The model uses the following parameters: application surface area (SA), a fitted application time, and the fitted Higuchi release constant to characterize the rate of passage of the active pharmaceutical ingredient from the ointment formulations into the tears in vivo.Parameter sensitivity analysis was performed to understand the impact of ointment formulation changes on ocular exposure. While application time was found to have a significant impact on the time of maximal concentration in AH, both the application SA and the Higuchi release constant significantly influenced both the maximum concentration and the ocular exposure.This initial model for ointment ophthalmic formulations is a first step to better understand the interplay between physiological factors and ophthalmic formulation physicochemical properties and their impact on in vivo ocular drug pharmacokinetic performance in rabbits.The online version contains supplementary material available at 10.1007/s11095-020-02965-y. The United States (US) Food and Drug Administration (FDA) may recommend in vivo and/or in vitro testing to establish the bioequivalence (BE) of ocular drug products with complex dosage forms. The selection of the method depends upon the most current understanding of FDA in relation to the information collected by the study, the analytical methods available, and the nature of the drug product. In general, BE testing should use the most accurate, sensitive, and reproducible approach that is able to capture differences between the test and reference products in terms of drug concentrations at the site of action . DiffereOintment formulations represents approximately 8% of the ophthalmic products in the US . BecausePhysiologically based pharmacokinetic models (PBPK) were first introduced in the 1970s to support drug development from preclinical to clinical trials as they can reduce the cost and attrition rate in drug development . PBPK moThe first physiologically based model describing ocular concentration-time profiles for different tissues in rabbits was developed in the late 1970s ,17. PBPKThe purpose of this article is to demonstrate the application of ocular PBPK and mechanistic absorption model to predict the impact of ophthalmic ointment characteristics on drug disposition in rabbit eyes using Dexamethasone (Dex) and Fluorometholone (Flm) as the model drugs.This study includes: 1) the verification of the developed mechanistic ocular absorption model (OCAT™) in rabbits using concentration-time profiles of Flm in different ocular tissues and plasma for multiple formulations with different product characteristics; and 2) the investigation of the impact of dose and administered volume on drug disposition in rabbit eyes for ointment formulations.®  was used for computer simulation of Flm and Dex biodistribution in rabbit ocular tissues. The Ocular Compartmental Absorption & Transit (OCAT™) model describing ocular drug absorption and disposition details is presented in supplementary materials GastroPlus® to describe the observed data in rabbit for both case studies. The ointment formulation is treated as a controlled release formulation with the release of API described by the Higuchi equation ::® to des®.Therefore, three parameters influence the behavior of this ointment formulation: the application SA, the Higuchi rate constant, and the application time. These parameters must be defined by the user in GastroPlusModel structure integrates an OCAT™ model  the administered dose is lost from the eye by overflow based on the maximum pre-corneal volume and the administered dose volume; 2) the particles of Flm suspension do not trigger excessive lacrimation; and 3) the drug is released from the ointment formulation into the tears where it can either be absorbed within both cornea and conjunctiva or be washed out the surface of the eye through drainage.®. Two verification steps were performed using published AH concentration time course for suspensions, with differences in dose and particle size (PS): three suspensions  with a mean PS of 2 μm for verification #1; and two suspensions (0.1% and 0.4%) with a mean PS of 10.4 μm and 6 μm, respectively, for verification #2  to rabbit . In vitration #2 ,34. For articles . Once thAn ocular model of Dex was previously developed and verified . For thi®.The impact of application time, application SA, and Higuchi rate constant on Flm AH concentration time course in rabbit were assessed using parameter sensitivity analysis (PSA) in GastroPlusThe initial model was developed against cornea and AH concentration time course data following the administration of 50 μL of a 4E-5 M Flm solution to rabbit . Figure ® so that the small amount of dissolved Flm (0.000775 mg) and the remaining mass of suspended particles could be administered at the same time. For these suspensions, the reported mean PS was 2 μm . These suspensions were modeled using the mixed multiple dose feature of GastroPluswas 2 μm . Figure For the second verification step, AH concentration-time courses following the administration of two suspensions of Flm with different doses administered (0.1 and 0.4%) and particle size  were extracted from literature . Figure 1/2 cm2).AH concentration-time courses following the administration of two similar 0.1% ointments (25 and 50 μL corresponding in two doses: 25 and 50 μg) were extracted from literature . ApplicaAs the Higuchi release constant is related to the ointment structure, the same value was used for the 25 μg dose. The volume administered was expected to have an impact on the overall application time; therefore, this parameter was adjusted for the 25 μg dose. Figure Based on this case study, it seems that the developed ointment model described the observed AH data for Flm ointment formulations. Although the information relative to ointment composition are not available, because data following the administration of two doses of the same ointment are published, the following observations can be made. The Higuchi release constant was linked to the ointment formulation and did not vary with the administered dose/volume. However, the application time was dependent on the total volume administered, and it decreased as the volume decreased from 50 to 25 μL. Due to blinking, the administered SA was assumed to be the entire cornea surface.1/2 cm2) allowed us to best describe the observed data  were extracted from literature . Applicaata Fig. . Therefoata Fig. , can be Using the verified model for Flm ointment in rabbit, a PSA was performed for the three main parameters influencing AH PK dynamic for ointment: application SA, Higuchi rate constant, and application time. Figure The understanding of ocular absorption mechanism is necessary for both the pharmaceutical industries and the regulatory agencies to support the development and the evaluation of new and generic drug products. Previous work presented the development and validation of an OCAT™-PBPK model by investigating the impact of physiochemical properties of ophthalmic suspensions on in vivo ocular drug absorption and disposition . This stOintments are an important type of semisolid dosage form for the ophthalmic delivery of APIs. These formulations vary in type depending on the choice of bases: oleaginous (hydrocarbon) bases, absorption bases, water-soluble bases, emulsifying bases, etc. APIs may be present inside the ointment in different states: either as a dispersion or as a molecular solution. It is expected, depending on the choice of ointment bases and state of the drug, its release from ointment may vary in both in vitro and in vivo conditions . Ophthalet al. provides an simple equation to estimate the Higuchi constant (SQRT(2*Cini*D*Cs)) where Cini is the initial concentration of the API in the ointment base, D is the reduced diffusion coefficient of the API in the ointment base, and Cs is the solubility of the API in the ointment base . However, that value is ~6.5 times greater than the fitted kHiguchi (2.7E-6 mg/(s1/2 cm2)). But the diffusion coefficient estimated in literature are for liquid compounds and it is known that Flm is present in solid state within the ointment base. This could decrease Flm diffusion coefficient. Also, it is expected the APIs would diffuse more freely in an anhydrous lanolin base as described by Wurster et al.,versus a petroleum base typically used in ointment formulations. Reducing the diffusion by two orders of magnitude helps to reconcile the calculated and fitted kHiguchi. To test the diffusion coefficient parameter that is extracted from literature, it was used for acyclovir ointment. The calculated kHiguchi for a 6% dose  is around 200 greater than the measured value in vitro . PSA analysis demonstrated the Higuchi rate constant is the major parameter influencing the ocular exposure for APIs administered as ointments. However, multiple release models have been identified in vitro for these formulations. The two main ones presented are the transient-boundary layer and Higuchi models ,11,12. Tent base . Wursterlin base . The difin vitro  are not The PSA for application/absorption SA demonstrates a significant impact on absorption rate in the AH. Although the OCAT™ model captured the AH concentration time course for Flm, it seems the absorption phase is slightly overpredicted compared to the observed data for both administrations Fig.  may provversus a suspension. The ointment will influence the residence time as the ointment base present at the surface of the eye can act as a reservoir for drug release in ocular tissues. Nevertheless, despite the improved exposure, most of the drug does not permeate in the ocular tissues and is eliminated from the surface of the eye, such as for suspensions and solutions. This elimination mechanism has not been clearly identified and may be linked to dynamic forces mediated by both the blinking process and the tears drainage. In the OCAT™ model, application time defines a very slow zero-order elimination driving the transfer of the unreleased fraction of the API through the nasolacrimal duct directly into the stomach. This parameter was necessary to capture the elimination part of the observed AH PK profiles that seems to happen in two phases. In the first phase the elimination is balanced by the presence of API in the ointment acting as a reservoir. Then in the second phase, once all the material had been removed from the ocular surface, the elimination rate is similar to the one simulated for solution and suspension. This seems to be confirmed by the Flm case study. Indeed, the 0.1% formulation was administered with two different volumes: 25 and 50 μL. To capture the AH PK profiles, the application