Keloids are dermal fibroproliferative tumors that arise beyond the boundary of the original wound edges and invades adjacent tissue. Keloids are characterized by the extensive production of extracellular matrix (ECM) and abnormal fibroblast proliferation. Chondroitin sulfate (CS) is one of the major structural components of cartilage and ECM. Recently, we reported the over-accumulation of CS in keloid lesions. Keloid-derived fibroblasts (KFs) and normal dermal fibroblasts (NFs) were incubated with CS. The fibroblast proliferation rate was analyzed using a tetrazolium salt colorimetric assay. The activation of the intracellular signaling pathway was analyzed by Western blotting. Wortmannin, a PI3K inhibitor, and anti-integrin antibodies were tested to investigate the mechanism of the CS-induced cell proliferation. CS strongly stimulated the proliferation of KFs, but not NFs. The analysis of the intracellular signal transduction pathway revealed that the stimulation effect of CS on KF proliferation was due to the activation of the protein kinase B (AKT) pathway and that integrin α1 was responsible for this phenomenon. We revealed that CS probably activates the AKT pathway through integrin to induce KF proliferation. CS may be a novel clinical therapeutic target in keloids. Keloids are benign dermal fibroproliferative tumors that grow beyond the boundary of the original wound edge and invade the adjacent tissue due to the extensive production of extracellular matrix and the abnormal proliferation of fibroblasts . KeloidsMultimodal treatments, including surgical excision, radiotherapy, corticosteroid injection, physical therapy, and combinations of these treatments, have been applied. Still, there is no satisfactory treatment. The mechanism underlying keloid formation remains unclear. Thus, revealing the etiology of keloids is a fundamental step in the development of clinical treatments for the condition.Recently, the formation of keloids has been recognized to occur due to the excess proliferation of fibroblasts with the over-accumulation of the extracellular matrix. Fibroblasts cultured from keloids (KFs) show altered behavior from normal dermal fibroblasts (NFs), including rapid proliferation, excessive extracellular matrix production, and increased migration activity, which may be a significant etiological factor in keloid development.To understand the behavior of KFs, we studied molecular signal transduction pathways, including growth factor-induced cell growth, apoptosis, and intracellular signal transduction ,3,4,5. KChondroitin sulfate (CS) is a glycosaminoglycan (GAG) composed of alternating, differently-sulfated residues of β-D-glucuronate and β-D-N-acetylgalactosamine . CS is oOur previous study showed that Chondroitin sulfate was over-accumulated in keloids  and thatWe explored the novel roles of CS in the positive regulation of KF proliferation by focusing on the promotion of the CS-mediated signaling pathway. This was directed by our previous study, which suggested that the over-accumulation of CS in keloid lesions may be the deteriorating etiological factor ,15. ReceThe following assays were conducted to evaluate CS-mediated cell proliferation. We used culture plates precoated with poly-D-Lysine (PDL), which is used as a substrate to support the maintenance of negatively charged CSs. Fibroblasts were harvested from tissue specimens using the explant method. We seeded fibroblasts onto PDL plates and incubated the plates for 24 h in serum-free media before CS stimulation. We analyzed five KFs and five NFs in total. A colorimetric tetrazolium salt assay revealed that CS significantly stimulated KF proliferation in comparison to the control (no CS) group . CS did To elucidate the effects of CS on KFs in cell proliferation, we next identified the canonical pathways that have been shown to alter the activation of KFs from recent studies ,20, suchWe then checked AKT activation. In KFs, incubation with CS elevated the phosphorylation level of AKT, in a time-dependent manner . In NFs,Next, we focused on the AKT pathway change caused by the CS stimulation. We examined downstream signal proteins related to the cell cycle. The cell cycle progression from G0 to S phase requires several cell-cycle regulating molecules in specific states, such as decreased p21, activated CDK2, and increased cyclin D. p21 is a cyclin-dependent kinase inhibitor (CKI) that decreases when the cell cycle progresses. A few hours after CS stimulation, we discovered the time-dependent phosphorylation of AKT, decreased p21, and CDK2 phosphorylation . In contTo reinforce our theory, we used wortmannin, a selective and irreversible PI3K/Akt inhibitor. We incubated KFs with wortmannin and obtained protein samples. Wortmannin successfully blocked CS-mediated activation of AKT and blocked p21 decrease , and inhFrom recent research  and our To determine which subtype of integrin was responsible for this mechanism, we selected and investigated several subtypes of integrin based on the relevant literature: integrin α1, integrin α2, integrin α4, integrin α5, integrin β1, and integrin Av. We measured the expression levels of these subtypes by RT-PCR. Integrin α1 (ITGA1) was highly expressed in KFs, relative to NFs . We also conducted an integrin expression assay to measure the integrin expression in tissue specimens. The expression of ITGA1 in keloids was three times greater than that in normal skin tissue . The ITGTo further investigate the role of integrin α1 in CS-induced KF proliferation, we used an anti-integrin α1 antibody and mouse anti-human IgG as a control. When incubated with anti-integrin α1 antibodies, the stimulation effect of CS on KF proliferation was successfully blocked . FurtherIn the present study, we revealed that the cell-proliferative effect of chondroitin sulfate (CS) in KFs in comparison to NFs. A tetrazolium salt assay and intracellular signaling pathway analysis revealed that the cellular proliferation of starved KFs was actively promoted. In keloids, growth factors, such as IGF-1 and TGFβ1, are closely associated with the pathogenesis of keloids, and fibroproliferation has been reported to be regulated—in part—by the ERK pathway, which is signaled by receptor tyrosine kinase phosphorylation by IGF-1. The PI3K-Akt signaling pathway was also found to be synchronously activated ,20,22. TWe propose that CS induces the activation of AKT in KFs via integrins . CS has Integrins are transmembrane receptors that activate intracellular signal transduction pathways, such as cell cycle regulation ,27, orgaFAK is a 125 kDa non-receptor protein kinase that directly binds to the cytoplasmic tail of β-integrin and which plays a significant role in integrin-mediated signaling. The biological importance of FAK-mediated signal transduction is understood by the fact that it plays a fundamental role in embryonic development, control of cell migration, and cell cycle progression . Recent Marlene et al. reported  that KFsSpecific types of CS have been reported to be responsible for the pathogenesis of diseases in neural cells ,39,40,41These observations suggest that CS plays a fundamental role in KF proliferation as a ligand of integrins. We showed that integrin α1 is overexpressed in keloids, which was consistent with the previous report . IntegriRather than as a growth factor reservoir or a scaffold of cell attachment, CS itself had an inductive role in the cellular proliferation of KF. Although we demonstrated that CS played a role in the proliferation of KFs, we could not rule out the possibility that another pathway (besides the integrins) produces intracellular proliferative signals, like contactin-1 in neural cells . The difRecently, Shida et al.  reportedIn conclusion, our data presented the mechanism responsible for the efficacy of CS degradation in keloid treatment . The actFive patients with keloids  and seven unrelated patients  undergoing surgical treatment between September 2014 and July 2016 were enrolled in this study. The present study was performed with approval from the Institutional Reviewing Committee in Kyoto University Faculty of Medicine , which adhered to the ethical standards as formulated in the Helsinki Declaration. Written informed consent was obtained from all patients. The skin tissue samples were obtained as surplus skin in plastic surgery. The sample information is shown in 3 pieces, placed in plastic tissue culture dishes, and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS), 10,000 U/mL penicillin G, and 10 mg/mL streptomycin sulfate. Cells were propagated at 37 °C, and semi-confluent cultures of fibroblasts were passaged by trypsinization up to two times before the analysis [Keloid fibroblasts and normal fibroblasts were extracted from surgical specimens by the explant method. Briefly, tissues were cut into 1 mm® Blot module (Thermo Fisher Scientific). The membrane was blocked with 5% non-fat dry milk  in Tris-based saline containing 0.05% Tween 20 (TBST) for 1 h at room temperature and then incubated with primary antibodies (at the dilution described in Trypsinized and counted cells were seeded in 6-well poly-D-Lysine (PDL)-coated cells at a density of 400,000 cells per well. After incubation with CS, fibroblasts were washed twice with ice-cold phosphate-buffered saline (PBS), and then harvested with RIPA buffer  containing phosphatase and protease inhibitors. The cell lysate was sonicated and cleared by centrifugation . The protein concentration of cell extracts was determined using a BCA protein assay kit . Cell lysate samples were separated by SDS-PAGE  and then blotted onto 0.20 µm nitrocellulose membranes  using the XcellTotal RNA was extracted from cells using RNeasy Micro Kits . First-strand cDNA was synthesized using a Prime Script RT reagent kit with a gDNA eraser . RT-PCR was performed with cDNA using a TaqMan Probe Assay (Thermo Fisher Scientific). Taqman Assay IDs Cyclin D1: Hs0076553_m1, integrin α 1: Hs00235006_m1, integrin α 2: Hs00158127_m1, integrin α 4: Hs00168433_m1, integrin α 5: Hs01547673_m1, integrin α v: Hs00233808_m1, integrin β 1: Hs01127536_m1, GAPDH: Hs02758991_g were used. The relative gene expression was normalized against an internal control, GAPDH. The expression of each gene was compared between depots using the ΔΔCt method.Serum-free DMEM containing KFs prepared as described above was added to 96-well PDL-coated plates. After 24 h of incubation, an anti-integrin α1 antibody  was added to the wells. Mouse anti-human IgG  was used as a control.Chondroitin sulfate  was obtained from Seikagaku Biobusiness Corporation  under a Material Transfer Agreement between Kyoto University and Seikagaku Biobusiness Corporation. We applied CS as a mixture of equal amounts of CS-A, CS-B, and CS-C, which was a similar composition to that observed in keloids, as we previously reported . All otht-test were used to analyze the results. P values of < 0.05 were considered to indicate statistical significance.Data were expressed as the mean ± SEM of at least three independent experiments. Unless otherwise stated, an ANOVA or two-tailed Student’s The present study showed that CS strongly stimulates the signaling to induce KF proliferation via the AKT pathway, which was successfully inhibited by blocking PI3K. Anti-integrin α1 antibodies blocked the proliferative effect of CS on KF. These findings provide a reasonable explanation for the rapid proliferation of KFs in vivo, where accumulated CS is abundant. We conclude that the excessive accumulation of CS and the elevated expression of α1 bata1 integrin and IGF-1R have a synergistic effect on KF proliferation and the overproduction of ECM components, such as CS, which plays an essential role in the etiology of keloids. The CS-enriched extracellular milieu may interact with KF and vice versa resulting in the deposition of CS into ECM. We consider CS to be a key molecule in the etiology of keloids. Further study is required because our experiment only used a 2-D in vitro model. The positive effect on fibroblast proliferation may increase in 3-D cultures.In conclusion, the KF-specific role of CS revealed in the present study represents a significant advance in the understanding of ECM-triggered proliferation, and has the potential to be applied in the development of treatments for keloids.


