1 VLPs C. After p = 0.0132) between L1:P18I10 VLP and Gardasil-9 immunization groups. The difference might be attributed to the unspecific adjuvanticity of L1:P18I10 VLPs according our modified formulation. After unpaired t test analysis, an extremely higher frequency of IFN-γ secreting splenocytes was observed in mice homologously immunized twice with chimeric L1:P18I10 VLPs, as compared to mice receiving HPV16 Gardasil-9 vaccines in response to P18I10 peptide-stimulated splenocytes (p = 0.0002)  E. These The development of an affordable, safe, and effective preventive vaccine against HPV and HIV is still an urgent need. The capacity of production and purification system to engineer preparative expression level, purity, and yield (recovery) of chimeric HPV:HIV (L1:P18I10) VLPs may facilitate the development of VLP-based vaccine against both viruses. In this study, (i) we demonstrated that the 293F expression system could be an alternative of BEVS/IC system to produce comparable expression level of L1:P18I10 VLPs for downstream purification; (ii) the chromatographic purification method could significantly increased L1:P18I10 VLP recovery (56%) approximately 6-fold higher than ultracentrifugal approaches (11%); (iii) both ultracentrifugation and chromatography-purified L1:P18I10 VLPs shared similar biophysical properties in vitro: stability, in vitro self-assembly and morphology. However, ultracentrifugation provided a milder purification condition for integral L1:P18I10 VLPs formation; (iv) both ultracentrifugation and chromatography-purified L1:P18I10 VLPs could be characterized by HPV16 L1 and HIV-1 V3 (P18I10) mAbs; (v) chromatography-purified L1:P18I10 VLPs were immunogenic after BALB/c mice immunization. We anticipated that this scalable chromatography-based purification procedures will reduce the time, cost, and labor involved in industrial-scale manufacturing of VLP-based vaccines. This work contributes towards developing an alternative platform for production and purification of a bivalent VLP-based vaccine against HPV and HIV-1, which is urgently needed in developing and developed countries.Mycobacterium bovis Bacillus Calmette-Guérin (rBCG) expressing HIVA immunogen and boosting with recombinant viral vector MVA.HIVA was safe and elicited HIV-1-specific T-cell immune responses in BALB/c mice [The P18I10 peptides derived from HIV-1 gp120 V3 loop are presented in HIV-infected cells by major histocompatibility complex (MHC-I) class I molecules . CD8+, cB/c mice ,65,66. TB/c mice . In our B/c mice . These f8 Sf9 cells in 80 mL Grace’s insect/TNM-FH medium was around 137.87 µg. Therefore, the overall yield per unit culture volume (mg/L) of our BEVS/IC-derived L1:P18I10 VLPs was calculated to be 1.72 mg/L. In our laboratory, the Sf9 cell density at 96 h of harvest could only reach approximately 1.2 to 1.5 × 106 cells/mL. This pattern match Merck’s application note indicating that the Sf9 cell density could expand from 1.0–1.2 × 106 cells/mL to 1.5–2 × 106 cells/mL through shake flask cultures to a bioreactor [6 cells/mL by using fed-batch bioreactors under tightly monitored culture conditions for large-scale manufacturing production [A comparison of the expression level of L1:P18I10 proteins between BEVS/IC and 293F expression systems is not always straightforward, since production is also affected by complexity of VLPs and different cell culture conditions . For insoreactor . However2 to 105 baculovirus particles during VLP purification [The BEVS/IC system was widely used in the pharmaceutical industry. Although both BEVS/IC and mammalian systems have post-translational modifications (PTM), the BEVS/IC system could only perform simpler glycosylation PTM, which is not in favor of enveloped VLP production . Anotherfication .8 293F cells in 30 mL FreeStyle 293 expression medium was 85.39 µg. Thus, the overall yield per unit culture volume of 293F-derived L1:P18I10 proteins was calculated to be 2.85 mg/L. Since mammalian cells tend to be lower VLP-producers [8 293F cells) was lower than BEVS/IC system (137.87 µg per 1 × 108 