time for the lowest volume is significantly different than the one obtained for the higher volume, demonstrating a clear impact of the administered volume on the residence time of the formulation on the ocular surface and therefore, the ocular exposure. As the elimination mechanism of the ointment base and the entrapped material is better understood, this process may need to be revised in future iterations of the OCAT™ model. However, it is not a limiting factor to capture the observed concentration, as well as investigating formulation changes impact on ocular exposure.For Flm and Dex, ophthalmic ointments result in a longer ocular exposure in AH compared to solution and suspensions. Indeed, for Flm, the AH AUC is increased by 100% when the drug is administered at the same dose but using an ointment vehicle In conclusion, we have successfully developed and verified an OCAT™ model for APIs administered as ointment formulations. We have been able to describe the AH concentration course for both Flm and Dex ocular ointments in rabbits. Based on our model simulations, the critical parameters influencing ocular PK metrics are application time, administration SA, and API release rate. Ideally, the Higuchi rate constant could be further broken down into various formulation factors, measurable in vitro, such as PS distribution, ointment base type and rheological characteristics. So far, the parameters included in the model should capture most of the formulation variants . This tool may support drug development and provide a better understanding of the impact of formulation modifications on the in vivo performance of ophthalmic ointments products. A deeper understanding of key physiological mechanisms influencing PK outcomes as well as the extrapolation from rabbit to human model are the next steps planned for this OCAT™ ointment model.ESM 1(DOCX 84 kb)


In order to investigate the effect of fluorination of graphene nanoflake on the dispersibility in polypropylene (PP) composites, fluorinated graphene oxide (FGO) was prepared by solvo-thermal reaction and applied as a filler of the PP nanocomposite. Due to the weakened inter-particle attraction among the graphene nanoflake and reduced surface energy difference between PP and the filler, PP/FGO composites showed better exfoliation and dispersion state of the filler compared with that of PP/graphene oxide (GO) or PP/reduced graphene oxide (RGO) composites. The improved exfoliation and dispersion of graphene nanoflake resulted in a significant reinforcement on the composites. The Young’s modulus and tensile strength of PP composites filled with 2 wt% of FGO increased by 31% and 15%, respectively, compared with those of PP. Polypropylene (PP) is one of the most abundant polyolefin and has a wide range of applications due to its low cost, good processability and recyclability. To satisfy the requirements for engineering applications, there is the need to enhance the mechanical properties of PP . PolymerThe dispersion of nanofillers is decisive in the mechanical properties of the polymer nanocomposites because the aggregated fillers will act as a large particle rather than nanoscale fillers . Due to Another approach to improve the dispersion of graphene nanoflake in PP matrix is lowering the surface energy of the nanofiller. The van der Waals interactions among nanofillers are so strong that they tend to agglomerate in polymer matrix owing to the large surface area per unit mass. Modifying the surface of a material with fluorine can reduce its surface energy and weaken its interaction with neighboring substances. If the inter-particle attraction of nanofillers is weakened, it is easier to separate the nanofillers in the mixing process . An examIn this study, we prepared PP nanocomposites filled with fluorinated graphene oxide (FGO) by melt blending. FGO was synthesized by the solvo-thermal fluorination of graphene oxide (GO) with hydrofluoric acid (HF) as a fluorine agent . For com2O) was obtained from Sigma-Aldrich . N-methyl-2-pyrrolidone  was purchased from Daejung Chemicals .PP was commercial product of Lotte Chemical with the trade name of SJ-160. GO was purchased from LS-Chem  with the trade name of GE-3550. Hydrofluoric acid  was added to 300 mL of NMP and sonicated at 40 °C for 4 h. HF (20 mL) was added to the GO dispersion and the mixture was maintained at 180 °C for 6 h, 12 h and 24 h in the Teflon-lined autoclave. The autoclave was cooled to room temperature. FGO dispersion was centrifuged at 10,000 rpm for 1 h and washed with ethanol. The final product was obtained after drying in a vacuum oven at 40 °C for 24 h. The graphene nanoflakes were designated FGO_6, FGO_12 and FGO_24, according to their reaction time. For comparison, RGO was also prepared by the solvo-thermal reduction. GO (1.0 g) was added to 300 mL of NMP and sonicated at 40 °C for 4 h. GO dispersion was maintained at 200 °C for 24 h and centrifuged at 10,000 rpm for 1h. RGO was obtained after washing with ethanol and drying in a vacuum oven at 40 °C for 24 h.The PP/graphene nanoflake composites were fabricated by melt blending using an internal mixer  with the rotor speed of 100 rpm at 200 °C for 10 min. After the melt blending, the composites were pelletized and dried at 40 °C. The amount of GO, RGO or FGOs added was 0, 0.5, 1.0 and 2.0 wt%.The thickness of RGO and FGO_12 was identified by using an atomic force microscopy  with tapping mode. The chemical composition was measured with an X-ray Photoelectron Spectroscopy  using monochromatic Al Kα radiation as the exciting source. Chemical structure of each filler was analyzed by a Fourier transform infrared spectroscopy  in attenuated total reflection mode. The wettability of surfaces of the graphene nanoflake films was measured using the water contact angle measurement system . Graphene nanoflake films were prepared by a filtering method . Graphen3. Scan data were measured with X-ray tube settings of 23 kV and 116 μA and the pixel size of 1.36 μm. The micro-CT image of the composites was obtained by reconstructing the projections.The fracture surface microstructure of the composites was examined with a field emission scanning electron microscope . The specimens were fractured under cryogenic conditions using liquid nitrogen. The dispersion state of the GNP in the composites was observed with a three dimensional non-destructive X-ray micro-computed tomography . The composite specimens were prepared by cutting to ca. 1.0 × 1.0 × 0.5 mm3 in dimension. The thermal stability of the composites was examined by thermo-gravimetric analysis  at temperatures ranging from 100 to 600 °C at a heating rate of 10 °C/min under a nitrogen atmosphere.The test specimens for measuring tensile properties were prepared using a micro injector machine . The cylinder and mold temperatures were set at 200 °C and 25 °C, respectively. The tensile properties of the composites were determined using a universal testing machine  at 25 °C and at the crosshead speed of 10 mm/min according to ASTM D-638 Type V method, which is for the specimen of 63.0 × 3.2 × 3.1 mm2)/C-C(sp3) at 284.8 eV, C-OH (hydroxyl) at 286.4 eV, C-O-C (epoxy) at 287.1 eV and C=O (carbonyl and carboxylic) at 288.8 eV [2 and CF2 at 285.8, 288.2, 289.9 and 292.1, respectively [GO, RGO, FGO_6, FGO_12 and FGO_24 were analyzed by XPS to determine the elemental composition and characteristics of the chemical bonds. The quantified atomic percentages of carbon, oxygen and fluorine are presented in ectively ,26. Thes−1 and 1625 cm−1, respectively [−1 and stretching peaks from C-O bonds of carboxyl, epoxy and hydroxyl groups were observed at 1380, 1200 and 1040 cm−1, respectively [−1. The same result was observed in other fluorinated graphene nanoflake studies because the C-F bonds vary from covalent bonds, through semi-ionic bonds, to ionic bonds. This is due to the large electronegativity difference between carbon and fluorine atoms [Fourier transform infrared spectroscopy (FT-IR) was used to further investigate the effect of reduction and fluorination and the results are shown in ectively . A peak ectively . In the ne atoms .The thickness of RGO and FGO_12 was identified by AFM tapping mode . Both th2, respectively. The similar result that C-F bonds reduced the surface energy of graphene nanoflake was reported [2, the surface energy of PP.Optical images of the probe droplets of PP, GO, RGO, FGO_6 and FGO_12 observed with the water contact angle measurement were shown in reported . FGO_12 The fracture surfaces of PP/GO, PP/RGO, PP/FGO_6 and PP/FGO_12 composites were examined. The content of graphene nanoflakes in the composite specimens was 2 wt%. SEM images of 5%) of PP, PP/GO, PP/RGO and PP/FGO_12 were 347, 383, 382 and 385 °C, respectively. The PP composites exhibited improved thermal stability at the initial stage of degradation compared with PP because the fillers act as heat sink that prevents heat from accumulating in PP. The temperatures at which the thermal degradation ends and there is no temperature change (Tend) of PP, PP/GO, PP/RGO and PP/FGO_12 were 427, 463, 468 and 484 °C, respectively. The difference in Tend between PP/FGO and PP/RGO was larger than the difference of T5% between PP/FGO and PP/RGO, which means the enhancement of the resistance to thermal degradation. This is because graphene nanoflakes also serve as transfer barrier and FGO with the improved dispersion more effectively hindered the volatile decomposed products [The thermal stability of PP and the composites filled with 2 wt% of graphene nanoflakes was examined by determining the weight loss during heating. Due to the high surface energy difference between PP and fillers, PP/GO and PP/RGO composites showed the poor dispersibility of graphene nanoflake. Thus, the composites showed little improvement in mechanical properties despite the increase of the filler content and a sharp drop in elongation at break. In order to reduce the surface energy of graphene nanoflake, fluorinated graphene nanoflake was prepared by solvo-thermal reaction between GO and HF. Through the observation of the morphology by SEM and 3D-micro CT, the improved exfoliation and dispersion of the filler were confirmed in the PP matrix. Since exfoliated graphene nanoflake had high aspect ratios, achieving effective stress transfer in polymer composites, PP/FGO composites exhibited better mechanical properties compared with those of PP/GO or PP/RGO composites. It was certain that the weakened inter-particle interaction of graphene nanoflake and reduced surface energy difference with PP achieved the improved dispersibility of the nanofiller and enhanced mechanical properties of the composites.