Sarcoidosis is a systemic, granulomatous, and noninfectious disease of unknown etiology. The clinical heterogeneity of the disease (targeted tissue(s), course of the disease, and therapy response) supports the idea that a multiplicity of trigger antigens may be involved. The pathogenesis of sarcoidosis is not yet completely understood, although in recent years, considerable efforts were put to develop novel experimental research models of sarcoidosis. In particular, sarcoidosis patient cells were used within in vitro 3D models to study their characteristics compared to control patients. Likewise, a series of transgenic mouse models were developed to highlight the role of particular signaling pathways in granuloma formation and persistence. The purpose of this review is to put in perspective the contributions of the most recent models in the understanding of sarcoidosis. NOD2 mutations were involved in the pathogenesis of sarcoidosis, as well as in Blau syndrome (BS) and early onset sarcoidosis (EOS) [FCRL1, IL23R) that could distinguish patients with or without Löfgren’s syndrome [Sarcoidosis is a systemic disease of unknown etiology that is characterized by the formation of immune granulomas in different organs, mainly the lungs, the lymphatic system, the skin, the eye, and the heart . The diais (EOS) ,5,6,7. Nis (EOS) . In a Swis (EOS) . Likewisis (EOS) . A gene–syndrome .The epidemiology and phenotype of sarcoidosis varies highly around the world across age, sex, ethnicity groups, environmental exposure, and socioeconomic status ,13,14,15Even if sarcoidosis is considered as a benign disease, it still represents an important issue considering that mortality is higher in patients with sarcoidosis than in the general population with an average age of death of 70.4 versus 76.2, respectively ,24. MortSarcoidosis presents a highly variable and unpredictable course. It is estimated that half of patients progress to spontaneous regression within a few years of diagnosis . The treSarcoidosis is distinct from other granulomatosis by the peculiarity of the granulomas location and distribution . LymphocM. tuberculosis, M. bovis, M. leprae, and M. avium [beryllium disease [Research models aim to be isomorphic , homologous (identification of the pathogenic mechanisms), and predictive . In sarcoidosis, the development of research models was initiated several years ago, with various degrees of success. Mainly in vitro and in vivo models brought some responses at the isomorphic and homologous levels, but they were not predictive yet. Generating sarcoidosis models is crucial considering excess mortality in sarcoidosis and the necessity to understand molecular mechanisms involved in sarcoidosis to develop alternatives therapies to corticotherapy. Developing experimental models is challenging for multiple reasons, including the following reasons: (1) causative agent(s) of the disease are unknown; (2) it is a systemic disease with different targeted organs for each patient and highly diverse presentations; (3) the multiplicity of cellular actors and their interactions; (4) genetic variability; and (5) the course of disease resolution is unpredictable amongst patients, indicating the influence of both environmental and genetic factors. Research models were developed using putative sarcoidosis causing agents, either antigens of infectious origin or inorganic particles. Several species of mycobacteria have been implicated as the origin of the immune reaction, notably M. avium . Exposur disease ,40,41,42 disease . Nonethe disease ,48,49,50 disease ,52,53,54The present review is not an exhaustive retrospective of all the literature on experimental granuloma formation, but it aims to put in perspective the contributions of the most recent models in the understanding of sarcoidosis.So far, in vitro models used peripheral blood mononuclear cells (PBMCs) i.e., a mixed population of peripheral monocytes and lymphocytes reproducing an immune milieu or isolated monocytes derived into differentiated macrophages. + monocytes, CD163+ mature macrophages, multinucleated giant cells (CD11b+), and T and B lymphocytes  generated granulomas-like structures composed of a mix of CD14iewed in ). AlthouThe multi-walled carbon nanotubes (MWCT) model was tested using non-adherent murine macrophages in 3D cultures to determine whether granulomas would arise independently of an immune context . Indeed,M. bovis bacillus Calmette–Guérin (BCG) [high Treg lymphocytes in granuloma formation [high FoxP3+ cells exhibited powerful antiproliferative activity, without completely inhibiting TNF-α production, indicating a role for Tregs in suppressing the early stages of autologous granuloma formation. However, functional differences were observed in CD4+CD25high cells from healthy subjects compared to those of patients with sarcoidosis. While healthy subjects’ CD4+CD25high cells accelerated granuloma formation, no changes in the time course of granuloma growth were observed with sarcoidosis patient cells, indicating a defect in the suppressive function of Tregs in sarcoidosis.Very few in vitro models were developed with PBMCs from patients with sarcoidosis. Taflin et al. cultured PBMCs from sarcoidosis patients with sepharose beads coated with in (BCG) . This stormation ,58. CD4+Epithelioid cells and multi-nucleated giant cells (MGC) derived from the monocyte–macrophage lineage are the main component cells intervening in the architecture of sarcoidosis granulomas. The differentiation of monocytes into MGC can be induced by various stimuli, including cytokines , growth factors, lectins, and muramyl dipeptide. Monocytes from patients with sarcoidosis stimulated by the supernatant of concanavalin A-stimulated mononuclear cells showed an enhanced potential to form MGCs compared to monocytes from healthy controls or patients with other granulomatous diseases . In the Mycobacterium tuberculosis (PPD)-coated polystyrene beads, or microparticles generated from Mycobacterium abscessus (MAB) cell walls. After 7 days of culture with the PPD model, granuloma-like multicellular aggregates were formed with the presence of macrophages in the center and peripheral lymphocytes. Crouser et al. showed that macrophages from sarcoidosis patients maintained a M2 phenotype in the presence of PPD beads compared to non-coated beads, compared to control patients for whom macrophages acquired M1 characteristics [IL13 gene expression profile in sarcoidosis patients compared to control patients in response to PPD beads [Recently, new in vitro models were developed using either PBMCs exposed to purified protein derivative of eristics . LikewisPD beads . In the PD beads . hi cells) were increased in sarcoidosis when compared with other ILD, whereas M2 macrophages (CD163 expressing cells) were similar [The polarization state of macrophages varies in various lung diseases and could depend on signals present within the microenvironment. While macrophage states are globally divided into classically (M1) and alternatively (M2) activated macrophages, their polarization appears as transient and reversible, depending on the milieu ,64. Data similar . On the  similar . Immunoh similar . Similar similar . Recentl similar . ObviousIn vivo models were developed in mice exposed to putative sarcoidosis-causing agents, either antigens of infectious origin or inorganic particles. In addition, two murine models of transgenic mice showed a spontaneous apparition of granulomatous with sarcoidosis-like aspects, indicating the potential role for particular genes/signaling pathways in sarcoidosis. (C. acnes) , which is a commensal strain of bacteria. Since sarcoidosis patients can be reactive to some mycobacterial proteins  [M. tuberculosis catalase-peroxidase (mKatG) and M. tuberculosis superoxide dismutase A (mSodA) induced pulmonary granulomatosis in mice [+ T lymphocytes with Th1 cytokine (IL-2 and IFNγ) production, and rare B lymphocytes. The presence of serum amyloid A (SAA), a highly inducible acute-phase reactant and amyloid precursor protein, was readily detected in CD68+ macrophages and multi-nucleated giant cells from sarcoidosis patient granulomas in various organs  and in mycobacterial KatG–bead–induced granulomas in mice [+ lymphocyte in part through a toll-like receptor 2 TLR2 signaling and correlated with tissue chronic inflammation and fibrosis. Huppertz et al. showed an increased expression of activated NLRP3 (NOD-like receptor (NLR) pyrin domain-containing protein 3) inflammasome components (caspase-1 and IL-1β) in lung granuloma, and increased IL-1β release of BAL cells from sarcoid patients [Mycobacterium tuberculosis, granuloma formation was decreased in -/-Nlrp3 mice and increased in -/-miR-223 (micro-RNA downregulating NLRP3) mice compared to wild-type mice. The two main in vivo models based on infectious agents followed different strategies: the use of mycobacterial antigens without provoking tuberculosis and the exposure to Cutibacterium acnes  or HSP)  and that or HSP) ,76,77,78 in mice ,80. Gran in mice . SAA exppatients . In a moC. acnes based on its detection in the lung and lymph nodes of patients [C. acnes-induced lung granulomatosis were published, each of them with differences in their experimental protocols [+T and CD45/B220+ B lymphocytes promoted by Th1 cytokines and chemokines  [C. acnes to induce pulmonary granulomatosis showed great variability concerning the mode of administration , the presence or not of adjuvant and the type of adjuvant used , the dose of C. acnes, the number of challenges, and the date of sacrifice. Several types of bacterial strains used in these experiments were either commercially provided (ATCC® 6919 and ATCC® 11828) [C. acnes in mice before immunization decreases pulmonary granulomatosis [C. acnes model, Werner et al. showed that the deletion of Myd88 or Cybb in mice increased the persistence of alive bacteria in the lung and enhanced granuloma formation, indicating that a defect of bacterial clearance through the impairment phagosome activity could participate in granuloma development [C. acnes [C. acnes induction of lung granulomas was reduced in both IL-17A gene knockout mice and mice receiving an IL-17A neutralizing antibody. To conclude, the difficulties in efficiently reproducing the C. acnes-induced pulmonary granulomatosis is probably dependent on the mechanism of repeated antigen recognition and the activation of the CD4 + lymphocyte toward a TH1 phenotype. This mechanism calls upon many factors, which are difficult to control despite the standardization of the conditions of animal facilities. In this sense, this model also responds to the random, multifactorial character described in sarcoidosis. The second model used patients ,83. Severotocols ,91,92,93IL12p70) ,91,92,93® 11828) ,90,91,92elopment , the TLRC. acnes . InteresC. acnes . C. acneBased on the possible association between carbon nanoparticles and sarcoid-like pulmonary granulomas, rodent models of chronic granulomatous inflammation were generated by the administration of MWCTs. After pulmonary exposure to MWCTs, a marked inflammatory response was present and associated with the development of granulomas and/or fibrosis ,96. DepeIn the pathology of sarcoidosis, alterations in circulating lipid levels are observed, notably the reduction in HDL-cholesterol levels and ApoA1 concentrations ,105,106.Peroxisome Proliferator-activated Receptor gamma) that controls the transcription of numerous genes implicated in differentiation, proliferation, and apoptosis. PPARγ was reduced in BAL lysates and nuclear PPARγ content in macrophages, suggesting that PPARγ could be an important negative regulator of inflammation in macrophages in sarcoidosis [Pparg specifically in the myeloid compartment. The loss of PPARγ expression in macrophages promoted a more pronounced granuloma formation and an elevated pro-inflammatory cytokine expression in mice [Pparg deletion in the myeloid compartment favored an increase in the Th1 cell population , and no changes in the Th2 population in BAL cells [EBF1) that could bind and activate the promoter of master regulators of adipocyte, including CCAAT-enhancer-binding proteins C/EBPα and PPARγ [IFNγ is a key mediator of granuloma formation in sarcoidosis. Barna et al. showed that this cytokine altered the expression of the transcription factor PPARγ  familial cases of sarcoidosis [de novo sarcoidosis after liver