Sf9 cells). However, the 293F cells in shake flask suspension cultures can grow until a defined density of 3.0 to 3.6 × 106 cells/mL, compared to Sf9 cell density of 1.2 to 1.5 × 106 cells/mL. When we changed the measure of the L1:P18I10 protein expression level (yield) from weight per unit cell (µg/1 × 108 cells) to weight per unit culture volume (mg/L), the overall L1:P18I10 protein yield using 293F expression system (2.85 mg/L) was higher than BEVS/IC system (1.72 mg/L). Therefore, we could re-clarify that the 293F expression system is capable of reaching an overall level of L1:P18I10 protein expression that could be comparable with BEVS/IC system.According to pre-studies, the expression level of recombinant proteins produced by using PEI-mediated transfection in 293E cells is around 22–50 mg/L ,73,74. Oroducers , the oveAlthough 293F cells provide a baculovirus-free platform to generate VLPs, the mechanistic understanding about polyethylenimine (PEI)-mediated plasmid DNA delivery is still unclear. The branched PEI-25K was demonstrated to be efficient for transient transfection . HoweverRegarding to VLP purification methods from former studies, the recovery of HPV16 L1 VLPs using 40% or 45% sucrose cushion (SC) is 27% and 18.1%, respectively, and the purity is ranged between 2.2 and 5.4% . Our ultPichia pastoris yeast. After chromatographic and ultracentrifugal purification process, the L1:P18I10 VLPs were recovered with 96% purity and 9.23% overall recovery [The recovery of L1:P18I10 VLPs during downstream purification is critical, because it affects overall costs in bioprocessing . To obtarecovery . Accordirecovery . Anotherrecovery . Since wrecovery . We showrecovery . Heparinrecovery ,83. Our recovery , removalIn our present study, we established optimum production and purification methods to engineer chimeric HPV:HIV (L1:P18I10) VLPs. Although the BEVS/IC system was widely used in licensed HPV prophylactic vaccine  manufacturing, low expression level of L1 capsid proteins remained challenging for the production of certain HPV types  or as ye2. The 293F cells (Gibco), derived from human embryonic kidney (HEK) 293 cells, were cultured in FreeStyle 293 expression medium (Gibco) supplemented with 5 mL/L of penicillin-streptomycin (Gibco) and incubated in a 37 °C incubator containing a humidified atmosphere of 5% CO2 on an orbital shaker platform rotating at 125 rpm.The insect Spodoptera frugiperda 9 (Sf9) cells  were grown in Grace’s insect medium (Gicbo), supplemented with 10% fetal bovine serum (FBS) (Sigma) and 100 U/mL of penicillin-streptomycin (Gibco), and incubated in a 27 °C incubator without a humidified atmosphere and COE. coli. DH10Bac contains a parent bacmid with a lacZ-mini-attTn7 fusion. When the transposition was successful, the Sf9 cells were transfected with isolated DNA to produce first generation of recombinant baculovirus. The viral titer of amplified recombinant baculovirus was determined by plaque assay. A density of 1 × 106/mL of Sf9 cells (~90% confluent) was seeded in 75 cm2 flask  with 10 mL Grace’s insect/TNM-FH medium and infected with recombinant baculovirus at a multiplicity of infection (MOI) of 1 to generate the chimeric L1:P18I10 proteins. After 96 h post-infection, the Sf9 cell density can be about 1.2 to 1.5 × 106 cells/mL with at least 20% viability.The HIV-1 P18I10 CTL peptide (RGPGRAFVTI) was inserted into the DE loop of HPV16 L1 capsid protein. HPV16 L1 DE loop sequence encoding 130–136 amino acids was replaced with P18I10I10 peptide. The recombinant baculoviruses were produced according to the manufacturer’s instructions of Bac-to-Bac BEVS/IC system with pFastBac kit . In brief, the chimeric L1:P18I10 DNA coding sequence was cloned into a baculovirus donor plasmid (pFastBac1) and transformed into competent 6/mL and transiently transfected with L1:P18 I10 pDNAs using the branched polyethylenimine with a MW of 25 kDa (PEI-25K)  at an optimized ratio of DNA to PEI 1:3 (w/w) and DNA to culture medium 1:1 (w/v) according to manufacturer’s instructions [6 cells/mL with around 50% viability.The