This is a theoretical investigation where Density Functional Theory (DFT) has been used in studying the phenomenon of Au intercalation within the 4H-SiC/graphene interface. The electronic structure of some carefully chosen morphologies of the Au layer has then been of special interest to study. One of these specific Au morphologies is of a more hypothetical nature, whilst the others are, from an experimental point of view, realistic ones. The latter ones were also found to be energetically stable. Band structure calculations showed that intercalated Au layers with morphologies different from a planar Au layer will induce a band gap at the Dirac point of graphene (with up to 174 meV for the morphologies studied in the present work). It should here be mentioned that this bandgap size is four times larger than the energy of thermal motion at room temperature (26 meV). These findings reveal that a wide bandgap at the Dirac point of graphene comes from an inhomogeneous staggered potential on the Au layer, which non-uniformly breaks the sublattice symmetry. The presence of spin-orbit (SO) interactions have also been included in the present study, with the purpose to find out if SO will create a bandgap and/or band splitting of graphene. One method, which is based on thermal annealing, uses a 4H-SiC (0001) substrate with an attached carbon buffer layer onto the Si-face of the substrate. Various types of metals have then been used with the purpose to intercalate these metal atoms between SiC and the buffer layers, thereby creating a monolayer (ML) of graphene with an intact Dirac point (DP)6. The few existing Si-C covalent bonds between the 4H-SiC (0001) substrate and the carbon buffer layer will then break . In relation to the majority of the buffer layer C atoms that are sp2-hybridized, those C atoms that bind to the SiC substrate are sp3-hybridized. This type of partial sp3-hybridization  makes graphene act as an insulator without any Dirac cone7. Moreover, when positioning heavy metal atoms under graphene, the spin-orbit coupling will increase that causes band splitting8 . A giant Rashba splitting is a very important property for a material to be used in the design of spintronic devices14. Perturbation theory has also earlier been used with the purpose to show that electrons within a pz orbital of graphene can be transferred to another pz orbital of graphene, either in pristine graphene or by using one of the d-orbitals, in the intercalated gold layer, as a bridge9.Intercalation processes have recently made it possible to produce large quasi free-standing graphene layers on different substrates20. Only a few of these studies resulted in the opening of a wide and controllable bandgap that is larger than the energy of thermal motion at room temperate. To the knowledge of the authors, the role of the morphology of the intercalated layer, on the Dirac point of graphene, has not yet been considered.In several earlier investigations, one has been trying to create a bandgap at the Dirac point of graphene by applying strain, spatial restriction, or by breaking the sublattice symmetryThe main goal of the present study has been to show how the morphology of the intercalated Au layer will affect the electronic properties of a monolayer (ML) of graphene. The possibility to induce a band gap at the Dirac point of graphene, by manipulating the staggered potential of a 4H-SiC/Au substrate, ha thereby been looked for. For this purpose, (i) several tilted Au layers, (ii) a zig-zag-shaped Au layer, and (iii) a mixed Au-Ag layer were used. These layers are monolayers, except (ii). This is a novel idea, which shows that the morphology of an intercalated layer can change the size of the bandgap at the Dirac point of graphene. The calculations were based on a highly accurate density functional theory (DFT) method, by which both the geometrical and electronic properties of the structures were obtained.23. (with distances larger than 3 Å). The theoretical work performed in these references were based on the same model as in the present investigation. However, they used the Projector Augmented Wave method (DFT-PAW) and the VASP software. This is different from the software and method that have been used in the present study ). Due to this difference in results, another theoretical parameter was tested . It must here be emphasized that the usage of different methods did not alter the Au/graphene distance to any important extent. The distance was still below 3 Å .The supercell models in the present study were based on monolayers of Au that were initially; i) flat, ii) slightly tilted, iii) built as a monoatomic step, or iv) intermixed with Ag within the monolayer Fig. . Model t4). This result is an indication of n-type doping, which has also earlier been observed for graphene21 (the nature of doping will be further explained in Section Zig-zag-sized Au layer). The difference between the here calculated position of DP and the experimental results is originating from different gold-graphene distances. As can also be seen in Fig. z orbitals of the C atoms (in graphene) and two types of 5d-orbitals (dz2 and dxy) from the Au atoms (in the gold layer). In order to visualize the hybridization of these orbitals, the projected band structure analysis was performed. The band structure was thereby projected onto the pz orbitals of the graphene carbon atoms, and onto the 5d-orbitals of the Au atoms. As can be seen in Fig. z orbitals of the C atoms hybridize very efficiently with the dz2 and dxy orbitals of Au. At the Γ point, the hybridization between pz and dz2 is stronger than the hybridization between pz and dxy, whereas the hybridization between pz and dxy is more substantial closer to the K point. Hybridization schemes involving the other Au d-orbitals  were observed to occur far from the DP .The band structure was calculated for the relaxed 4H-SiC/Au/graphene model, with a planar Au monolayer, including also spin-orbital interactions see Fig. . It can 24. Hence, it can be concluded that spin-orbit coupling will not result in a bandgap opening in graphene.It was at this stage very interesting to outline the underlying causes of the bandgap opening at the DP of graphene. To achieve a deeper understanding of this phenomenon, the band structure calculations had to be remade, but without any spin-orbit interactions. As a result, the value of the bandgap did not noticeably change; from 14.7 meV (with spin-orbit interactions) to 13.9 meV (without spin-orbit interactions)  might be the cause of the observed bandgap opening. The background to this hypothesis was, that a closer analysis of the geometrical structure of the initially flat Au monolayer revealed that minor changes in the relative z-positions had occurred as a result of the geometry optimization of the 4H-SiC/Au/graphene model. More precisely, the positions of Au2 and Au3, as indicated by 2 and 3 in Fig. The band structures of these artificial structures were accordingly calculated, and the results confirmed that by increasing the tilting angle in the Au layer , the size of the bandgap at the Dirac point of graphene will increase to a quite large extent. Even tilting at a smaller degree, with a Δz value of 0.35 Å for Au3, was found to create a wide bandgap of size 118 meV  at the Dirac point of 2ξ (ξ is a parameter which controls the proximity-induced energy gap). Moreover, the orbital dispersion, HD, is equal to 26, the term for the staggered potential, HSP, can break the A and B sublattice symmetry in graphene, and thereby create a bandgap at the DP of graphene. This is due to a breakage of the space inversion symmetry, and a chemical equivalence to these A and B lattice sites27 are observed for a graphene monolayer adhered to an h-BN (0001) sheet28. Hence, a monolayer of graphene being adhered to an Au monolayer, which is the situation in the present investigation, will ultimately play a very important role in the calculated results in the present study. However, another factor  will also affect the bandgap size at the Dirac point of graphene, and this factor is the inhomogeneity in the gold-graphene interactions within the x-y plane. As an explanation of what this means for the present model systems, by altering the degree of morphology  of the intercalated Au layer, the staggered potential (HSP) will also be altered, and in an identical manner. In short, by breaking the sublattice symmetry in graphene in an asymmetry way, a sizable band gap at the Dirac point of graphene will be formed. If we compare Fig. A look at the Tight Binding (TB) Hamiltonian of graphene can be very helpful in describing the bandgap opening. Here, Eq. To further illuminate the importance of an inhomogeneous staggered potential in creating the bandgap, a flat atomic layer that consists of both Au and Ag atoms has been considered Fig. . The ban4. Due to the strong effect of the Au-graphene distance on the bandgap opening, the given band structures in Fig. Since the distance between the gold layer and graphene was, in several experimental studies, reported as larger than 3 Å, the band structure of the 4H-SiC/Au/graphene model, given in Fig. After these observations, it was quite natural to continue the investigation by also introducing a more realistic model of an asymmetric 4H-SiC/Au/graphene structure . Within the supercell, an Au layer with a monoatomic step was thereby formed. The geometry optimized structure thereof can be seen in Fig. The calculated band structure for this 4H-SiC/Au/graphene structure, with monoatomic thick steps on the intercalated Au layer, showed similar results as for the other Au morphologies, but to a larger extent. The resulting size of the bandgap at the DP of graphene was 174 meV see Fig. , which iz2 orbitals on the Au atoms.To verify the dynamic stability of the SiC/Au/graphene system, with a monoatomic step on the intercalated Au layer, the phonon band structure was also calculated. As can be seen in Fig. 29.The values of the bandgap and band splitting, as well as the position of the Dirac point for each considered 4H-SiC/Au/graphene structure, are summarized in Table Moreover, it is also interesting to study the effect of inhomogeneity length on the bandgap opening at the DP of graphene. Additional calculations were thereby made, using a larger supercell of SiC/Au/graphene  of the graphene monolayer was not observed in the present study. There are two plausible explanations for this circumstance. One of these relates to the very strong intra-bonds  within graphene, which is to be compared with the much weaker Au-graphene bonds. The other explanation is the very large concentration of step-edges on the Au substrate. There is, thus, not enough length  of the lower Au surface, for it, with its weak attraction force towards the graphene monolayer, to pull the graphene sheet closer to itself. The maximum lower-surface length in the present study was 3.28 nm, while it was 60 nm in the Ir example30. Simulation of such a large model is, though, practically impossible when using DFT techniques.It has earlier been reported that when experimentally depositing a monolayer of graphene onto a step-layer of Ir (111), the graphene layer was observed to follow the substrate shape. Hence, it was bent at the step edges on the Ir substrate, maintaining a constant Ir-graphene distanceBy depositing more than one intercalated gold layers, it will be possible to control the bandgap at