transplantation, the use of rapamune, an immunosuppressor that binds to the specific cytosolic protein FKPB-12 inhibiting mTOR activation, improved dramatically the patient status with both normal chest imaging and liver biopsy [A second model of transgenic mice spontaneously developing sarcoid-like granulomas was generated with the deletion of the inhibitor tuberous sclerosis complex 2 (Tsc2) in the myeloid lineage in mice. This model demonstrated a major role of a Tsc2 downstream target, rapamycin (mTOR) complex 1 (mTORC1) and its signaling pathway in the initiation and persistence of granulomas . Mice expatients  and an ecoidosis . Interesr biopsy .So far, the multiplicity of research model tried to replicate both pathological and histopathological features of sarcoidosis. However, very few were used as a predictive tool to test the toxicity/efficacy of potential new pharmaceutical molecules or combination of existing treatments.A major predictive model would be to test corticosteroids (CS) resistance. Failure to respond to CS therapy is a common feature of chronical inflammatory diseases, including sarcoidosis, asthma, inflammatory bowel diseases, rheumatoid arthritis, organ transplant rejection, and chemotherapy ,130,131.Recently, Zhang et al. evaluated the anti-inflammatory properties of α-melanocyte stimulating hormone (α-MSH) in the MAB model . α-MSH iRosiglitazone is a selective nuclear receptor agonist of PPARγ and is an antidiabetic agent of the class of thiazolidinediones. McPeek et al. used rosiglitazone to maintain high alveolar macrophage PPARγ levels in MWCNT-induced pulmonary granulomas. Mice receiving rosiglitazone had lower MWCNT-induced pulmonary granulomas in association with a reduced pro-inflammatory response, and sustained ABCG1 expression in macrophages, supporting the concept that PPARγ deficiency could play a role in sarcoidosis pathogenesis .In a different model of granulomatosis induced by Schistosoma Mansoni infection, Soliman et al. used resveratrol (RSV), a polyphenolic stilbenoid that is present in several plants . ResveraFinally, new perspectives of treatment should be considered in regard to the potential role of the NLRP3 inflammasome pathway in sarcoidosis. The use of MCC950, a potent and selective inhibitor of the NLRP3 inflammasome or anti-IL-1β antibody, reduced granuloma formation . Actual in vitro and in vivo models seek to understand the pathophysiology of the complex disease that is sarcoidosis. The need for models concerns in particular patients with poor outcomes. Future models should focus on the identification and the understanding of the mechanisms influencing the switch between spontaneous sarcoidosis resolution and treatments necessity as well as resistance/insensitivity to treatments.To date, research models allowed identifying and dissecting some potential interesting actors and their molecular pathways involved in antigen-driven cell aggregation/granuloma formation . We have-No spontaneous in vitro model of granuloma formation exists, and antigenic stimulation is required. Granuloma formation (in vitro and in vivo) is induced in response to agents  that are probably implicated in sarcoidosis, without certainty. In addition, certain models are more representative of foreign body granulomas (nanotube model) than sarcoidosis granulomas.-No experimental study was done on sarcoidosis patient cells that takes into consideration the epidemiological characteristics of sarcoidosis  to form distinct experimental sub-groups inside the “sarcoidosis group”. Exploration of the late stages of the disease is also missing, notably the comparison of different stages of sarcoidosis . The generation of cell lines and/or transgenic mice expressing polymorphisms identified in sarcoidosis patients may represent great tools to understand the influence of genetic variants in the pathology.-In vitro models are short-term studies due to the limited life span of certain cell types.-Granuloma dynamics cannot be studied in vitro as new cell recruitment from systemic origin does not occur.-Influence of the local environment  remains unknown.-Murine models of “sarcoidosis-like” granulomatosis present slightly different characteristics in terms of temporality and fibrotic evolution. It is interesting to note that exposure to sarcoidosis-causing agents generates only lung phenotypes, while genetically modified mice developed multiple sites of granulomatosis . In mice, local pulmonary exposure is not sufficient to spread the disease in other physiologic systems, suggesting that either the murine immune system is more efficient than humans to neutralize and eliminate pathogens, or that sarcoidosis patients are chronically exposed to one or multiple triggers, promoting granuloma development and persistence.-Most in vivo models are restricted to a pulmonary phenotype that does not recapitulate neither the human lung disease (no PAH has been observed) nor certain extrapulmonary lesions . Notably, no models of cardiac sarcoidosis or neurosarcoidosis exist, although they represent the two more severe organ complications after pulmonary damages.-Fibrotic lesions were rarely observed in the various in vivo models. In sarcoidosis, fibrosis begins at the periphery of sarcoid active granulomas. Chronic inflammation is probably responsible for fibrosis extension, resulting in larger collagen deposition, and fibroblasts/myofibroblasts differentiation causing parenchymal destruction. Recently, studies have identified several MMPs in BAL fluid and the granulomas of sarcoid patients ,158,159,-In silico experimentation involves the combination of biological data and mathematical and computer-based representations to granuloma models. Often, biological data are not sufficient or precise enough to establish proper mathematical models. Carrying computer-based experiments in combination with in vitro/in vivo research could facilitate not only the understanding of the disease but also the testing of new therapeutics. Humanized mice  are potential preclinical animal models for the study of human diseases. The engraftment of hematopoietic stem cells from sarcoidosis patients would address not only patient uniqueness  but also experimental differences between human and murine immune systems. The engraftment of microdissected human granulomas would allow studying granuloma biodynamics in an in vivo context and potentially drug testing. Ultimately, the use of humanized mice may lead to the achievement of personalized medicine. Very few in silico models were proposed so far ,165. In However, the generation of a valid and widely accepted experimental model that responds to all expectations is probably impossible. Models described so far have a number of weaknesses that would need to be addressed in futures studies:Although a great effort has been put into the development of various sarcoidosis models, there is still a lack of in vitro/in vivo models of sarcoidosis that recapitulate the human disease. A financing program was initiated by the Foundation of Sarcoidosis Research (FSR) to fund research projects developing novel experimental models to explore sarcoidosis pathogenesis and potentially the development of new therapies. Ideally, the molecular features of model granulomas would be conformed with human sarcoidosis tissue granulomas. In addition, the model should be relatively inexpensive, high-throughput, and easily manipulated experimentally. This type of sarcoidosis research model should benefit the testing of new pharmaceutical strategies intended to target a large number of patients. Nowadays, three lines of therapy are sequentially considered ,167. Cor


This study aimed to bring together people who smoke or vape, people who do not smoke and healthcare professionals to identify and agree priorities for electronic cigarette research in the UK. We carried out a priority setting partnership, guided by the methodology developed by the James Lind Alliance involving five key stages: initiation, consultation, collation, prioritisation and dissemination. A total of 765 people submitted 1887 questions that they wanted answered by research. Questions were organised into themes, merged and rewritten as summary questions, with 52 unique questions going forward to the prioritisation survey. Participants then ranked their top 10 questions. Following this ranking exercise, the top 26 were identified by selecting the most frequently prioritised questions adjusting for representative stakeholder group. These were put forward for discussion in the final prioritisation workshop, whereby the top 10 electronic cigarette research questions were agreed. The list of priorities identified will be of interest to researchers and funders of electronic cigarette research and will hopefully direct future research and funding calls. These priorities provide insight into the questions that matter to people who are using or concerned about e-cigarettes, including frontline professionals. Smoking is the leading cause of preventable morbidity and premature death. In the UK, 15% of the population smoke , and appE-cigarette research is a relatively new topic; therefore, it is essential to identify and address research priorities that are relevant to people who smoke or vape, and healthcare and public health professionals (HCPHP). Typically, academic researchers decide the questions that are addressed and investigated, but their priorities may not match those of patients and carers. There is often a mismatch between the types of interventions patients and clinicians wish to see evaluated. There is a need to ensure research is informed and guided by the needs of patients to avoid waste in terms of inadequate focus and outcomes . Given tThe Cochrane Tobacco Addiction Group (TAG) conducted a priority-setting stakeholder engagement project to identify where further research is needed in the areas of tobacco control and smoking cessation . E-cigarThe e-cigarette PSP took place between June-2018 and September-2019. The JLA method (outlined in the JLA guidebook ) is openThe people who take part in the surveys and priority setting stages of the work are not research participants. Thus, there is no requirement for ethics approval. The University of Nottingham Research Ethics Committee deemed this work not to require ethical approval. Completion of the surveys implied consent for submitted information to be included. Principles of ethical research were followed such as confidentiality.A steering group was convened with ”patients” (representing people who smoke and/or vape) and HCPHP. A typical PSP uses the term ”patient”; however, the steering group felt it was more appropriate to refer to people who smoke and/or vape as consumers. The steering group comprised four HCPHPs ), five consumers, three academics, and was chaired by a JLA advisor who supported the group throughout the process to understand the steps required. Each member of the Steering Group completed a ‘Steering Group Interests and Privacy form’, to create a culture of transparency in the group and help the JLA Advisor manage potential bias. Each member of the steering group brought with them knowledge of the topic, an understanding of the patient and clinician populations and access to networks of patients and clinicians. The role of the steering group was to coordinate the PSP and was responsible for a number of tasks, including publishing the initiative, overseeing the checking and collating of uncertainties and taking the final priorities to research funders. The steering group met face-to-face on three occasions with another four teleconferences during key stages of the study.”What questions about electronic cigarettes would you like to see answered by research?” In order to keep the survey as succinct as possible to encourage completion, we did not include further questions such as educational background or participants’ health literacy or understanding of research. It was felt that this information would not be necessary as we did not ask them to consider how a question would be answered or how a study may be designed, only to consider what the priority questions would be. Participants could submit as many questions as they wanted. Participants could contact the lead investigator if they wanted to be involved in the subsequent prioritisation stage.Uncertainties about e-cigarettes were collected via a survey which consisted of an explanation