HIV-1 P18I10 CTL peptide (RGPGRAFVTI) was inserted into the DE loop of HPV16 L1 capsid protein. HPV16 L1 DE loop sequence encoding 130–136 amino acids was replaced with P18I10I10 peptide. The L1:P18I10 DNA coding sequence was modified with Kozak sequence, optimized with human codon, flanked by the restriction enzyme sites of HindIII and XbaI and cloned into pcDNA3.1(+) vector by using GeneArt gene synthesis services . The recombinant plasmid DNA (pDNA) was transformed into DH5α competent cells (Invitrogen) for amplification and extracted by using plasmid Maxi kits . The 293F cells were cultured with 30 mL FreeStyle 293 expression medium in a 125 mL Erlenmeyer flask (Corning) to a density of 1.0 × 10ructions . The 2935 Sf9 cells) from a total of 1.2 × 107 infected Sf9 cells in a 75 cm2 flask (10 mL culture medium/flask). Additionally, we collected 100 μL culture medium  from ~1.1 × 108 transfected 293F cells in a 125 mL Erlenmeyer flask (30 mL culture medium/flask). The cells were permeabilized on the glass slide with 100% cold acetone. Subsequently, the fixed cells were probed with anti-HPV16 L1 antibody CAMVIR-1  and captured with anti-mouse IgG-FITC . Immune-stained cell monolayers were thoroughly washed with PBS and covered with mounting medium with DAPI (Abcam). The immunofluorescence images were inspected under an inverted microscope at 40× magnification. Transfection efficiency was determined by the ratio of FITC (green)-positive cells to DAPI (blue)-stained cells.We collected 100 μL culture medium  infected Sf9 cells in eight 75 cm2 flasks (10 mL culture medium/flask) or 1.0 × 108 (~1.1 × 108) transfected 293F cells in a 125 mL Erlenmeyer flask (30 mL culture medium/flask) were collected by centrifugation at 1500 rpm for 5 min and washed twice with PBS. The pellet was re-suspended with cell lysis buffer formulated with 20 mM Tris (pH = 7.1), 20 mM NaCl, 1% Triton X-100, protease inhibitor (1:100) , and Benzonase (25 U/mL) (Millipore) and 2 mM MgCl2 and was then incubated at 4 °C for 24 h for complete lysis and DNA degradation. The crude cell lysates were centrifuged at 13,000 rpm for 10 min at 4 °C, and, subsequently, clarified by the 0.45 µm syringe filter (Millipore) to remove cell debris and clumps.A total of 1.0 × 10w/v) were prepared in PBS . The clarified Sf9 cell lysate adjusted in PBS was carefully layered on the top of two-step (20% and 70%) sucrose cushion in a thin wall ultracentrifugation tube . After ultracentrifugation at 40,000 rpm  for 4 h at 4 °C, the tube was placed on ice to avoid the interface layer being re-suspended. The practically purified and concentrated L1:P18I10 VLP sample was located by UV light and collected through a puncture using a 1 mL sterile syringe.The 20% and 70% sucrose cushions (SC) (g) for 16–24 h at 4 °C. After ultracentrifugation, the tube was placed on ice to avoid the layer being re-suspended. The CsCl gradient was fractionated from the top of the tube (400 μL per fraction). The signal of L1:P18I10 VLPs in each fraction was detected by dot blot, using anti-HPV16 L1 mAb CAMVIR-1.The SC-purified L1:P18I10 VLP sample was mixed with 40% CsCl solution in PBS and scattered with the sonicator . The L1:P18I10 VLP sample was ultracentrifuged at 40,000 rpm . Since we used HiLoad Pump P-50 (GE), pH and conductivity of flowthrough (FT), eluates or eluents were determined by pH papers (Sigma) and the EC/salinity meter. The clarified 293F cell lysate were adjusted to a volume of 5 mL with the starting buffer and loaded on to the column at a flow rate of 1 mL/min. After washing with 10 mL of the starting buffer, the CEC-captured L1:P18I10 VLPs were one-step eluted with 5 mL of elution buffers  at a flow rate of 1 mL/min. The column was regenerated by washing with 5 mL of 2 M NaCl in 20 mM Tris-HCl buffer at pH 7.1 to remove remaining ionically bound proteins.The HiTrap Capto SP ImpRes column  was washed with 5 mL of ddH2O and equilibrated with 10 mL of the running buffer . The CEC-purified L1:P18I10 VLP sample was adjusted to a volume of around 20 mL with the running buffer and