Graphene and its derivatives have been a burning issue in the last 10 years. Although many reviews described its application in electrochemical detection, few were focused on food detection. Herein, we reviewed the recent progress in applying graphene and composite materials in food detection during the past 10 years. We pay attention to food coloring materials, pesticides, antibiotics, heavy metal ion residues, and other common hazards. The advantages of graphene composites in electrochemical detection are described in detail. The differences between electrochemical detection involving graphene and traditional inherent food detection are analyzed and compared in depth. The results proved that electrochemical food detection based on graphene composites is more beneficial. The current defects and deficiencies in graphene composite modified electrode development are discussed, and the application prospects and direction of graphene in future food detection are forecasted. It has Graphene oxide (GO), an oxygen-containing derivative of graphene, belongs to a branch of graphene research. Generally, graphene oxide is a material obtained by the multistep chemical or thermal reduction of graphene  Dikin e. ChemicaReduced graphene oxide (rGO) is an ideal support material for metal nanoparticles, which can be produced at a low cost and large scale. rGO sheets possess higher conductivity than GO sheets due to restoring the conjugated network in the rGO sheets. The oxygen-containing functional groups on rGO render it with an extremely high specific surface area, superior electronic conductivity, excellent mechanical strength, and elasticity . rGO is Graphene quantum dots (GQDs) are attractive nanomaterials consisting of a monolayer, or a few layers, of graphene with excellent and unique properties. Unlike graphene sheets, GQDs are 0D graphene segments that exhibit bandgap responsible for their unique optical and electrical properties. Due to their small size, GQDs display a quantum effect when produced using carbon-rich precursors such as fullerene, glucose, graphite, graphene oxide (GO), CNTs, and carbon fibers (CFs). Two main GQD synthesis methods are followed, namely, top–down and bottom–up methods, as shown in 2 substrate. Their research showed that the resulting graphene structure was more homogenous than when using other methods.Graphene sheets are removed directly from the surface of large graphite crystals by mechanical force and transferred to carriers such as silica. Monolayer graphene sheets can be obtained on substrates such as silica. This is the first known method to synthesize graphene using a top–down technique, by which layered graphene is split into several layered graphene sheets through repeated peeling. The obtained flakes varied greatly in size and thickness, and the product size could not be controlled. Therefore, it could not meet the industrial requirements of graphene. Entani et al. developed and modified these methods by synthesizing graphene on a SiO6 Pa), silica atoms are volatilized, and the residual carbon atoms are arranged on the SiC surface to form graphene layers throughout the structure.Under high vapor pressure, high temperature , and ultra-high vacuum pressure  are used to treat the raw graphite. Small molecules of a strong acid are inserted in between the graphite layers, followed by strong oxidants such as KMnO4 and KCIO4  . Currentnd KCIO4 . Finallybtain GO . The degbtain GO .via CVD is of high quality and is expected to meet the application requirements of transparent conductive films. It is widely used in the large-scale industrial preparation of semiconductor materials for thin-film transistors.The graphene prepared As a branch of chemistry, electrochemical analysis has been verified by many practical and theoretical experiments. It is an analytical method to determine the composition and content of an object by directly measuring different electrical signal parameters, such as the current potential conductance, according to the electrochemical characteristics of different substances. Electrochemical detection methods have the advantages of low cost, small working space, fast speed, high sensitivity, and high accuracy when compared with other food detection methods. Therefore, the research, application, and promotion of electrochemical detection in food quality and related areas can play an essential role in promoting future food quality detection and standards. As an eminent factor in materials science in the 21st century, graphene has been widely used according to its excellent properties . ApplyinThe synthetic pigments that commonly appear in food are azo pigments . Azo dyes account for more than half of the global synthetic dye production. Azo pigments exhibit excellent color performance, and desired colors can be obtained by adjusting the type and proportion of the azo components, creating a wide variety of colors. The excessive intake of azo dyes may be harmful to human health. For example, sunset yellow and tartrazine contain groups such as azo and aromatic rings in their structure, which can induce allergies, asthma, migraines, and cancers . Existin2, to develop a rapid and low-cost method without any sample pre-treatment required to detect sunset yellow and tartrazine yellow colorants, with the detection limits of 6.0 and 8.0 nM(2 nanorods (MnO2 NRs) with a modified glassy carbon electrode (GCE) surface. When compared with standard GCE, a well-defined sunset yellow (SY) oxidation peak was observed at the MnO2 NR-ERGO/GCE. It had a detection limit of 2.0 nM and a good linear response to the SY in the ranges of 0.01–2.0 µM, 2.0–10.0 µM, and 10.0–100.0 µM. This method was applied to detect SY in soft drinks and obtained satisfactory results . Palanisamy et al. fabricated graphene/β-CD/PtNP-modified electrodes through platinum nanoparticles (PtNPs) decorated with graphene-β-cyclodextrin (graphene/β-CD)–modified electrodes. Studies on the electrode cyclic voltammetry determination of different modified electrodes on the electrochemical behavior of Sudan I indicated that the electric catalytic activity of graphene/β-CD/PtNP-modified electrodes on Sudan I was higher than that of other modified electrodes. With a detection linear response range of 0.005–68.68 µM and a detection limit of 1.6 nM, the sensor was used for monitoring Sudan I–adulterated food samples  and achieved satisfactory results in practical tests 6] 3+/Nafion via the drop coating method on a glassy carbon electrode (GCE), which successfully determined trace amounts of Pb(II), Cu(I), Cu(II), Sn(IV), and Hg(II) in canned food samples. Pb2+, Cd2+, and Cu2+ exhibited the highest sensitivity by anodic stripping voltammetry (ASV). There was an excellent linear relationship between heavy metal concentration and peak current, and the detection limit of Pb2+ was 0.74 ppb, that of Cd2+ was 37 ppb, and that of Cu2+ was 945 ppb  added to food samples. Karthik used electron microscopy (HR-SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and UV–Vis-NIR Fourier-transform infrared spectroscopy (FT-IR) to analyze cobalt-doped zinc oxide/reduced GO (Co: ZnO/RGO) nanorods, obtained by chemical co-precipitation  100–2,500 μM, with a sensitivity of 0.8587 μAμM−1 cm2. The detection limit was 0.5 nM (S/N = 3). The method successfully determined methyl parathion in ugli and tomato fruits, beetroot, and broccoli, indicating its practical applicability (Organophosphate pesticides (OPs) have been widely used in agricultural production due to their wide control range, low cost, and high insecticidal efficiency. Organophosphates are neurotoxins that inhibit acetylcholinesterase activity, causing acetylcholine accumulation and neurotoxicity . In the cability .In the early 1950s, methylcarbamate was introduced as a pesticide and is still used today . This is2) nanocomposite. As-synthesized GOS@CuFeS2 nanocomposite film screen-printed carbon-modified electrode (SPCE) displays excellent electrocatalytic ability toward MOX. Under optimized working conditions, the modified electrode provides a linear response range from 0.073 to 801.5 µM. The detection limit was obtained as 4.5 nM. The sensor displayed outstanding sensitivity at 17.97 μA μM−1 cm−2 35. The GOS@CuFeS2 nanocomposite–modified electrode shows greater real-time practicality in actual vegetable samples (Methyl paraoxon (MOX) is a highly toxic organophosphate pesticide. It has been recently reported that MOX can enter the human body through ingestion, inhalation, or dermal penetration. Due to its high nondegradability, it can bind to fruits and vegetables. When consumed, it can impose sub-chronic and chronic diseases by inhibiting acetylcholinesterase in human metabolism. Umamaheswari et al. reported the detection of non-enzymatic electrochemical sensors based on the 3D porous phase graphene oxide sheet–encapsulated chalcopyrite –coated glass substrates through electrophoretic deposition. The AFB1 monoclonal antibody was covalently fixed on the deposition electrode GQDs/ITO , improved detection limit (0.23 ng ml−1), and good stability (5 weeks) in label-free detection (via electrophoretic deposition. The sensing results of the anti-AFB1/RGO/ITO––based immunoelectrode obtained as a function of aflatoxin concentration showed high sensitivity (68 mA ng−1 ml cm2) and an improved detection limit (0.12 ng ml−1) (One of the most harmful food contaminants is aflatoxin (AFT) . AflatoxGQDs/ITO . The det in food . Srivastetection . Pumera etection . Reducedetection . Srivastng ml−1) .n = 6), and stability. Subsequently, it has been successfully applied in maltol analysis in various foods . Maltol is banned in children’s food by both the FDA and China’s national food safety standards because of its potential to harm health and is also banned in Europe . Therefous foods . Gan’s sus foods .3O4/polyDOPA/RGO composite material using a green method, and its electrocatalytic activity on nitrite oxidation was excellent  and high relative recovery (82.70–105.97%) (Acrylamide (2-propenamide) is found in carbohydrate-rich food cooked under high temperatures. Nodeh et al. (2018) successfully cleaned and measured acrylamide in various foods using a Fe105.97%)  (Nodeh e105.97%) .2Te3@g-C3N4 BNs to quantify food toxic chemicals in meat samples. As modified, the Bi2Te3/g-C3N4 BN-modified electrode exhibits excellent electrochemical activity toward food toxic ractopamine (RAC) with high-sensitive (L.R: 0.015–456.4) and nanomolar detection limit (LOD: 1.77 nM)  synthesized a super-active electrocatalyst of Bi1.77 nM) .2 NSs). Features including (a) a wider linear range (0.05–490 μM), (b) a nanomolar detection limit (0.03 μM), and (c) high sensitivity (29 μA μM−1 cm−2) have been recorded. It also yields high accuracy and good recovery  developed a sensitive electrochemical  sensor for the determination of coccidiostat drug (roxarsone) based on the use of an SPCE (screen-printed carbon electrode) modified with tungsten disulfide nanosheets . The electrode has a high level of selectivity in the large excess concentrations of interfering species. In addition, the modified electrode offers satisfactory repeatability, reproducibility, and stability (4 embedded on sulfur-doped–graphitic carbon nitride composites (SrMoO4/SGCN) using green methods for the electrochemical detection