of the study, basic demographic questions , self-reported smoking status , whether they were a HCPHP and an open-ended question: The survey was circulated via steering group members, partner organisations , Action on Smoking and Health, National Centre for Smoking Cessation and Training, Royal College of Physicians, New Nicotine Alliance), and was available on the UK Centre for Tobacco and Alcohol Studies (UKCTAS) website. It was promoted on Facebook and Twitter. Efforts were made to disseminate the survey to target groups—for example, paper versions of the survey were taken to a local homeless centre. In addition to submitted uncertainties, we searched reviews of e-cigarettes for highlighted research uncertainties.Uncertainties gathered in the consultation stage were reviewed, and out-of-scope questions were removed. A list was created of indicative uncertainties . The data were categorised using themes identified in the raw data. This process was managed by the lead investigator (with input from four steering group members) to ensure consistency in dealing with interrelated questions. Those that were difficult to assign to a theme were discussed with the steering group. Uncertainties that could be answered by published systematic reviews were removed from the process; these were identified by searching the Cochrane Library, Prospero and NHS Centre for Reviews and Dissemination databases. Uncertainty was confirmed if there was no review, an out-of-date review (over three years old) or a recent review with an equivocal answer. The collation process was closely monitored by the JLA advisor to ensure transparency and minimization of bias.www.optimalworkshop.com) was used to create an online survey whereby the list of indicative questions was presented in a randomised sequence for each participant. Participants read all questions and selected (drag and drop) the 10 questions they thought were most important for research, and ranked them in order of preference. To facilitate the process of identifying their top 10 questions, an intermediate ”questions of interest” box was included as a holding pen for questions they wished to consider. Participants could swap and change questions as often as they liked before submitting their final top 10. Participants could contact the lead investigator to express an interest in taking part in the final workshop.The long list of indicative uncertainties was reduced into a more manageable list by asking participants to rank the questions according to those most important to them. Interactive card sorting software  consumers/public, 168 (22%) HCPHPs) submitted 1887 uncertainties at the consultation stage. These could be anything they deemed important, including safety, effectiveness, different populations, supply or accessibility of e-cigarettes, environmental issues and related products such as snus and heated tobacco. Submitted questions per person ranged from one to 16. Participants ranged in age from 17 to 88 years old (mean age 47 years). Just over half of the participants were male (57.1%). Participants indicated their smoking status see  and coulHCPHP represented 168 participants. A fifth of these were stop smoking advisors. Other professions included nursing and midwifery, public health, medicine, dentistry, healthcare workers, mental health, pharmacy, physiotherapy, paramedics, occupational therapy, optics, audiology and social work.(“does vaping cause popcorn lung?”). This left 52 indicative uncertainties that progressed to the ranking stage, representing 12 broad themes . The subthemes for each of these overarching themes can be seen in Some submissions were just comments or statements with no underlying question, and these were excluded. After removal of out-of-scope submissions, nonquestions or statements, 1632 uncertainties remained. These were represented by 53 indicative uncertainties; however, the steering group agreed to remove one of these as it was not considered to be a true uncertainty based on the evidence The steering group agreed this was a suitably representative and manageable sized list to take forward to the next stage of the process. No other uncertainties were resolved by evidence of recent published systematic reviews.The ranking stage involved 415 participants  prioritising their top 10 from the list of 52 indicative uncertainties. Again, participants indicated their smoking status see . This tiFor the interim prioritisation, participants chose their top 10 questions from the list of 52 and ranked them in order of preference. These 52 questions and the frequency they were highly prioritised can be seen in It was important to ensure the final indicative questions for the prioritisation workshop were representative of all groups—i.e., of equal significance to votes from across both stakeholder groups. The top 15 questions for HCPHPs, and the top 15 questions for consumers/public gave us a top 21 as nine of these questions were common in both groups. Each group had six questions that did not feature in the other group’s top 15. The steering group then voted for their top five questions from the next 10 most popular questions. This gave us a total of 26 questions for discussion in the final prioritisation workshop see .The final workshop to agree the top 10 research priorities  had 23 pDuring the workshop, each participant was given the opportunity to express their views on which questions they thought should be prioritised and to hear other people’s perspectives. Participants were able to debate and change their minds throughout the day. The research team observed that participants were keen to include as many topics as possible within the top 10 priorities—for example, ensuring there was a question related to pregnancy, and a question related to mental health within the top 10.“Will prescribing e-cigarettes to pregnant smokers encourage smoking cessation, and reduce risk of relapse?”. This word change was agreed with the lead investigator who felt this wording still represented the original respondents’ intent.One change to the wording of a question was made following discussion and agreement from the workshop participants: the word ”prescribing” was changed to ”providing” in the question: The top 10 priorities, , were diThis PSP was conducted according to an independent and transparent process to identify and prioritise questions for research in which people who smoke or vape, people who do not smoke and HCPHPs were successfully brought together. To our knowledge, this is the first prioritisation exercise focusing specifically on e-cigarettes as a therapeutic aid.The top ten research priorities include questions on long term safety; effectiveness for quitting smoking; policy; risks, benefits, barriers and facilitators to use in different populations including pregnancy and those with mental health issues; ingredients and testing; and the effects of second hand vape. This process has demonstrated numerous uncertainties surrounding e-cigarettes; such widespread uncertainty is likely to have an impact on the consistency of advice and information given by HCPHPs, and therefore patients’ confidence in this advice, as evidenced by the literature ,11,12,13Most of the top 10 priorities of this PSP also align with those identified by the Cochrane Tobacco Addiction Group (TAG) . Their pCRUK  have alsIt is reassuring, in this new field of research in e-cigarettes, consumers, HCPHPs and the research establishment are all on the same page when it comes to identifying priorities for future research. However, the 10th priority question in our PSP concerned the safety around second hand vape. This was not identified as a priority for TAG or CRUK, but it highlights the concerns the public and HCPHPs have and are perhaps seeking more assurance and evidence that second hand vape is harmless.Our PSP has shown that there is a lack of research on e-cigarettes and many safety concerns among HCPHPs and consumers . HCPHPs There were no questions on supply or availability of e-cigarettes. Within the original uncertainties submitted, there were questions on how people access vaping equipment, including children and young people, and what the preferred sources are. However, these questions were ranked very low in the prioritisation survey, and therefore, were not discussed in the final workshop. However, the supply of e-cigarettes still remains an uncertainty, and preferred sources for accessing vaping equipment warrant further research.With regard to other products such as heated tobacco, Swedish snus or shisha, there was one question asking what the risks and benefits of these products are and whether they should be treated the same as e-cigarettes (question asked by a vaper). However, this question was not ranked high enough in the prioritisation survey to be discussed in the workshop.All submitted questions were verified against the evidence, but were still considered to be uncertainties, except one question on popcorn lung. The steering group agreed there was enough evidence that there is no risk of popcorn lung from vaping . HoweverFor the other questions, there was often no systematic review evidence, or where there was a systematic review it lacked relevant studies to answer the question. For some questions, there were individual randomised controlled trials, and some participants in the final workshop argued that a question had already been answered because of available data from one trial. However, the JLA process requires a stronger evidence base, such as systematic review data, because a single trial can vary in quality and context, and a subsequent trial may report different results.In order to keep the survey succinct to encourage responses, we did not ask many personal questions, so it is not possible to ascertain if we had responses from a wide range of ethnic populations, from areas of deprivation, or from specific populations—e.g., pregnant women, homeless population, prison population, those with mental health conditions or people suffering from other health conditions such as chronic obstructive pulmonary disease or lung cancer. However, efforts were made to ensure participants were as representative as possible to achieve a wide range of questions. The survey was distributed via the steering group’s broad network, to heads of maternity services, and to the general population via social media. Efforts were made to try to reach different populations—e.g., we succeeded in visiting a homeless shelter, but we faced difficulty reaching the prison population. Furthermore, we did not notice any difference in the types of questions submitted based on smoking status, besides the fact that some vapers asked questions specific to the devices, whereas non-smokers and smokers did not.Additionally, this PSP was concerned with questions relevant to the UK population and organisations. However, many individuals from around the world submitted uncertainties, with 25 other countries represented. These questions were included if they were applicable to the UK. Nonetheless, it is likely that many of the questions will be applicable to an international audience—e.g., questions about safety and effectiveness including in different populations, long-term effects, effects on physical health and second hand vape. However, it is worth noting that there are wide variations in regulatory environments for e-cigarettes around the world. At least 14 countries have banned the sale and use of e-cigarettes, including Thailand, Brazil and Singapore. In the USA, at least five states have banned flavoured e-cigarettes . Therefore, some questions may be dependent on national policy or regulatory environments and may vary by country . Nonetheless, all questions in the top 10 will be relevant to an international audience but may need to be answered differently according to each country’s regulatory environment.A recent publication has highlighted how JLA PSPs have transformed research, and the impact they have had . One sigThe list of priorities identified here will be of interest to researchers and funders of e-cigarette research and will hopefully direct future research and funding calls. These priorities provide insight into the questions that matter to people who are using or concerned about e-cigarettes, including front line professionals. Furthermore, many of these priorities also align with the issues identified by Cochrane TAG and CRUK.