loaded on to the column at a flow rate of 0.6 mL/min. The first and second fractions (10 mL per fraction) containing the SEC-purified L1:P18I10 VLPs were collected through washing with the running buffer. Finally, a CIP procedure was performed again to clean the column.Before using HiTrap Capto Core 700 column , cleaning-in-place (CIP) was performed to remove the bound impurities. The column was washed with 5 mL of ddH2O and equilibrated with 10 mL of the binding buffer . The SEC-purified L1:P18I10 VLP sample (10 mL) was loaded on to the column at a flow rate of 0.5 mL/min. After washing with 10 mL of the binding buffer, the heparin-bound L1:P18I10 VLPs were one-step eluted with 5 mL of elution buffers  at a flow rate of 1 mL/min. The column was regenerated by washing with 5 mL of 2 M NaCl in 20 mM Tris-HCl buffer at pH 7.1 to remove remaining impurities. The eluates were subsequently diafiltrated 10-folds with Tris-HCl  by using 100 kDa Ultra-4 centrifugal filter devices .The HiTrap Heparin HP column  was washed with 5 mL of ddHThe L1:P18I10 VLPs purified by ultracentrifugal and chromatographic methods were mixed with 2× Laemmli sample buffer  in the presence or absence of 20 mM dithiothreitol (DTT) and reacted at room temperature (RT) for 15 min. Samples were separated by 8–16% TGX stain-free protein gels (BIO-RAD). Then, the gels were transfer to PVDF membranes. The membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate  at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit (Bio-Rad). The blot images were acquired by using Odyssey Fc imaging system.L1:P18I10 VLPs purified from both methods were treated or un-treated with 20 mM DTT for 15 min and were then filtered out through 1000 kDa  or 100 kDa (Amicon) molecular weight cutoff (MWCO) ultrafiltration devices. The retentates were reconstituted to the original volume and collected from the filter device sample reservoir, while the filtrates were collected at the bottom of the centrifuge tube. The L1 signal was measured by using dot blot probed with anti-HPV16 L1 mAb and detected by anti-mouse IgG-peroxidase conjugate (Sigma-Aldrich). Images were acquired using Odyssey Fc Imaging System at a chemiluminescence channel.After charging the carbon-coated copper grids (Sigma-Aldrich) under ultraviolet light for 5 min, purified L1:P18I10 VLPs were absorbed on grids for 1 min and rinsed 3 times by miliQ water. The L1:P18I10 VLPs purified by ultracentrifugation in PBS  were negative-stained with 2% phosphotungstic acid (PTA) at pH 7.0 (Sigma-Aldrich) for 1 min. The L1:P18I10 VLPs purified from chromatography in Tris-HCl  were negative-stained with 2% uranyl acetate at pH 4.5 (Sigma-Aldrich) for 1 min. Excess staining agents were removed by Whatman qualitative filter paper (Sigma-Aldrich). Grids were placed in a dehumidifier chamber at least 2 h before observation. Images were acquired using a transmission electron microscopy  at magnification SA270K (50 nm), SA59000 (200 nm) and SA529500 (400 nm), respectively.The band intensity of L1 from the Western blot was quantified by densitometric assay using Image Studio Lite version 5.x software. The purified L1:P18I10 VLPs were quantified by indirect ELISA. The band intensity of total host cellular proteins (HCPs) from Coomassie-stained SDS-PAGE was quantified by densitometric assay using Image Studio Lite 5.x software. The HCPs were also quantified by BCA protein assay kit (Thermo Fisher). 2 mg/mL of bovine serum blbumin Standard (Thermo Fisher) were used to construct a standard curve plotting concentration versus absorbance. The total protein from each purification step was extrapolated from this standard curve to determine the actual amount of HCPs by using a NanoDrop 2000 spectrophotometer.Equal amounts (200 ng) of HPV16 L1 protein (Abcam) and L1:P18I10 VLPs purified from both methods were mixed with 2× Laemmli sample buffer containing 5% 2-ME and boiled at 95 °C for 5 min. Samples were separated by 8–16% TGX Stain-free protein gels and were then transferred to a PVDF membrane (Millipore) using a Semi-Dry transfer device (Bio-Rad). The