of CAP. The sensor can be used in river water samples, urine, and human blood serum with a high recovery rate. SrMoO4/SGCN/GCE impedance shows a lower resistance charge transfer (Rct), which helps favor the superior electrochemical detection of CAP. The SrMoO4/SGCN/GCE exhibits an ultralow detection limit of 1.5 nM for an extensive concentration range of 0.005 1316.8 M and high sensitivity is 9.619 AM/cm2 using the amperometric method  over a range of 0.02 374.4 μM, with a nanomolar detection limit of 1.2 nM   array electrochemical sensor was used to detect sulfadiazine (SM2) residues in food. Under optimized conditions, a wide linear range of 0.2–1000 ng/ml and a detection limit of 0.169 ng/ml (S/N = 3) were obtained. The recovery rate of the sensor is 92.3–102.23%, and the relative standard deviation is 2.27–4.10%  . Wei’s s27–4.10% . He and 27–4.10% .2Te3) decorated graphitic carbon nitride (GCN) nanostructures as a modified electrode for electrochemical sensing. A nanomolar limit of detection (1.36 nM) was calculated in a 0.05-M phosphate buffer (PB) supporting electrolyte (pH 7.0) using differential pulse voltammetry. The linear dynamic ranges concerning salbutamol concentration were 0.01 892.5 μM, and the sensor’s sensitivity was 36.277 μA μM−1cm2.Rajaji et al. developed a rapid detection of the feed additive drug  using bismuth telluride (BiHz/60 W). As-prepared ZnS NPs@RGO hybrid was applied toward the electrochemical determination of caffeic acid (CA) in various food samples. The sensor can be used in red wine and soft drink samples with a high recovery rate. The proposed electrochemical caffeic acid sensor produces a wide linear range of 0.015–671.7 µM with a nanomolar level detection limit (3.29 nM)  encapsulated on reduced graphene oxide (RGO) hybrid by an ultrasonic bath (50 k3.29 nM) .Graphene is an excellent new 2D material with an extensive application range . HoweverAt present, the biodegradation of graphene materials mainly focuses on enzyme degradation. During body metabolic processes, the biological effect of hydrogen peroxide during substance oxidative decomposition is combined with active enzymes in the body to achieve the biodegradation of graphene materials. Differences in graphene (and its derivatives) structure, properties, and composition affect their degradation behavior. Using polyphase atom doping and surface functionalization, the physical and chemical properties of the materials are changed to affect the degradation process to regulate the enzymatic degradation of graphene materials. Some methods of obtaining graphene cannot meet research specifications due to dangerous pollution levels and low purity. Therefore, the development of green and environmentally friendly preparation methods is needed.Based on existing electrochemical detection models, adding modified functional graphene-like materials to develop new, more reliable, rapid, and accurate detection methods and improving detection stability and limits has always been an essential topic in food detection. Although the commercialization of graphene materials is still in its infancy, the related long-term safety and environmental issues still need to be addressed. The physical and chemical properties of graphene have a decisive influence on biological outcomes, such as the effects of size, shape, surface charge, chemical composition, and surface modification on the toxicity of biofilm, which are still unknown. Furthermore, the active mechanisms are still unclear. Research on the mechanisms and biology of graphene interaction is still in its infancy at home and abroad. The physiological and biochemical processes caused by graphene’s impact on organisms have not been thoroughly studied. Therefore, it is urgent to study further the toxic effects and functional mechanisms of graphene biofilms.


Pmmn borophene. We also show that smooth electron waveguides in tilted Dirac materials can be used to manipulate the degree of valley polarization of quasiparticles travelling along a particular direction of the channel. We examine the particular case of the hyperbolic secant potential to model realistic top-gated structures for valleytronic applications.We show that if the solutions to the (2+1)-dimensional massless Dirac equation for a given one-dimensional (1D) potential are known, then they can be used to obtain the eigenvalues and eigenfunctions for the same potential, orientated at an arbitrary angle, in a 2D Dirac material possessing tilted, anisotropic Dirac cones. This simple set of transformations enables all the exact and quasi-exact solutions associated with 1D quantum wells in graphene to be applied to the confinement problem in tilted Dirac materials such as 8- A broad class of quasi-1D potentials can also be solved quasi-exactly by transforming the 2D Dirac equation into the Heun equation2 or one of its confluent forms5, or via the application of Darboux transformations11. These exact and quasi-exact bound-state solutions have direct applications to electronic waveguides in 2D Dirac materials13, such as graphene, where the low-energy spectrum of the charge carriers can be described by a Dirac Hamiltonian14, and the guiding potential can be generated via a top gate20. Recent advances in device fabrication, utilizing carbon nanotubes as top gates, has enabled the detection of individual guided modes21, opening the door to several new classes of devices such as THz emitters13, transistors12, and ultrafast electronic switching devices22. These advances in electron waveguide fabrication technology make the need for analytic solutions all the more important, since they are highly useful in: determining device geometry, finding the threshold voltage required to observe a zero-energy mode12, calculating the size of the THz pseudogap in bipolar waveguides13, as well as ascertaining the optical selection rules13 in graphene heterostructures.It can be shown using supersymmetric methods that whenever the Schrödinger equation can be solved exactly for a one-dimensional (1D) potential, there exists a corresponding potential for which the two-dimensional (2D) Dirac equation admits exact eigenvalues and eigenfunctions23. There are only a handful of 2D electronic systems that have been predicted to host these tilted cones33, one of which is 8-Pmmn borophene34, which has attracted considerable attention. In general, boron-based nanomaterials are a growing field of interest37; indeed, exploring the tilt of 2D Dirac cones in the context of 8-Pmmn borophene has recently led to a plethora of theoretical works spanning many fields of research84 from optics to transport, and many more; for a comprehensive review, see Ref.85. The spectacular rise of borophene, and the growing interest in tilted Dirac materials, has led to the revisiting of several well-known problems in graphene, e.g., Klein tunneling47 and transport across quasi-1D heterostructures86, within the context of tilted Dirac materials. As mentioned previously, methods such as supersymmetry and reducing the Dirac equation to the Heun equation, utilize solutions of known problems, to generate solutions to new ones. This begs the question: does a simple mapping exist which would allow us to harness the large body of exact and quasi-exact solutions for 1D waveguides in graphene and then apply them to materials with tilted Dirac cones?In extension to the well-known case of graphene, Dirac cones can in general possess valley-dependent tilt14, for the same potential, but of modified strength, effective momentum, and modified energy scale. After outlining the transformation, we study the particular case of the hyperbolic secant potential, which in graphene is known to admit quasi-exact solutions to the eigenvalue problem; but nevertheless, the whole spectrum can be obtained via a semi-analytic approach12. We use this known graphene waveguide spectrum to generate the corresponding tilted waveguide spectrum, which is verified using a transfer matrix method. Finally, we discuss valleytronic applications.In what follows, we show that the differential equations governing guided modes in an anisotropic tilted Dirac waveguide (orientated at an arbitrary angle) can, via a simple transformation, be mapped onto the graphene problem, i.e., transformed into the massless 2D Dirac equationK and Pmmn borophene23, 2B:Pmmn borophane87, and 25. In what follows, we set The Hamiltonian describing the guided modes contained within a smooth electron waveguide in a tilted Dirac material can be written asHence, the wave vector operators in the non-rotated coordinate system A and B sublattices of the tilted Dirac material. These coupled first-order differential equations can be recast into the same equations used to describe guided modes propagating along a smooth electron waveguide in graphene:L is a constant, associated with the effective width of the potential. We define the tilt and anisotropy parameters as The Hamiltonian, Eq. , acts onentclass1pt{minimat with T with It should be noted that to perform the same transformations for the other chirality  used in the mapping. In the same figure, we also plot the numerical solutions to the tilted Dirac problem obtained via a transfer matrix method . We show in blue crosses the exact solutions given in Eq. , smooth electrostatic potentials in tilted Dirac materials can be utilized as a means to achieve valley polarization. The majority of studies have focused on tunneling across electrostatically-induced potential barriers, and valley filtering and beam splitting have been demonstrated86. It has also been shown that the allowed transmission angles through a potential can be controlled using magnetic barriers86. We propose a change in geometry: rather than studying chirality-dependent transmission across barriers, we shift the focus to studying guided modes along quasi-1D confining potentials. The conductance along such a channel can be measured by placing one terminal at each end. According to the Landauer formula, when the Fermi level is set to energy E (by modulating the back-gate voltage21), the conductance along the waveguide is simply K and It has been suggested that the valley quantum number can be used as a basis for carrying information in graphene-based devices21. Full valley polarization can be achieved for energies less than the lowest-lying supercritical state , for a 2D Dirac material hosting anisotropic, tilted Dirac cones. Therefore, all the rich physics associated with guiding potentials in graphene, e.g., THz pseudogaps in bipolar waveguides, can be revisited in the context of tilted Dirac materials, but with the distinct advantage of knowing the eigenvalues and eigenfunctions. We have also shown that in stark contrast to smooth electron waveguides in graphene, valley degeneracy can be broken in tilted 2D Dirac materials for a broad range of waveguide orientations, anisotropy and tilt parameters. The degree of valley polarization along the waveguide can be controlled by varying the potential strength of the top gate, and also by changing the back-gate voltage. Tilted 2D Dirac materials, such as 8-The Supplementary Information contains a full description of the transfer matrix method used to calculate the band structure of guiding potentials in 2D Dirac materials with tilted Dirac cones.Supplementary Information.