Four possible azimuth steering angles were designed for −26.7°, 0°, 13°, and 42.4°. Measured far-field patterns show good agreement with simulation results.Low-cost and conformal phased array antennas (PAAs) on flexible substrates are of particular interest in many applications. The major deterrents to developing flexible PAA systems are the difficulty in integrating antenna and electronics circuits on the flexible surface, as well as the bendability and oxidation rate of radiating elements and electronics circuits. In this research, graphene ink was developed from graphene flakes and used to inkjet print the radiating element and the active channel of field effect transistors (FETs). Bending and oxidation tests were carried out to validate the application of printed flexible graphene thin films in flexible electronics. An inkjet-printed graphene-based 1 × 2 element phased array antenna was designed and fabricated. Graphene-based field effect transistors were used as switches in the true-time delay line of the phased array antenna. The graphene phased array antenna was 100% inkjet printed on top of a 5 mil flexible Kapton Digital beamforming phased array antennas (PAAs) with distributed control and processing electronics offer numerous advantages for radio frequency (RF) communications, such as electronically controllable beamforming and steering, high average transmitted power and efficiency, flexible sub-arraying to provide multiple communication links simultaneously, reconfigurable high-gain patterns, and increased reliability through reconfiguring redundant array elements. Low-cost and conformal PAAs on flexible substrates are of particular interest in many applications, such as space communications, train antennas, car radio antennas, and cellular base station antennas. The major deterrent to producing flexible PAA systems is the difficulty in integrating antenna and electronics circuits on the flexible surface. One of the major integration methods is to attach and interconnect electronic chips onto the antenna surface . Due to Previously, a prototype PAA was designed, using carbon nanotubes (CNTs) as the active material in the digital switching device and nanosilver ink as interconnect and antenna element material ,5. This Graphene is a 2D material for electronics and photonics, with remarkable electronic, optical, mechanical, and thermal properties. Graphene is being investigated and applied in many fields ,13,14,15High-performance graphene powder suitable for inkjet printing has been grown through chemical vapor deposition (CVD) . A singlCompared to conventional printing techniques, such as offset lithography, gravure printing, flexography, and screen printing, inkjet printing is an ideal solution for high-resolution, low-cost, high-speed printed electronics, due to low material consumption, low equipment and setup costs, and the possibility for mass customization. It is also a non-contact printing technique which does not require any template preparation. The wide application of printed electronics includes organic photodiodes, photovoltaic cells, high-frequency micro-electro-mechanical-switches (MEMS), electronic paper displays, foldable thermochromic displays, and high-performance organic thin-film transistors ,25,26,27Traditional rigid microstrip antennas mainly use copper as the radiating material, due to the cost. However, inks made of nanocopper particles are not mature, and special equipment is needed for the sintering process. The majority of current printed flexible antennas is based on nanosilver particle inks. In this paper, a fully printed 1 × 2 graphene-based PAA is presented. Graphene FETs are used as switches on the TTD lines with on/off ratio of 38 and on-current of 32 µA. Patch antenna elements were also printed with graphene ink. The far-field patterns are characterized and agree well with simulation results. The performance of the graphene-based antenna is compared with the silver-based counterpart. The devices were produced in a room-temperature environment, with thermal curing processes used to anneal the inks which can be substituted with a room-temperature photosintering process. Such a low-temperature processing method permits us to use any flexible substrate with regular transparency, as demonstrated in this paper.To prepare the ink, terpineol was added to cyclohexanone in a volumetric ratio of 15:85. Then, ethyl cellulose was added to the mixture and thereafter placed in an ultrasonic bath (Branson model 2510), which provided mild mechanical vibration and a steady temperature environment for the reaction until all the white powder was completely dissolved. This process takes 1–2 h. Next, the graphene nanoflakes were dispersed in the mixture. For uniform and homogenous dispersion, the nanoflakes were added gradually, at intervals, while the mixture was still in an ultrasonic bath. After the addition of the graphene nanoflakes, the mixture was kept under mild vibration, in the ultrasonic bath, for 10–20 h, and, thereafter, the mixture was centrifuged at 5000 RPM for 20 min, to obtain a homogeneous solution . Finally, the homogeneous dispersant was collected, and the residues were recycled. R2 = 0.9967, which is above the minimum regression coefficient of 0.98 that we required for reliable viscosity measurements. This 5.51 cP viscosity at room temperature is compatible with the inkjet printer we used for the whole fabrication process which can jet out ink or solution only if the viscosity of the ink/solution is within the range of 2–12 cP.After the preparation of the homogeneous solution of graphene, the viscosity of the solution was measured at different shear rates, using a RheoSense m-VROC viscometer  In the conjugated software module (version: 3.1.4), we entered the sample size in mL and an estimated viscosity for an ink from the literature. Then the software provided an estimated calculation for minimum and maximum flow rate. We set five different flow rates within the displayed range and measurement times, and then we dispersed the sample through the measurement sensor. ® flexible substrate. After the viscosity measurement, the contact angle and surface tension between graphene ink and the substrate were also checked. A KRÜSS drop-shape analyzer (DSA-1000)  was used to determine these two parameters. Contact angle is a crucial characterization to check before any printing technique, including inkjet, aerosol jet, or spray coating. The result of contact angle determines how much resolution we can achieve with an ink, and surface tension determines the bonding between ink and substrate. It is recommended to have a surface tension between 25 and 45 mN/m to achieve good bonding between materials in printing techniques. ® substrate. The contact angle was found to be between 20 and 21 °C. This contact angle also represents the wettability of the ink with the substrate. The surface tension was found to be 28.75 mN/m, meaning that the ink has a good bonding with flexible KaptonA DoD Fujifilm Dimatix (model DMP-2800) inkjet printer  was used to print all the thin films and devices. It was equipped with a Dimatix materials cartridge (DMC-11610) . The cartridge head has 16 piezoelectric jetting nozzles with a diameter of 21.5 µm each. The nozzles can dispense a droplet of the nominal volume of 10 pL from the cartridge head. The optimum drop spacing for the graphene ink was 30 µm, and six nozzles were used to print these inks. There was no delay between subsequent printing passes, except for a 0.3 s purge in every ten printing passes. Additionally, to avoid the coffee-ring effect, the platen temperature was set at 60 °C ,30. The The selection of an appropriate waveform is important in order to achieve a good printed pattern. Graphene ink is still a new ink concept, and so there are few waveforms available from different vendors, and they do not work perfectly all the time. Thus, the default waveform of Fujifilm Dimatix Model Fluid is a good waveform to start with for any newly formulated ink. After investigating the jetting of the nozzles, 5–8 nozzles were chosen to print the antenna. To avoid nozzle clogging, nozzles were cleaned after every 30 min of printing. The final voltage setting for each nozzle was 25 V with 4.5 meniscus setting point. Printing frequency can be selected as low as 1 kHz and as high as 5 kHz. Lower printing frequency increases the printing time, and higher frequency decreases the printing time. However, higher printing frequency is also responsible for significantly increasing the ink spreading all over the substrate. Thus, in our work, the cutoff printing frequency was selected to be 2 kHz, to avoid the ink spreading. The printer head was set at 1.5 mm above the platen, to provide sufficient clearance after placing the substrate. Due to the very low percentage (less than 1%) of solid loading of graphene ink, it was necessary to use multiple printing passes. In this work, it was found that, after a minimum of 40 printing passes, graphene could create enough percolation network with good coverage to be conductive. To achieve a good conductive film, thermal annealing was performed after every 10 layers. The current–voltage measurements of the transistors were performed with the Keysight B1500A semiconductor device analyzer . In the far-field pattern measurements, the under-test antenna (UTA) was mounted onto a rotational stage, and a standard gain horn antenna was mounted onto a stage with adjustable angles with far-field distance between the two antennas. A vector network analyzer was used to determine S-parameters and provide the RF signal to the UTA. While the UTA was rotated by the rotational stage, a microwave signal analyzer was used to detect the signal from the receiving standard horn antenna, to get the radiation pattern. Graphene thin film was used to substitute for the widely used silver nanoparticles thin film, due to graphene’s flexibility and anti-oxidization. Graphene nanopowder (Product NP-AO3)  of 12 nm lateral size was used to prepare printable graphene ink . The priThe flexibility of the printed graphene and silver patches with the same thickness of one micron were investigated by performing resistance measurements on a homemade bendable system. Valley bend of 90° for 2000 bending cycles was conducted first, followed by 2000 bending cycles of >135° valley bend, 1000 bending cycles of 90° mountain bend, and 500 bending cycles of >135° mountain bend. The resistance of the silver patch increased around 1993% after 5500 bending cycles, while the printed graphene patch increased around 15%. ® substrate. However, the measured resistance data show less of an effect of bending on graphene film. These results indicate that this kind of printed graphene holds a possible pathway for flexible and bendable electronic devices.The oxidation test was carried out on a hot plate, in air. Each patch was first heated to 120 °C for one hour. Resistance measurements were taken, and then the process was repeated for 10 °C hourly increases up to 290 °C. θ is the beam steering angle. The delay line length interval for adjacent antenna elements can be calculated as follows [The time delay between adjacent elements of a PAA can be calculated as follows :(1)t = d follows :LintervaUsing the above equations, we designed a true-time delay network for a PAA radiating at 4 GHz (within S band). Element distance was designed as half wavelength, to avoid grating lobes. ® FPC polyimide film, source and drain electrodes were inkjet printed, using a Fujifilm Dimatix DMP-2800 materials deposition system. Silver nanoparticle ink   was used for electrodes. The printed electrodes were followed by thermal annealing at 130 °C for 30 min. Then, the graphene active layer, prepared as in 2 monolayer/multilayer dispersion [The top-gate schematic structure of the proposed graphene FET is shown in spersion . Ion gelspersion . To demonstrate the azimuth beam steering, ® FPC polyimide film, using a Fujifilm Dimatix DMP-2831 inkjet-printing system. Using electronic grade graphene solution, we fabricated an FET with an on–off ratio of 38. The true-time delay lines incorporated with the FET switches were proven to work well through the beam-steering experiments of the PAA. The devices were produced in a room-temperature environment, with thermal curing processes used to anneal the inks which can be substituted with a room-temperature photosintering process. The fully printed graphene PAA technology will greatly help the advancement of conformal communication antennas.In this study, an all-inkjet-printed graphene-based 2-bit 2-element PAA was fabricated on a DuPont™ KaptonCompared with the mainstream of silver-based printed antennas, graphene antennas take a longer time to be fabricated. Due to very low percentage (less than 1%) of solid loading of graphene ink, it was necessary to continue 40 printing passes in this work, while only three printing passes were needed for our previous silver-based antenna system. To achieve a good conductive graphene film, we performed thermal annealing after every 10 layers, further increasing the fabrication time and process complexity.