the Dirac point of graphene.31. .By increasing the thickness of the gold layer steps, it will both be possible to control the bandgap at the DP of graphene, as well as shifting the DP to a positive value. This brings p-type doping in grapheneThe here presented results clearly show that it is possible to control the width of the bandgap by controlling the surface morphology of the intercalated gold layer. Even though a truly experimental 4H-SiC/Au/graphene system has not been modeled, with all of its plausible defects and asymmetric morphologies, it is possible to draw some major conclusions from the present results;The effect by the morphology of an intercalated Au layer on the bandgap at the Dirac point of graphene has here been investigated for the 4H-SiC/Au/graphene system by using first principle DFT calculations. The gold layer was thereby used as an intercalated layer within the 4H-SiC (0001)/graphene interface. It was shown that wide bandgaps can be created by the formation of different types of Au morphologies of nanosized dimensions. The Dirac cone near the K point of graphene was, however, observed to be intact, and did thus not vary with the different nanostructures of the gold layer. The inhomogeneous staggered potentials that were created by either a nano-structured Au intercalating layer or by a mixed Ag-Au intercalating monolayer, confirmed that a uniformly staggered potential at the 4H-SiC/Au surface cannot induce a wide gap at the Dirac point of graphene. Instead, the breakage of the graphene sublattice symmetry to an asymmetric form of the 4H-SiC (0001)/Au substrate potential, is the key parameter for creating this type of bandgap.z orbitals on the C atoms (in graphene) with the dz2 and dxy orbitals on the Au atoms (in the gold layer), are the main causes to this type of effect. However, the spin-orbit interactions were not observed to have any important effect on the size of the bandgap at the Dirac point of graphene. We also found a strong connection between the size of the bandgap at the Dirac point of graphene and the distance between the gold layer and the graphene sheet. The size of the gap became reduced by increasing the Au-graphene distance. The here presented results can be useful, not only in diverse scientific fields  but also within future electronics industries that are based on 2D materials.Within the present study, the most important nanostructured Au layer was constructed with monoatomic thick steps (in the supercell) on the surface. It is therefore regarded to be an experimentally feasible surface, and calculations of the phonon band structure verified the stability of this specific SiC/Au/graphene structure. This type of zig-zag-formation  resulted in an inhomogeneous staggered potential, that in turn acted upon the adhered graphene monolayer and caused the bandgap opening. In addition to these observations, spin-orbit interactions also resulted in a band splitting at the Dirac point of graphene. The hybridizations between the p33, using a pseudopotential in addition to a linear combination of atomic orbitals (LCAO). . The pseudopotential used in the calculations was the norm-conserving pseudopotential (SG15) which includes multiple projectors, semi-core states, and a non-linear core correction with fully relativistic features36. Also, to describe the Van der Waals forces between graphene and the gold layer, DFT-D2 was added to the calculations37. Moreover, a dense Monkhorst-Pack k-point sampling (35 × 35 × 1) was used in calculating the band structure and the projected band structure (as proposed in ref. 38). This number of k-points was here regarded to be accurate enough in the description of the present types of model systems, including both d-orbitals within Au, as well as the Dirac point of graphene38. To avoid malicious effects (such as eggbox effects39), the density mech cut-off was set to a quite large value; 300 Ha (8163 eV)40. Since the spin-orbit interaction is an intrinsic effect, the Fermi-Dirac occupation method, with a broadening of 50 K (0.0043 eV), was used in describing the electronic properties of the system with high precision. To evaluate the choice of basis set, the band structures were calculated by using also a larger basis set  in the DFT calculations; SG15-High-BasisSet. No significant changes were thereby observed, both with and without the inclusion of spin-orbit interactions.The DFT calculations were based on a generalized gradient approximation (GGA)21 was used. To match the graphene monolayer to the underneath positioned SiC/Au structure, a tensile strain of 8.4% was considered . This value is in agreement with previous theoretical investigations41. In this matching procedure, a (2 × 2) unit cell of graphene was adjusted to the unit cell of 4H-SiC (0001) /Au/graphene interface, the method proposed in ref./Au/graphThe Au adlayer on the Si face of 4H-SiC 0001), and beneath the buffer C layer, was thereafter formed (as shown in Fig. , and benDataset information.


The interfacial friction performances of graphene covered and hydrogen-terminated diamond surfaces were investigated comparatively by first-principles calculations within density functional theory (DFT). Both systems exhibit similar excellent lubricating effects under small load, but the graphene covered interface presents small friction than that of hydrogenated system for the larger load. The calculated interfacial friction between two sheets of graphene covered diamond surface increases slowly than that of hydrogenated system in a wide range of pressure scale, and the friction difference between the two systems increases with increasing external pressure, indicating that graphene has flexible lubricating properties with high load-carrying capacity. This behavior can be attributed to the large interlayer space and a more uniform interlayer charge distribution of graphene covered diamond interface. Our investigations suggest that graphene is a promising candidate as solid lubricate used in diamond film, and are helpful for the understanding of interfacial friction properties of diamond film. Diamond coatings, from diamond single crystal (DSC) film to amorphous diamond-like carbon (DLC) film, have attracted great interest in both industrial application and scientific research fields due to its low friction, high wear resistance properties . SlidingCompared to amorphous DLC films, DSC films have much simpler geometric structure, which is helpful for researchers to study contributions of special mechanism to complex friction phenomena of diamond coating . As the and wear ,20,21,22and wear . Sen et ructures . More imructures ,16. So iDue to its superior physical and chemical properties, graphene has been extensively studied in various fields in recent years . EspeciaTo understand the lubricant mechanism of graphene on diamond film, the interfacial interaction and friction between graphene and C(111) surface were investigated by using first-principles calculations in this paper. Our calculations show that the graphene covered diamond interface exhibits a smaller friction than that of the hydrogenated one in a wide range of loads. The smaller friction can be attributed to large interlayer space and a more uniform interlayer charge distribution. We also found that graphene coating has flexible lubricating properties with high load-carrying capacity when used in the diamond film system.The Vienna Ab-initio Simulation Package (VASP) code armed with the projector augmented-wave (PAW) method was used in our calculations ,46. The S6=0.75 . An enerS6=0.75 . The conTo investigate the effects of graphene coating on tribological properties of diamond film, we first considered the interaction energy per unit area,  wsep=ΔE . By consposition .The existing experimental and theoretical results have confirmed that graphene can attach to the C(111) surface and form an interfacial structure ,21,22,25The stable structure of graphene adsorbing on C(111) film was calculated firstly. It should be noticed that the interlayer distance between the first and second layers is only 0.44 Å in the relaxed C(111) film, which approaches to one plane. The distance between the second and third layer was enlarged form 1.43 to 1.45 Å. The above results agreed well with the previous results that the diamond surface has a tendency of graphite. There are three high symmetry stackings for the Gra-C(111) structure. One is the top stacking, in which all of the C atoms in graphene located on the top of C toms in the first and second layers of C(111) film. The interaction energy as a function of interlayer distance between graphene and C(111) film for the three stackings are shown in We next considered interfacial friction between graphene and C(111) film. Three kinds of sliding models were constructed to reflect the influence of diamond substrate on friction of graphene. For the first model, we put one layer of graphene on the C(111) substrate (Gra-C(111)) and then draw it across the diamond film substrate a. To refNext, we explored the shielding effect of graphene on diamond film in the view of interfacial friction. From the comparison between In this part, the lubrication of graphene in the diamond interface was investigated. Two sheets of Gra-C(111) films were put against to simulate the role of reducing friction (Gra-C(111)/Gra-C(111)), as shown in The PESs under free-load were constructed for the two systems, as shown in To reflect the effect of load on lubrication more directly, the shear strength ated see , in whic results . These rTo understand the lower friction behavior of Gra-C(111)/Gra-C(111) system, charge density distribution and charge density difference of the two systems were calculated, as shown in To further understand the reason why Gra-C(111)/Gra-C(111) system exhibits flexible lubrication, the dependence of interaction energy on interlayer distance was calculated, as shown in Based on the first-principles calculations, the interfacial friction properties of graphene on diamond surface have been investigated. Our results show that diamond film substrate has little influence on the interfacial friction properties of bilayer graphene, which indicates that the graphene can provide an effective shielding to the diamond surface. The friction properties between graphene covered diamond surface and hydrogenated terminated diamond surface have been investigated comparatively. Our results demonstrate that both systems exhibit similar small friction under the situation of free-load. However, with the increase of normal pressure, the sliding barrier in H–C(111)/H–C(111) system increases faster than that of Gra-C(111)/Gra-C(111) system, and the shear strength in H–C(111)/H–C(111) system is several times larger than that of later system. These low friction can be attributed to the large interlayer distance and more uniform interlayer charge distribution of graphene covered diamond interface. As diamond films are always used for devices operating in high-pressure environments, such as cutting tool, critical engine parts, artificial joints, and micro or nanoelectromechanical systems (MEMS/NEMS), this kind of flexible lubrication with high load capacity is meaningful for the practical application of diamond. This study suggests that graphene can be used in diamond film as a kind of excellent coating, and has a certain reference significance for the wear resistance modification of diamond film and surface.