In sarcoidosis, peripheral lymphopenia and anergy have been associated with increased inflammation and maladaptive immune activity, likely promoting development of chronic and progressive disease. However, the molecular mechanisms that lead to reduced lymphocyte proportions, particularly CD4+ T-cells, have not been fully elucidated. We posit that paradoxical peripheral lymphopenia is characterized by a dysregulated transcriptomic network associated with cell function and fate that results from altered transcription factor targeting activity.Messenger RNA-sequencing (mRNA-seq) was performed on peripheral blood mononuclear cells (PBMCs) from ACCESS study subjects with sarcoidosis and matched controls and findings validated on a sarcoidosis case-control cohort and a sarcoidosis case series. Preserved PBMC transcriptomic networks between case-control cohorts were assessed to establish cellular associations with gene modules and define regulatory targeting involved in sarcoidosis immune dysregulation utilizing weighted gene co-expression network analysis and differential transcription factor involvement analysis. Network centrality measures identified master transcriptional regulators of subnetworks related to cell proliferation and death. Predictive models of differential PBMC proportions constructed from ACCESS target gene expression corroborated the relationship between aberrant transcription factor regulatory activity and imputed and clinical PBMC populations in the validation cohorts.We identified two unique and preserved gene modules significantly associated with sarcoidosis immune dysregulation. Strikingly, increased expression of a monocyte-driven, and not a lymphocyte-driven, gene module related to innate immunity and cell death was the best predictor of peripheral CD4+ T-cell proportions. Within the gene network of this monocyte-driven module, TLE3 and CBX8 were determined to be master regulators of the cell death subnetwork. A core gene signature of differentially over-expressed target genes of TLE3 and CBX8 involved in cellular communication and immune response regulation accurately predicted imputed and clinical monocyte expansion and CD4+ T-cell depletion.Altered transcriptional regulation associated with aberrant gene expression of a monocyte-driven transcriptional network likely influences lymphocyte function and survival. Although further investigation is warranted, this indicates that crosstalk between hyperactive monocytes and lymphocytes may instigate peripheral lymphopenia and underlie sarcoidosis immune dysregulation and pathogenesis. Future therapies selectively targeting master regulators, or their targets, may mitigate dysregulated immune processes in sarcoidosis and disease progression. Sarcoidosis is a systemic inflammatory disease characterized by dysregulated immune processes . The intHistorically, lymphopenia has been considered the product of hypersplenism, bone marrow infiltration, or T-cell sequestration , 9–11. HDespite these implications, the underpinnings of peripheral lymphopenia in sarcoidosis remain unclear and comprehensive assessment of the genetic repertoire associated with this phenomenon has been limited . To dateBased on evidence from our prior studies, we hypothesized that a dysregulated transcriptomic network characterizes circulating immune cells in sarcoidosis and aimed to discern master regulator transcription factors of aberrant gene expression to gain insight into the molecular mechanisms underlying paradoxical peripheral lymphopenia in sarcoidosis , 16, 43.in-silico cellular deconvolution. Further details pertaining to study cohorts, mRNA-seq library preparation and annotation for the ACCESS cohort, processing of raw microarray expression data for the UCSF cohort, and mRNA-seq library preparation and annotation for the UIC STAR cohort are described at length in the data supplement. All raw sequencing data for the ACCESS cohort has been deposited to GEO, series record GSE155644. Study approval was obtained through the UIC institutional review board (Protocol #s: 2019-0452 and 2016-0063). Briefly, upon messenger RNA extraction from peripheral blood mononuclear cells (PBMCs), gene expression data obtained from whole transcriptome shotgun sequencing (mRNA-seq) was investigated in treatment naïve Caucasian subjects newly diagnosed with sarcoidosis (n=14) and matched controls (n=14). All 28 subjects were enrolled through the RNA core laboratory in “A Case Controlled Etiologic Study of Sarcoidosis”  as previously described and utilized as a reference cohort in this study given that the gene expression profiles from cases were deemed to represent baseline immune dysregulation in sarcoidosis . InclusiWGCNA) to identify gene modules or clusters and their relationship with sarcoidosis and other clinical features  test or linear regression models and considered significant if p-value < 0.05. Differential expression of genes between sarcoidosis cases and controls within modules significantly associated with disease in the ACCESS cohort was determined utilizing a moderated t-statistic at a predetermined false discovery rate (FDR) of 10%  (https://david.ncifcrf.gov)  was utilized to determine whether specific PBMC subset enrichment was associated with gene expression within modules of interest . Further details regarding network construction, module preservation, and cell subset enrichment are provided in the data supplement.Module eigengenes, obtained from specific gene expression of standardized modules between the ACCESS and UCSF cohorts, were extracted as representations of modular gene expression. Associations between gene modules and clinical features  were evaluated by Spearman’s correlation and ue <0.1) . Subsequcrf.gov) , 51. Biointerest , 53. DifMonster algorithm  within a gene regulatory network. To determine DTFI that underlie immune regulatory networks in health and disease, ACCESS cohort control samples (n=14) were defined as the baseline (reference) state and sarcoidosis samples (n=14) as the perturbed  state. DTFI significance was established if the false discovery rate (FDR) p-value based on the Monster Z-score method was <0.1.To identify master regulator transcription factors capable of driving the changes in PBMC transcriptional programming (cell state transitions) from the healthy to the dysregulated immune response observed in sarcoidosis we applied the lgorithm . MonsterMonster algorithm were extracted separately for the ACCESS cohort sarcoidosis cases and controls. Then bipartite networks were filtered to include significant DTFI and differentially expressed target genes with greater predicted edge weight in sarcoidosis compared to controls. Additional subnetwork reduction was performed to evaluate significantly enriched GO biologic processes associated with cellular proliferation or death assumed to alter PBMC subset proportions. Specifically, only target genes related to GO biologic processes involved in cell “aging/senescence,” “apoptosis,” “arrest,” “autophagy,” “cycle,” “death,” “differentiation,” “division,” “exhaustion,” “phagocytosis,” “proliferation,” and “quiescence” comprised the final subnetworks. Upon subnetwork construction, the Laplacian centrality measure for directed and weighted networks was utilized to perform topological analysis and determine transcription factor relevance  of receiver operating characteristic (ROC) curves determined by the model’s ability to accurately predict “high” or “low” PBMC subset proportions in comparison to negative controls using Venkatraman’s test  scores were calculated and utilized to determine the discriminatory power of each feature within PLS-DA models. Individual target genes with high VIP scores (>1) were considered the most relevant predictors of cell subset variability in sarcoidosis  were constructed using data from the ACCESS cohort for model calibration and externally validated . To corrn’s test . Lastly,coidosis , 60.Our aim was to examine gene expression and transcriptional regulation patterns that might contribute to altered peripheral immunity in sarcoidosis. We obtained PBMCs from subjects with sarcoidosis within 6 months of diagnosis and their matched controls (n=14 per group) in the ACCESS cohort and performed mRNA-seq and network-based analyses. Baseline characteristics of the ACCESS cohort are outlined in 2 test p-value >0.05). Significant differences were found with regards to age between cohorts. Specifically, UCSF cohort cases and controls were found to be significantly older than their ACCESS cohort counterparts  with the majority of subjects in age groups 4 and 5 . UCSF cohort sarcoidosis cases included 14/31 (45%) cases with low lung function defined by the study’s original parameters as forced expiratory volume in one second (FEV1) or forced vital capacity (FVC) percent predicted <80%. Individual subject data pertaining to other variables  was not publicly available. Nonetheless, in contrast to the ACCESS cohort, the original GSE19314 cohort included only 2/38 cases with a positive smoking history, 10/38 with Scadding stage 4 sarcoidosis, and at least 32% on systemic therapy for sarcoidosis.A publicly available PBMC gene expression data set (GSE19314) was used to validate expression and transcriptional