EGFR-mutated non-small cell lung cancer (NSCLC) to EGFR-targeted therapy. We retrospectively evaluated patients who received first-line treatment with EGFR-TKIs for advanced NSCLC with EGFR mutations. Pre-treatment plasma concentrations of PD-L1 and on-treatment (1 month after treatment initiation) plasma concentrations of PD-L1 were measured using the R-plex Human PD-L1 assay. The association between the sPD-L1 level and the clinical outcome was analyzed. Among 66 patients who were eligible for the study, patients with high pre-treatment or on-treatment sPD-L1 levels had decreased objective response rate (ORR) compared with that of patients with low sPD-L1 levels . A high baseline sPD-L1 level was associated with a shortened progression-free survival (PFS) rate . Both univariate and multivariate analyses showed that a high baseline sPD-L1 level was an independent factor associated with the PFS . Our study revealed that the sPD-L1 level was strongly related to the outcome of EGFR-TKIs in NSCLC patients harboring EGFR mutations.Programmed cell death ligand 1 (PD-L1) expressed on tumor tissues is a vital molecule for immune suppression and its impact on the response to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has been reported. The significance of soluble PD-L1 (sPD-L1) for lung cancer patients remains unknown. This study investigated whether sPD-L1 could predict the response of  EGFR mutations  pathway, which leads to exhausted T-cells and continuous cancer growth, has been identified as the most critical mechanism of tumor evasion . PD-1/PDhibitors . Recent hibitors . High lehibitors , 14. TheEGFR mutations.Evaluating PD-L1 expression in tumor tissue is challenging. First, it is not easy to obtain sufficient tumor samples for analysis from inoperable patients. Furthermore, the test results of PD-L1 expression may differ according to the anti-PD-L1 antibodies applied . And theEGFR mutation (defined as 19DEL or L858R), a treatment-naïve status regarding EGFR-TKIs and a thorough documentation of the response evaluation for patients. The treatment response was evaluated every 2–3 months using computerized tomography according to the Response Evaluation Criteria in Solid Tumors version 1.1.  according to the manufacturer's instructions. All the samples were tested in duplicate.Continuous data were summarized as medians and ranges. When assessing changes in sPD-L1 levels, for each patient with available blood sample, we estimated the difference between levels at baseline and at 1 month after initiating an EGFR-TKI. Patients with a change in sPD-L1 level that was lower than the median difference for the entire population were considered to have a reduction in sPD-L1 levels, whereas others were considered to have no reduction in sPD-L1 levels. For pre-treatment and on-treatment sPD-L1 levels, values that were lower than the median concentration for the entire population were considered to be low, whereas those above or equal to the median concentration were considered to be high.t-test or Mann-Whitney U-test was used for comparing continuous data according to the data distribution determined by the Kolmogorov-Smirnov test. Kaplan-Meier curves and the log-rank test were used to compare survival times across different patient groups. The Cox proportional hazards regression analysis was performed, and HRs and 95% confidence intervals (CIs) were calculated to determine the survival difference. Variables were included in the multivariate analysis if they were statistically significant (p < 0.10) in the univariate analysis. All statistical analyses were performed using GraphPad Prism software  and SPSS statistical software . Results were considered statistically significant at a two-sided p < 0.05.The relationship between categorical parameters was determined using a chi-square test or Fisher's exact test. The student's In total, 66 patients met the inclusion criteria and were enrolled in this study. On-treatment blood samples were collected for 46 of these patients. The distributions of pre-treatment and on-treatment plasma sPD-L1 concentrations are shown in n = 62) and had an ECOG PS status of 0–1 . Fifty-six patients (84.8%) were at stage IIIB to IV at the time of diagnosis, and 10 patients (15.2%) were with recurred disease. A majority of patients were non-smokers , and 16 patients (24.2%) were current or former smokers. Regarding the baseline EGFR mutation status, 33 patients (50.0%) harbored the exon 19 deletion and 33 patients (50.0%) had the exon 21 L858R point mutation . A majority of patients were treated with first-line EGFR-TKIs  and two patients received afatinib treatment. As for the metastasis status, there were 22 patients (33.3%) with brain metastases, 24 patients (36.4%) with bone metastases, and four patients with liver metastases at diagnosis. Among all patients, 38 patients received the EGFR T790M test at progression; 22 of these patients harbored an EGFR T790M mutation when they became resistant to first-line EGFR-TKIs.The demographic and clinical characteristics are demonstrated in EGFR mutation status, type of EGFR-TKI treatment received, metastasis status or T790M mutation at progression between the low sPD-L1 expression group and high sPD-L1 expression group.The patient cohort was divided into two groups based on the level of sPD-L1 before treatment had been initiated. There were no significant differences in gender, age, histological status, ECOG PS status, stage, smoking status, p = 0.026). Meanwhile, a higher on-treatment sPD-L1 level was also associated with a poor response to EGFR-TKIs. The ORR was 73.9% in patients with low on-treatment sPD-L1 levels, but the ORR was only 43.5% in patients with high on-treatment sPD-L1 levels. There were no differences in the treatment response between patients with or without a reduction of sPD-L1 levels. Other clinical characteristics, including gender, age, ECOG PS score, tumor stage, smoking status, EGFR status, and type of EGFR-TKI received were not associated with the therapeutic response.Clinical characteristics of patients and distributions of sPD-L1 concentrations according to the therapeutic response to EGFR-TKIs are listed in We next compared both the pre-treatment and on-treatment sPD-L1 concentrations in patients who achieved a PR and patients who had a best response of stable disease (SD) or progressive disease (PD). The PR group demonstrated significantly lower levels of pre-treatment plasma sPD-L1 compared with the SD+PD group. As for the on-treatment plasma sPD-L1 levels, although the finding was marginally significant, the PR group also showed a lower level of sPD-L1. In whole patient group and subgroups divided by treatment response, the levels of sPD-L1 were not significantly changed by EGFR-TKIs treatment .p-value was 0.005. Although it was not statistically significant, a shorter PFS rate was also observed in patients with higher on-treatment sPD-L1 concentrations . The change in sPD-L1 levels was not correlated with the PFS rate of patients treated with EGFR-TKIs, however.The median progression-free survival (PFS) in the whole patient group was 12.5 months (95% CI: 9.7–15.2 months). As demonstrated in EGFR driver mutation type, brain metastasis status, sPD-L1 concentration, and T790M status at progression were included in the Cox regression analysis. A worse outcome for patients with high sPD-L1 levels before EGFR-TKI treatment was also found for the PFS rate in the Cox regression model with an HR of 2.56 . No clinicopathological factors were associated with the PFS rate .To further evaluate the potential impact of clinical variables on the therapeutic efficacy of treatment with first-line EGFR-TKIs, we performed both univariate and multivariate analyses on the whole patient cohort. Typical factors of age, sex, smoking history, stage, PFS rate , but theEGFR-mutant NSCLC patients with low plasma sPD-L1 levels than in those with high sPD-L1 levels. Furthermore, a prolonged PFS rate was significantly associated with a lower pre-treatment sPD-L1 level. Our results suggested that the plasma PD-L1 concentration could be a promising marker for determining the efficacy of EGFR-TKIs for NSCLC patients harboring EGFR mutations.A growing number of studies have demonstrated that sPD-L1 might play a crucial role in the prediction of the treatment response of PD-1/PD-L1 inhibitors and also the prognosis of cancer patients , 21, 24.The underlying mechanisms of generation and regulation of the soluble forms of PD-L1 are still unclear. One possible source is spliced variant. Zhou et al. showed that alternative splicing of PD-L1 occurred in all melanoma cell lines and splice variants could result in the secretion of sPD-L1 . BesidesEGFR-mutant lung adenocarcinoma patients, PD-L1 represented a favorable biomarker for the response to EGFR-TKIs and outcomes of these patients  for the publication of any potentially identifiable images or data included in this article.YJ, XL, and CZho designed this study and drafted the manuscript. YJ, XL, CZha, FZ, and JL reviewed the patient record, collected patient samples, and conducted the relevant experiments. YJ, CZha, GG, and WL performed the statistical analyses. SR and CS provided critical comments and revised the manuscript. All authors read and approved the final version of the manuscript.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.