Efficient excitation of surface wave (SW) remains one of the most challenging considerations in the photonics and plasmonics areas. Inspired by recent investigations of metasurfaces, we propose a hybrid metal-graphene transmitarray converting incident propagating wave (PW) to SW, as a solution for SW excitations–a meta-coupler. The structure comprises ultra-thin four-layer transparent metasurfaces in which H-shaped etched metal films together with graphene patches are employed, and also all four layers are identical. Full-wave simulations demonstrate that the suggested meta-coupler possesses an efficiency of 46% and a directivity of 19 dB, which is promising in the terahertz (THz) range. At the same time, in light of unique graphene characteristics, the proposed device is tunable and easily reconfigurable, i.e., the direction of converted SWs can be electrically switched from right to left and vice versa. We believe that this system responds to emerging applications such as THz communications and sensing, and furthermore the employed architecture introduce electrostatically tunable building blocks being able to develop graphene plasmonic components effectively. Two extraordinary and unique features of SPPs, subwavelength confinement and local field enhancement, have opened up next-generation applications, such as super-resolution imaging, biosensing, and integrated plasmonic circuits5. Although the behavior of metals does not lead to those extraordinary properties by themselves at lower frequencies (from GHz to mid-infrared range), some certain structured metals may provide them in light of spoof SPPs2. Therefore, same applications can be feasible at lower frequencies. As concerns SW generation developments, both efficient and unidirectional SW excitation is demanding and indeed challenging7, which originates from an incident propagating wave (PW). The main reason for this is the momentum mismatch between incident PWs and SW modes. Prism and grating couplers have conventionally resolved this issue forbidding direct coupling between PWs and SWs. Challenges in achieving an efficient and unidirectional coupling, nevertheless, have remained in these schemes. To the best of recent researches7, metasurface-based SW couplers, i.e., meta-couplers with their novel functionality would overcome these challenges in PW-SW conversion8.Excitation and manipulation of surface waves (SWs), including surface plasmon polaritons (SPPs) and spoof SPPs which are the low-frequency counterparts of SPPs, have attracted ample interests in photonics and Terahertz (THz) scopes11. Metasurface elements are usually subwavelength resonators that enable control over EM waves behavior through an specific boundary. These elements, so-called meta-atoms, can locally manipulate phase, amplitude, and polarization. Graphene, a monolayer of carbon atoms arranged in a honeycomb lattice12, has been incorporated recently into metasurfaces15 to make them more promising, operative and applicable in different scopes due to its fascinating optoelectronic properties, e.g., tunability and ability to support plasmons from mid-infrared to THz frequencies16. Consequently, tunable and reconfigurable devices and components with diverse capabilities can be realized22, one category of which is meta-couplers23. One class of graphene-based devices is featured by the metal-graphene hybridization where the EM coupling between metal structures and graphene is tailored24. This configuration provides various applications, where light-matter interactions can be controlled, e.g., ultrafast and spatial light modulation, electromagnetically induced transparency, and beam steering for THz and mid-infrared ranges28.To now, researches on metasurfaces, planar artificial surfaces modulating electromagnetic (EM) boundary conditions, have been growing owing to their promising functionalitiesf  = 3 THz), which comprises four layers of an ultra-thin metasurface. Not only does our meta-coupler overwhelm the issues related to both efficiency and unidirectionality, but it also is reconfigurable, i.e., we can select the direction of the coupled SW. The reconfigurability–and tunability–of the meta-coupler is achieved by virtue of graphene patches participation in our design process. In this regard, our device can switch between two states corresponding two opposite directions for the SW propagations along the target waveguide. In our system, a dielectric-coated metal sheet–grounded dielectric slab– has been chosen as the target waveguide supporting SWs, and geometrically identical meta-atoms have constituted the meta-coupler. Based on the full-wave simulations, the proposed device behaves satisfactorily in terms of coupling efficiency (46%) as well as directivity (19 dB).In this work, we propose a novel system for realizing a meta-coupler at THz range  as much as possible. Transparent windows and metasurfaces would be an unquestionable choice, which leads us to this goal39, and, on the other hand, the graphene shows plasmonic effects. In this way, the hybrid metal-graphene configurations in which the boundaries of metal and graphene touch each other may be exploited to develop emergent THz components, e.g., meta-couplers. Concerning our proposed unit cell structure, the electromagnetic interactions between the graphene patch and the H-shaped etched metal films play the main role in the transmission response. Consequently, the transmission response of the proposed single-layer unit cell can be adjusted by tuning the Fermi level of graphene patches, and this outcome can be seen from Fig. At low THz frequencies, the conductivity of metals is high enough to approximately model them as PECsz-axis direction. Figure d = 17 μm for the distance between every two adjacent layers as in our design procedure.Now, the single-layer graphene-based metasurface upon which we compose the transmitarray is specified. We extract the shunt admittance of this single-layer metasurface from full-wave simulations, and then make use of it in our design which is based on the circuit theory. Using transmission line models and that obtained admittance, we developed a four-layer metasurface whose layers are equally spaced with respect to the normal direction–As plotted in Fig. Additionally, we plot the amplitude and phase of the transmission response as a function of frequency. As manifest in Fig. 40, the dielectric is made of SiO2, the thickness of which is It is time to comprise our meta-coupler based on those picked Fermi level values according to their responses explained previously. The supercell of our geometrically uniform configuration comprises five meta-atoms for which the phase change step is chosen Throughout our device development, we keep the geometric features of our meta-coupler elements identical through the whole structure to, firstly, have a more straightforward design procedure, and secondly, make our device easily reconfigurable by the Fermi level of graphene patches, although this consideration limits our choices. Hence, the meta-coupler can provide either positive or negative phase gradient, and finally, we would be able to control the guiding direction of the coupled SW, which may be either in the t, for the unit cell of the biasing structure where polysilicon pads are considered is almost the same as the results in Fig. In this regard, an architecture is developed to control the Fermi level of graphene patches electrically. The employed architecture is shown in Fig. As the final step in our design procedure, we find the optimum gap distance Designing and analyzing transmitarray, we compose the whole configuration where the meta-coupler performance investigated by means of full-wave simulations. Regarding that the meta-coupler in states I and II are the mirrored version of each other, it is required to investigate one of them, and thus the other state has the same performance. In this regard, we consider the meta-coupler operating in state I, whose 3D schematic as well as functionality were illustrated in Fig. see Fig. , whose dx-polarized Gaussian beam with beam waist y-component of the magnetic field pattern obtained by full-wave simulation at a working frequency of 3 THz is depicted in Fig. The proposed structure is normally illuminated by an Figure 45. Nevertheless, higher coupling efficiency have been reported in microwave or optical regimes46. But, it should be noted that our THz device is reconfigurable and tunable and it has reasonably high efficiency.As an endpoint in the investigation of our meta-coupler performance, the achieved efficiency is larger than many recently proposed PW-SW converters or grating couplersIn summary, as a starting point, we studied a hybrid graphene-metal metasurface as transparent windows upon which an efficient and reconfigurable meta-coupler is achievable. A transmitarray was developed by cascading four identical layers of those transparent metasurfaces in light of circuit theory to cover ∼360° transmission phase with an adequate amplitude. As a consequence, we designed a meta-coupler which solves PW-SW conversion issues, unwanted reflection and decoupling effects. The meta-coupler’s meta-atoms having electrical tunability granted by graphene patches in its structure is the cornerstone of our arrangement, where the intra-coupling between the graphene and the H-shaped etched metal film play the main role. Our proposed meta-coupler would convert the normally illuminated PW to the SW in the desired direction with efficiency of 46% and directivity of 19 dB just by electrostatic biasing of five graphene patches in each supercell. The versatility of the hybrid graphene-metal unit cell reveals a broad range of emerging applications in tunable, real-time, and integrated THz/optoelectronic systems such as more functional meta-couplers enabling polarization and wavelength splitting/controlling or even modulation (by nonlinear optics in the visible range).x- and y- directions and Floquet ports in the z-direction. The unit cells transmission responses are obtained when these structures are normally illuminated by an x-polarized plane wave.The proposed configurations are numerically calculated using the commercial package CST Microwave Studio. For unit cell structures simulations, to consider the inter-element coupling, we apply the unit cell boundaries as periodic boundary conditions along the 47:S] is the scattering matrix, [U] is the unit, or identity, matrix, and η0 is the intrinsic impedance of vacuum. Considering the admittance matrix for the single-layer metasurface, then, we are able to calculate the transmission coefficient of the transmitarray unit cell in light of transmission line theory, whose circuit model was depicted in Fig. For the multi-layer unit cell analysis based on circuit theory, we firstly obtain the admittance matrix of the single-layer metasurface from the scattering parameters which are extracted based on full-wave simulations, asx-direction are terminated using ports, and moreover, the periodic boundary condition as well as perfectly matched layers (PMLs), are applied in the As to the full structure simulation, in order to terminate the whole domain, the begining and the end of the target waveguide in the 48.The dielectric substrate used in our structures is made of 39.The carved thin metal films placed on each layer of the meta-coupler are modeled as (2D) PEC boundaries since metals, e.g., silver, nearly behave as PECs in the low THz region49, the conductivity is given by:The graphene is modeled as a conductive sheet with surface conductivity