After having investigated the dispersion behaviour of biochar in different organic solvents, a biochar-based screen-printed electrode was prepared with the drop-casting technique. In order to understand the electrochemical potentiality and performances of the biochar/sensor tool, different electroactive species, i.e., ferricyanide, benzoquinone, epinephrine, ascorbic, and uric acids, were used. The results were compared with those of the same electrodes that were modified with commercial graphene, confirming that the proposed electrode showed improved electrochemical behaviour in terms of resolution, peak-to-peak separation, current intensity, and resistance to charge transfer. Furthermore, a tyrosinase biosensor was developed by direct immobilisation of this enzyme on the biochar/screen printed electrode, as an example of the potential of biochar for disposable biosensor development. The efficiently occurred immobilisation of the biochar on the screen printed electrode’s (SPE’s) surface was demonstrated by the observation of the working electrode with a scanning electron microscope. The detection was performed by measuring the current due to the reduction of the corresponding quinone at low potential, equal to −0.310 V for epinephrine. The experimental conditions for the tyrosinase immobilization and the analytical parameters, such as applied potential and pH of buffer, were studied and optimized. Under these conditions, the electrochemical biosensors were characterized. A linear working range of epinephrine was obtained from 0.05 up to 0.5 mM. The detection limit was 2 × 10 From an agronomic point of view, biochar can improve agricultural productivity, particularly in low-fertility and degraded soils, reducing losses of nutrients, and improve the water-holding capacity of soils 3− + 1e− ⇆ [Fe(CN)6]4− were calculated, using the Equation (2):D0 and RD are the diffusion coefficients for the ferricyanide (D0) and ferrocyanide (RD), ν is the scan rate (Vs−1), n is the number of electrons involved in the process, F is the Faraday constant (mol−1), T is the temperature (K), R is the universal gas constant (JK−1mol−1), and α the dimensional transfer coefficient  versus log [EP] (the logarithm of the substrate concentration), obtaining 0.89 for biochar/SPE biosensor (r2 = 0.990) and 1.01 for graphene/SPE biosensor (r2 = 0.999), respectively. The h parameter, calculated from the corresponding Hill’s plot, was close to 1, demonstrated that the kinetics of the enzymatic reaction fitted into a Michaelis–Menten kinetics model [S] is the concentration of the substrate, I the cathodic current at −0.32V, and maxI the steady-state current. Using the Lineweaver–Burk equation and representing 1/I versus 1/[EP], the apparent Michaelis-Menten constant was calculated from the slope and the maxI from the intercept.Using these results, the Hill coefficient (h) can be calculated as the log[cs model . The enzcs model :(4)1I=1IFrom the data shown in the To study the repeatability of Ty/biochar and graphene based SPEs, amperometric measurements for epinephrine detection were carried out with a 0.25 mM EP solution in PB, using the same biosensors devices for 10 times. The relative standard deviation (%RSD) for the Ty/biochar/SPEs was equal to 6%, while for Ty/graphene platform it was 4%.The stability of the biosensor was studied by monitoring the amperometric response of the same biosensor at regular intervals of 24 hours for 20 days. Between measurements, the biosensor was stored in a refrigerator at 4 °C in dry condition. The amperometric responses of the biosensor with and without biochar remained quite constant in the first 7 days of storage. In particular, biochar-based biosensor maintained 98.2% of its initial current response with a decrease to 87.6% after two weeks, while the Ty/graphene-SPE showed an initial 98.5%, decreasing to 92.2% after two weeks. After this time, the biosensor’s amperometric response constantly decreased, until 55.7% (biochar) and 75.6% (graphene) of the initial signal in 20 days. Therefore, both graphene and biochar-based platforms can be considered “ready to use” and stored at 4 °C within the first 7–10 days, obtaining good results for EP determination.In the present study, brewers’ spent grain biochar was investigated as an innovative, eco-friendly graphene-alternative for the production of screen-printed electrodes.From SEM investigation, it is evident that the biochar powder we produced was made of irregularly shaped macro- and micro-particles with a sponge-like structure, characterized by the presence of several pores of different sizes and many hollow channels, suitable for enzymes’ immobilization and electrochemical applications. Following this approach, the biochar obtained from spent grain was used to improve the performances of screen-printed electrodes through the modification of their surfaces with the proposed material by drop casting. Moreover, the good and uniform distribution of biochar on the SPE surface was demonstrated by observation at SEM, as well as the tyrosinase immobilization in the case of Ty/biochar/SPE. The Ty/biochar/SPEs exhibited similar characteristics to graphene/SPEs, widely used for biosensor development, in terms of the electron transfer kinetics for electroactive compounds. Large peak potential separation and high peak current could be obtained using CV on the developed electrode. Good sensitivity and detection limit for epinephrine promote the Ty/biochar/SPE to be an effective sensor for direct determination of this molecular target in real samples.