membrane was blocked with 5% skim milk in TBST. Then, the membranes were probed with the anti-HPV16 L1 CAMVIR-1 mAb at a dilution of 1:4000 and anti-HIV1-V3 loop mAb  at a dilution of 1:500, respectively. After that, the membranes were incubated with anti-mouse IgG Peroxidase Conjugate (Sigma-Aldrich) at a dilution of 1:4000. The signal was developed and visualized by chemoluminiscence using Western Blot ECL substrate kit (Bio-Rad). The blot images were acquired by using Odyssey Fc imaging system.n = 4 and female n = 4 per group). In the group A, BALB/c mice were immunized intramuscularly (i.m.) with 10 μg of L1:P18I10 VLPs, respectively by following a homologous prime-boost regime. In the group B, positive control mice were offered Gardasil-9 prime followed by Gardasil-9 boost intramuscularly with 10 μg of HPV16 L1 VLPs. The prime-boost interval was 2 weeks. Mice were sacrificed on day 28. Blood samples were collected from the heart of mice. Sera were recovered by centrifugation and stored at −20 °C for ELISA assay. Murine spleens were removed and pressed individually through a cell strainer  with a 5 mL syringe rubber plunger. Following the removal of red blood cells with ACK lysing buffer (Lonza), splenocytes were washed and resuspended in lymphocyte medium R10 , penicillin-streptomycin, 20 mM HEPES and 15 mM 2-ME) at a concentration of 2 × 107 cells/mL.Chromatography-purified L1:P18I10 VLPs were emulsified with an equal volume of (225 μg per each 0.5 mL dose) aluminum hydroxyphosphate sulfate (Thermo Fisher), to ensure a similar formulation to the licensed Gardasil-9 HPV vaccine . All mou2SO4. The OD of each well was measured at a wavelength of 450 nm by using ELx800 absorbance microplate reader.To measure the VLP-induced antibodies in the sera of BALB/c mice, the microtiter plates were coated with 50 µL of 2 µg/mL HPV16 L1 VLPs and HIV-1 P18I10 peptide , respectively, with 50 mM carbonate–bicarbonate buffer (pH = 9.6). The plates were incubated at 4 °C overnight. Plates were blocked with the blocking buffer (5% skim milk in TBST) at 37 °C for at least 2 h. At the same time, sera collected from group A and B immunized mice were two-fold serially diluted with 5% skim milk in TBST from a ratio of 1:50 to 1:800. After washing twice with TBST, the plates were added with the diluted sera and incubated at 37 °C for 2 h. After washing 3 times with TBST, the plates were added with recombinant protein G HRP conjugate at a dilution of 1:4000 in blocking buffer and incubated at 37 °C for 1 h. TMB was used to develop the ELISA signal and stopped with 50 µL of 2 M H5 fresh splenocytes were added to each well. Subsequently, the cells from group A and B were stimulated with 2 μg/mL of HPV16 L1 VLPs and HIV-1 P18I10 peptides, respectively, and the plates were incubated at 37 °C with 5% CO2 for 24 h. After emptying the cells by washing 5 times with PBS, the plates were added with biotinylated anti-IFN-γ detection mAb diluted to a concentration of 1 μg/mL in PBS containing 0.5% FCS and incubated for 2 h at RT. After washing 5 times with PBS, the plates were added with diluted Streptavidin-ALP (1:1000) in PBS-0.5% FCS and incubated for 1 h at RT. After the final wash, the alkaline phosphatase conjugate substrate (BIO-RAD) was added to the plate until distinct spots emerge. Color development was stopped by washing extensively in tap water, and the count spots were inspected using an ELISpot reader .The ELISpot assay was performed according to the manufacturer’s instructions . The PVDF plates  pre-treated with 70% EtOH were coated with anti-mouse IFN-γ capture mAb (15 μg/mL) in PBS and incubated at 4 °C overnight. After removing excess antibody by washing 5 times with PBS, a total of 2.5 × 10t test to determine the statistical significance between two groups.Statistical analysis was performed using Prism 6 GraphPad software . The line graph of ELISAs were analyzed by simple linear regression test to compare the slope of the two lines together and to confirm two data sets were significant different. ELISpot data were tested by unpaired Six to eight-week-old BALB/c mice were purchased from Envigo  and approved by local authorities  and the Universitat Autònoma de Barcelona Ethics Commitee. The animal welfare legislation strictly conformed to the Generalitat de Catalunya. All experimental works were approved by the local Research Ethics Committee .