Seasonality of respiratory diseases has been linked, among other factors, to low outdoor absolute humidity and low relative humidity in indoor environments, which increase evaporation of water in the mucosal layer lining the respiratory tract. We demonstrate that normal breathing results in an absorption-desorption cycle inside facemasks, where super-saturated air is absorbed by the mask fibers during expiration, followed by evaporation during inspiration of dry environmental air. For double-layered cotton masks, which have considerable heat capacity, the temperature of inspired air rises above room temperature, and the effective increase in relative humidity can exceed 100%. We propose that the recently reported, disease-attenuating effect of generic facemasks is dominated by the strong humidity increase of inspired air. Respiratory viral infections are perhaps the most common type of illness. They range from influenza, colds, and measles to, most recently, COVID-19. The common cold encompasses more than 200 different viruses [The seasonality of COVID-19  is now iOther factors that relate seasonality to disease concern how the host reacts to the viral infection. Iwasaki and co-workers demonstrated in mice that low humidity increases disease severity following a respiratory challenge with the influenza A virus . This efBreath droplet formation results from transient occlusion of the small airways that can occur upon expiration , 22. Subd ~0.7 μM) appears too low for this inhibition to be effective. Instead, we propose its benefits may derive from its ability to enhance mucociliary clearance through hydrolytic depolymerization of mucus protein fibers as well as increased hydration of the epithelial surface, which both relate to its use in general clinical practice as a secretolytic expectorant. Interestingly, the anti-asthma drug budesonide, used as an inhaled rather than a systemic corticosteroid [Bromhexine, an over-the-counter medication, has shown remarkably positive results for limiting disease severity in recent clinical trials , 24. Altosteroid , also isosteroid ). AlthouAlthough much debate remains about the relative importance of the various season-related factors on the transmissibility and severity of the COVID-19 disease, the correlation between increased disease severity and low humidity of inhaled air appears strong. Equally striking are recent reports that link the use of face coverings to reduced disease severity in wearers of generic face masks , 28, butWe propose that the attenuating effect of face masks on coronavirus-2019 (COVID-19) disease severity , 28 is d2 levels were mounted close to the geometric center of the box. A 10-cm fan (flow rate 100 L/min), positioned at the bottom of the box and pointed upward at a 45° angle, served to keep the contents of the box homogeneous and to maximize airflow over the three types of sensors. The response to a step change in humidity was found to depend on temperature , defined by exponential time constants of 15, 5, and 2.8 s at 8, 22, and 37 °C, respectively. At the top rear end of the box, an inflatable polyethylene bag was mounted to accommodate the changes in total gas volume at ambient pressure upon breathing. This bag also served as a visual gage to control the volume of the exhaled air.Measurements were made by breathing into a sealed steel box with dimensions 38.74 × 38.74 × 63.50 cm (width/height/depth), for a total volume of 95.3 L . The side walls of the chamber were factory-powder-coated, resulting in minimal moisture adherence. The rear wall, originally bare steel, was oil-base painted for minimal moisture adherence. The high thermal conductivity and heat capacity of the box reduced temperature increases upon expiration of warm breath into the chamber to less than 0.6 °C. The acrylonitrile-butadiene-styrene front panel contains a sealable opening, shaped to accommodate the chin, mouth, and nose of the breathing volunteer, with the edges of the opening lined with high density foam rubber to make a tight seal with the facial skin of the volunteer. The front panel also contains a second, sealable, 10 cm diameter hole for refreshing the contents of the box with environmental air by using a high capacity (300 L/min) fan mounted in front of this opening between measurements, with the breathing opening serving as the channel for air exhaust. Sensors to record temperature, humidity, and CO2 uptake when the CO2 level in the chamber increased to >6000 ppm during the breathing maneuver. The breathing was synchronized with an audible timer, and relative humidity and temperature data were monitored by video and manually entered into a computer for analysis. Under each condition, each measurement was repeated three times, with raw values reported in SI Table S1.All data reported here were recorded under conditions aimed to simulate tidal breathing at a rate of 10 breaths per minute, and a volume of 0.95 L per breath. Although this volume is 10–50% higher than often used for tidal breathing, it was adjusted for optimal comfort of the volunteer and accounts for the decreased OMeasurements were carried out in a laboratory cold room, at 7.7 ± 0.1 °C, with dry air (relative humidity (RH) 13 ± 0.5%) used to vent the box prior to measurements; indoors at room temperature ; and in a cell culture room at 37.4 ± 0.5 °C; RH 14.3 ± 0.3%. Four types of masks materials were evaluated: N95 ; standard, disposable 3-ply surgical (NIH stockroom); a two-ply cotton-polyester blend mask (NIH stockroom); and an online-purchased all-cotton mask . Results were compared to the humidification of the box interior obtained for the same breathing maneuver without a mask.mabs(H2O)mask, was calculated by subtracting the mass of water vapor added to the chamber when wearing the mask, Δmchamber(H2O)mask, from the mass added when not wearing the mask, Δmchamber(H2O)none:Relative humidity was measured using a Honeywell HIH-5030 humidity sensor powered by a regulated 5-V power supply. The measured voltages were converted to relative humidity using the formulas given in the sensor’s datasheet. Relative humidity  measurements were converted to absolute humidity  values according to:p is the volume of the chamber, q is the volume of exhaled breath, and AH(n) is the absolute humidity of the chamber after n breaths. Recursive application of n successive breaths yields:For mixing a single breath with the contents of the chamber, the absolute humidity after this breath is given by,p and q are known, the absolute humidity of exhaled breath is then obtained fromProvided The above equations assume the air is well mixed, aided by the fan mounted inside the chamber, by the time the air is inspired from the chamber. At 22 °C, the absolute humidity of exhaled breath obtained using mabs(H2O)mask, stored and released in the mask during n breathing cycles, corresponds toThe mass of water, q, and the number of breathing cycles, the increase in absolute humidity of inspired air over the average absolute humidity in the chamber is given by mabs(H2O)mask/(rq), where r is the number of breath cycles, and mabs(H2O)mask is converted to an increase in inspired RH by using Knowing the volume of each breath, The current measurements of the effect of masks on the humidity of inhaled air were motivated by observations made while testing various types of face coverings for blocking speech and breath particles, using laser light scattering observation , 34, 35.Measurements were carried out at three different temperatures, from 8 to 37 °C, which covers the most relevant range from a work and living environment perspective. The ability of various types of synthetic and natural fibers to absorb water depends strongly on temperature –38. NatuFor testing, the temperature and humidity of the mask were equilibrated on the face of the volunteer for at least 10 minutes prior to the start of the measurements. After a few cycles of optimizing and controlling the rate and quantity of breathing, measurements proved highly reproducible and self-consistent. In particular for the measurements at room temperature, where it was possible to keep room temperature and humidity within a very narrow range, the three repeats of each measurements were frequently within the digital readout of the humidity sensor, corresponding to 0.3% change in RH .ca 35% was observed for the surgical mask. Under the same conditions, both the N95 mask and the polyester-cotton mask increased the humidity of inspired air by 55–60% (ca 30%) in the chamber was about 90%. At first sight, this apparent increase of inspired air humidity to above 100% may defy intuition. However, the explanation is grounded in simple physics. The exhalation of breath warms up the mask (and the water stored in it) to well above the environmental temperature. Indeed, with the air temperature at 8 °C, rapidly removing the mask after exhalation during tidal breathing, folding it double while clamping a thermocouple sensor near its center-fold, followed by rolling up the doubled mask, consistently yielded temperature readings of 30±1 °C. The same measurement after inspiration yielded 27±1 °C. Presumably, this 30±1 °C temperature represents a lower limit for the actual temperature at the face side of the mask at the start of inspiration, allowing the humidity of inspired air to reach values that are well above 100% at 8 °C. Even if only 20% of the mask surface was involved in the actual passage of air, this fraction of the mask corresponds to about 4 grams of cotton with a total heat capacity of 20 J/K, considerably higher than the heat capacity of ca 1.2 J/K for one liter of inspired air. Hence, the high heat capacity of the mask causes it to function like a rudimentary heat and humidity exchanger, warming and humidifying the inspired air to values much closer to those in the lower respiratory tract than of room air.All four different types of mask were found to strongly decrease the build-up of humidity in the chamber with breathing, but to different extents. At room temperature, the smallest increase in apparent RH of inspired air over the average RH of the chamber by y 55–60% . Surprisca 27 mg/cm2, the N95 mask increases the humidity of inspired air comparably to the ca two-fold heavier, polyester-cotton mask. It seems likely that the large surface area of the extremely fine fibers in N95 masks that carry out most of the actual filtering [The considerably lower increases seen for the lighter masks are consistent with this general concept, although the filtering material itself also appears to play a significant role. For example, despite its low mass of iltering , togetheAt low temperature (8 °C), the humidifying effect of all masks strongly increases. It is important to note, however, that these very large RH increases at 8 °C (150–300%) correspond to less dramatic increases in absolute humidity of 12–24 mg/L. Once heated inside the respiratory tract, this corresponds to changes in relative humidity at 36 °C of 29–58%. By contrast, even while the RH changes of inspired air are much smaller for the measurements at 37 °C, this increase directly translates into the relative humidity change of air inspired into the lungs. As a consequence, from the perspective of respiratory tract dehydration, the effect of each mask type is fairly constant across the entire 8–37 °C range. At all temperatures, the humidification resulting from the heavy cotton mask is about double that of a surgical mask, with the N95 and cotton-polyester cloth masks falling in between.Variola) prior to the availability of a vaccine [variolation, where the virus was applied to a small scratch on the skin, inhaled airborne SARS-CoV-2 virions will be distributed randomly across the epithelial surface of the respiratory tract. This makes any requirement for overload of the innate immune system, a concept that applies locally, highly unlikely.Over the past year, it has become abundantly clear that facemasks offer an effective tool for containing the spread of the COVID-19 pandemic. Remarkably, mask usage correlated not only a protective effect in terms of disease transmission but also to a striking decrease in disease severity, the latter effect attributed to the infected person receiving a lower dose of the disease-causing virus . The eff vaccine . Althoug vaccine , we also vaccine , the dos vaccine  or Indep vaccine , meaning vaccine . Even wh vaccine . In contincreased disease severity relative to infection of the upper respiratory tract [There is a second problem with the proposal that reduced COVID-19 disease severity of mask wearers results from lower exposure. It is well recognized that only particles smaller than a few microns can enter the small airways and cause infection of the lungs, commonly associated with ry tract , 43, 44.Here, we propose that the increased humidity of air inspired through face masks is responsible for the lower disease severity of mask wearers , 28. In The increased humidity of inspired air associated with wearing a face cover is perhaps well recognized by the public and contributes to the general feeling of mugginess, in particular when the weather is humid. Our measurements confirm that the increased humidity of inspired air is real and quite large.It is important to note that our measurements were carried out in the absence of air leakage around the mask edges. For non-N95 masks, such leakage is often significant and will proportionately lower the effective humidity increase of inspired air. So, in practice the cotton mask humidification efficiency will drop somewhat, to values comparable to the tight fitting N95 mask. The surgical and polyester-cotton blend masks, which also will be subject to leakage, then are expected to perform somewhat lower than the N95 and all-cotton masks.