The identification of a heritable tumor predisposition often leads to changes in management and increased surveillance of individuals who are at risk; however, for many rare entities, our knowledge of heritable predisposition is incomplete.Families with childhood medulloblastoma, one of the most prevalent childhood malignant brain tumors, were investigated to identify predisposing germline mutations. Initial findings were extended to genomes and epigenomes of 1,044 medulloblastoma cases from international multicenter cohorts, including retrospective and prospective clinical studies and patient series.GPR161) gene in six patients with infant-onset medulloblastoma . GPR161 mutations were exclusively associated with the sonic hedgehog medulloblastoma (MBSHH) subgroup and accounted for 5% of infant MBSHH cases in our cohorts. Molecular tumor profiling revealed a loss of heterozygosity at GPR161 in all affected MBSHH tumors, atypical somatic copy number landscapes, and no additional somatic driver events. Analysis of 226 MBSHH tumors revealed somatic copy-neutral loss of heterozygosity of chromosome 1q as the hallmark characteristic of GPR161 deficiency and the primary mechanism for biallelic inactivation of GPR161 in affected MBSHH tumors.We identified heterozygous germline mutations in the G protein-coupled receptor 161 (GPR161 mutations and characterized by MBSHH in infants. Additional studies are needed to identify a potential broader tumor spectrum associated with germline GPR161 mutations.Here, we describe a novel brain tumor predisposition syndrome that is caused by germline  Thus, understanding the molecular function of these genes can lead to new therapeutic concepts in both sporadic and heritable tumors. In children and adolescents with cancer, tumor predisposition as a result of germline mutations is found in at least 7% to 10%.4 Medulloblastoma (MB), a tumor that originates in the cerebellum and dorsal brainstem, has a peak incidence in childhood and makes up a large proportion of embryonal brain tumors. Consensus molecular subgroups of MB include WNT, sonic hedgehog (SHH), group 3, and group 4, each showing distinct transcriptional and epigenetic profiles.5 Recent advances in deep molecular phenotyping suggest additional MB subtypes with distinct somatic driver mutations and epigenetic signatures.8 The SHH subgroup (MBSHH) is characterized by a constitutive transcriptional and genomic activation of the SHH pathway9; driver mutations in PTCH1, SUFU, SMO, TERT, DDX3X, TP53, and KMT2D; and structural aberrations that affect most frequently chromosomes 9, 10, and 17. Most MBs are thought to develop sporadically, but inherited forms also exist, most often in children with MBSHH.4 Heritable predisposition to MB is observed in Li-Fraumeni syndrome,10APC-associated polyposis,11 subtypes of Fanconi anemia,13 and Gorlin syndrome . The latter is associated with germline mutations in SUFU16 and PTCH1,18 which play crucial roles in the SHH pathway. Active SHH signaling is a key element of embryonic development and cell differentiation.19 Here, we describe the SHH regulator G protein-coupled receptor 161 (GPR161) as a novel brain tumor predisposition gene in children.Cancer predisposition syndromes are defined by germline mutations that result in a highly or moderately increased tumor risk in affected individuals. Knowledge of these syndromes has a profound impact on patient care and the prevention of malignant disease through the provision of genetic counseling and careful surveillance of families who are at risk.20Written informed consent was obtained from study participants after approval from the institutional review boards at the participating institutions (Uniklinik RWTH Aachen: EK302-16). Consent was obtained according to the Declaration of Helsinki. The study had access to international multicenter MB studies, including retrospective cohorts , and prospective cohorts from clinical studies or patient series .21Whole-exome sequencing (WES) of the index patient (M20769) was done at the Institute of Human Genetics, Uniklinik RWTH Aachen, using the Nextera Rapid Capture Exome kit . The library was sequenced on a NextSeq500 Sequencer with 2 × 75 cycles on a high-output flow cell. For sample MB11_06, WES data for germline and tumor samples were generated at the German Cancer Research Centre  using Agilent SureSelect Human All Exon V5  without untranslated regions and sequenced on an Illumina HiSEq 4000 System (paired-end mode). For sample MB13_03, whole-genome sequencing data for germline and tumor samples were generated at the German Cancer Research Centre and sequenced on the Illumina HiSeq X Ten System (paired-end mode). For samples SJMB335, SJMB303, and SJMB054, WES data for germline and tumor samples were generated at St Jude Children’s Research Hospital  using the Agilent SureSelect Human All Exon V5 kit. For the detection of single-nucleotide variations (SNVs), small insertions and deletions (indels), fusions, and copy number aberrations, the customized enrichment/hybrid capture–based next-generation sequencing gene panel analysis covering 130 genes of particular relevance in brain tumors was applied, as previously described.https://dockstore.org/containers/quay.io/pancancer/pcawg-dkfz-workflow). In brief, reads were aligned to the phase II reference human genome assembly of the 1000 Genomes Project, including decoy sequences (hs37d5), using BWA-MEM (version 0.7.15). Somatic SNVs were called with the previously described SAMtools-based German Cancer Research Centre pipeline adjusted for ICGC PCAWG settings, and short somatic indels were called using Platypus.23 The CNVkit library24 and ACEseq25 were used to infer and visualize somatic genome copy number from the WES and whole-genome sequencing data. Loss of heterozygosity (LOH) analysis for tumor-only panel sequencing data was based on known germline variants (1000 Genomes Project) in target and off-target regions, 20× sequencing coverage, and one or more read support for the reference and alternative allele. Germline genomes and exomes were analyzed using the freebayes (version 1.1.0) pipeline with ICGC PCAWG settings26 and deleterious germline variants—SNVs, multi-nucleotide variants, indels, and complex variants—were inferred using automated pipelines and manually curated. For sequencing data of the index case, demultiplexing and fastq file generation were performed using bcl2fastq2  and read alignment and variant calling using the automated SeqMule pipeline (v1.2.6).27 For germline variant detection in the index patient, different variant callers were used  and variants shared by at least one pair of variant callers were written to the final variant file. Variant annotation and bioinformatics prioritization were performed using KGGSeq .28 Synonymous variants and variants with a minor allele frequency greater than 0.75% in public databases, that is, gnomAD, ExAC, 1000 Genomes Project, and National Heart, Lung, and Blood Institute Exome Sequencing Project, were excluded.Sequencing data were processed using a standardized alignment and somatic variant calling pipeline, which was developed in the context of the ICGC Pan-Cancer Analysis of Whole Genomes (PCAWG) project  as previously described.29DNA from tumor tissue with tumor cell content greater than 80% by histopathologic evaluation was extracted from formalin-fixed, paraffin-embedded tissue using the automated Maxwell system with the Maxwell 16 formalin-fixed, paraffin-embedded Plus LEV DNA Purification Kit . We performed DNA methylation profiling of all samples using the Infinium MethylationEPIC (850k) BeadChip (Illumina) or Infinium HumanMethylation450 (450k) BeadChip (Illumina) array, as previously described.SHH subgroup. According to the revised WHO classification of tumors of the CNS 2016, the tumor classifies as desmoplastic/nodular, SHH-activated, TP53 wild-type MB (WHO grade IV).30 At age 16 years, the patient developed her first basal-cell carcinoma (BCC) and has subsequently gone on to develop 10 more BCCs, all within the radiation field and all amenable to surgical removal. By age 18, the patient underwent a total thyroidectomy for a multinodular goiter, and at 23 years old, the patient underwent the excision of a rectal tubular adenoma with low-grade dysplasia, a low-grade intraepithelial neoplasia in the stomach, and several hyperplastic serrated polyps. A meningioma in the right temporal region was removed at age 24. The patient, currently age 29 years, has a microcephaly and a mild frontal bossing and in summary fulfills some, but not all, criteria of Gorlin syndrome  with an extensive history of neoplasia that began with the diagnosis of MB at the age of 12 months. Histologic evaluation showed a highly cellular, undifferentiated small-cell neoplasm with increased mitotic activity. Cells had hyperchromatic nuclei and a scant cytoplasm. Silver impregnation demonstrated an increased density of argyric fibers and fiber-rich areas of ensheathed fiber-free islands of tumor cells. Cells expressed the neural marker MAP2 and the SHH target protein p75-NGFR . Consistsyndrome .4; no somatic copy-number alterations affecting MBSHH hallmark chromosomes 9, 10, and 17; and no somatic mutations in MBSHH driver genes . Two patients (MB11_06 and SJMB335) shared the same frameshift mutation as that of the index patient; kinship analysis demonstrated that all three patients were unrelated.To further corroborate the role of utations . Clinica20 revealed that all six patients with GPR161 mutations developed MBs that belonged to the SHH subgroup  and infant (age < 4 years) patients