regulation analyses performed in the ACCESS cohort Table 2 2 test p-value >0.05) but UIC STAR cohort subjects tended to be older than ACCESS counterparts . Significant differences in smoking history were not observed between the UIC STAR and ACCESS cohorts . Furthermore, pulmonary function between the UIC STAR and ACCESS cohorts was similar with only FEV1% predicted showing a trend towards significantly lower values in the UIC STAR cohort  despite the fact that the majority of subjects in the UIC STAR cohort had chronic disease (>3 years duration), 50% (15/30) had severe disease, and only 20% (6/30) were off of systemic therapy. Other differences between the UIC STAR cohort and UCSF cohort could not be assessed due to lack of publicly available data.Unlike the Caucasian case-control cohorts, the UIC STAR cohort (n=30) consisted of a predominantly African American (66.7%) case series of subjects with sarcoidosis as detailed in the data supplement Table 3.WGCNA, 12 distinct modules of gene expression were identified within the 28 subjects sampled in the ACCESS cohort  and one trended towards significance     but had no association with disease status. Thus, expression of unique genes within the modules significantly associated with disease was considered independent of age, gender, and smoking history.To analyze disrupted patterns of gene expression in sarcoidosis, mRNA-seq data obtained from the ACCESS cohort and microarray data from the UCSF cohort were filtered and normalized to identify 12,047 genes commonly expressed within both datasets. Utilizing BH.adj p-value <0.1; respectively) and likely attributable to clinical, demographic, and geographic heterogeneity between the cohorts  gene ontology terms that characterized the magenta module were consistent with fundamental notions of sarcoidosis and included: “immune system processes (GO:0002376),” “defense response (GO:0006952),” “immune response (GO:0006955),” “regulation of immune system processes (GO:0002682),” and “cell activation (GO:0001775)”. In contrast, the blue module was characterized by significant enrichment of “RNA metabolic processes (GO:0016070),” “nucleic acid metabolic processes (GO:0090304),” “gene expression (GO:0010467),” “nucleobase-containing compound metabolic processes (GO:0006139),” and “cellular aromatic compound metabolic processes (GO:0006725)” suggesting that sarcoidosis is not only marked by immune dysregulation but may also be a result of altered metabolic pathways. Other significantly enriched ontology terms related to multiple innate immunity and cellular immunity pathways and genes were also found in the magenta and blue modules, respectively  , 51. TheTo determine if module-specific gene expression was driven by distinct immune cell types, absolute immune signal (ABIS) deconvolution was performed utilizing the Human Protein Atlas (HPA) and demonstrated that the magenta module was significantly associated with protein coding genes characteristic of monocytes and dendritic cells. The magenta module was therefore renamed the monocyte activity module (MAM) , 53. TakMonster to independently infer master regulators driving cell state transcriptional programs and their target genes in MAM and LSM within the ACCESS cohort in a comprehensive and unbiased manner , may be central players in aberrant processes associated with sarcoidosis , 43. In d manner . The resMonster analysis yielded a transcription factor to target gene network consisting of 106,029 edges in sarcoidosis cases and a corresponding network with differing strengths of interaction (edge weights) in controls from 81 transcription factors identified within 1,379 total genes in MAM. Examination of DTFI in MAM identified 11 transcription factors (13.58%) with significantly different targeting patterns in sarcoidosis compared to controls (FDR < 0.1)  and in the UCSF (24.48%) cohorts defined the “low” lymphocyte groups. Conversely, CD14+ monocytes greater than or equal to 31.96% and 31.84% median percentages defined the “high” monocyte groups in the ACCESS and UCSF cohorts, respectively. Surprisingly, the LSM derived calibration model pertaining to transcription factors ARID1B, CDK2, CRY1, and KMT2A resulted in 5 misclassifications and <70% predictive accuracy for both cell subsets; while all other calibration models were capable of predicting subjects with low CD4+ T-cells and high CD14+ monocytes accurately and were considered for validation  in a large cohort of healthy adults that was inclusive of African American and Caucasian subjects and corroborated reduced CD4+ T-cell proportions in all subjects within the “low” group  and CD4+ T-cell proportions of PBMCs was ascertained. Imputed and clinical cell proportions in the UIC STAR cohort were highly and significantly correlated and demonstrated coefficients  in the CD4+ T-cell model and 37 in the CD14+/total monocyte model. Similarly, 40/87 and 34/87 target genes had high VIP scores in the corresponding CBX8 models. These genes represented the most influential determinants of PBMC proportions and likely comprise a core gene signature of sarcoidosis immune dysregulation , 61–63. Aberrant systemic T-cell activity is crucial in the immunopathogenesis of sarcoidosis and peripheral lymphopenia is of clinical significance , 68, 69.By analyzing gene expression regulation, we sought to discern disrupted regulatory pathways driving changes in lymphocyte activity and survival. Traditionally proposed mechanisms of peripheral lymphopenia uphold that it is a consequence of T-cell sequestration, infiltrated bone marrow, or impaired survival and function of regulatory T-cells , 13, 14.We characterized two significant and preserved gene co-expression modules of sarcoidosis and the transcription factors that influence immune network dysregulation related to PBMC function and survival. The best predictor of low CD4+ T-cell abundance was not LSM, which included lymphocyte activity genes, such as CD28, CD40LG, and LEF1, necessary for T-cell effector function, but MAM. Within MAM, aberrancies inciting transcriptional up-regulation of monocyte activity and cell death related processes may be instrumental to the development of paradoxical peripheral lymphopenia. Specifically, significantly altered regulatory activity of transcription factors CBX8, ETV6, SPI1, TLE3, and XRCC3 was central to the cell death subnetwork. The gene targets of ETV6, SPI1, and XRCC3 did not satisfy our criteria to consider these transcription factors significantly impactful as master regulators on circulating lymphocyte and monocyte proportions. However, it is worth noting that these transcription factors have established roles in DNA damage repair, cell proliferation, cell differentiation, and TNF and interferon inflammatory pathways in malignancies and infections previously associated with sarcoidosis , 70–80. CBX8 and TLE3 were determined to be master regulators within the MAM cell death subnetwork. Unlike the target genes of XRCC3, ETV6, and SPI1, the target genes of CBX8 and TLE3 were predictive of CD4+ T-cell and CD14+ monocyte proportions in the UCSF cohort and their predictive ability was substantiated on the UIC STAR cohort. CBX8, a polycomb protein, is involved in DNA damage repair and has been linked to cell proliferation and differentiation through epigenetic modifications , 82. Intvia different mechanisms. Consequently, aberrant expression of distinct key determinant genes related to specific phenotypes adversely impact monocyte-lymphocyte interaction pathways and contribute to maladaptive peripheral inflammatory signaling which in turn is associated with development of lymphopenia and sarcoidosis progression.Our findings in the MAM cell death subnetwork indicate that immune cell imbalances are significantly related to an aberrantly expressed core gene signature modulated by altered CBX8 and TLE3 regulatory activity. Within this core signature, preserved differential over-expression was found between the ACCESS cohort and 14 genes in the UCSF cohort and between the ACCESS cohort and 31 genes in the UIC STAR cohort. These genes exhibited high discriminatory capacity and were considered the most influential predictors and key determinants of CD14+/total monocyte expansion and CD4+ T-cell depletion. Despite accurate prediction of cell proportions in both the UCSF and UIC STAR cohorts, little overlap was observed between key determinants. This finding was not surprising given the variability that is observed among differential expression gene signatures in sarcoidosis and the fact that our predictive models were calibrated on treatment naïve subjects early in the course of their disease from the ACCESS cohort and validated on demographically and clinically heterogenous cohorts , 28–34. in-silico methodologies to determine transcription factor involvement and impute immune cell populations and despite the congruency observed on cell proportions from clinical tests in the UIC STAR cohort in-vitro or in-vivo validation will still be required in future studies to further substantiate these findings. With respect to the in-silico methods, Monster and Cibersort, we note that both make use of input datasets to derive associations with transcription factors and cell types, and neither dataset was developed