Detection of both inducible and stable DNA methylation indicates two different epigenetic modes of salinity adaptation. Gasterosteus aculeatus) along a salinity cline. Wild populations differing in salinity tolerance revealed differential methylation (pop-DMS) at genes enriched for osmoregulatory processes. A two-generation experiment demonstrated that 62% of these pop-DMS were noninducible by salinity manipulation, suggesting that they are the result of either direct selection or associated genomic divergence at cis- or trans-regulatory sites. Two-thirds of the remaining inducible pop-DMS increased in similarity to patterns detected in wild populations from corresponding salinities. The level of similarity accentuated over consecutive generations, indicating a mechanism of transgenerational plasticity. While we can attribute natural DNA methylation patterns to the two information channels, their interplay with genomic variation in salinity adaptation is still unresolved.Epigenetic inheritance has been proposed to contribute to adaptation and acclimation via two information channels: (i) inducible epigenetic marks that enable transgenerational plasticity and (ii) noninducible epigenetic marks resulting from random epimutations shaped by selection. We studied both postulated channels by sequencing methylomes and genomes of Baltic three-spined sticklebacks ( Recent advances in epigenetics challenge our understanding of inheritance and adaptive evolution  from three populations that are locally adapted to different salinities  ] and whole genomes of three-spined sticklebacks  Stickleback populations from different salinities  show differentially methylated CpG sites (hereafter referred to as pop-DMS). (ii) Such pop-DMS include both types of methylation sites: experimentally stable sites  and experimentally inducible sites . (iii) Upon transgenerational salinity acclimation, inducible DNA methylations become more similar to the patterns of natural populations at corresponding salinities. When associated with beneficial phenotypic effects and increased relative fitness, the latter would be evidence for a mechanism underlying adaptive transgenerational plasticity , corresponding to ~4% of all CpG sites in the stickleback genome. Among pairs of wild-caught populations, we detected 1470 (comparison of 20 versus 6 PSU) and 1158 (20 versus 33 PSU) pop-DMS. The distribution of these sites was random with regard to the genomic features  and chromosomal regions (fig. S1A). Among these pop-DMS, 1098 (20 versus 6 PSU) and 871 (20 versus 33 PSU) were located close to [<10 kb from transcription start sites (TSS)] or within genes and thereby associated with 655 and 510 genes, respectively. Many of these genes are involved in fundamental biological processes such as DNA repair and strand renaturation, as well as chromosome condensation and separation (fig. S2). Of particular relevance is the enrichment in genes associated with osmoregulatory processes such as ion transport and channel activity, renal water homeostasis and absorption, and urine volume regulation , 33 PSU; Kiel (KIE), 20 PSU; Nynäshamn (NYN), 6 PSU; gulation . Genes aq ≥ 0.0125). On the other hand, if a pop-DMS was differentially methylated between at least one of the acclimation groups  compared to the control group , then this site was considered inducible. Pop-DMS with a significant q value not exceeding the threshold of differential DNA methylation were treated as a separate category (hereafter referred to as inconclusive). After two generations of salinity acclimation, we found that most of the pop-DMS remained stable, regardless of the direction of salinity change . A smaller number of pop-DMS (13%) were inducible, as they showed a significant change in CpG methylation upon experimental salinity decrease (198 pop-DMS) or increase (148 pop-DMS). An additional 24 and 27%  were inconclusive. The number of inducible pop-DMS (13%) derived from comparisons between natural populations was much higher than expected from a random subset of CpG sites across the genome . This means that pop-DMS are enriched for sites that plastically respond to salinity change, which is expected for populations from different salinities.To assess the proportion of inducible DNA methylation, we conducted a two-generation salinity acclimation experiment with laboratory-bred sticklebacks from the mid-salinity population that was subjected to either increased or decreased salinity . We consGene functions associated with stable pop-DMS  were enriched not only for a number of fundamental biological processes such as DNA repair and chromosome separation (fig. S2) but also for osmoregulatory functions  and 148  inducible pop-DMS, 130 (66%) and 101 (68%), respectively, became more similar to methylation levels of wild population to the corresponding salinity (hereafter referred to as “expected” direction). Conversely, at 68  and 47  inducible pop-DMS, experimental fish showed methylation changes in the opposite direction, reducing the similarity to methylation levels observed in the natural populations (hereafter referred to as “opposite” direction).FST value (±5-kb window) between populations. In line with our hypothesis, the populations from KIE (20 PSU) and NYN (6 PSU) were genetically more differentiated at opposite inducible pop-DMS than at expected sites , a well-described gene involved in lateral plate formation  can be identified in natural populations, focusing on salinity adaptation among populations of three-spined sticklebacks. Consistent with expectations for selection-based DNA methylation sites . This analysis revealed a negative correlation between population genetic differentiation and the propensity of the experimental population to approach the methylation level of the low salinity population (NYN) under salinity decrease (FST) and methylation direction was inconclusive, suggesting that a combination of environmental cues shaped DNA methylation levels among wild populations at these sites. Overall, these findings emphasize the importance of the genomic background for interpreting DNA methylation patterns.The genetic background is considered to be an important source for epigenomic variation via cis- and trans-regulatory mechanisms . All catches were performed under legal authorization issued by the German Ministry of Energy Transition, Agriculture, Environment, Nature and Digitalization in Schleswig-Holstein (MELUR: V242-7224.121-19), the Danish Ministry of Food, Agriculture and Fisheries of Denmark (case no: 14-7410-000227), the Estonian Ministry of the Environment (Keskkonnaministeerium - eripüügiluba nr 28/2014), and the Swedish Sea and Water Authority (Havs och Vattenmyndigheten). Ethical permission for the experiments required by German law was given by the MELUR: V312-7224.121-19, and the study is also in line with the Institutional Animal Care and Use Committee guidelines.G. aculeatus; 31.68 ± 14.25 mm) from three different salinity regimes inside and outside the Baltic Sea  in September 2014. Fish were immediately euthanized using tricaine methane sulfonate solution (MS222), photographed, measured , and stored in RNAlater solution . A cut along the ventral side ensured that the RNAlater solution diffused into all tissues. Conserved specimens were later dissected in the laboratory, and gill tissue was separated as the main osmoregulatory organ in fishes. For the acclimation experiment, we collected adult fish from KIE (20 PSU), which were crossed in our facilities at GEOMAR to obtain 10 F1 laboratory-bred families, herein referred to as “parental generation”. At 9 months after hatch, we split each family into three salinity treatment groups of 10 fish each: one at 33 PSU, one at 6 PSU, and one control group at 20 PSU. The salinity transition was performed within 10 days by 3-PSU steps every second day. Over the entire time, each group was fed ad libitum and kept in a 20-liter aquarium connected to one of three filter tanks per salinity treatment. After 5 months under treatment conditions, six pure crosses per salinity treatment group were performed in vitro, herein referred to as “offspring generation” (F2). Offspring and parental generations were kept at 18°C water temperature and a 15:9 light/dark (L/D) cycle. During the past 8 weeks before the F2 crosses, the F1 generation underwent an artificial winter to trigger reproduction . Upon fertilization, clutches were split and separated into different treatments (n = 16), Letipea , and Barsta .For the field survey, we collected juvenile three-spined sticklebacks , which is a proxy for energy reserves in the form of glycogen storage. We analyzed the effect of treatment  was performed using the DNeasy Blood and Tissue Kit (QIAGEN). Further purification of the extracted DNA was done with NucleoSpin gDNA Clean-up (Macherey-Nagel). For laboratory-bred F2 offspring of the two-generation acclimation experiment, dual extraction of whole RNA and DNA was performed from gill tissue . Paired-end sequencing of the quality-controlled and multiplexed libraries was performed on the Illumina HiSeq 4000 platform (2 × 150–bp reads).For whole-genome sequencing (WGS), the TruSeq Nano DNA (Illumina) library preparation kit was used according to the manufacturer’s protocol by the Sequencing Facility of the IKMB, University of Kiel. Ultrasonication was conducted with a Covaris E220 (Covaris) to shear the input DNA (100 ng per sample and 350-bp insert size). Before the enrichment with a PCR step (8 cycles), fragmented and bead-purified DNA was ligated with adenylate at the blunt 3′ ends (end repair and A-tailing) and with indexing adapters. Fragments were cleaned with MagSi-NGSThe command line tools of Picard version 2.7.1 (Broad Institute 2016) was used to (i) reformat the Fastq to uBAM file format and to add further values  to the SAM header using FastqToSam, (ii) mark the location of adapter sequences using MarkIlluminaAdapters, and (iii) reconvert the sequences to Fastq format with SamToFastq. The stickleback genome (Broad/gasAcu1) was indexed with bwa index and used as a reference for the mapping with bwa mem  of balanced sex ratio were DNA-sequenced at an average of 19.8 ± 3.5 million reads for experimental fish and 11.4 ± 2.1 million reads for wild-caught fish (table S4). Demultiplexed Fastq files were quality-checked using FastQC version 0.11.5 . BisulfiF124,1 = 2.611, P = 0.109). However, the principal components analysis revealed a bias in methylation pattern by families over all experimental groups. Therefore, to identify differentially methylated CpG sites (DMS) between treatment groups, we performed pairwise comparisons (table S5) fitting a logistic regression model per CpG site with calculateDiffMeth in methylKit using family as covariate for the experimental groups. A chi-square test was applied to assess significance levels of DMS, and P values were corrected to q values for multiple testing using the sliding linear model method . Ultimately, CpG sites were considered to be differentially methylated with a q < 0.0125 and a minimum weighted mean methylation difference of 15%. To ensure that the DMS obtained are not laboratory artifacts, we used calculateDiffMeth implemented in methylKit and compared the wild population from KIE to the experimental control group (KIE population from 20 PSU at 20 PSU). The resulting 11,828 DMS were excluded from the DMS obtained by the pairwise comparisons mentioned above (table S5). DMS were plotted across the genome for the comparison between KIE versus NYN  and KIE versus SYL  using ggplot2 . On the other hand, pop-DMS were considered inducible when at least one of the acclimation groups was differentially methylated compared to the control group . pop-DMS with a significant q value not exceeding the threshold of differential DNA methylation (15%) will be referred to as inconclusive hereafter. We used a randomization test to ensure that the number of inducible sites obtained did not occur by chance. To this end, we randomly sampled 1470 (KIE-NYN) and 1158 (KIE-SYL) pop-DMS from the complete dataset (1000 replicates). A chi-square test was used to assess whether our observed number of inducible, stable, and inconclusive sites differs from a random set of sites (averaged over replicates). Last, we tested whether the weighted mean methylation difference  between wild populations matches the inducible methylation difference by subtracting the “meth.diff” in the experiment (exp) from the meth.diff between wild-caught populations (wild)By comparing wild-caught individuals from the mid-salinity population  to the populations sampled at low  and high  salinity in the field, we obtained 1470 (KIE-NYN) and 1158 (KIE-SYL) pairwise pop-DMS. We first tested whether these pop-DMS distinguishing natural populations are inducible or stable at the respective salinity in the experiment. A pop-DMS was considered stable when the within- and the transgenerational acclimation groups did not significantly differ in methylation to the control group  and increased  salinity.P ≤ 0.05) with the Ensembl stickleback annotation dataset “gaculeatus_gene_ensembl,” and all genes that were associated to any sequenced CpG site were used as universe. We identified overrepresented biological processes, molecular functions, and cellular components using the packages GOstats version 2.5  from WGS data of the exact same individuals with vcftools version 0.1.15 (FST difference between the two groups (expected and opposite)To evaluate the genetic differentiation up- and downstream  of the pop-DMS position, we calculated the mean FST values and calculated a P value by dividing the proportion of values smaller than the true difference by the number of bootstraps. Figures were produced using ggplot2 version 3.2 (We plotted the 10,000 delta mean