with MBSHH was 3.4% (six of 178) and 5.5% (five of 91), respectively  and prospective  pediatric MBSHH cohorts (GPR161 (3.4%), PTCH1 (4.5%), and SUFU (5.6%) was comparable among pediatric patients with MBSHH.Classification of 872 human MBs into four consensus MB subgroupsop MBSHH . Overallectively . GPR161  cohorts . Of noteSHH into TP53 mutated and wild type, respectively.31 We assessed TP53 mutation status across all MBSHH and observed that all GPR161-associated MBSHH were TP53 wild type and represented 4.1% (six of 147) of pediatric patients with MBSHH,TP53-wt. Recently, MBSHH tumors have been further classified into four molecular subtypes.8 These include two infant subtypes SHH-γ (low risk) and SHH-β (high risk), which are also equivalent to infant-SHH-I and infant-SHH-II in another study.32 Classification of these infant GPR161-associated MBSHH tumors into molecular subtypes demonstrated that the majority (five of six) belonged to the MBSHH-β subtype; however, this was not statistically significant when considering all infant patients with MBSHH in our series (P = .09). Finally, germline GPR161 protein-truncating variants were significantly enriched in pediatric patients with MBSHH compared with the general population .The latest edition of the WHO CNS tumor classification divides MBGPR161 germline mutation carriers revealed LOH at the GPR161 locus, unexceptionally leading to loss of the wild-type allele in all affected patients . Patient SJMB335 had a somatic SUFU missense mutation, but clonal evolution analysis did not provide evidence for an SUFU-associated tumor as a result of low mutation frequency. Finally, the MB of the index patient showed low-level copy number gain at the GLI2 locus, but this was not a true gene amplification and the tumor lacked a loss of 17p and a TP53 mutation that usually accompanies a prototypical MB with GLI2 amplification. In summary, biallelic GPR161 inactivation occurred in all six patients with MB that otherwise did not harbor any other driver events that affect the SHH pathway , which suggests that 1q cnLOH is a hallmark for GPR161 deficiency and a molecular marker for the identification of patients with GPR161 mutations.Remarkably, five of six patients with MB demonstrated an inactivation of n status . This anGPR161 reported here developed MBSHH before the age of 3 years. Overall, we estimate that GPR161 mutations are responsible for approximately 5% of infant MBSHH cases. All GPR161 mutations that were identified within our patients with MBSHH were also found to be present at a low carrier frequency in the general population , and analysis of two available parent-offspring trios (M20769 and MB13_03) demonstrated parental transmission in both affected patients. Of note, the frequency of germline GPR161 protein-truncating variants in the general population is six in 10,000 individuals, and extrapolated across the global population one would anticipate approximately four million people worldwide to be affected (Data Supplement).Recognition of tumor predisposition syndromes helps to optimize tumor surveillance programs and to improve clinical outcome. Recent progress in high-throughput genomics and access to larger patient cohorts enables the identification of novel tumor predisposition syndromes. In the current study, we have identified a novel tumor predisposition syndrome with SHH-activated and early-onset MB as the primary clinical manifestation. Carriers of a protein-truncating germline mutation in GPR161 mutations (genomic location: 1q24.2) showed recurrent LOH of GPR161 as a second hit in the tumor. This primarily resulted from segmental cnLOH of 1q, an event that is completely absent among GPR161 wild-type MBSHH and can thus be considered a hallmark characteristic of GPR161-associated MB. cnLOH and the unmasking of germline mutations have previously been shown to be of importance in the inactivation of tumor suppressor genes and are described in a variety of different tumor types.33 The 1q cnLOH event most likely originates from mitotic recombination as a result of DNA double-strand breaks that necessitate the second, homologous chromosome as a template for DNA repair. Such double-strand breaks tend to be more frequent at common fragile sites, which are prone to breakage upon DNA replication stress.34 A common fragile site (FRA1F) is also present at the 1q cnLOH breakpoints in GPR161-associated MB (1q21.1 to 1q21.3). Moreover, the lack of 1q loss events among GPR161 wild-type MBSHH tumors and the preferential mode of GPR161 inactivation, either focal or via cnLOH, suggests that 1q harbors dose-sensitive genes that might restrict MBSHH tumor development. Future studies could therefore explore whether genes on 1q might serve as a substrate for novel treatment options based on synthetic lethal interactions. Of note, analysis of somatic mutations in known and putative MB driver genes revealed biallelic inactivation of GPR161 as the sole shared event across the six patients, indicating that loss of GPR161 is sufficient for tumor growth, which is consistent with results of recent Gpr161 knockout studies in mice.35Patients with truncating germline 38 GCs of the cerebellum are by far the most numerous neurons in the brain and MBSHH originates from their progenitors (GC progenitors [GCPs]).39 Proliferation of GCPs is critically regulated by the mitogenic activity of the SHH ligand. SHH secretion from cerebellar Purkinje cells at late stages of embryonic development boosts the postnatal mitotic rate of GCPs before differentiated cells exit the cell cycle to become postmitotic neurons.40 Consequently, constitutive activation of SHH signaling increases the proliferation rate and neoplastic transformation of GCPs.41 As Gpr161 acts as a negative regulator of the SHH pathway37 and prevents GCP overproduction, a loss of GPR161 activity is consistent with MB pathogenesis, which was recently demonstrated in a mouse model with a neural stem cell–specific Gpr161 deletion.35 The timing of the GPR161–SHH interplay at the stage of GPC differentiation is in line with a vulnerable phase for MB development in early childhood. Similar to PTCH1- and SUFU-associated MBSHH in Gorlin syndrome, patients in this study were typically diagnosed with MB during infancy.GPR161 function is essential for several aspects of embryonic development and, among other things, is also relevant for granule cell (GC) proliferation.GPR161 germline mutation, has died of an adenocarcinoma of the colon at age 55 years. In a second family (SJMB335), both the maternal aunt and grandmother had ovarian cancer, but were not available for additional genetic testing. Other potential manifestations of GPR161-associated tumors are clearly an avenue for additional studies, not least because of their fundamental importance for genetic screening programs. In summary, the GPR161 disorder defines a novel MB predisposition syndrome.In our study, patients were mainly children and other tumors could potentially occur at later stages of life. The oldest patient of the study  developed recurrent BCCs and a meningioma at age 24 in the radiation field. She also developed multiple hyperplastic GI polyps and a tubular adenoma with low-grade dysplasia. Her father, likewise a carrier of the


This study aims to propose a capacitance-to-digital converter (CDC) based on a third-order cascade of integrators with a feed-forward (CIFF) incremental sigma-delta modulator for smart humidity sensor application. Disguised zoom-in technology was proposed to enlarge the measurable range of the CDC. The input range of the CDC was 0–388 pF. The proposed CDC was realized using 0.18 μm complementary metal-oxide-semiconductor technology. Results show that the CDC performs a 13-bit capacitance-to-digital conversion in 0.8 ms. The analog system consumes 169.7 μA from a 1.8 V supply, which corresponds to a figure of merit (FOM) of 3.0 nJ/step. The proposed CDC was combined with a HS1101 humidity sensor to demonstrate its incorporation in an overall system design. The resolution was 0.7% relative humidity (RH) over a range of 30%–90% RH. Relative humidity is an important factor in industrial processes, human health, and preservation of perishable food and cultural relics ,2. It isA low-cost thermal-conductivity-based humidity sensor has been previously reported , having A capacitance-to-voltage front-end CDC has been proposed  which reA time-based CDC that performs capacitance-to-time conversion followed by time-to-digital conversion has also been reported . HoweverHowever, these sensors did not meet the market’s requirements of low power consumption, large capacitance input range, and fast measurements.Herein, a novel CDC is proposed to address the abovementioned issues. A new method called disguised zoom-in technology was employed to enlarge the measurable range to 0–388 pF. Low power consumption and a short conversion time were also focused on in the design. The proposed CDC’s total power consumption was 305.46 uW, and the measurements only took 0.8 ms. The measurable relative humidity range was 30%–90%.The proposed CDC combined with a humidity sensor system could be applied to the industrial production of a relative humidity controller and to monitor humidity in daily life.This article is organized as follows. eq represents the capacitance to be measured and Cref denotes the reference capacitance. The CDC structure is essentially a charge balance that uses a sigma-delta modulator. A third-order modulator, as shown in DD charges Ceq first. It is determined by the previous bitstream such that Vref or −Vref charges Cref. When the previous