The aim of the study was to investigate clinical significance of soluble PD-L1 (sPD-L1) serum level in head and neck cancer and to evaluate its role as a possible prognostic and predictive biomarker.A prospective analysis of sPD-L1 levels in 60 patients diagnosed and treated due to malignant and non-malignant lesions in the region of head and neck was performed in peripheral blood by an ELISA test.p =  0.006) depending on the histopathological advancement of the lesions, 0.704 ± 0.349 and 0.512 ± 0.177 respectively in the malignant and benign group. The separate analysis of laryngeal lesions confirmed statistical difference in sPD-L1 (p =  0.002) for the malignant lesions (0.741 ± 0.353) compared with the benign (0.489 ± 0.175). The sPD-L1 level of 0.765 ng/mL or higher, revealed 35% sensitivity and 95.5% specificity for the diagnosis of head and neck malignant lesions . The 1-year DFS was 83.3% in the group of patients with low sPD-L1 levels (< 0.765 ng/mL) and 53.8% in patients with high sPD-L1 (≥0.765 ng/mL). The 2-year OS were 68% and 69.2% respectively in both groups. The log-rank test confirmed statistically significant prognostic value of sPD-L1 level for 1-year DFS .The range of sPD-L1 in the study group was 0.16–1.63 ng/mL, mean 0.64 ± 0.32. There were no differences in the mean sPD-L1 regarding patients’ age, sex, and the localization of the lesion. Statistically significant difference was revealed in the average sPD-L1 level (sPD-L1 is a promising prognostic and early recurrence predictive biomarker for head and neck cancers, most significantly for laryngeal lesions.3. Head and Neck Cancers (HNCs) include malignancies of oral cavity, oropharynx, larynx, hypopharynx, salivary glands, nasopharynx and paranasal sinuses. HNCs are one of the most common malignancies worldwide, accounting for 5.3% of all cancers with 890,000 incidence, and 5.3% of cancer mortality with 507,000 deaths reported annually.Programmed Death protein-1 (PD-1) is an inhibitory receptor expressed on various immune response cells, including activated T-cells, B-cells, natural killer T-cells, activated monocytes and dendritic cells.Wide range of malignant tumors are known to express PD-L1 to successively escape immune response. The main form of PD-L1 is membrane-bound PD-L1 (mPD-L1), present on the cell surface. Its soluble form (sPD-L1) can be detected circulating in the blood flow of patients with solid tumors, autoimmune diseases and some other chronic conditions.One of the causes for current attention to PD-1/PD-L1 signaling pathway are monoclonal antibodies, that target it with remarkable clinical outcomes. Pembrolizumab and Nivolumab registered in the treatment of HNCs are antibodies directed at PD-1.11In recent years a growing number of studies focused on sPD-L1 is evaluating its role as a potential biomarker. Detection of this protein is convenient, fast, repeatable and objective. So far few meta-analyses have proven that high sPD-L1 level is correlated with poor survival outcomes in numerous solid tumors.17In this study we investigate the pretherapeutic levels of sPD-L1 in serum of patients with different sites of HNCs, dysplastic lesions and benign tumors. The data is analyzed and correlated with clinical features, histopathological staging and treatment outcomes.The research was approved by the local Ethics Committee, and written consent was obtained from each patient. All procedures were conducted in accordance with the ethical standards of the Declaration of Helsinki.The prospective study included 60 patients hospitalized and treated in the Department of Otorhinolaryngology, Head and Neck Surgery between June and December 2018 due to malignant and non-malignant tumors in the region of head and neck. The inclusion criteria