Sarcoidosis is a systemic disease of unknown etiology characterized by the presence of noncaseating granulomas that can occur in any organ, most commonly the lungs. Early and accurate diagnosis of sarcoidosis remains challenging because initial presentations may vary, many patients are asymptomatic, and there is no single reliable diagnostic test. Prognosis is variable and depends on epidemiologic factors, mode of onset, initial clinical course, and specific organ involvement. From a pathobiological standpoint, sarcoidosis represents an immune paradox, where an excessive spread of both the innate and the adaptive immune arms of the immune system is accompanied by a state of partial immune anergy. For all these reasons, the optimal treatment for sarcoidosis remains unclear, with corticosteroid therapy being the current gold standard for those patients with significantly symptomatic or progressive pulmonary disease or serious extrapulmonary disease. This review is a state of the art of clinical presentations and immunological features of sarcoidosis, and the current therapeutic approaches used to treat the disease. Sarcoidosis is a systemic granulomatous disorder that remains an enigma and challenges both researchers and clinicians due to its unknown cause, heterogeneous clinical presentation, and unpredictable outcome, occasionally severe and even lethal, often with no therapy able to change the course of the disease . This huThe frequency of organ involvement at the time of diagnosis is summarized in Even though the thorax is the most common site of disease, skin involvement occurs in at least 25% of patients and is often overlooked. Indeed, cutaneous sarcoidosis is often attributed to other causes, mainly due to its wide range of manifestations, which include erythema nodosum, maculopapular lesions, hyper- and hypo pigmentated areas, keloid formation, and subcutaneous nodules , often dEye involvement may precede the diagnosis of sarcoidosis by years or even decades and occurs in up to 25% of patients depending on race . For exaCardiac involvement in sarcoidosis is much more common than clinically appreciated, and it can be present without the involvement of other organs. The three major manifestations of cardiac sarcoidosis are: conduction abnormalities, ventricular arrhythmias, and congestive heart failure. Granulomatous infiltration of the myocardium is responsible for arrhythmias and cardiomyopathy . In partNeurosarcoidosis is reported in 5–10% of sarcoidosis patients and appears to be equally frequent across ethnic groups. Any part of the nervous system can be affected, and among all parts of the central nervous system (CNS), cranial neuropathy and meningeal involvement are the most common manifestations. Furthermore, II, VII, and VIII cranial nerves are the most commonly involved, even if all cranial nerves have been reported to be potentially affected. Meningitis accounts for 10% to 20% of cases of neurosarcoidosis, although the reported frequency of subclinical leptomeningeal damage is much higher . FinallyThe clinical presentation of sarcoidosis is highly heterogeneous, and the onset can be acute or subacute/chronic. It has been hypothesized that different immunologic features underlie these diverse presentations. From both clinical and immunological standpoints, sarcoidosis is mainly classified in Löfgren’s syndrome (LS) and non-Löfgren’s syndrome. In more than half of the cases of LS, the disease resolves spontaneously within 2 years, on average ,20. AfteThe typical combination of symptoms with acute onset such as fever, erythema nodosum, and arthritis accompanied by bilateral hilar lymphadenopathy at chest X-ray is defined as LS  21]. LS. LS21]. The non-acute form, or non-LS sarcoidosis, is much more heterogeneous than LS and presents with a wide range of nonspecific symptoms and with an insidious/subacute onset . SubacutProgression to irreversible fibrosis. Pulmonary fibrosis (PF) represents a rare (20% of chronic forms) yet poorly studied phenotype of sarcoidosis . ChronicPatients with sarcoidosis have a lower survival rate than the general population , with a Sarcoidosis is characterized by non-infectious non-caseating granulomas, comprised primarily of macrophages that differentiate to epithelioid cells, which subsequently fuse to form multinucleated giant cells. CD4+ T helper cells, the specificity of which is unknown, are interspersed in the granuloma while CD8+ T cells, regulatory T cells (Tregs), fibroblasts, and B cells surround the periphery . This heThe involvement of both the innate and the adaptive immune cells calls into question the nature of the antigen/s involved in the pathogenesis of sarcoidosis. Thus far, apart from the tuberculin known to trigger the formation of the sarcoid granuloma, many have been the candidate antigens associated with the onset and the progression of the disease. Bacterial DNA has been identified in sarcoidosis lesions  as well Overall, the immune cascade triggered by the antigen/s is much better known compared to the nature of the antigen itself. We here describe the role of innate and adaptive immune responses in the onset and the progression of sarcoidosis. γ), which is highly expressed in the BALF  of sarcoidosis patients [γ inhibits the expression of macrophage peroxisome proliferator-activated receptor γ (PPARγ), a negative regulator of inflammation. Under normal physiological conditions, macrophages constitutively express PPARγ. PPARγ promotes macrophage IL-10 production, which inhibits the release of TNFα, IL-12, and matrix metalloproteinases (MMPs) from dendritic cells (DCs). In sarcoidosis, PPARγ activity is deficient in AMs [α, IL-12, and MMPs, which cause lung damage and fibrosis and induction of T-cell chemotaxis [α and decreased IL-10 expression liberate DCs from the inhibition by macrophages, initiating a self-amplifying inflammatory loop (see below)Alveolar macrophages (AMs) are involved both in innate and adaptive immune responses in sarcoidosis . AMs propatients . IFNγ int in AMs , leadingemotaxis . MoreovePeripheral blood DCs from patients with sarcoidosis are less immunostimulatory than normal blood DCs and are less capable of mounting a delayed-type hypersensitivity (DTH) response . Consistγ in CD4+ T cells becomes more pronounced. In response to IL-12, CD4+ T-cell production of IL-4 and IL-13 (cytokines that facilitate the fibroproliferative response) is inhibited. IL-12 and IL-18 act synergistically to promote the formation of sarcoid granulomas. As detailed above (see AM section) in sarcoidosis, the deficient PPARγ activity in AMs [T-cells, particularly activated CD4+ T-cells, play a key role in the inflammation in sarcoidosis. Different subsets of CD4+ helper T cells participate in the immunopathogenesis of sarcoidosis. The main feature of the acute disease is represented by a Th1/Th17/regulatory T cells (Tregs)-driven inflammatory process involving macrophages both as antigen-presenting cells and key effectors. As discussed above, when triggered by factors as yet unidentified, APCs such as DCs release cytokines   and othey in AMs  attractsy in AMs . Pulmonay in AMs . Other sy in AMs . Th17 cells that express IL-17 and a specific master transcription factor, known as RORc , have a Failure of immune regulatory mechanisms to limit the duration of Th1–Th17 inflammation has been suggested to contribute to persisting granulomatous responses in sarcoidosis. T regulatory cells (Tregs) are vital for the suppression of cell-mediated immune responses. However, Tregs in the sarcoid granulomas  that have also been found elevated in BALF from patients with sarcoidosis undergo extensive amplification and are therefore impaired in their ability to repress immune responses . On the Little is known about B cell immunity in sarcoidosis . HypergaIn sarcoidosis, the decision to initiate treatment is not straightforward, mainly due to the highly variable and unpredictable course of the disease and the lack of evidence-based management guidelines. Broadly, the treatment is selected based on the presence of life- or organ-threatening disease and very poor quality of life. CO (Diffusion Capacity for Lung Carbon Monoxide) decline of ≥20% over three to six months); or major progression of radiographic abnormalities  [Systemic glucocorticoids (GCs) are the first-line therapy for pulmonary and extrapulmonary sarcoidosis, as they inhibit macrophage and lymphocyte activation and modulate several cytokines that participate in granulomatous inflammation. However, they do not alter the course of the disease, and their long-term use is associated with significant safety and tolerability issues. Therefore, the pros and cons of initiating GC treatment should be carefully weighed, bearing in mind that approximately half of the patients have self-remitting or non-progressive disease, which does not require therapy ,76. In pcombing) . Some pacombing) ,78. The combing) ,80. The majority of patients are able to discontinue treatment after one year; however, relapse is common during tapering or after GC discontinuation, with as many as 30% of patients requiring longer-term therapy . OverallImmunosuppressants: for patients experiencing relapse/disease progression despite GC treatment or intolerable side effects of GCs, a step-up approach to second-line agents is recommended. Methotrexate (MTX) is the drug of choice for sarcoidosis selected in several consensus statements . MTX is 18F-FDG PET (Fluorodeoxyglucose Positron emission tomography) as maximum standardized uptake value (SUVmax). Notably, change in lung function significantly correlated with the level of disease activity. Adalimumab is a fully human anti-TNF-α monoclonal antibody. In an open-label, single-center study, 11 patients with refractory pulmonary sarcoidosis received adalimumab for 45 weeks [Biologicals: TNF-α antagonists are generally reserved for patients with active/progressive disease despite being treated with GCs and at least one second-line immunosuppressant agent, particularly in cases of organ- or life-threatening disease. Infliximab is a humanized monoclonal antibody that neutralizes TNF-α. In a phase II, randomized, double-blind, placebo-controlled study, 138 patients with chronic pulmonary sarcoidosis were randomized to receive i.v. infusions of infliximab or placebo at baseline and at weeks 2, 6, 12, 18, and 24 . Inflixi45 weeks . At the 45 weeks . Severe 45 weeks . At pres45 weeks ,96,97.n = 10) [Besides TNF-α inhibitors, rituximab, a chimeric anti-CD20 monoclonal antibody that induces B cell depletion is the only biologic agent for which there is some evidence of efficacy. Sweiss and colleagues conducted a prospective, open-label, phase I/II trial of rituximab at baseline and two weeks later in patients with refractory pulmonary sarcoidosis (n = 10) . At weekOther novel therapies: several drugs targeting the immune system are currently being evaluated as future therapeutic strategies for sarcoidosis. The active inflammatory form of sarcoidosis is characterized by an exaggerated production of TNF-α and INF-γ. Fontolizumab, a humanized monoclonal antibody against INF-γ currently tested in Crohn disease , has theTransplant: for highly selected patients with advanced fibrotic lung disease, severe pulmonary hypertension, or both, lung transplantation may be the only therapeutic option for prolonging survival and improving quality of life . BilaterSarcoidosis is a heterogeneous disease with diverse clinical presentations that likely mirror the wide umbrella of underlying immunological endotypes. Thus, a “one size fits all” approach to treating sarcoidosis is not applicable, as some patients may benefit from therapies targeting the excessive spread of the inflammatory responses, while others might benefit from therapies boosting their immune and/or regulatory responses. Further studies aimed at identifying the clinical phenotypes associated with specific blunted and/or exaggerated immune responses are needed in order to shed light on the possible therapeutic approaches for this multifaceted disease.