bitstream is positive, −CrefVref is added to CeqVDD. Otherwise, CrefVref is added to CeqVDD. The two parts of the charge are added to each other and transmitted to an integrator, the output of which flows to the comparator, generating a second bitstream. The polarity of the first bitstream after the reset does not impact the final result substantially.Let us assume that the modulator has completed the reset operation; thus, it is ready to perform a new round of measurements. During each conversion cycle, VThe integrators of the modulator are reset before each measurement to prevent interference from any residual charge from the previous conversion ,12. The entire charge balancing process can be represented by Equation (1), where μ is expressed by Equation (2).eq. Then, CDC superimposes the charge that is controlled by the weight of the bitstream to balance it. The residual charge is sent to the modulator until it is finally balanced.According to the above description of CDC operation, the CDC first converts the capacitance into the charge via charging Ceq to Cref. Thus, Ceq can be calculated, as long as the density of the bitstream is processed, according to Equation (2).The CDC uses the ratiometric measurement principle for ADC conversion. The density of the bitstream is the ratio of CThe output bitstream was saved in the form of a text document using the Data Acquisition (DAQ) Assistant and then processed in MATLAB R2013b.The disguised zoom-in module and sigma-delta modulator are described in detail below.Humidity capacitance sensors were implemented off-chip because of process limitations. CDC was implemented on-chip. The HS1101 relative humidity sensor was adopted as the humidity capacitor. The humidity sensitive capacitor can operate between −40 °C and 125 °C, and the range of relative humidity operation is 0%–100%. The polynomial response is presented by Equation (3) . The humidity sensitive capacitor has a capacitance of 162–199.44 pF at a relative humidity of 0%–100% at 25 °C. As the dynamic capacitance of the sensor is 37.44 pF and the baseline capacitance is as large as 162 pF, it exceeds the measurement range of most CDCs reported. Therefore, to enable normal measurement, the disguised zoom-in technique was proposed.The disguised zoom-in technology contains two important processes.sensor denotes the humidity sensor, and R1 and R2 are resistors for voltage proportional amplification. Cin is the port where the CDC accesses the capacitance to be tested. VA and VB are used to charge the capacitance to be measured for the subsequent charge balancing of the CDC. The output voltage of the op amp is VC.The baseline capacitance and dynamic capacitance are both beyond the CDC measurable range; therefore, the humidity sensitive capacitor must first be scaled, which is the first step in disguised zoom-in technology. This process is shown in B is the inverse signal of VA, and Equation (4) can be simply obtained by including the character of a virtual short in the op amp. Note that Vx represents the equivalent capacitance after being scaled.Equation (6) can be obtained using the principle of conservation of charge. Therefore, the equivalent capacitance can also be expressed by Equation (7), where Cx, in parallel with the compensation capacitor, Coff. Using zoom-in technology, the adverse effects of baseline capacitance can be eliminated. This process is shown in The zoom-in technology connects the capacitor to be tested, Ccharge. During the sample phase, x, and the compensation capacitor, Coff, are connected in parallel to the interface. Equation (8) is obtained using the principle of conservation of charge, wherein the equivalent capacitance to CDC is given by Cx − Coff. Ceq represents the final equivalent capacitance in the disguised zoom-in technology and the result is shown in Equation (9).During the integrating phase, off, is controlled by a binary toggle switch, where the toggle has been designed off-chip. Through the four toggle switches of S0000–S1111, Coff can be controlled between 0–7.5 pF using individual steps of 0.5 pF.The compensation capacitor, CThe baseline capacitance of the humidity sensitive capacitor is eliminated in the second process of the disguised zoom-in technique. In addition, the scaling process of the disguised zoom-in technique further scales the dynamic capacitance of the humidity sensitive capacitor such that the entire dynamic range falls within the input range of the CDC.As the capacitance of the humidity sensor changes relatively slowly with ambient humidity and the capacitance is converted to the form of a charge input, the input amount is approximately direct current (DC) voltage for the ADC. Therefore, an incremental sigma-delta ADC with a third-order cascade of integrators and a feed-forward structure was selected using a one-bit quantizer ,15.eq represents the humidity sensor capacitance after application of the disguised zoom-in technology. Cref denotes a reference capacitor used in the design. Some quantization errors are generated during the quantization process; these are marked as “Error” in In The signal transfer function and noise transfer function are denoted as STF(z) and NTF(z), respectively. Equations (10) and (11) can be easily obtained by inspecting 1, a2, and a3 are 0.16, 0.5, and 0.2, respectively.Usually, the zeros of the noise transfer function are concentrated at z = 1. If the zeros of the noise transfer function are evenly distributed in the signal baseband by some mathematical software, a better attenuation effect on the quantization noise can be achieved ,17. WithTo achieve a higher resolution in the case of the ADC, the architecture and its corresponding coefficients need to carefully be considered and the specific circuits that constitute the internal structure must carefully be designed. A good circuit structure can improve accuracy, reduce power, and increase speed. Therefore, the circuits that constitute the ADC are discussed in detail in this section.The first-stage op amp that constitutes the CDC must withstand operation at very high or low voltages, which are caused by the initial input voltage when the capacitance to be tested is large or the charge-balancing circuit starts to work and the feedback negative voltage is too large. This results in a higher or lower input voltage of the first-stage integrator. Therefore, we use rail-to-rail inputs in our design to accommodate such harsh conditions. For the output of the first-stage op amp, a class AB structure is adopted, which can improve the current-utilization efficiency. The specific circuit of the first-stage op amp is shown in The performance requirements of the second- and third-stage op amps are not as high as those of the first-stage system, so the power consumption is reduced under the premise of satisfying the basic performance (medium gain and transconductance). A current-starved op amp, which greatly reduces power consumption, is used . The corThe first-, second-, and third-stage op amps consume 161, 0.8, and 0.4 µA. The reason why the first-stage op amp consumes the most current is that a larger bandwidth and higher transconductance are required to quickly establish the integrator signal. The second- and third-stage op amps consume less current because the requirements for the op amp are relaxed and the current-starved op amp improves the current utilization efficiency.For the comparator design, a combination of a preamplifier and static latch is used. The preamplifier can pre-amplify the signal to be compared, thus increasing the speed of comparison. The use of preamplifiers with latches prevents kickback noise . Using a2. To flexibly handle and reduce the area occupied by the digital circuit, the decimation filter was implemented off-chip.The proposed CDC for a smart humidity sensor was implemented in 0.18 µm CMOS technology provided by Semiconductor Manufacturing International Corporation (SMIC). The CDC is powered by a 1.8 V supply voltage with a sampling frequency of 250 kHz. The analog circuit consumes 169.7 µA of current. The measurement time of the CDC is 0.8 ms. The resolution of the CDC is 13 bits. Furthermore, the figure of merit (FOM), which is defined in Equation (12), is 3.0 nJ/step. In similar studies ,21, the Fast Fourier transform was performed on the bitstream acquired in the text. The output spectrum is shown in off, the input range could be adjusted. Using the proposed disguised zoom-in technology the whole input range was enlarged to 0–388 pF.By controlling the capacitance of CThe packaged chips were placed in a climate chamber for testing to quantitatively analyze the performance of the design. Owing to the deviation in the foundry process, a slight deviation is still observed in chips from the same batch. Calibration through the digital back-end can reduce the influence of the deviation to some extent . Under certain relative humidity, the capacitance can be obtained using Equation (3). The relationship between Dout and capacitance as long as relative humidity is shown in The accuracy of the capacitance measured by the CDC will affect the accuracy of the relative humidity after conversion. The comparison between the measured and actual relative humidity has been done. As listed in Herein, a CDC for a humidity sensor was proposed, wherein the use of disguised zoom-in technology enlarged the measurable range of the CDC. The error in the relative humidity was also minimized to a value between +3.2% and −2.7%. Thus, the proposed CDC combined with a humidity sensor system is applicable to a wide range of humidity monitoring applications.