were age above 18 years and primary tumor diagnosis. Patients with the history of another malignancy, acute inflammatory condition, chronic inflammatory disease, systemic immunotherapy were excluded. Each participant enrolled was subjected to a surgical treatment of the lesion and was observed for minimum of two years.The final diagnosis and staging were based on histopathological examination, conducted according to World Health Organization (WHO) classification for each type of malignancy. Laryngeal lesions were staged into adequate categories: non-dysplastic, Low-Grade Dysplasia (LGD), High-Grade Dysplasia (HGD) and invasive cancer. The study population included 17 female and 43 male participants; the mean age was 63.47 years . The site of a lesion was in 46 cases larynx, in 9 cases oral cavity, in 3 cases salivary gland, and in 2 cases neck . For statistical analysis the patients were divided into two groups based on a histopathological advancement. The malignant lesions group included invasive cancers (n = 34) and HGD (n = 6), and the benign lesions group consisted of non-dysplastic lesions (n = 18) and LGD (n = 2). All malignant lesions were squamous cell carcinomas Neither of the malignancy has positive expression of human papilloma virus. The non-dysplastic group clinically presented with laryngeal leukoplakia and hypertrophic mucosal changes, adenolymphoma and pleomorphic adenoma of salivary gland and two lateral neck cysts. The demography and clinical characteristics of the study population are summarized in The blood samples were obtained from peripheral vein with an interval of 0–2 days before the surgical treatment. The samples were centrifuged at 4 °C to retrieve serum and further stored at −80 °C in the biological resource repository at the Department of Laboratory Diagnostics. The national standards and protocols for collecting, processing, and storing human tissues were maintained. The serum samples were thawed shortly before determination of human Programed Death Ligand-1 (PDL-1) expression by enzyme-linked immunosorbent assay (PDL-1/B7-H1/CD274 Sunred Bio ELISA kit). The ELISA detection was performed according to manufacturer’s instruction. Individual serum concentration was reported in ng/mL.p-value lower than 0.05 was considered statistically significant for all analyses. For each statistical test, the assumptions for the tests were checked, and depending on the results, the parametric independent two-sample t-tests was used. For the graphical presentation of the results, box plots were created showing the mean, Standard Error (SE) and Standard Deviation (SD) values. Using the ROC curve analysis, the level of sPD-L1 with the highest sensitivity and specificity for differentiation between malignant and benign lesions was determined. The next part of the analysis was conducted only for malignant lesions (HGD + invasive cancer). The Kaplan-Meier curves for DFS and OS were determined depending on the critical level of sPD-L1. Log-rank test was calculated for all groups. The Cox proportional hazards model was used to quantify risk factors for recurrence and death.Parameters were evaluated using IBM SPSS Statistics 26.0 and Statistica 13.3. A t-test, statistical difference was revealed in the average sPD-L1 level (p = 0.006) depending on the histopathological staging of the lesions. The separate analysis of patients with laryngeal site also confirmed a statistical difference in the serum levels of sPD-L1 (p = 0.002) for the malignant group (0.741 ± 0.353) compared with the benign origin group (0.489 ± 0.175). The analysis of correlations between sPD-L1 levels and clinical staging of malignant lesions did not reveal significant differences, neither for early and advanced T-stages , nor for the nodal advancement (N0 vs. N1‒N3 stage). There were no significant differences in the mean serum level of sPD-L1 in regard to histopathological tumor grading, however the data distribution was in favor of G2 (n = 20) and the grading was not provided for five patients. The comparison of sPD-L1 level with smoking status and