on sarcoidosis patients. Lastly, other limitations arise from the depth and accuracy of information stored in the databases that were utilized to generate the input datasets and determine functional annotation which can be addressed by re-analyzing the data in the future as information within public databases is expanded with information derived from more advanced technologies.In this study, we leveraged gene expression data from various high-throughput “-omic” platforms and clinically heterogenous cohorts to determine an immune regulatory network and gene signature associated with lymphopenia in sarcoidosis and provide a methodology to incorporate data from multiple studies to improve our understanding of the immunopathogenesis of sarcoidosis. Even though there are advantages to utilizing a well-defined, matched, and treatment naïve case-control cohort of sarcoidosis with mixed phenotypes, we recognize that there are several limitations to our study that will need to be addressed in the future. As noted, by utilizing the ACCESS cohort as a reference data set, our study was possibly limited in its ability to identify pertinent findings that may be held in other gene modules such as the darkred module that demonstrated a trend towards an association with smoking and the red module that may represent a distinctly preserved signature of immune dysregulation in sarcoidosis that is influenced by differences in race, treatment, or other features that contributed to cohort heterogeneity. A larger study utilizing a different analytical approach or different parameters to contrast specific sarcoidosis phenotypes with other granulomatous and lymphopenic diseases, in addition to health, might have been better able to target transcription factors and pathways of interest in sarcoidosis and increase the statistical power and specificity of our findings. Additionally, though our findings on the UIC STAR cohort suggest that our gene signature is applicable to African Americans with sarcoidosis, our findings are likely to be more representative of immune dysregulation in Caucasians with sarcoidosis. It is crucial that subsequent studies focus on African Americans to better elucidate specific mechanisms underlying disease pathogenesis unique to this population and equitably address disparities. Furthermore, this study utilized data obtained from different “bulk” transcriptomic methodologies and demonstrated preservation of findings; however, it should also be noted that despite efforts to rigorously normalize data and utilize statistics that are agnostic to type of “-omic” platform utilized (such as the Z-summary statistic and PLS-DA models) the possibility that gene selection bias was introduced into our signature by a specific platform cannot be fully discounted. By utilizing “bulk” transcriptomic methodologies our study was also limited in its ability to directly assess involvement of specific PBMC subsets. To address this, in-depth single-cell transcriptomic analyses utilizing the same methodology could be employed to provide a comprehensive assessment of cell-specific dysregulation. Our analytical strategy also relied on In conclusion, we determined master regulator transcription factors and target genes of immune dysregulation that are linked to characteristic paradoxical lymphopenia in sarcoidosis. Notably, we identified that differential transcriptional activity in monocytes was the best predictor of low lymphocyte counts. Crosstalk between monocytes and lymphocytes possibly involving the TNF pathway and Fc-γ receptors, as well as other features of innate immunity were the most likely dysregulated pathways involved with lymphopenia. Previous studies have established roles for both monocyte hyperactivity and T-lymphocyte dysfunction in sarcoidosis pathogenesis, but those analyses were limited in their assessment of the functional relationship between monocytes and lymphocytes , 68, 124The datasets presented in this study can be found in online repositories and the Supplementary Material. The names of the repository/repositories and accession numbers can be found in the article/This study was reviewed and approved by the University of Illinois Institutional Review Board (Protocol #s: 2019-0452 and 2016-0063). Written and informed consent was not required for protocol 2019-0452 in accordance with the national legislation and the institutional requirements. Written and informed consent was obtained from all subjects who participated in protocol 2016-0063 in accordance with the national legislation and the institutional requirements.A Case Control Etiologic Study of Sarcoidosis (ACCESS) Research Group Clinical Centers:Beth Israel Deaconess Medical Center: Steven E. Weinberger, M.D.; Patricia Finn, M.D.; Erik Garpestad, M.D.; Allison Moran, R.N.Georgetown University Medical Center: Henry Yeager, Jr., M.D.; David L. Rabin, M.D.; Susan Stein, M.A.Case Western Reserve University - Henry Ford Health Sciences Center: Michael C. Iannuzzi, M.D.; Benjamin A. Rybicki, Ph.D.; Marcie Major, R.N.; Mary Maliarik, Ph.D.; John Popovich, Jr., M.D.Johns Hopkins University School of Medicine: David R. Moller, M.D.; Carol J. Johns, M.D.*; Cynthia Rand, Ph.D.; Joanne Steimel, R.N.Medical University of South Carolina: Marc A. Judson, M.D.; Susan D’Alessandro, R.N., Nancy Heister, R.N.; Theresa Johnson, R.N.; Daniel T. Lackland, Dr.P.H.; Janardan Pandey, Ph.D.; Steven Sahn, M.D.; Charlie Strange, M.D.Mount Sinai Medical Center: Alvin S. Teirstein, M.D.; Louis DePalo, M.D.; Sheldon Brown, M.D.; Marvin Lesser, M.D.; Maria L. Padilla, M.D.; Marilyn MarshallNational Jewish Medical and Research Center: Lee S. Newman, M.D., M.A.; Cecile Rose,M.D., M.P.H.; Juliana Barnard, M.A.University of Cincinnati Medical Center: Robert P. Baughman, M.D.; Elyse E. Lower, M.D.; Donna B. WingetUniversity of Iowa College of Medicine: Geoffrey McLennan, M.D., Ph.D.; Gary Hunninghake, M.D.; Chuck Dayton, B.S.Pharm.; Linda Powers, M.S.University of Pennsylvania and Medical College of Pennsylvania - HahnemannMedical Centers: Milton D. Rossman, M.D.; Eddy A. Bresnitz, M.D.; Ronald Daniele, M.D., Jackie Regovich, M.P.H.; William Sexauer, M.D.National Heart, Lung, and Blood Institute: Robert Musson, Ph.D.; Joanne Deshler; Paul Sorlie, Ph.D.; Margaret Wu, Ph.D.Study Chairman: Reuben Cherniack, M.D.Study Co-Chairman: Lee Newman, M.D.Clinical Coordinating CenterClinical Trials & Surveys Corp.: Genell L. Knatterud, Ph.D., Michael L. Terrin, M.D., Bruce W. Thompson, Ph.D., Kathleen Brown, Ph.D., Margaret Frederick, Ph.D., Frances LoPresti, M.S., Patricia Wilkins, B.S., Martha Canner, M.S., Judy DotsonCentral Repository:McKesson Bioservices : Steve Lindenfelser BBI-Biotech Research Laboratories : Mark Cosentino, Ph.D.* Deceased.Central Laboratories:DNA Core Laboratory: Mary Maliarik, Ph.D.BAL Central Laboratory: Robert Baughman, M.D.HLA Class II Typing Laboratory: Milton Rossman, M.D.; Dimitri Monos, Ph.D.; Chung Wha Lee, Ph.D., Boyana Cizman, Ph.D.Etiologic Antigen in Kveim Reagent Laboratory: David Moller, M.D.Immunogenetics Laboratory: Janardan Pandey, Ph.D.L-Forms Core Laboratory: Peter Almenoff, M.D.; Ian Brett, Sheldon Brown, M.D.; Marvin Lesser, M.D.Pathogenic T Cells Laboratory: Lee Newman, M.D.; Brian Kotzin, M.D.Ribosomal DNA Core Laboratory: Geoffrey McLennan, M.D., Ph.D., Gary Hunninghake, M.D.RNA Core Laboratory: Patricia Finn, M.D.Random Digit Dialing Interview Group:Telesurveys Research Associates: Richard D. Jaffe, M.A.Executive Committee:Reuben Cherniack, M.D. (Chair)Robert P. Baughman, M.D. (9/1/98-8/31/99)Joanne DeshlerMichael C. Iannuzzi, M.D.; Marc A. Judson, M.D. (9/1/96-8/31/97); 9/1/00-6/30/01)Genell L. Knatterud, Ph.D.Geoffrey McLennan, M.D. (9/1/97-8/31/98)David R. Moller, M.D. Robert A Musson, Ph.D.Lee S. Newman, M.D.Milton D. Rossman, M.D. Alvin S. Teirstein, M.D. (9/1/97-8/31/98)Michael L. Terrin, M.D. M.P.H.Steven E. Weinberger, M.D. (9/1/97-3/31/98)Henry Yeager, Jr., M.D. (9/1/98-8/31/99)Data Safety and Monitoring Board:William Martin, M.D. (Chair)Takamaru Ashikaga, Ph.D.David B. Coultas, M.D.Gerald S. Davis, M.D.Fred Gifford, Ph.D.James J. Schlesselman, Ph.D.Diane Stover, M.D.Ex Officio:Reuben Cherniack, M.D., Genell L. Knatterud, Ph.D., Robert Musson, Ph.D.Lee Newman, M.D.CA, YH, CS and BT conceived, designed experiments, and performed experiments. Specimens and clinical data were collected by the ACCESS Research Group. CA, YH, and NE performed ACCESS clinical database abstraction. WW indexed, annotated and quantified transcriptomic libraries. CA, CAS, YH and BT analyzed data. DP, PF contributed reagents and materials. CA, CAS, YH, NS, DP and PF wrote the manuscript. All authors contributed to the article and approved the submitted version.Support for this study was provided by NIH grant numbers R01 HL138628-01A1S1, T32 HL082547, and F30 HL137267 for the University of Illinois at Chicago Division of Pulmonary, Critical Care, Sleep and Allergy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.This manuscript was prepared using A Case Controlled Etiologic Study of Sarcoidosis (ACCESS) Research Materials obtained from the NHLBI Biologic Specimen and Data Repository Information Coordinating center and does not necessarily reflect the opinions or views of the ACCESS or the NHLBI.