Pulmonary sarcoidosis involves the deposition of granulomas within the lung. These granulomas may affect lung function and lead to pulmonary symptoms, pulmonary dysfunction, functional impairment, and worsening of quality of life. Corticosteroids are generally highly effective in resolving the granulomatous inflammation of sarcoidosis. However, despite the effectiveness of corticosteroids, many corticosteroid-responsive patients continue to experience significant problems because of the development of fibrosis from previously active or active smoldering granulomatous inflammation, inflammatory effects from sarcoidosis unrelated to granuloma deposition in lung tissue (parasarcoidosis syndromes), and the development of significant corticosteroid-related side effects. For these reasons, the decision to treat pulmonary sarcoidosis and endpoints to measure meaningful outcomes may extend beyond considerations of pulmonary granulomatous inflammation alone. In this article, we propose a conceptual framework to describe the mechanisms by which pulmonary sarcoidosis significantly impacts patients. This conceptual framework suggests that indications for the treatment of pulmonary sarcoidosis and endpoints to assess treatment depend on the specific mechanisms that are causing functional or quality-of-life impairment (or both) in patients with pulmonary sarcoidosis. We believe that these concepts are important to clinicians treating pulmonary sarcoidosis and to clinical researchers designing pulmonary sarcoidosis trials. The decision to treat pulmonary sarcoidosis is complex because (a) the disease may cause neither symptoms nor permanent organ damage2; (b) a small percentage of cases, maybe 10 to 20%, are progressive or cause fibrosis  or both3; (c) there are no reliable biomarkers to predict whether the natural course of pulmonary sarcoidosis will be benign or lead to severe impairment4; and (d) treatment of pulmonary sarcoidosis with corticosteroids and other therapies is associated with significant toxicity5. These issues force clinicians, clinical researchers, and pharma to carefully select appropriate pulmonary sarcoidosis patients who have the potential to receive impactful benefits from interventions and to select the appropriate methods to assess therapeutic responses. Clinical trial design for pulmonary sarcoidosis has been problematic, and major trials have been criticized as not adequately accounting for the natural history of the disease in order to determine the benefits of study drugs6. This article will explore the indications for treatment of pulmonary sarcoidosis and methods to assess the effects of therapy. This exercise will involve the construction of a proposed conceptual framework outlining how pulmonary sarcoidosis leads to outcomes that are clinically impactful to patients or clinicians or both. This conceptual framework will suggest that patients in pulmonary sarcoidosis trials require partitioning into disease profiles that each require distinct clinical endpoints to optimally assess the effects of therapy.Sarcoidosis is a multisystem granulomatous disease of unknown cause. Granulomas consist of conglomerations of inflammatory cells, predominantly lymphocytes and macrophages. In sarcoidosis, these granulomas may deposit in any organ but most commonly develop within the lung, where they are found in more than 90% of sarcoidosis cases7.Because sarcoidosis may affect any organ in the body and its severity is highly variable, it is problematic to define specific criteria for treatment. Most sarcoidosis clinicians agree with Athol Wells’s distillation of the treatment indications for sarcoidosis to just two: situations of danger and significant quality of life (QOL) impairment (“Wells’s law”)9. Interestingly, the treatment of most of these dangerous situations involves therapy other than anti-granulomatous therapy, although most clinicians and sarcoidosis drug trials focus on obliterating the sarcoid granuloma.Sarcoidosis is a systemic disease, and situations of danger are not confined to the lung. Cardiac sarcoidosis may cause sudden death, eye involvement may result in blindness, and neurosarcoidosis may result in coma and permanent neurologic impairment. In the case of pulmonary sarcoidosis, situations of danger develop in a minority of patients and are listed in10. Mechanisms proposed to be involved in the development of sarcoidosis-associated fibrosis include alveolar macrophage-induced fibrosis11, transition from a T helper 1 to T helper 2 signature12, transforming growth factor-beta13, upregulation of profibrotic genes that may “transmit” signals for fibrosis through the blood compartment14, and M2 polarization of alveolar macrophages15.The immunopathogenesis of fibrosis in sarcoidosis is not well understood. It is not known whether the fibrosis is triggered by profibrotic inflammatory events, by an inherent predisposition toward fibrosis in a subset of patients, or by an exaggerated wound-healing response to uncontrolled, chronic inflammation16. This conjecture is further supported anatomically through chest imaging studies17  positron emission tomography demonstrated significant pulmonary FDG uptake in 22 (85%) of 26 patients with fibrotic pulmonary sarcoidosis19. These data suggest that the granulomatous inflammation of sarcoidosis and the development of fibrosis often go hand in hand20. The amount of fibrosis that develops is variable, from negligent to copious amounts. As previously mentioned, a significant fibrotic response from the granulomatous inflammation of sarcoidosis occurs in, at most, only 20% of cases9. Herein lies a key dilemma in preventing the granulomatous-induced fibrosis of sarcoidosis: since there is no available biomarker to predict which 10 to 20% of patients with pulmonary sarcoidosis will develop significant fibrosis, does the clinician (a) treat all pulmonary sarcoidosis with anti-granulomatous therapy that will prevent the development of fibrosis in 10 to 20% but subject 80% to 90% to potential drug toxicities from corticosteroids and other medications or (b) withhold anti-granulomatous therapy that will result in serious complications in the 10 to 20% who develop fibrosis? Clearly, a reliable biomarker that can accurately predict the development of significant pulmonary fibrosis from sarcoidosis is an unmet need. Effective anti-fibrotic therapy would also be very useful for this form of sarcoidosis, although none is available at present.It is conjectured that the fibrosis in pulmonary sarcoidosis results from the granulomatous inflammation. This conjecture is supported histologically where the majority of the fibrosis from sarcoidosis develops within or around the granuloma, resulting in a “hyalinized granuloma”dies17  as well 21. One could argue that sarcoidosis therapy is used primarily to improve physiology or organ function impaired by granulomatous inflammation or to decrease the granuloma burden , but a critical examination of this issue suggests that this is not the case22. Sarcoidosis-induced granulomatous inflammation may not cause a significant physiologic derangement or result in a significant impairment of QOL23. A common clinical situation where this is the case is with asymptomatic bilateral hilar lymphadenopathy24. Even when the granulomatous inflammation of pulmonary sarcoidosis results in physiologic abnormalities, they are often minor25 and do not invariably lead to appreciable symptoms25. In addition, the correlation between pulmonary dysfunction and pulmonary symptoms is poor in pulmonary sarcoidosis27. Therefore, the presence of granulomatous inflammation or mild physiologic derangements from pulmonary sarcoidosis does not mandate anti-sarcoidosis therapy unless the patient’s QOL is significantly impaired or significant organ dysfunction occurs22.Because the dangerous situations associated with pulmonary sarcoidosis are not common, the more common of the two indications suggested by Wells’s law to treat pulmonary sarcoidosis is sarcoidosis-induced impairment of QOL. A survey of 1842 European patients with sarcoidosis found that QOL and functionality were judged as the most important disease outcomes29. In most instances, they work quickly and reliably. However, they are associated with a number of potential side effects that may impair QOL. These include weight gain, mood swings/psychological disturbances, diabetes, osteoporosis, hypertension, glaucoma, cataracts, edema, acne, increased skin and vascular fragility, and increased risk of infection. Corticosteroid therapy has been clearly linked to the development of significant toxicities in patients with sarcoidosis30. If the clinician focuses primarily on reducing the granuloma burden or improving physiology, a medication-induced impairment of QOL may be undetected such that the clinician assesses the patient as improved while the patient feels worse off. This scenario may be relatively common, as it has been shown that those receiving corticosteroids at three major university sarcoidosis clinics in the US had statistically and clinically significant reductions in health-related QOL (HRQOL) compared with sarcoidosis patients not receiving corticosteroids27, and those receiving more than 500 mg or prednisone equivalent per year had a statistically and clinically worse HRQOL than those on a lower total yearly dose31.QOL assessment in pulmonary sarcoidosis is also significantly impacted by the potential toxicity of therapy, particularly corticosteroids. Corticosteroids are the drug of choice for the treatment of pulmonary sarcoidosis32. The HRQOL status of a patient may be assessed informally through unstructured historical questioning. Such an assessment may be thorough and used to make treatment decisions, but in the reality of the current era of patient care where time is compressed, such a detailed evaluation rarely takes place22. For this reason, many have advocated that the assessment of HRQOL in patients with sarcoidosis be assessed by patient-reported outcome measures (PROs), which are patient-administered reports of how they function or feel about their health condition and the effects of therapy34. HRQOL PROs provide a quantitative assessment of QOL and functional status in order to assess potential therapeutic effects. In addition, HRQOL PROs may be performed outside of patient clinic visit time and even serially between visits to assess the effects of therapy. However, there are several limitations of using HRQOL PROs on which to base pulmonary sarcoidosis treatment decisions. First, although some sarcoidosis disease–specific PROs have been developed and validated37, most HRQOL PROs used in patients with sarcoidosis are derived from other disease cohorts. However, an HRQOL PRO derived from non-sarcoidosis cohorts may be used in sarcoidosis provided that there is no plausible rationale that patients with sarcoidosis would assess the trait being measured in a different way than the cohort in which the PRO was validated23. Second, although PROs can reliably assess HRQOL in large clinical cohorts, most PROs have inadequate resolution to be useful in individual patients. PROs developed with modern methodology, such as item response theory (IRT), may have adequate resolution to accurately assess HRQOL in individuals40. Finally, QOL and HRQOL are not synonymous, as many high-priority QOL issues do not pertain to a medical condition or its treatment. It is often problematic to distinguish HRQOL issues from non-health-related QOL issues using HRQOL PROs. Because of these concerns, the