BC from wood burning (BCwb) was significantly higher at non-EJ communities. Correlation analysis indicated that the six HAPs were predominantly from fossil fuel combustion sources, not from wood burning. The HAPs were moderately variable across sites (coefficient of divergence (COD) range of 0.07 for carbon tetrachloride to 0.28 for m- and p-xylenes), while BCff and BCwb were highly variable . The BCwb was well correlated with levoglucosan (R2 of 0.68 to 0.95), indicating that BCwb was a robust indicator for wood burning. At the two permanent monitoring sites, wood burning comprised 29–39% of the fine particulate matter (PM2.5) on nights when PM2.5 concentrations were forecasted to be high. Phone survey data were consistent with study measurements; the only significant difference in the survey results among communities were that non-EJ residents burn with indoor devices more often than EJ residents.Ambient air monitoring and phone survey data were collected in three environmental justice (EJ) and three non-EJ communities in Sacramento County during winter 2016–2017 to understand the differences in air toxics and in wood smoke pollution among communities. Concentrations of six hazardous air pollutants (HAPs) and black carbon (BC) from fossil fuel (BC The resultant high-concentration PM2.5 events over the course of an evening can have acute health impacts [Wood smoke from residential burning is the largest source of wintertime particulate matter with aerodynamic diameter less than 2.5 microns , secondarily formed organic mass, and a wide range of gaseous species ,9,10,11.wb) and from fossil fuel combustion (BCff) [While there are thousands of chemicals emitted from residential biomass burning, unique chemicals are emitted from the combustion of wood lignin, namely, levoglucosan and other anhydrous sugars; therefore, levoglucosan was used as the main tracer for wood burning emissions. Levoglucosan is emitted during combustion of wood cellulose , and itsn (BCff) ; this men (BCff) ,27,28,292.5 and cancer risk. This study focused on three such communities—South Natomas, Arden, and South Sacramento—and paired them with nearby non-EJ communities—T Street, Del Paso Manor, and Colonial Heights. Since wood smoke from residential burning is the largest single source of wintertime PM2.5 in Sacramento, and is typically due to local rather than urban- or regional-scale sources, we investigated the levels of PM2.5, hazardous air pollutants (HAPs), and wood smoke contributions to HAPs in these EJ and non-EJ communities, as well as the burning behavior of residents in these communities.Pollution can be higher in environmental justice (EJ) areas which are typically lower-income areas with a higher proportion of minority residents compared to non-EJ areas ,31,32,332.5 was used as the environmental index which in EJ Screen is a fusion of annual average monitored data and modelled data. The PM2.5 concentration in each census block is estimated by the EPA’s Office of Research and Development using a Bayesian space–time downscaling fusion model approach [Ambient air was monitored in three EJ and three non-EJ communities. The EJ communities were determined using the EPA’s EJ Screen tool . In EJ Sapproach ,35. Fina2.5 in the highest 10% nationally as EJ locations, and those in the lowest 50% EJ Index as non-EJ locations; see 2.5. The nearby community (within 5 miles) of Colonial Heights was selected as the non-EJ community to pair with the South Sacramento community. Sacramento Metropolitan Air Quality Management District (SMAQMD) then conducted public outreach through local community groups, neighborhood associations, and its Board Members and staff in a search of volunteers to host monitoring equipment.This results in a rank of the EJ Index for each US census block relative to all other census blocks. A higher EJ index indicates more potential for exposure/risk/proximity to certain facilities and/or a higher percentage minority population. Here, we identified communities with an EJ Index for PMThe HAPs and BC were measured at one site in each of the six communities. Quartz fiber filter samples for quantifying levoglucosan were collected at Del Paso Manor, T Street, and South Sacramento. PM was measured at all sites using low-cost sensors; details can be found in Mukherjee et al. . Details2.5 concentrations would be high on a particular day. Daytime (06:00 or 18:00) and nighttime (18:00 to 06:00) samples were taken, since mobile source emissions are relatively higher in daytime, and residential wood burning emissions are higher in nighttime [The HAPs were collected at a site in each community using five-liter Summa stainless-steel canisters at each site. Samples were collected for four sequential 12 h durations starting at either 06:00 or 18:00 PST on days when forecasts indicated that PMighttime ,26,37. T2.5, and BC data to assess whether there were any covariates that would indicate similar emissions sources.Canisters were shipped to Eastern Research Group (ERG) for analysis by EPA Method TO-15  using a Quartz fiber filters  were collected at Del Paso Manor, T Street, and a South Sacramento site on a forecast basis, generally at the same time as the nighttime HAP samples . Filters were analyzed for organic carbon (OC), elemental carbon (EC), and levoglucosan. Two daytime (06:00 to 18:00 PST) filters were also collected at each of these three sites during January 2017 to evaluate the difference between daytime and nighttime wood smoke concentrations, since residential wood smoke emissions generally occur in the evening when ambient temperatures begin to decrease. At the Del Paso Manor and T Street sites, Thermo Scientific 2025i sequential filtration samplers were used, with a sampling rate of 16.7 L/min. At the South Sacramento site, an AirMetrics mini-vol sampler was used, with a sampling rate of 5 L/min. Sample flow was evaluated before and after each sampling event. Five collocated filters were collected at South Sacramento to evaluate precision. Prior to sampling, filters were pre-baked for 12 h at 500 °C to remove residual organic compounds.Levoglucosan was determined using a Dionex DX-500 series ion chromatograph with detection via an ED-50/ED-50A electrochemical cell. This cell includes two electrodes: a pH-Ag/AgCl (silver/silver chloride) reference electrode and a “standard” gold working electrode. For the separation, a sodium hydroxide gradient and a Dionex CarboPac PA-1 column (4 × 250 mm) were used. The complete run time was 59 min with an injection volume of 100 µL. More details on the method can be found in Sullivan et al. ,44,45.wb and BCff. This method assumes that there are no other sources of BC such as coal dust. In the Sacramento emissions inventory, there is little to no impact from coal combustion emissions, so mobile sources and wood burning emissions are the main sources of BC.Magee Scientific model AE33 Aethalometers were deployed at a site in each community to measure BC. The AE33 collects ambient aerosol onto a filter tape, and then every five minutes measures the absorbance of the material deposited on the tape at 7 wavelengths, ranging from 370 to 960 nm. The BC concentration is from the 880 nm channel. While BC absorbs uniformly across all wavelengths at which absorbance is measured, brown carbon, which is produced by combustion of wood or other biomass, absorbs primarily in the lower wavelengths of the measured range ,47. A cowb and BCff, the model described by Sandradewi et al. [wb and BCff are then calculated by multiplying the BC value from the 880 nm channel by the percentages obtained in the previous step. The calculation is based on a model in which the total BC can be divided into pure black and pure brown carbon using the spectral dependence of the absorbing properties of each material. The spectral dependence is described by the Angstrom exponent (α). From the Beer–Lambert Law, the following equations can be obtained: abs(λ)x is the absorption coefficient for the BC type x (wb or ff) at wavelength λ (470 or 950).To calculate BCi et al.  and summi et al.  was usedwb measurements were compared to levoglucosan to assess the quality of the BCwb calculation.The Sandradewi method requires input values for the Angstrom exponents for pure black and pure brown carbon. For this study, an Angstrom component of 1 for the pure black carbon and a value of 2 for the pure brown carbon were used. These are the default values used by the Aethalometer ,49 and h2.5 mass and meteorological data were obtained from EPA’s air quality system (AQS) for the pre-existing routine monitoring sites at Del Paso Manor and T Street. At both sites, a MetOne beta attenuation monitor (BAM) 1020 collected hourly PM2.5 data, and an R&P 2025 sequential filter sampler was operated as the 24 h Federal Reference Method (FRM) for PM2.5 mass. The FRM filters were collected daily, and PM2.5 mass was determined by gravimetric analysis. Temperature, RH, wind speed, and wind direction data were collected at both sites using R.M. Young instrumentation. Dew point was calculated from temperature and RH measurements. Instrumentation and operations at these sites followed national guidance set by EPA on operations and methods. Wind and temperature data were used for the analysis of BC data. Each site with Aethalometer and canister measurements was assigned meteorological data from the weather station closest to that monitoring site. The Arden and Del Paso Manor communities were closest to the Del Paso Manor weather station, while the T Street, South Natomas, South Sacramento, and Colonial Heights communities were closest to the T Street weather station.Hourly PMp-value of a Student’s t-test is less than 0.05, i.e., significant at a 95% confidence level.Statistical methods used here included conditional bivariate probability function plots ,52, notcff, and BCwb existed across communities, a Kruskal–Wallis rank sum test was performed on HAP concentrations among the communities. The Kruskal–Wallis test was selected to assess how similar the distribution of data was among sites. Post-hoc Nemenyi tests were applied for pairwise comparisons of BC data between each community to determine which communities’ measurements differed significantly from each other; this test is useful for determining whether multiple groups of data are similar or not when there is a large amount of data [To determine whether significant differences in measured concentrations of BC, BC of data .m is the number of samples with a concentration greater than the specified percentile, n is the total number of samples, θ is a wind direction sector, and j is the wind speed interval, the following equation is used to determine CBPF.To identify potential sources of black carbon, a conditional bivariate probability function (CBPF) analysis  was usedThe CBPF is able to determine whether certain combinations of wind speed and wind direction have a high probability of producing concentrations above the specified percentile of data. By providing a greater degree of granularity to the analysis, CBPF facilitates the identification of sources that might not be detected by traditional CPF and can also be used to evaluate the characteristics of a source.wb, and BCff concentrations based on the 80th percentile of the concentrations of each pollutant. The calculations, including the 80th percentile threshold, were performed individually for each site, and radial plots displaying the probability of exceeding the 80th percentile relative to wind speed and direction were created.The polarPlot function available in the openair R package ,54 was uwb) and fossil fuel  combustion [x- and y-axis, e.g., benzene/2,2,4-trimethylpentane versus toluene/2,2,4-trimethylpentane. We examined all possible combinations of the 12 h HAPs relative to the wood smoke indicator BCwb or the mobile source indicator 2,2,4-trimethylpentane to determine under which indicator species the HAPs varied the most. Differences in how ratios change depending on the indicator species in the denominator can indicate which source  most affects the species in the numerator [Ratio:ratio plots were used to assess how