This paper describes a method for patterning the graphene layer and gold electrodes on a ceramic substrate using a Nd:YAG nanosecond fiber laser. The technique enables the processing of both layers and trimming of the sensor parameters. The main aim was to develop a technique for the effective and efficient shaping of both the sensory layer and the metallic electrodes. The laser shaping method is characterized by high speed and very good shape mapping, regardless of the complexity of the processing. Importantly, the technique enables the simultaneous shaping of both the graphene layer and Au electrodes in a direct process that does not require a complex and expensive masking process, and without damaging the ceramic substrate. Our results confirmed the effectiveness of the developed laser technology for shaping a graphene layer and Au electrodes. The ceramic substrate can be used in the construction of various types of sensors operating in a wide temperature range, especially the cryogenic range. For the last ten years, there has been increasing interest in the use of graphene in electronic devices , transparent conducting electrodes and circuitry, nanocomposites, supercapacitors, and sensors ,6,7,8,9.Higher pattern resolution is important for producing nanoscale graphene electronic devices. Such structures take advantage of the lateral confinement of electrons within graphene nanoribbons, which introduces an energy gap, and have been achieved by applying ion beam lithography ,23,24,25Compared to the graphene patterning techniques outlined above, processes using a laser beam have several advantages, chief among them are high efficiency, short exposure times, low energy consumption, lack of catalysts, repeatability, scalability, and extensive process control capabilities ,33. Thre2 have been patterned using a Nd:YAG continuous-wave (cw)laser (λ = 532 nm) to heat Ni foil in the process of synthesizing graphene from a mixture of CH4 and H2 [2 using direct laser writing (λ = 780 nm) in a co-sputtered Ni/C film [2 laser, λ = 10.6 μm) [2) that was next covered with a thin Cu film in a CVD process. The catalytic film was melted at 700 °C, forming a liquefied Cu network, on which graphene was locally synthesized. Patterned graphene synthesis can be performed using two methods: first, a laser beam is used as an energy source, which is applied directly during the process of laser-induced CVD (LCVD) synthesis; second, the laser beam creates patterns in the catalyst layer, allowing for synthesis in localized areas . Areas o4 and H2 . Ribbons4 and H2 . Grapheni/C film . Laser-ai/C film . A simili/C film  was used10.6 μm)  allowed 10.6 μm) . Laser b2 laser has been shown to enable the patterning of supercapacitor devices [2 graphene-like structure, which needs heat. Generally, a single-beam treatment has been applied for the laser reduction of GO and patterning. However, simultaneous reduction and patterning of nanostructures in GO multilayers using the interference of two laser beams with wavelengths of 355 nm and pulse durations of 10 ns has also been performed [Various types of lasers have been used for the laser reduction of GO on different substrates. Using a femtosecond laser beam (λ = 790 nm), lines can be produced with widths of about 200 nm  or below devices . Compreh devices . This prerformed .In the above-mentioned works ,47,48,492/Si and on glass substrates, were patterned using an excimer KrF laser  [Direct laser patterning of graphene layers is associated with two main problems. On one hand, there is the need to ensure the resolution, fidelity, and dimensional accuracy of the pattern, and on the other, there is the difficulty of completely removing the graphene without damaging the substrate. Achieving these tasks is possible by choosing the appropriate type of laser  and ablation process parameters. Extremely short laser pulses are considered an excellent tool for micromachining graphene layers. Nanometer-scale patterning (width about 400 nm) has been performed using a femtosecond laser  and a laser beam converted into a Bessel beam . Using a 248 nm) . In the  248 nm) . Interdi 248 nm) .2/Si [2/Si [Summarizing the literature and issues discussed above, two conclusions can be made. First, a variety of methods and substrates can be used for graphene synthesis and to support the graphene layer: the chemical vapor deposition of graphene on various materials, such as metallic foil  ,17, SiC 2/Si ,20,22, o2/Si ; transfeSi [2/Si ,25,52,54Si [2/Si , or on SSi [2/Si ; laser-iSi [2/Si ,35; grapSi [2/Si ,58; direSi [2/Si ,38; spinSi [2/Si ,42,43,45Si [2/Si ,49, or oSi [2/Si ; and reaSi [2/Si . Second,3N4 [2O3 [3 [2O3) using a Nd:YAG nanosecond fiber laser. To the best knowledge of the authors, there are no previous reports regarding the transfer of high-strength metallurgical graphene (HSMG®) to a ceramic substrate. The transfer of the graphene layer from a metallic substrate was performed using PMMA as the graphene-supporting material. In terms of using graphene on a ceramic substrate as a sensor layer, it would be very useful to develop a method that allows for graphene to be removed either from ceramics or from an electrode layer (Au), or the graphene and electrode simultaneously. The main goal of the research was to determine the optimal conditions for direct laser recording in these three cases and to examine the efficiency, precision, and purity of the process. The areas of the samples with and without the laser treatment were studied via microscopic examination and Raman spectroscopy. Measurements of the electrical properties of graphene on the ceramic substrate were performed. This enabled the impact of the laser processing on electrical parameters of the samples to be identified via comparison with previous results. The electrical conductivity was measured across a wide temperature range (293–15 K). The results are particularly important for the construction of cryogenic sensors  that are based on graphene.Previous studies in the literature relating to graphene on a ceramic substrate have essentially focused on the process of synthesis. Graphene films have been synthesized using a CVD process on Si3N4 , Al2O3 [3N4 [2O3 , on a hi [2O3 [3 , or with [2O3 [3 . This pa® supported on a ceramic substrate with gold electrodes. This configuration enables various types of sensors to be built with HSMG® as the active layer. The ceramic substrate parameters are important for the construction of sensors (resistance temperature detectors (RTDs), Hall sensors, biosensors, etc.) and other devices based on graphene . All the samples were designed with graphene transferred onto an Al2O3 ceramic substrate. Alundum ceramics can be used in devices operating over a wide temperature range, including cryogenic temperatures and temperatures over 1300 K. In addition, the ceramic substrate provides very good thermal, mechanical, and dielectric parameters. The thermal expansion of Al2O3 over a wide temperature range is comparatively small (≈8.2 × 10−6 K−1) and its thermal conductivity (≈35 W/mK at 293 K) is larger than that of typical polymers or dielectric composite materials. The Al2O3 ceramic substrate used in this study was extremely pure (>99.5%), with small crystallites (10 μm) and without open porosity. Both surfaces were polished to a roughness value Ra = 0.08 μm. The graphene layer was transferred to the ceramic substrate from a metallic substrate using PMMA as the graphene-supporting material. Before the transfer process, physical vapor deposition (PVD) was used to make flat, gold, nano-thickness (150–200 nm) electrodes on the ceramic surface.We studied samples based on a layer of HSMG® layer and on the gold nano-thickness electrodes. The design of sensors requires high-fidelity mapping of the sensory layer. The precision of this process determines the repeatability of the parameters and the accuracy of the sensor. Laser processing was used to form structures of different geometries and dimensions, both in the HSMGA fiber laser was used to generate a one-mode pulsed beam in the nanosecond range. Shaping of the structures could thereby be performed in a controllable ablation process. When the laser beam was focused on a spot of 26 μm, the Gaussian distribution of the power density provided good accuracy for the ablation process. The experiments were performed using laser pulses of various durations in the range of 15–35 ns, with repetition frequencies from 290 to 80 kHz. The main goal was to remove the graphene layer or the thin gold layer without damaging the ceramic substrate. The pulse energy was therefore finely adjusted according to the pulse duration, repetition frequency, and velocity of beam scanning. The pulse energy varied from 12 μJ to 40 μJ.The same uniform distribution of the laser beam power was obtained independently of the repetition frequency due to an appropriate adjustment of the scanning velocity . A pulseTo confirm the effectiveness and correctness of the process, samples were tested before and after laser patterning. Only part of each sample was laser treated. The studies included microscopic observations of samples, Raman spectroscopy, and tests of electrical properties over a wide temperature range (15–295 K).® graphene transfer. The well-known PVD method was applied to produce gold electrodes on the ceramic substrate. The rectangular shape of the electrodes (8 × 4 mm) was the effect of the masking process and the PVD method. A modified transfer procedure of HSMG® graphene was used. A thin film of PMMA was used as a graphene-supporting material. Variations of the applied method are commonly used for the transfer of 2D materials (graphene) onto substrates of any kind. Wrinkles and cracks were observed on the graphene after the transfer process, the explanation for which has been discussed in a previous work [All the tested samples were designed according to the same method. The first step in the design process was substrate partitioning . Laser cous work . It shouStrips of silver thin foil (35 μm) were soldered to the gold electrodes on the substrate surface to make metallic, elastic electrical leads. Indium soldering was used and the obtained joints were protected against oxidation by using polyurethane resin. Each of the samples had two pairs of the same electrodes, which allowed for the electrical parameters to be measured before and after the laser treatment . AlthougThe areas of the samples with and without laser treatment were studied via microscopic examination and Raman spectroscopy. Due to the nature of graphene, SEM, TEM, and AFM techniques are preferable for the examination of graphene structures, although optical microscopy can also be useful. In our research, the quality of the patterning of the graphene structures was assessed using optical microscopy . To obtain good quality SEM images, the vacuum in the microscope chamber was decreased to 100 hPa.