Sarcoidosis is a heterogeneous disease with various treatment indications. Although it affects mainly the lungs, sarcoidosis can affect every organ, especially when the disease course is chronic and protracted. Regular patient follow-up is recommended for early recognition of active, ongoing organ-specific granulomatous inflammation to avoid progression to irreversible fibrosis. In this review, we elaborate on treatment indications and various anti-sarcoidosis regimens proven useful in clinical trials. We also review specialized treatment of specific disease manifestations, with a focus on cardiac sarcoidosis. We also report on treatment for special conditions such as fatigue and small fiber neuropathy. Treatment for sarcoidosis is an emerging landscape, with new data complementing the existing knowledge. Cardiac sarcoidosis (CS) occurs in 5% of all sarcoidosis cases, but autopsy and imaging studies reveal clinically silent CS in 25% of cases in the United States.3 CS manifests with atrioventricular blocks, arrythmias, heart failure, and sudden cardiac death, accounting for up to 25% of total sarcoidosis deaths; therefore, prompt recognition and treatment is crucial.4 CS affects all races, has a mean age presentation of 50 years, and may be isolated without evidence of extracardiac sarcoidosis in 25% of those who have it.4 In this review, we address the principles and indications of sarcoidosis treatment, regimens and disease-modifying drugs, and treatment of special conditions.Sarcoidosis is a multisystem granulomatous disease of yet unknown etiology. It has great predilection for the lungs, but multiorgan involvement is common, especially when the disease course is chronic and protracted. Timely recognition and treatment is important as inflammation may progress to fibrosis, leading to irreversible organ damage. Older as well as recent guidelines for follow-up include identification of symptoms, history and physical examination, pulmonary function testing, and blood tests .5 Also, certain disease manifestations  may be treated only locally. Conversely, a recent British Thoracic Society report states that a significant proportion, up to 30%, of patients develop severe and worsening chronic disease requiring ongoing systemic treatment. Mortality is significant among this subgroup of patients, ranging up to 25%.6Not all sarcoidosis patients need systemic immunomodulatory treatment. This variation occurs because in roughly 30% of cases, the disease may subside by itself without ever being treated.2 In other words, treatment is warranted when there is danger of permanent debilitating organ damage or dysfunction with associated morbidity and/or a significantly compromised health-related quality of life on patient-reported outcomes.6 Thus, the goal of treatment is to prevent irreversible evolution of active granulomatous inflammation to fibrosis.7 Disease activity has to be demonstrated before initiation of treatment because already established organ dysfunction should not be treated; disease activity is not sufficient by itself for treatment initiation when organ dysfunction is absent or mild.The decision to treat with disease-modifying agents requires symptomatic, ongoing, and gradually worsening organ involvement (pulmonary or extrapulmonary) as well as certain asymptomatic but significant extrapulmonary manifestations, namely CS, neurosarcoidosis, posterior uveitis, and hypercalcemia.9 Isolated CS may be diagnosed clinically in the absence of cardiac and extracardiac histology when positive PET scan combines with clinical features with or without positive CMR.10Diagnosis of CS with high probability requires abnormal findings in electrocardiogram and/or Holter monitoring and/or echocardiography with abnormal advanced imaging features at cardiac magnetic resonance (CMR) and/or cardiac fluorodeoxyglucose positron emission tomography (FDG-PET).12 The ideal regimen and medication doses in sarcoidosis are not always straightforward, because randomized double-blind placebo-controlled trials are generally lacking in sarcoidosis due to disease complexity, heterogeneity, and ethical considerations to withhold established treatments (with corticosteroids). Table 1 summarizes a treatment algorithm of CS.14 FDG-PET seems to be useful to monitor active myocardial inflammation.15 Treatment has not shown to alter the natural course of the disease longitudinally, as granulomas often recur or progress. The most efficient drug against sarcoidosis is undisputedly corticosteroids. The other two categories of drugs with anti-inflammatory effect are antimetabolites and biologic agents.Recent studies have identified key risk factors for increased mortality in pulmonary sarcoidosis, including decreased diffusion capacity of carbon monoxide (DLCO), presence of pulmonary fibrosis in high-resolution CT, and pulmonary hypertension.20 Note that oral corticosteroids may not have an effect on pulmonary physiology as expressed by forced vital capacity (FVC) and diffusing capacity for carbon monoxide. Furthermore, inhaled corticosteroids, commonly used, have proven ineffective in clinical trials.21Corticosteroids remain the mainstay first-line treatment of sarcoidosis. They have been proven to improve overall disease control, symptoms, quality of life, and pulmonary radiology and/or delay disease deterioration in patients with symptomatic pulmonary sarcoidosis. These effects are short-term, lasting for a period of 1 to 2 years, and fade after treatment withdrawal.24 In recent studies, these relatively moderate doses are shown to manage sarcoidosis manifested as acute disease or as flares of chronic disease, and they are currently recommended by major societies  as an initiation dose with specific exceptions.14 These guidelines consider the significant toxicity of treatment with corticosteroids associated with both cumulative dose and duration of treatment. Common side effects including weight gain, diabetes, glaucoma, osteoporosis, and depression have been frequently observed in sarcoidosis patients.25Studies show that an initiating dose of prednisone at 20 mg daily is not inferior to higher doses in clinical efficacy, control of the disease, physiology, or quality of life parameters.When initiating corticosteroids in a patient with sarcoidosis, close follow-up every 3 months is mandatory. The initial dose is tapered after 3 to 6 weeks, usually by 5 mg, targeting a maintenance dose of 5 to 10 mg of prednisone by approximately 6 months after initiation of treatment. The maintenance dose is required by the majority of patients for a period of 6 to 24 months.7Based on existing literature, several principles apply when considering treating pulmonary sarcoidosis with corticosteroids: (1) acute disease and flares of chronic disease require the same prednisone doses, (2) the chronic maintenance dose should be the lowest possible, and (3) treatment with corticosteroids should be fairly prolonged to allow clearance of the etiologic antigen and adequate suppression of granulomatous reaction.Decision to discontinue corticosteroids is based on disease activity and end organ damage. When adequate control of the disease is accomplished, they should be tapered off. Optimal duration of treatment is not known. Factors that may facilitate decisions in clinical practice are the appropriate selection of high-risk cases, PET-CT scan, and quality of life parameters. Up to 70% of patients experience sarcoidosis relapse during corticosteroids tapering or after treatment withdrawal. Relapses are treated with the same corticosteroid dose as in acute disease.26Addition of a second anti-inflammatory agent is deemed necessary when disease is severe and continued, when serial relapses occur, when corticosteroids are unable to be tapered below 10 to 15 mg of prednisone, when toxicity is significant, and when disease does not respond to corticosteroids.29 MTX has also shown improvement in radiology and in FVC but only in observational or retrospective studies.29 Additionally, MTX has proven effective in neurosarcoidosis, cardiac disease, skin, eye, and disrupted calcium metabolism.1 It is given at a total dose of 15 mg weekly, divided in one or two doses. The most common side effects are blood (cytopenias) and liver toxicities, so these parameters should be monitored closely. To prevent bone marrow toxicity, concomitant treatment with folic acid is imperative. Less than 10% of patients have to stop MTX because of toxicity, and relapses after MTX withdrawal may occur.28 When initiating MTX, males and females of child-bearing age should be warned to prevent fertility due to fetal toxicity during pregnancy. Based on the existing evidence and acceptable toxicity profile, MTX is the recommended first option for a steroid-sparing second-line agent when treating sarcoidosis.26The most commonly studied second-line agent in sarcoidosis is antimetabolite methotrexate (MTX). MTX has exhibited a higher efficacy rate among second-line options managing disease control and steroids tapering in about 65% to 85% of patients with persistent pulmonary sarcoidosis.30 However, these data come from retrospective or observational studies only. Additionally, AZA has significant side effects, mainly gastrointestinal issues, myelodysplasia, malignancies, and secondary infections, with treatment cessation more frequent than MTX. Recent European Respiratory Society (ERS) guidelines made no recommendation for its use as a steroid-sparing agent in sarcoidosis.14Azathioprine (AZA) is a second antimetabolite that has long been used for rheumatic and hematologic diseases and historically has been used in sarcoidosis. Its efficacy is considered less than that of MTX, ranging between 20% to 80%.32 Leflunomide is typically used when a need arises for a second agent with a better safety toxicity profile than MTX. However, leflunomide has adverse effects, most significantly interstitial pneumonia and peripheral neuropathy.34Leflunomide is a cytotoxic drug used in rheumatoid arthritis. Small series have shown efficacy of leflunomide at a dose of 10 to 20 mg daily in pulmonary and extrapulmonary sarcoidosis to improve pulmonary physiology, quality of life, extrapulmonary organ response, and as a steroid-sparing agent.35 HCQ has shown efficacy in cutaneous sarcoidosis and hypercalcemia.37 In these conditions, it is usually given as an adjunct to low-dose corticosteroids or monotherapy. One randomized trial showed some efficacy in pulmonary sarcoidosis and physiology.38 Considered less potent than MTX or other second-line agents, HCQ may be considered in refractory cases when drug toxicity is significant and in combination with one or two other drugs.Chloroquine and hydroxychloroquine (HCQ) are antimalaria drugs used to treat sarcoidosis. Recently, HCQ is used more often due to its lower toxicity profile, although retinopathy remains a significant concern, prompting ophthalmology evaluation upon symptoms.41 Its specific position in the treatment pyramid, likely as a second-line agent, must be further elucidated.Mycophenolate mofetil (MMF) is a powerful inhibitor of lymphocyte proliferation, used to prevent allograft rejection after transplant. Used in various rheumatic diseases, its role in sarcoidosis remains unclear. Retrospective studies showed that MMF has a steroid-sparing effect in chronic sarcoidosis and neurosarcoidosis but is probably less effective than other second-line agents (mainly MTX), with higher relapse rates and a more favorable toxicity profile.43 Infliximab is