role of HRQOL PROs in determining treatment decisions for individual patients with pulmonary sarcoidosis has not been standardized. There is uniform agreement that the clinician should not make treatment decisions in isolation without some insight into the patient’s HRQOL status, preferences, goals, and wishes.The assessment of QOL in pulmonary sarcoidosis is problematic. HRQOL refers to the extent that the physical, social, mental/emotional, cognitive, or spiritual domains (or a combination of these) are affected by a medical condition or its treatment41, and they may be responsible for significant QOL and functional impairment. Examples of parasarcoidosis syndromes are erythema nodosum42, small fiber neuropathy43, fatigue syndromes44, pain syndromes45, cognitive decline46, and vitamin D dysregulation47. It has been postulated that these syndromes result from systemic release of mediators associated with the granulomatous inflammation of sarcoidosis48. Although these parasarcoidosis syndromes may not result in appreciable pulmonary symptoms, they may be the result of the granulomatous inflammation from pulmonary sarcoidosis. Parasarcoidosis syndromes may respond to anti-granulomatous therapy50, although the decision to use such therapy must weigh the benefits versus the side effects of treatment in relation to the impact of the parasarcoidosis syndrome22. However, the etiology of parasarcoidosis syndromes is probably more complex than simply a result of granulomatous inflammation, as there may be several non-granulomatous contributions to some of these disorders, such as pain, fatigue, and cognitive impairment syndromes related to the psychological impact of the presence of a chronic disease or decreased function/mobility or both52.Sarcoidosis may also cause symptoms or organ dysfunction that is not related to tissue deposition of granulomas or the development of fibrosis. These entities are collectively known as parasarcoidosis syndromesWe have proposed a conceptual framework of how pulmonary sarcoidosis impacts patients through the relationships of granulomatous inflammation, physiologic impairment, fibrosis, parasarcoidosis syndromes and the impact of anti-sarcoidosis therapy . This coThis conceptual framework is directed at immunologic and physiologic factors of pulmonary sarcoidosis that impact patient health. It reflects relevant issues concerning the evaluation of these drugs in clinical pulmonary sarcoidosis trials, including the establishment of study entry criteria and clinically relevant endpoints. However, issues concerning the management of patients with pulmonary sarcoidosis extend beyond these immunologic and physiologic factors. For example, psychosocial and socioeconomic concerns are important considerations of therapy. Such concerns include the cost of care, including medication costs, access to care, social support, employment issues, emotional and psychological issues, and ease in maintaining a healthy lifestyle.53. Obviously, anti-granulomatous therapy is a rational approach to the treatment of this condition. As depicted by the black arrows inAcute pulmonary sarcoidosis is a form of the disease that causes worsening pulmonary symptoms and dysfunction over weeks to months and is thought to result from the formation of new granulomas within the lung or growth of existing pulmonary granulomas58. However, as previously mentioned, because the granulomatous inflammation of sarcoidosis may not progress or even spontaneously resolve without therapy and therapy is associated with significant toxicity, treatment of acute pulmonary sarcoidosis also requires that the condition has resulted in a significant impairment of QOL.Obviously, patients with pulmonary sarcoidosis should be considered for anti-granulomatous therapy only if they have evidence of active granulomatous inflammation.59.The endpoints for the treatment of acute untreated pulmonary sarcoidosis should include all three of the following: (a) improvement/resolution of granulomatous inflammation, (b) improvement in pulmonary physiology, and (c) improvement in function status or QOL or both. The rationale for this is explained in60.61. For pulmonary sarcoidosis, these corticosteroid-sparing agents include anti-metabolites such as methotrexate63, azathioprine65, and the tumor necrosis factor-alpha (TNF-α) antagonists infliximab56 and adalimumab66. These agents usually are used as second- or third-line agents that are added on to corticosteroid therapy because they either take weeks to months longer than corticosteroids to be effective65 or—in the case of the TNF-α antagonists—are much more expensive than corticosteroids.Because corticosteroids are highly efficacious for pulmonary sarcoidosis, additional anti-granulomatous agents are often used as corticosteroid-sparing agents in order to lower the corticosteroid dose requirement. Additional agents can also be used to provide further anti-granulomatous therapy on top of corticosteroids, although often the additional benefit of such agents is minimal in pulmonary sarcoidosis patients receiving more than 10 to 15 mg of prednisone per day56.Entry criteria for anti-granulomatous therapy trials for patients with chronically treated pulmonary sarcoidosis should include evidence of granulomatous inflammation, physiologic impairment, and functional/QOL impairment at study entry or—if a corticosteroid withdrawal phase of the trial is planned—historical evidence of these abnormalities when the corticosteroid dose is lowered.For these reasons, the endpoints of trials for acute untreated pulmonary sarcoidosis are not highly relevant to evaluate anti-granulomatous drugs used in chronically treated pulmonary sarcoidosis patients. There is often little granulomatous inflammation left to treat, and the issue is whether the drug will continue to suppress granulomatous inflammation when the corticosteroid dose is lowered. The same could be said for the physiologic endpoint: the issue is often not whether the additional drug will improve pulmonary physiology but rather whether it will allow the pulmonary physiology to be maintained as the corticosteroid dose is lowered. The QOL endpoint is still highly relevant, especially in terms of a reduction of corticosteroid side effects.As previously mentioned, it is conjectured that the fibrosis in sarcoidosis is the result of granulomatous inflammation. The relationship of granulomatous inflammation, pulmonary fibrosis, and treatment of pulmonary sarcoidosis is depicted in68, bronchiectasis70, and pulmonary mycetoma72. The indications for treatment of these conditions as well as the endpoints to monitor these forms of disease are well covered elsewhere75. Anti-granulomatous therapy is rarely useful for sarcoidosis-associated pulmonary hypertension and is often detrimental for bronchiectasis and pulmonary mycetoma as it may cause serious infectious complications. As end-stage pulmonary fibrosis is a disabling and life-threatening condition, lung transplantation should be considered in fibrotic pulmonary sarcoidosis patients with limited chance for improvement and minimal extrapulmonary comorbidities76.Therapy for fibrotic pulmonary sarcoidosis is currently aimed at either (a) treating the complications of pulmonary fibrosis or (b) preventing the progression of further lung fibrosis. The complications of pulmonary fibrosis are listed in19. It is thought that such “smoldering” granulomatous inflammation is the nidus for the development of further fibrosis. Therefore, evidence of granulomatous inflammation would be a prerequisite for instituting anti-granulomatous therapy for fibrotic pulmonary sarcoidosis.In terms of therapy to prevent the development of further fibrosis, it has already been mentioned that the majority of patients with fibrotic pulmonary sarcoidosis have evidence of granulomatous inflammation79.An additional issue when considering treatment of sarcoidosis-associated pulmonary fibrosis is the rapidity of its development. It is thought that fibrosis develops slowly in pulmonary sarcoidosis, such that the cumulative toxicities of long-term anti-sarcoidosis medications, especially corticosteroids, would limit their long-term use. Certainly, evidence of rapid development of pulmonary fibrosis with a concomitant rapid decline in physiology or functional status/QOL would be a strong impetus to consider anti-granulomatous therapy. Because of the toxicity of corticosteroids and other anti-granulomatous therapies, another approach would be to use “downstream” medications targeting the development of fibrosis itself. Such medications include pirfenidone and nintedanib, which have been shown to decelerate the development of lung fibrosis in idiopathic pulmonary fibrosis and other interstitial lung diseases57. The ablation of granulomatous inflammation may improve QOL by reducing symptoms derived from parasarcoidosis syndromes that may be indirectly derived from granulomatous inflammation (see below). However, other useful endpoints for the treatment of fibrotic pulmonary sarcoidosis may reflect a lack of deterioration of patient status.In terms of endpoints for the treatment of fibrotic pulmonary sarcoidosis, improvements in pulmonary function and functional status/QOL from anti-granulomatous therapy may be significant yet are unlikely to be large because improvement in the patient’s pulmonary function is unlikely to be greatThe physiologic impairment resulting from the granulomatous inflammation of pulmonary sarcoidosis is highly variable. Even more variable is the effect of these physiologic derangements on the QOL of the patient with pulmonary sarcoidosis. This granulomatous inflammation can also adversely affect the QOL of the patient with sarcoidosis by systemic mechanisms other than direct deposition of granulomas into specific organs (“parasarcoidosis syndromes”). Additionally, this granulomatous inflammation may lead to the development of pulmonary fibrosis that causes irreversible pulmonary dysfunction and, potentially, disabling and life-threatening situations. Finally, anti-granulomatous therapy not only may improve the QOL of the patient with pulmonary sarcoidosis by effectively treating the disease but also may worsen it by causing serious drug side effects. We have proposed a conceptual framework to outline how these many factors interact to cause physiologic and QOL impairments in pulmonary sarcoidosis. We believe that this conceptual framework may be useful in constructing a clinical drug trial for pulmonary sarcoidosis that reflects the concerns of patients and clinicians.Because of numerous aspects of pulmonary sarcoidosis and its therapy, it is not surprising that developing treatment indications and endpoints for the therapy of the disease is challenging. We have made a case for partitioning patients into three treatment groups when considering indications for therapy as well as therapy endpoints: untreated patients with active granulomatous inflammation, patients with chronically treated disease, and those with significant lung fibrosis. It is hoped that this proposed patient partitioning will stimulate debate and constructive criticism that will eventually lead to therapy for pulmonary sarcoidosis that is better directed in treating the true concerns of patients.


This article has been retracted due to the authors' unlicensed useof The Morisky Medication Adherence Scale, otherwise known as the Morisky Scale(MMAS-8), which was copyrighted in 2006.  Additionally, the scoring and coding of the MMAS-8is incorrect, thus invalidating the results.