−1 in the spectral range of 1100–3200 cm−1. Acquisition times varied between 120 and 200 s . The signals were collected from nine points: zero (in the middle of the laser cutting line) and eight points at distances of 50 μm (points 1 and 5), 60 μm (points 2 and 6), 75 μm (points 3 and 7), and 100 μm (points 4 and 8) from the zero line. The spectra were deconvoluted in Peakfit 4.11 software .For the graphene quality analysis, a Renishaw inVia Raman spectrometer  was used. The experiments were carried out using a 532 nm laser with a 50× objective lens . The Raman spectra were analyzed at a spectral resolution of 1 cmAll measurements of the electrical properties of graphene on the ceramic substrate were performed in a helium closed-cycle DE-210 cryostat . The samples were placed in a vacuum chamber and cyclically cooled from 293 K to 15 K at a rate of about 4 K/min . The tesThe four-probe method was used to measure the resistance of the samples. Resistance measurements were conducted using a Keysight 34420A Micro-Ohm Meter . Two-stage tests of the samples were performed. In the first stage, the electrical properties of the samples were measured, followed by laser patterning. The results showed the parameters of the original, primary samples, with possible defects and impurities resulting from the transfer process. In the second stage, the laser-processed samples were tested using the same method. This measurement procedure enabled the impact of the laser processing on the electrical parameters of the samples to be identified via comparison with the previous results. Additionally, measurements across a wide temperature range enabled the type of electrical conductivity to be identified, as well as the associated phenomena, such as the phonon scattering and defects. These measurements allowed us to observe the interaction of the graphene layer with the ceramic substrate, particularly at cryogenic temperatures, and the effect of the substrate on the parameters of the layer. The results are particularly important for the construction of cryogenic sensors  based on graphene .p = 15 ns, repetition frequency 290 kHz, at a scan velocity of 2500 mm/s, and with a hatching of 10 μm. Further research focused on determining the threshold value for the pulse energy. The pulse energies needed to achieve complete ablation were 18 μJ for graphene on alumina ceramic, 24 μJ for graphene on Au electrodes, and 34 μJ for simultaneous ablation of the graphene layer and Au film. Microscopic pictures of the three cases of graphene ablation are shown in The influence of the patterning process parameters on the effectiveness of the removal of the graphene layer from the alumina ceramic surface and Au electrodes was examined experimentally. Tests were performed at a constant pulse overlap ratio of 1:3. The effectiveness of graphene ablation was determined using measurements of electrical conductivity on the ablated path. In the absence of electrical conductivity, the possible presence of graphene fragments was additionally investigated using Raman spectroscopy. In our preliminary studies, the highest edge quality was obtained with pulses of the shortest duration tBoth optical microscopy a,b and s2O3 substrate, the ablated path width was ≈240 μm. In the area on the Au layer, the path width was ≈225 μm. The path width was greater than the assumed value (200 μm) due to the laser micro-machining method used  was defined at the intensity level Imax/e2, which means in practice that the laser beam–surface interaction occurred on an area with a larger diameter. In particular, in thin-layer micromachining, the diameter of the ablation area is greater than d0. The reduction in path width on the Au layer area may be explained similarly. The overlapping of pulses “inside” the ablation path provided an averaging of the absorbed energy  occurred at a lower threshold value of absorbed energy than the ablation of the Au layer . Changes in the sizes of the paths were more noticeable when they had smaller widths.In the microscopic images b,d, therd energy . The eff2O3 substrate and Au layer was effective. This was confirmed by the absence of graphene and other carbon forms in the middle area of the path (below ± 50 µm from the centerline) . This may indicate that some portion of laser energy was used in the oxidation process of graphene. To determine the degree of oxidation, XPS tests would be required; however, in the presented work, further considerations about the oxidation degree of graphene were omitted.The Raman analysis of a laser-ablated path is presented in terline) . The firnterline b, where nterline a at poin2O3 substrate. The absence of additional peaks suggests that the graphene structure was not disordered. The 2D and G band intensities did not depend on the distance from the center of the ablation path. Therefore, it can be concluded that the graphene was not affected by laser irradiation. The results of the Raman spectroscopy revealed G and 2D peaks that are typical for graphene structures, with a higher intensity corresponding to the 2D band. The intensity relation of IG/I2D indicates that the investigated graphene was a multilayer structure. According to the literature, an IG/I2D relation of ≈0.2–0.8 indicates one to six graphene layers . In our 2O3 substrate, the paths after the laser cutting were graphene-free at distances of 50 μm above and below the centerline, while a peak D could be observed in the Raman spectrum for the laser-ablated graphene on the Au layer. The appearance of the D band is related to defects occurring in the graphene structure [In the case of the Altructure ,69.2/Si substrates, where local heat accumulation is observed, which is associated with the thermal properties of the substrate. The thermal conductivity of the substrate plays an especially important role in the thermal processes. It should be noted that the thermal conductivity of SiO2/Si is about twenty times lower than that of Al2O3, while the specific heat of both is similar, which determines the dynamics of the thermal processes. The thermal parameters of the graphene layer are insignificant in this process because the layer is very thin; therefore, the longitudinal heat flux in the layer is negligible. The same thermal processes occur in the Au layer (thickness around 150 nm). This means that the substrate is generally responsible for heat accumulation and dissipation during the laser treatment process. Disturbances to the graphene structure occurring after laser treatment near the edges of the ablation area may also be the result of the deposition of Au decay products on the graphene. The Raman spectra showed different quantities of defects, expressed by the ID/IG intensity ratio . The intensity ratio depends on the distance from the centerline but the number of defects was random a. Our obThe dependence of sample resistance on temperature was investigated using samples before and after the laser treatment. The treatment consisted of cutting a and cut2O3 and the effective shaping or partitioning of graphene on the Au layer. Our results show that it is possible to remove graphene from the Au layer on an Al2O3 substrate without damaging the Au layer (A+BR) was slightly higher than the initial resistance of the sample (beforeR). This was the effect of a small loss of material as a result of the laser processing. The second studied sample, like the first, was divided into two parts and the sizes of the graphene layer and gold electrodes were also reduced  eliminated the impact of pressure changes during the cryogenic tests. An encapsulation process was therefore necessary to obtain samples with repeatable and stable parameters. Effective technology is currently under development.It should be noted that all the studied samples were tested in a vacuum environment but the pressure inside the cryostat was not constant or stabilized. Changes in pressure are a natural result of changes in the temperature of the rest gases and it was impossible to stabilize the pressure inside the cryostat. To minimize and standardize the impact of the vacuum on the parameters of the samples (opened samples without encapsulation), they were placed in a vacuum 24 h before testing. Our previous studies showed that samples based on HSMGR(T) is important for the design of the signal conditioning system; however, a small TCR value can be a source of additional errors in the potential temperature sensor. In our opinion, the presented method has notable application potential and can be used for both the production and trimming of sensors based on graphene on a ceramic substrate.The most important advantage of the presented method is the ability to simultaneously shape the graphene and Au layers on a ceramic substrate without causing significant damage, especially on the graphene layer in the sensory area, i.e., graphene on ceramics. The usage of laser processing ensures the high speed and repeatability of the process and allows for the shaping of any simple geometries without the masking process. All presented results  confirmed the effectiveness of the proposed method. In addition, the use of a ceramic substrate ensures the optimal thermal parameters of the sensor, i.e., high thermal conductivity, low thermal expansion, and high electrical resistivity. The local nature of the laser ablation process provided good shape mapping and did not cause significant defects. However, the sample preparation method required miniaturization and encapsulation of the layer to stabilize the parameters. A drawback of the presented solution is that the temperature range is limited to cryogenic temperatures. The limitation is directly related to the electrode assembly technique , not to the properties of the graphene layer and ceramic substrate. Studies in the range above room temperature require changes in the sample preparation method mentioned earlier. The linear relationship