with the history of gastroesophageal reflux disease did not reveal significant differences . The 54.8% sensitivity and 93.3% specificity for identification of malignant laryngeal lesion were established for the serum sPD-L1 concentration of 0.67 ng/mL ; ; .Figure 2Disease Free Survival (DSF) and Overall Survival (OS) analysis with the Kaplan-Meier curves and the Cox proportional hazards models were performed to determine a prognostic utility of sPD-L1 in head and neck malignant lesions. The cut-off value of sPD-L1 was 0.765 ng/mL, as this level of biomarker had the highest predictive and prognostic value. The follow-up time for the study group was 24 months .Figure 3p-value = 0.035).The 1-year DFS was 83.3% in the group of patients with low sPD-L1 level (<0.765 ng/mL) and 53.8% in patients with high sPD-L1 level (≥0.765 ng/mL). The 2-year DFS in both groups were 64% and 46.2%, respectively. For the whole study population 1-year DFS amounted 73%, and 2-year DFS ‒ 57.9%. The 1-year OS valued 88% among patients with low sPD-L1 level and 69.2% among patients with high sPD-L1 level. The 2-year OS were 68% and 69.2% in both groups, respectively. The OS for study population were 81.6% for one year and 68.4% for two years. The log-rank test was calculated for all 1- and 2-years DFS and OS, but only in case of 1-year DFS the difference was statistically significant  and probability of death on average 6.68 times . The remaining variables did not have a statistically significant effect on the endpoints, although it should be noted that the sPD-L1 value was at the borderline of the significance level  for DFS  was considered statistically significant and compared to the regional stage (N-), it increased the probability of recurrence on average 3.93 times  delivered very promising results. The level of sPD-L1 was significantly related to grade, clinical stage and lymph node advancement.Clinical importance of proven and reliable prognostic biomarkers in oncological setting cannot be overestimated. They enable individualized, reasonable intensification or de-escalation of the therapy and the most appropriate follow-up. So far, a few meta-analyses have proven that high sPD-L1 level is correlated with poor survival outcomes in solid tumors,Although our study reveals some encouraging results, it is not free of limitations. The study group was fairly small. Moreover, the distribution of cancer localization within the head and neck region was not sufficient, however the reason for that was the consecutive inclusion into the study group. The representation of patients with LGD was deficient and therefore it was not possible to assess the predictive role in regard to the histopathological staging. Another limitation of the study is the lack of comparison of serum sPD-L1 level to tissue expression of PD-L1. This analysis could be conducted retrospectively, after securing grant for further investigations. The bias regarding the prognostic role of PD-L1 may be involved with the type of curative therapy, what in the future will require analysis of large and comparable in advancement cohorts.Presented results encourage further cohort-based and multicenter studies regarding predictive and prognostic role of serum sPD-L1 in head and neck cancers. Future research concerning comparison of sPD-L1 level with a tissue expression in this group is required for more comprehensive understanding and individualized treatment options.sPD-L1 is a promising predictive and prognostic biomarker for head and neck cancers, particularly for laryngeal lesions. Further research concerning this protein is required to fully understand its significance in HNCs.Conceptualization: AR, MMM, MŻ; methodology AR, MMM, MŻ; validation, AR, MMM, MŻ, IK; investigation MMM, AR, MŻ, IK; data curation MMM, MŻ, AR; writing-original draft preparation, MMM; writing-review and editing, AR, MŻ; visualization AR, MMM, MŻ; supervision, KN, UD; project administration AR, MMM, MŻ. All authors have read and agreed to the published version of the manuscript.The authors declare no conflicts of interest.