further proven effective in lupus pernio (efficacy 77%), cutaneoussarcoidosis, and neurosarcoidosis .45 Clinicians must be aware of side effects, ie, infections and tuberculosis.47 Adalimumab may be an alternative in case of infliximab intolerance, as shown (at least partly) in small open-label studies.48 Conversely, neither golimumab nor ustekinumab (anti-IL12/23) showed efficacy in refractory pulmonary sarcoidosis in one randomized trial.49Anti-tumor necrosis factor-α (anti-TNF-α) agents are considered effective third-line drugs in sarcoidosis  by blocking TNF-α, a significant cytokine in granulomatous inflammation. In particular, infliximab (and biosimilars) have shown improvement in FVC in two double-blind randomized studies and was granted a conditional recommendation for use in refractory pulmonary sarcoidosis under CS with or without a second-line agent.52 Rituximab is an anti-CD20 B-lymphocytes antibody that has shown partial efficacy in small case series of advanced pulmonary, eye, and neurosarcoidosis, with a favorable tolerance profile, and may be considered a third-line option.53 Antimycobacterial treatment (CLEAR regimen) for 4 months failed to demonstrate clinical or physiological improvement in chronic pulmonary sarcoidosis.54 Repository corticotropin injection (RCI) was approved in the 1950s for sarcoidosis. Originally considered a steroid analog, RCI may have additional immunological properties and a steroid-sparing effect, although its action is currently limited.55 Janus kinase inhibitors are a drug family recently introduced in sarcoidosis treatment. Among them, tofacitinib showed 60% steroid-sparing efficacy in chronic sarcoidosis in a small proof-of-concept study.56 An overview of drugs used, dosages, and respective monitoring events, with a focus on CS, is presented in Table 2.Various other drugs have been tested in sarcoidosis. Phosphodiesterase inhibitors, pentoxifylline, roflumilast, and apremilast have shown to reduce disease flares, improve quality of life, and improve cutaneous disease, respectively, in small studies.Depending on organ involvement, extrapulmonary sarcoidosis may warrant local treatment, systemic treatment escalation, or multimodality interventions.57 Continued disease should be treated with an additional agent. Hydroxychloroquine and methotrexate are currently used and have shown efficacy in small studies.58 Randomized studies provide support for the use of infliximab in severe skin disease.59 Thalidomide, on the other hand, is proven ineffective compared with a placebo in skin sarcoidosis.60Cutaneous sarcoidosis should initially be treated with local or intralesional corticosteroid injection. Cosmetically important disfiguring lesions (such as lupus pernio) should be treated with oral corticosteroids to achieve control in two-thirds of cases.61 Mycophenolate mofetil and infliximab may be used as well, depending on course and individual toxicities.Anterior uveitis should be treated locally with steroidal eye drops. Posterior uveitis, however, carries risk for blindness and requires systemic treatment, which includes corticosteroids and/or methotrexate in the majority of cases.36 Osteoporosis should be treated carefully in sarcoidosis, as exogenous vitamin D supplementation may lead to hypercalcemia and is only indicated when 1,25(OH)2 vitamin D levels are normal or reduced. When 1,25(OH)2 vitamin D is elevated, osteoporosis is treated with bisphosphonates or denosumab.62Hypercalciuria is treated initially with dietary dairy and restricted sun exposure while hypercalcemia requires systemic treatment. Corticosteroids and/or HCQ are effective in treating hypercalcemia, the latter as sole treatment particularly in cases of unacceptable corticosteroids toxicity.65 When no indication for ICD exists, performing an electrophysiology study is reasonable. Anti-arrhythmic drugs, pacemaker placement in high A-V blocks, and catheter ablation have been of modest value.66Treatment of CS is two-fold. First, based on current guidelines and cardiology consultation, patients at increased risk for sudden cardiac death should be recognized and given an implantable cardioverter defibrillator (ICD), as in all arrhythmiogenic cardiomyopathies. Such patients include those with episode of arrest, left ventricle ejection fraction (LVEF) ≤ 35%, and sustained ventricular tachycardia but also when LVEF is > 35% along with certain physiological or functional features.70 Ongoing studies are underway to evaluate prospectively the impact and dose of corticosteroids in CS . Regarding second- and third-line agents, functional benefit is shown with MTX, MMF, infliximab, and adalimumab.74 In most of these studies, adding a second agent was superior to treatment with corticosteroids alone. Anti-TNF agents were not associated with heart failure deterioration in these studies of CS patients. Unanswered questions remain as per the duration of treatment, the value of adding a second agent from the beginning or later in the course of the disease as well as the management of clinically asymptomatic  cardiac involvement.75 FDG-PET seems a promising biomarker to serially evaluate cardiac inflammation and response to treatment.15Second, patients with clinically relevant cardiac sarcoidosis should initiate anti-inflammatory treatment with corticosteroids. Studies have shown long-term benefit of early initiation of immunosuppressives in CS. Corticosteroids in particular have shown survival and functional benefit, albeit not in randomized trials; lower doses of prednisone (no more than 30 mg) are not inferior to higher doses and are preferred.76 When SAPH is suspected, right heart catheterization is necessary for confirmation and differentiation of multiple SAPH etiologies . Precapillary SAPH carries a 3-year mortality of 30%. Worse outcomes relate to reduced DLCO < 35%, reduced 6-minute walk distance < 300 m, and preserved FEV1/FVC ratio.77 In a small series, epoprostenol and combined ambrisentan with tadalafil showed hemodynamic and functional improvement.79 As to randomized trials, bosentan improved pulmonary hemodynamics at 4 months of treatment, while riociguat significantly delayed time to clinical worsening and improved exercise capacity at 1 year of treatment.81Sarcoidosis-associated pulmonary hypertension (SAPH) is encountered in 5% to 20% of sarcoidosis patients. SAPH along with pulmonary fibrosis constitute an advanced sarcoidosis phenotype, ie, sarcoidosis with a risk of significant loss of organ function or death.12 Transplant referral is indicated when sarcoidosis progresses to respiratory failure despite treatment. Transplant has been performed in patients with fibrosis or obstructive or mixed defects with or without pulmonary hypertension. Ensuing 5-year survival rates were 69%, with age and fibrosis being the worst prognostic factors.82Pulmonary fibrosis extending > 20% of the lungs in high-resolution CT implies advanced disease with high mortality risk.83 A study exploring pirfenidone in fibrotic sarcoidosis is currently underway.Antifibrotic treatment with nintedanib in sarcoidosis with pulmonary fibrosis has shown to ameliorate vital capacity decline, as it did with other progressive fibrosing interstitial lung diseases, in the IN-BUILD trial and may be considered pretransplant.85 Although data are derived from retrospective studies and meta-analyses, they support initiation of corticosteroids versus no treatment, with efficacy rates of corticosteroids alone up to 70%.85 The dose usually administered in clinical practice is prednisone ≥ 0.5 mg/kg daily. A second-line agent will probably be required in continued or relapsing disease, with MTX exhibiting superiority versus MMF, azathioprine, and HCQ (response in 55-70% of cases).86 Lastly, about a third of patients with neurosarcoidosis may require a third-line agent concomitantly. Infliximab is mostly efficient as shown in two studies and less toxic than cyclophosphamide, but relapses tend to occur after treatment discontinuation.87Occurring in 5% to 20% of sarcoidosis, neurosarcoidosis manifests mainly as cranial nerve palsy, parenchymal masses, hydrocephalus, or aseptic meningitis and is associated with a 10-year mortality of 5% to 10%.Figure 1 depicts lines of treatment with available regimens and, on the right-hand side, specific considerations in advanced disease.88 Inspiratory muscle training, a component of pulmonary rehabilitation, may add benefit.89 Pulmonary rehabilitation should be offered in sarcoidosis, as with all interstitial lung diseases. Dexmethylphenidate hydrochloride and armodafinil improved fatigue in 10 and 15 patients with sarcoidosis, respectively, but their use is restricted by side effects such as insomnia and anxiety.91 Lastly, in a randomized trial of sarcoidosis subjects without indication for immunosuppressant treatment, 1 mg dexamethasone daily for 12 months improved fatigue parameters along with serum inflammatory markers; however, this intervention needs confirmation.92Fatigue affects ≥ 60% of patients with sarcoidosis, compromising quality of life, yet it is not directly related to disease activity of the granulomas. Fatigue may be multifactorial due to diabetes, thyroid disease, sleep apnea, depression, and neuropathies. Interventions to alleviate fatigue include pulmonary rehabilitation, neurostimulants, and low-dose corticosteroids. Pulmonary rehabilitation has shown in several studies to improve physical activity, exercise capacity, or just the levels of fatigue.93 Often under-recognized, diagnosis requires large fiber disease exclusion and is facilitated by skin biopsy and sudomotor testing.94 Since there is no established treatment, palliative treatment on a case-by-case basis may include tramadol, anticonvulsants, topiramate, gamma-aminobutyric acid analogues, and intravenous immunoglobulin.95Small fiber neuropathy relates to many diseases, is characterized by debilitating neuropathic symptoms—mainly pain and dysautonomia—and occurs in up to 60% of sarcoidosis patients.Treatment of a patient with sarcoidosis should take into account not only physiologic impairment of the organ but also quality of life and patient-reported outcomes. Corticosteroids, the first-line treatment for acute and chronic disease, should be employed at the lowest affordable dose. To allow this, steroid-sparing agents, in particular methotrexate, are useful and effective. Infliximab, among anti-TNF agents, is effective in refractory pulmonary, cutaneous, and neurosarcoidosis. Patients with advanced disease, pulmonary fibrosis, pulmonary hypertension, cardiac sarcoidosis, and neurosarcoidosis should be thoroughly identified and treated accordingly. Fatigue and small fiber neuropathy impose significant burdens to patients, and efforts should be made to relieve them.Treatment goals in sarcoidosis are preventing permanent end-organ dysfunction and preserving quality of life.Corticosteroids remain the first-line treatment, although with significant cumulative side effects.Second- and third-line agents may be used in refractory or relapsing disease or when corticosteroids cause unacceptable toxicity; methotrexate and infliximab have shown important efficacy with acceptable safety profile in cardiac sarcoidosis.Advanced sarcoidosis refers to high mortality disease manifestations  and necessitates treatment.Cardiac sarcoidosis requires immunosuppressive treatment and recognition of patients at risk for sustained arrythmias, which have to be managed thoroughly.Long-term maintenance therapy remains controversial.