individual species relationships changed as a function of known tracers of wood smoke  assess wood burning behavior; (2) evaluate wood burning activity by type of device used to burn; and (3) compare wood burning activity between EJ and non-EJ communities. Telephone interviews were completed with 900  Sacramento County residents who owned a wood or pellet burning device (other than an outdoor barbecue) either inside or outside their home. Addresses in each community were matched with listed landline and mobile phone numbers, then selected at random for interviewing.The margin of error associated with a sample of 900 completed interviews is ±3.3% at the 95% confidence level. That is, there is a 95% chance that the true population parameters lie within 3.3% of the sample statistics. For example, if a response category to a question was chosen by 50% of sample respondents, there is a 95% chance that if the entire county population were surveyed, that same response category would be selected by 46.7–53.3% of all residents (50% ± 3.3%). For the EJ communities, with 444 completed interviews, the margin of error at the 95% confidence level is ±4.7%, while that for the non-EJ communities, with 456 completed interviews, is ±4.6%.Most of the questions were asked in a closed-ended format which were categorized for quantitative analysis; responses to open-ended questions were not used in quantitative analysis. Interviews took approximately 8 min on average to administer. Respondents were screened for age (18+) and ownership of a wood or pellet burning device, and to confirm residency in Sacramento County. Interviewing took place from 2 December through 19 December 2016, and from 6 January through 22 January 2017. Surveys were not conducted on holidays because response rates are typically lower on those days. A full list of the survey questions is provided in the The precision of HAP measurements was determined using ten collocated  samples at T Street. Precision was calculated using the following formula, where A and B are the concentrations from the two collocated samples.R2 value of the collocated measurements when plotted on a scatter plot was 0.99, indicating high precision.The National Air Toxics Trends Station’s (NATTS) goal for high-precision data is ±15% . As showwb compared to PM2.5 from wood burning estimated from levoglucosan measurements. If BCwb has a strong relationship with levoglucosan, it can be used to determine wood burning PM concentrations from Aethalometer measurements. Levoglucosan was compared to BCwb concentrations when the filters were collected as shown in wb (R2 = 0.95). There were moderate correlations between BCwb and levoglucosan concentrations at the South Sacramento (R2 = 0.68) and T Street (R2 = 0.80) sites.Levoglucosan is a unique tracer for wood burning. It is typically collected via multi-hour filter measurements, so it cannot be measured continuously; significant labor and equipment are required to collect and chemically analyze the filters. The Aethalometer provides a calculated value of wood burning on an hourly basis and can run with little maintenance at multiple sites but requires validation that the wood burning calculation is correct. With both filter and Aethalometer measurements collected at the Del Paso Manor, South Sacramento, and T Street sites, we assessed how Aethalometer BC2.5 at the three sites where filters were collected. Filter results are summarized by site in 2.5 from wood burning was also calculated using hourly PM2.5 measurements. Median levoglucosan concentrations across sites ranged from 0.86 μg/m3 at T Street to 1.84 μg/m3 at Del Paso Manor; the highest measured levoglucosan concentration was 5.8 μg/m3 at Del Paso Manor on the night of 19 December, when PM2.5 averaged 69 μg/m3 during the 12 h of sampling. Overall, levoglucosan and wood burning PM concentrations were highest at the Del Paso Manor site; concentrations at T Street and South Sacramento sites were similar. At the Del Paso Manor site, 39% of the PM2.5  during the filter sampling periods was from wood burning. At the T Street site, wood burning accounted for 29% of the PM concentrations . At the South Sacramento site, the median concentration of wood burning PM2.5 was similar to that at the T Street site (6.8 μg/m3 at the South Sacramento site and 6.3 μg/m3 at the T Street site), and about half of the median concentration at the Del Paso Manor (13.5 μg/m3) site. These results suggest that wood burning emissions are higher around the Del Paso Manor site than around the other sites but are still responsible for a significant amount of the PM2.5 at T Street and South Sacramento.Levoglucosan was used to estimate wood burning PM2.5 is well distributed across these three sites during the daytime and is elevated around Del Paso Manor during the nighttime.Nighttime concentrations were compared to two daytime samples collected at each site. As discussed later in the results of the phone survey, residential burning occurs predominantly in the evening or nighttime, not during the day, so levoglucosan found during the daytime is likely carried over from the prior night. On 28 and 29 January 2017, samples were collected at 06:00 and 18:00 PST at each site to provide a rough comparison of daytime concentrations to nighttime concentrations. p < 0.05) at EJ communities compared to non-EJ communities. For example, benzene concentrations were 0.65 µg/m3 in EJ communities and 0.58 µg/m3 in non-EJ communities, on average. There was no significant difference between individual EJ/non-EJ site pairs for any species  when comparing individual site locations. The HAPs that were significantly higher in EJ communities were typically from mobile sources, including 2,2,4-trimethylpentane, an indicator for mobile sources [2.5 in the highest (worst) decile of the entire United States, predominantly due to the fact of mobile source emissions.Concentrations of 2,2,4-trimethylpentane, acrolein, benzene, ethylbenzene, m- and p-xylenes, and toluene were significantly higher , a very long-lived HAP that is no longer emitted in the US, were examined. It was phased out as part of the Montreal Protocol to ban chlorofluorocarbons (CFCs) to repair the ozone hole; because it has a very long residence time in the atmosphere, it is very homogeneous, and concentrations should be approximately 0.085 ppb everywhere in the United States [First, the day/night trends of carbon tetrachloride  rather than consistent robust differences in emissions on Wednesday and Thursday compared to other days.In Sacramento, the T Street site has a typical urban diurnal pattern with elevated morning BC, while the other sites have a nighttime peak in BC that is higher than the BC morning peak. For all sites, BCwb, and BCff measurements were combined with meteorological data to try to identify the likely source of these pollutants. The CBPF was used to identify wind speeds and directions associated with the highest quintile of concentrations. For all sites in this study, the highest quintile of concentrations of BC, BCff, and BCwb occurred at very low wind speeds associated with stable and stagnant conditions . This finding indicates that wood burning had little relation to HAPs at any site. As expected, carbon tetrachloride had little correlation with any other HAP (R2 < 0.12), since its concentration levels are representative of global background concentrations. The HAPs that are typically from fossil fuel combustion  had high correlations among themselves at every site (R2 > 0.70). This means that all of these species were from the same type of source, i.e., mobile source emissions.The association of individual HAPs with BCmbustion ,74,75,762.5. The relative importance of the two sources can be found by plotting the ratio of each species relative to tracers of wood smoke (represented by BCwb) or fossil fuel combustion . High correlation in a ratio:ratio plot indicates that there is little impact from the applied tracer on the species in question, whereas low correlation indicates a high effect from the tracer. Examples are given in wb, the HAPs were highly correlated on the ratio:ratio plots, but when divided by 2,2,4-trimethylpentane, the results clustered together tightly. These relationships indicate that a singular source of emissions dominated the species shown in the figures. Similar results were seen for most of the hydrocarbons . However, the oxygenated, nitrogenated, and chlorinated compounds, such as acetonitrile and acrolein, did not display this characteristic relationship when divided by either BCwb or 2,2,4-trimethylpentane, indicating that these compounds were not associated with BCwb or 2,2,4-trimethylpentane or were not measured due to the difficulty in measuring such compounds via canisters.Ratio:ratio plots were used to help understand the relative contribution of wood smoke and fossil fuel combustion to HAPs, OC, EC, and PMOverall, the survey results show no significant differences between EJ and non-EJ communities in the type of device owned, fraction of homes burning with fireplaces, fraction of homes burning day and night, fraction of homes burning only at night, or fraction of homes with EPA-certified burning devices. The only significant differences are that homes in non-EJ communities burn with an indoor device more often than homes in EJ communities do and, thus, have a higher relative number of burn days than EJ communities. Specifically, the most commonly owned device was an indoor fireplace, owned by 79–80% of all respondents in non-EJ and EJ communities; other devices, such as pellet stoves, accounted for less than 10% of responses. Those respondents who do burn with any device do so between one and two days per week. This frequency increases with the more devices a respondent owns. Of all respondents owning an indoor fireplace, those in non-EJ communities burn significantly more than those in EJ communities . Among respondents who burn with fireplaces, those in non-EJ communities are burning significantly more often than respondents in EJ communities .wb results. Non-EJ communities have more burning device usage than EJ communities and, thus, a significantly higher relative number of burn days. This difference in the number of burn days likely means there are more emissions from wood burning in non-EJ communities which corresponds with the significantly higher observed BCwb in non-EJ communities. However, HAPs have an opposite relationship; for example, in the EJ communities where less wood burning occurs than in non-EJ communities, concentrations of multiple HAPs are higher. These results support all other analyses of the ambient data showing that wood burning has little impact on ambient HAP concentrations.ff were significantly higher at EJ communities than at non-EJ communities. Even though wood smoke emissions can contribute to HAPs, results from this study consistently showed that wood burning has little influence on the ambient levels of HAPs and that fossil fuel combustion was the main source of HAPs. The BCwb had a very high correlation with collocated measurements of the wood burning tracer levoglucosan (R2 across three sites of 0.68 to 0.95), on the high end of reported literature values, indicating that BCwb was a robust indicator for wood burning in Sacramento. Thus, Aethalometer BCwb data could be used from all sites to assess wood burning contributions. Wood burning accounted for 29–39% of the nighttime PM2.5 at the two sites with hourly PM2.5 and filter measurements (T Street and Del Paso Manor). The BCwb concentrations were significantly higher at non-EJ communities than at EJ communities. As shown in the phone survey results, the higher BCwb concentrations in non-EJ communities were mostly likely due to the more burning with indoor fireplaces in the non-EJ communities. Wood burning was not a significant source of HAPs. The HAPs and BCff were higher in EJ communities compared to non-EJ communities, indicating mobile source emissions were likely higher in EJ communities.In this first detailed community monitoring study during the winter in Sacramento County, we found that concentrations of six HAPs  and BC