Carbon nanoelectrodes with nanogap are reliable platforms for achieving ultra-small electronic devices. One of the main challenges in fabricating nanogapped carbon electrodes is precise control of the gap size. Herein, we put forward an electroburning approach for controllable fabrication of graphene nanoelectrodes from preprocessed nanoconstriction arrays. The electroburning behavior was investigated in detail, which revealed a dependence on the size of nanoconstriction units. The electroburnt nanoscale electrodes showed the capacity to build molecular devices. The methodology and mechanism presented in this study provide significant guidance for the fabrication of proper graphene and other carbon nanoelectrodes. An approach for the efficient fabrication of graphene nanoelectrodes through the combination of dash-line lithography and electroburning is demonstrated in detail. For instance, electron-beam (e-beam) lithography, as a principle patterning method, can achieve a resolution of sub-50 nm and even sub-10 nm with the aid of specific resists or equipment,9,10 which is, however, still far from the atomic scale. Therefore, the key point of a fabrication strategy is to produce a target nanostructure under patterning precision. In this regard, different kinds of methodologies, including ion/electron beam etching,11,12 selective plasma etching,1,2 and electroburning,3,13,14 have been developed to fabricate nanogapped carbon electrodes. Among them, electroburning is a widely applied method with the advantage of simplicity, controllability, and a lack of dependence on special equipment. In addition, electroburning benefits from the extra capability to control the formation of uncut graphene nanoconstrictions or cracked nanoelectrodes by controlling the burning degree. Hereinto, the former has been applied in the investigation of quantum effects15–17 and the fabrication of functionalized devices,18–20 whereas the latter acts as source/drain electrodes for molecular electronic devices, as discussed above.Nanogapped carbon electrodes are pairs of extremely close carbon materials such as carbon nanotubes or graphene. Because of their advantages including easy chemical processing, strong organic material affinity, low atomic mobility, high carrier mobility, and low screening of the gate electric fields, nanogapped carbon electrodes have attracted extensive interest and have been widely applied in molecular electronics2 Compared to a single unit, they have more potential binding sites and a higher possibility in an array to find a unit with a suitable interval for particular molecules that is beneficial for molecular bridging. This method is described in detail in the Experimental section as well as in literature  process was transferred to silicon wafers with a layer of thermally-grown 300 nm SiO2. Then, graphene was tailored into a 40 μm wide ribbon by a standard photolithographic and selective oxygen reactive ion etching (RIE) method. Metallic electrodes (Cr 8 nm + Au 60 nm) were further thermally evaporated on graphene as source and drain electrodes. A 5 nm wide dash line (d1) and spaces (d2) was patterned on a polymethylmethacrylate (PMMA) mask by a standard e-beam lithographic process. Oxygen RIE was applied to selectively etch graphene from the dash-line windows. The shape of the etching holes (r), which mainly results from the radius of the electron beam, the corrosion of resist development, and the etching of RIE. It is worth noting that for each unit, graphene is processed into a nanoconstriction if 2r < d2 .terature .2 In genash line  with altng holes  inset in 2r < d2  and a gr  were immersed in acetic acid for 30 min at room temperature to remove copper oxide and other impurities. High-quality single-layer graphene was grown on copper films by a chemical vapor deposition (CVD) method. In this procedure, the furnace was heated to 1050 °C in a 14 cm min2.2/Si substrate; subsequently, PMMA was totally removed by boiling acetone. A graphene sheet (40 × 2200 μm) was fabricated by the combination of photolithography and selective oxygen reactive ion etching (RIE) . Finally, high-density patterned metallic electrodes (Cr 8 nm/Au 60 nm) were deposited onto the graphene sheet through photolithography and thermal evaporation.After spin coating  of the polymethylmethacrylate (PMMA) supporting polymer , the PMMA–graphene–copper layers were put into a solution of ferric chloride to etch copper substrates. The PMMA–graphene layers were rinsed with abundant ultrapure water and transferred to a 300 nm thick SiO3.d1 = 150 nm and d2 = 40 nm in the dash line; 40 μm width of graphene sheets), the number of graphene nanoconstriction units in one array was calculated to be ∼210  was applied to the device until the current dropped in magnitude to ∼pA.After spin coating  with a PMMA layer , we ran a DesignCAD file with a 5 nm-width dash line between each pair of electrodes to obtain 50 μm-length windows by e-beam lithography. The development of resist was performed in a mixture of ultrapure water/isopropanol (1 : 3) at 5 °C with the aid of ultrasonication. Considering the routine condition (4.2 gas. The I–V characteristics were determined using an Agilent 4155C semiconductor characterization system.The devices were immersed in a pyridine solution containing a mixture of amino-terminated molecules (0.1 mM) and the dehydrating/activating agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (1 mM) under the protection of argon. After reacting for 72 h, the devices were removed from solution, rinsed with acetone and isopropanol, and completely dried with a stream of N−1) was applied to the graphene nanoconstriction arrays, and the corresponding current was determined at 10 Samples per s (Sa per s). I–V) characteristics of the electroburning process. Generally, the current increases along with the voltage up to a critical point at which electroburning is induced and the current rapidly drops to ∼pA with one . For SiO2 substrates, J = ∼108 A cm−2 according to numerous results.3,15,21,22 A classical theoretical model was built to show the relationship between the critical current and voltage of each graphene nanoconstriction unit .To understand this regime, we initially regard graphene as a classical two-dimensional resistor obeying the law of resistance :1which hd2 is in the range of 2r, the units that 2r > d2 are cracked by RIE, leaving the rest units as uncut nanoconstrictions. This inhomogeneity was proved in regular SEM characterization  can be evaluated according to the Ohm's law:U is the applied voltage, n is the unit number, Rele is the resistance of the graphene electrode, and Runit is the resistance of each nanoconstriction unit. It is easy to find that Iunit increases when n decreases; this means a positive feedback: the initial breakdown of several units results in the increase of the resistance of the multiplied nanoconstriction array as well as the corresponding voltage drop, and thus, results in the current increase in each rest units, which further promotes electroburning. This regime explained why electroburning events occurred in bursts after the critical point. It is worth noting that the positive feedback ratio (C) equalsRunit/n » Rele. Experimental results indicated that the ratio between the resistance of the nanoconstriction arrays (nearly Runit/n) and the corresponding resistance of the electrode Rele was from ∼0.6 to ∼12 in Region 1 and from ∼9 to ∼2500 in Region 2 , we deem that this current decay derives from the electroburning process of the surviving one or several units in Device 2, which are gradually narrowed by the oxidation from the edge. However, in Device 1, the feature size of each nanoconstriction is larger. Due to the large range of the hot zone and the limited proportion of edge atoms, electroburning is unlikely to proceed only from the edges. In contrast, oxygen can react with graphene with the aid of phonons or random defects,21,28,29 which may cause a rapid, simultaneous oxidation in the full hot zone (and thus the rapid current drop). Specifically, the inelastic scattering of transport electrons may randomly motivate higher vibration levels, namely phonons, of chemical bonds in graphene. The higher vibration energy makes it more favourable for reaction, which allows initial oxidation inside graphene. Both electroburning regimes have been supported by previous reports.12,28 SEM images of Devices 1 and 2  are located in the bottom of Region 2, where the critical voltage is 2–3 V and the critical current is <1 μA. In this area, the devices were almost open circuit at low bias voltages (<1.5 V), and then, the current sharply increased near the critical voltages . In the transition region, there may be a competitive process between two regimes. There are two reasons to explain the existence of the transition region. First, the quantum size effect is gradual along with the shrinkage of the feature size; this means that the control of the quantum effect is continuous. Second, the specific properties of particular devices are not strictly identical; this may cause a broadening in statistics.2I–V characteristics of a device before oxygen etching , before electroburning , after electroburning  and after molecular immobilization . Especially, the current recovered to some extent after molecular immobilization, which is similar to the behavior of a direct cut device (To demonstrate the capacity of electroburnt graphene nanoelectrodes for the application, we attempted to use these devices to fabricate single-molecule junctions. In particular, amine-terminated molecules can be immobilized to nanogapped graphene electrodes, which are carboxyl functionalized at the edges due to oxidation by RIE through amide bonds (t device ; this inIn conclusion, we developed an efficient route to fabricate nanogapped graphene electrodes in combination with dash-line lithography and electroburning. The electroburning behaviors from different graphene nanoconstriction arrays were systemically investigated, which demonstrated that the regime of electroburning depends on the feature size of nanoconstriction units. This relationship between the feature size of nanoconstriction/nanoelectrode units and the corresponding electroburning behavior is promising to offer new opportunities for future device fabrication with nanogapped electrodes.The authors declare no conflicts of interest.RA-008-C7RA13106B-s001